Pathology_Robbins

Pathology_Robbins. Plasma Membrane: Protection and Nutrient Acquisition Biosynthetic Machinery: Endoplasmic Reticulum and Golgi Apparatus Waste Disposal: Lysosomes and Proteasomes Modular Signaling Proteins, Hubs, and Components of the Extracellular Matrix Proliferation and the Cell Cycle Pathology literally translates to the study of suffering (Greek pathos = suffering, logos = study); as applied to modern medicine, it is the study of disease. Virchow was certainly correct in asserting that disease originates at the cellular level, but we now realize that cellular disturbances arise from alterations in molecules (genes, proteins, and others) that influence the survival and behavior of cells. Thus, the foundation of modern pathology is understanding the cellular and molecular abnormalities that give rise to diseases. It is helpful to consider these abnormalities in the context of normal cellular structure and function, which is the theme of this introductory chapter.


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Pathology_Robbins. It is unrealistic (and even undesirable) to condense the vast and fascinating field of cell biology into a single chapter. Consequently, rather than attempting a comprehensive review, the goal here is to survey basic principles and highlight recent advances that are relevant to the mechanisms of disease that are emphasized throughout the rest of the book. The sequencing of the human genome at the beginning of the 21st century represented a landmark achievement of biomedical science. Since then, the rapidly dropping cost of sequencing and the computational capacity to analyze vast amounts of data promise to revolutionize our understanding of health and disease. At the same time, the emerging information has also revealed a breathtaking level of complexity far beyond the linear sequencing of the genome. The potential for these new powerful tools to expand our understanding of pathogenesis and drive therapeutic innovation excites and inspires scientists and the lay public alike.


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Pathology_Robbins. The human genome contains about 3.2 billion DNA base pairs. Yet, within the genome there are only roughly 20,000 protein-encoding genes, comprising just 1.5% of the genome. The proteins encoded by these genes are the fundamental constituents of cells, functioning as enzymes, structural elements, and signaling molecules. Although 20,000 underestimates the actual number of proteins encoded (many genes produce multiple RNA transcripts that encode distinct protein isoforms), it is nevertheless startling that worms composed of fewer than 1000 cells— and with genomes 30-fold smaller—are also assembled from roughly 20,000 protein-encoding genes. Perhaps even more unsettling is that many of these proteins are recognizable homologs of molecules expressed in humans. What then separates humans from worms?


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Pathology_Robbins. The answer is not completely known, but evidence supports the assertion that the difference lies in the 98.5% of the human genome that does not encode proteins. The function of such long stretches of DNA (which has been called the “dark matter” of the genome) was mysterious for many years. However, it is now clear that more than 85% of the human genome is ultimately transcribed, with almost 80% being devoted to the regulation of gene expression. It follows that whereas proteins provide the building blocks http://ebooksmedicine.net Euchromatin Nucleus (dense, inactive) (disperse, active)


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Pathology_Robbins. Fig. 1.1 The organization of nuclear DNA. At the light microscopic level, the nuclear genetic material is organized into dispersed, transcriptionally active euchromatin or densely packed, transcriptionally inactive heterochromatin; chromatin can also be mechanically connected with the nuclear membrane, and nuclear membrane perturbation can thus influence transcription. Chromosomes (as shown) can only be visualized by light microscopy during cell division. During mitosis, they are organized into paired chromatids connected at centromeres; the centromeres act as the locus for the formation of a kinetochore protein complex that regulates chromosome segregation at metaphase. The telomeres are repetitive nucleotide sequences that cap the termini of chromatids and permit repeated chromosomal replication without loss of DNA at the chromosome ends. The chromatids are organized into short “P” (“petite”) and long “Q” (“next letter in the


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Pathology_Robbins. alphabet”) arms. The characteristic banding pattern of chromatids has been attributed to relative GC content (less GC content in bands relative to interbands), with genes tending to localize to interband regions. Individual chromatin fibers are composed of a string of nucleosomes—DNA wound around octameric histone cores—with the nucleosomes connected via DNA linkers. Promoters are noncoding regions of DNA that initiate gene transcription; they are on the same strand and upstream of their associated gene. Enhancers are regulatory elements that can modulate gene expression across distances of 100 kB or more by looping back onto promoters and recruiting additional factors that are needed to drive the expression of pre-mRNA species. The intronic sequences are subsequently spliced out of the pre-mRNA to produce the definitive message that includes exons that are translated into protein and 3′- and 5′-untranslated regions (UTR) that may have regulatory functions. In addition to the enhancer


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Pathology_Robbins. re-mRNA to produce the definitive message that includes exons that are translated into protein and 3′- and 5′-untranslated regions (UTR) that may have regulatory functions. In addition to the enhancer, promoter, and UTR sequences, noncoding elements are found throughout the genome; these include short repeats, regulatory factor binding regions, noncoding regulatory RNAs, and transposons.


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Pathology_Robbins. and machinery required for assembling cells, tissues, and organisms, it is the noncoding regions of the genome that provide the critical “architectural planning.” The major classes of functional non–protein-coding DNA sequences found in the human genome include ( Fig. 1.1 Noncoding regulatory RNAs. Of the 80% of the genome dedicated to regulatory functions, the vast majority is transcribed into RNAs—micro-RNAs and long noncoding RNAs (described later)—that are never translated into protein, but can regulate gene expression Mobile genetic elements (e.g., transposons). Remarkably, more than one-third of the human genome is composed of such “jumping genes.” These segments can cruise around the genome, and are implicated in gene regulation and chromatin organization. Special structural regions of DNA, including telomeres (chromosome ends) and centromeres (chromosome “tethers”)


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Pathology_Robbins. Special structural regions of DNA, including telomeres (chromosome ends) and centromeres (chromosome “tethers”) Importantly, many genetic variations (polymorphisms) associated with diseases are located in non–proteincoding regions of the genome. Thus, variation in gene regulation may prove to be more important in disease causation than structural changes in specific proteins. Another surprise that emerged from genome sequencing is that any two humans are typically >99.5% DNA-identical (and are 99% sequence-identical with chimpanzees)! Thus, individual variation, including differential susceptibility to diseases and environmental exposures, is encoded in <0.5% of our DNA (importantly, this still represents about 15 million base pairs). The two most common forms of DNA variation in the human genome are single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs).


Pathology_Robbins

Pathology_Robbins. The two most common forms of DNA variation in the human genome are single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). SNPs are variants at single nucleotide positions and are almost always biallelic (only two choices exist at a given site within the population, such as A or T). More than 6 million human SNPs have been identified, with many showing wide variation in frequency in different populations. The following features are worthy of note: SNPs occur across the genome—within exons, introns, intergenic regions, and coding regions. Roughly 1% of SNPs occur in coding regions, which is about what would be expected by chance, because coding regions comprise about 1.5% of the genome. SNPs located in noncoding regions can occur in regulatory elements in the genome, thereby altering gene expression; in such instances the SNP may have a direct influence on disease susceptibility.


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Pathology_Robbins. SNPs located in noncoding regions can occur in regulatory elements in the genome, thereby altering gene expression; in such instances the SNP may have a direct influence on disease susceptibility. SNPs can also be “neutral” variants with no effect on gene function or carrier phenotype. Even “neutral” SNPs may be useful markers if they happen to be coinherited with a disease-associated gene as a result of physical proximity. In other words, the SNP and the causative genetic factor are in linkage disequilibrium. The effect of most SNPs on disease susceptibility is weak, and it remains to be seen if the identification of such variants, alone or in combination, can be used to develop effective strategies for disease prediction or prevention.


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Pathology_Robbins. CNVs are a form of genetic variation consisting of different numbers of large contiguous stretches of DNA; these can range from 1000 base pairs to millions of base pairs. In some instances these loci are, like SNPs, biallelic and simply duplicated or deleted in a subset of the population. In other instances there are complex rearrangements of genomic material, with multiple alleles in the human population. CNVs are responsible for several million base pairs of sequence difference between any two individuals. Approximately 50% of CNVs involve gene-coding sequences; thus, CNVs may underlie a large portion of human phenotypic diversity.


Pathology_Robbins

Pathology_Robbins. It is important to note that alterations in DNA sequence cannot by themselves explain the diversity of phenotypes in human populations; moreover, classical genetic inheritance cannot explain differing phenotypes in monozygotic twins. The answers to these conundrums probably lie in epigenetics—heritable changes in gene expression that are not caused by alterations in DNA sequence (see later). Even though virtually all cells in the body have the same genetic composition, differentiated cells have distinct structures and functions arising through lineage-specific programs of gene expression. Such cell type–specific differences in DNA transcription and translation are regulated by epigenetic modifications that consist of several changes that profoundly influence gene expression, including: Fig. 1.2 ). Genomic DNA is packed into nucleosomes, which are composed of 147 DNA (1.8 turns, about 150 base pairs)


Pathology_Robbins

Pathology_Robbins. Fig. 1.2 Chromatin organization. (A) Nucleosomes are comprised of octamers of histone proteins (two each of histone subunits H2A, H2B, H3, and H4) encircled by 1.8 loops of 147 base pairs of DNA; histone H1 sits on the 20 to 80 nucleotide linker DNA between nucleosomes and helps stabilize the overall chromatin architecture. The histone subunits are positively charged, thus allowing the compaction of the negatively charged DNA. (B) The relative state of DNA unwinding (and thus access for transcription factors) is regulated by histone modification, for example, by acetylation, methylation, and/or phosphorylation (so-called “marks”); marks are dynamically written and erased. Certain marks such as histone acetylation “open up” the chromatin structure, whereas others, such as methylation of particular histone residues, tend to condense the DNA and lead to gene silencing. DNA itself can also be also be methylated, a modification that is associated with transcriptional inactivation.


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Pathology_Robbins. base pair DNA segments wrapped around a central core of proteins called histones. Nucleosomes resemble beads joined by short DNA linkers; the entire structure is generically called chromatin. Importantly, the winding and compaction of chromatin in any given cell varies in different genomic regions. Thus, nuclear chromatin exists in two basic forms (visualizable by standard histology): (1) histochemically dense and transcriptionally inactive heterochromatin and (2) histochemically dispersed and transcriptionally active euchromatin. Because only euchromatin permits gene expression and thereby dictates cellular identity and activity, there are a host of mechanisms that tightly regulate the state of chromatin (described below).


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Pathology_Robbins. DNA methylation. High levels of DNA methylation in gene regulatory elements typically result in chromatin condensation and transcriptional silencing. Like histone modifications (see later), DNA methylation is tightly regulated by methyltransferases, demethylating enzymes, and methylated-DNA-binding proteins. Histone modifying factors. Nucleosomes are highly dynamic structures regulated by an array of nuclear proteins and post-translational modifications: Chromatin remodeling complexes can reposition nucleosomes on DNA, exposing (or obscuring) gene regulatory elements such as promoters.


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Pathology_Robbins. Chromatin remodeling complexes can reposition nucleosomes on DNA, exposing (or obscuring) gene regulatory elements such as promoters. writer” complexes carry out more than 70 different covalent histone modifications generically denoted as marks. These include methylation, acetylation, and phosphorylation of specific histone amino acid residues: Histone methylation of lysines and arginines is accomplished by specific writer enzymes; methylation of histone lysine residues can lead to transcriptional activation or repression, depending on which histone residue is “marked.” Histone acetylation of lysine residues (occurring through histone acetyl transferases) tends to open up chromatin and increase transcription; histone deacetylases (HDAC) reverse this process, leading to chromatin condensation. Histone phosphorylation of serine residues can variably open or condense chromatin, to increase or decrease transcription, respectively.


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Pathology_Robbins. Histone marks are reversible through the activity of “chromatin erasers.” Other proteins function as “chromatin readers,” binding histones that bear particular marks and thereby regulating gene expression. The mechanisms involved in the cell-specific epigenetic regulation of genomic organization and gene expression are undeniably complex. Despite the intricacies, learning to manipulate these processes will likely bear significant therapeutic benefits because many diseases are associated with inherited or acquired epigenetic alterations, and dysregulation of the “epigenome” has a central role in the genesis of benign and malignant neoplasms (Chapter 6). Moreover—unlike genetic changes—epigenetic alterations (e.g., histone acetylation and DNA methylation) are readily reversible and are therefore amenable to intervention; indeed, HDAC inhibitors and DNA methylation inhibitors are already being used in the treatment of various forms of cancer.


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Pathology_Robbins. Another mechanism of gene regulation depends on the functions of noncoding RNAs. As the name implies, these are encoded by genes that are transcribed but not translated. Although many distinct families of noncoding RNAs exist, only two examples are discussed here: small RNA molecules called microRNAs and long noncoding RNAs >200 nucleotides in length. • Micro-RNAs (miRNAs) are relatively short RNAs (22 nucleotides on average) that function primarily to modulate the translation of target mRNAs into their corresponding proteins. Posttranscriptional silencing of gene expression by miRNA is a fundamental and evolutionarily conserved mechanism of gene regulation present in all eukaryotes (plants and animals). Even bacteria have a primitive version of the same general machinery that they use to protect themselves against foreign DNA (e.g., from phages and viruses).


Pathology_Robbins

Pathology_Robbins. Even bacteria have a primitive version of the same general machinery that they use to protect themselves against foreign DNA (e.g., from phages and viruses). The human genome contains almost 6000 miRNA genes, only 3.5-fold less than the number of protein-coding genes. Moreover, individual miRNAs appear to regulate multiple protein-coding genes, allowing each miRNA to coregulate entire programs of gene expression. Transcription of miRNA genes produces a primary transcript (pri-miRNA) that is processed into progressively smaller segments, including trimming by the enzyme Dicer. This generates mature single-stranded miRNAs of 21 to 30 nucleotides that associate with a multiprotein aggregate called RNA-induced silencing complex (RISC; Fig. 1.3 ). Subsequent base pairing between the miRNA strand and its target mRNA directs the RISC to either induce mRNA cleavage or to repress its translation. In this way, the target mRNA is posttranscriptionally silenced.


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Pathology_Robbins. Taking advantage of the same pathway, small interfering RNAs (siRNAs) are short RNA sequences that can be introduced into cells. These serve as substrates for Dicer and interact with the RISC complex in a manner analogous to endogenous miRNAs. Synthetic siRNAs that can target specific mRNA species are therefore powerful laboratory tools to study gene function (so-called knockdown technology); they also are promising as therapeutic agents to silence pathogenic genes, e.g., oncogenes involved in neoplastic transformation. Long noncoding RNA (lncRNA). The human genome also contains a very large number of lncRNAs—at least 30,000, with the total number potentially exceeding coding mRNAs by 10-to 20-fold. lncRNAs modulate gene expression in many ways (


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Pathology_Robbins. Fig. 1.4 ); for example, they can bind to regions of chromatin, restricting RNA polymerase access to coding genes within the region. The best-known example of a repressive function involves XIST, which is transcribed from the X chromosome and plays an essential role in physiologic X chromosome inactivation. XIST itself escapes X inactivation, but forms a repressive “cloak” on the X chromosome from which it is transcribed, resulting in gene silencing. Conversely, it has been appreciated that many enhancers are sites of lncRNA synthesis, with the lncRNAs expanding transcription from gene promoters through http://ebooksmedicine.net Unwinding of miRNA duplex


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Pathology_Robbins. Unwinding of miRNA duplex Fig. 1.3 Generation of microRNAs (miRNA) and their mode of action in regulating gene function. miRNA genes are transcribed to produce a primary miRNA (pri-miRNA), which is processed within the nucleus to form premiRNA composed of a single RNA strand with secondary hairpin loop structures that form stretches of double-stranded RNA. After this premiRNA is exported out of the nucleus via specific transporter proteins, the cytoplasmic enzyme Dicer trims the pre-miRNA to generate mature double-stranded miRNAs of 21 to 30 nucleotides. The miRNA subsequently unwinds, and the resulting single strands are incorporated into the multiprotein RISC. Base pairing between the single-stranded miRNA and its target mRNA directs RISC to either cleave the mRNA target or to repress its translation. In either case, the target mRNA gene is silenced posttranscriptionally. A. Gene activation Ribonucleoprotein transcription complex Decoy lncRNAB. Gene suppression Gene suppression


Pathology_Robbins

Pathology_Robbins. A. Gene activation Ribonucleoprotein transcription complex Decoy lncRNAB. Gene suppression Gene suppression C. Promote chromatin modification Methylation, D. Assembly of protein complexes Fig. 1.4 Roles of long noncoding RNAs (lncRNAs). (A) Long noncoding RNAs (lncRNAs) can facilitate transcription factor binding and thus promote gene activation. (B) Conversely, lncRNAs can preemptively bind transcription factors and thus prevent gene transcription. (C) Histone and DNA modification by acetylases or methylases (or deacetylases and demethylases) may be directed by the binding of lncRNAs. (D) In other instances lncRNAs may act as scaffolding to stabilize secondary or tertiary structures and/or multisubunit complexes that influence general chromatin architecture or gene activity. (Adapted from Wang KC, Chang HY: Molecular mechanisms of long noncoding RNAs, Mol Cell 43:904, 2011.) a variety of mechanisms (


Pathology_Robbins

Pathology_Robbins. (Adapted from Wang KC, Chang HY: Molecular mechanisms of long noncoding RNAs, Mol Cell 43:904, 2011.) a variety of mechanisms ( Fig. 1.4 ). Ongoing studies are exploring the role of lncRNAs in diseases like atherosclerosis and cancer.


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Pathology_Robbins. Exciting new developments that permit exquisitely specific genome editing stand to usher in an era of molecular revolution. These advances come from a wholly unexpected source: the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas (or CRISPR-associated genes). These are linked genetic elements that endow prokaryotes with a form of acquired immunity to phages and plasmids. Bacteria use this system to sample the DNA of infecting agents, incorporating it into the host genome as CRISPRs. CRISPRs are transcribed and processed into an RNA sequence that binds and directs the nuclease Cas9 to a sequences (e.g., a phage), leading to its cleavage and the destruction of the phage. Gene editing repurposes this process by using artificial guide RNAs (gRNAs) that bind Cas9 and are complementary to a DNA sequence of interest. Once directed to the target sequence by the gRNA, Cas9 induces double-strand DNA breaks.


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Pathology_Robbins. Repair of the resulting highly specific cleavage sites can lead to somewhat random disruptive mutations in the targeted sequences (through nonhomologous end joining [NHEJ]), or the precise introduction of new sequences of interest (by homologous recombination). Both the gRNAs and the Cas9 enzyme can be delivered to cells with a single easy-to-build plasmid ( Fig. 1.5 ). However, the real beauty DNA with random mutation DNA with specific mutation


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Fig.

Pathology_Robbins. Fig.


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Pathology_Robbins. 1.5 Gene editing with clustered regularly interspersed short palindromic repeats (CRISPRs)/Cas9. In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of about 20 bases. The constant regions of gRNAs bind to Cas9, permitting the variable regions to form heteroduplexes with homologous host cell DNA sequences. The Cas9 nuclease then cleaves the bound DNA, producing a double-stranded DNA break. To perform gene editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest. Coexpression of the gRNA and Cas9 in cells leads to efficient cleavage of the target sequence. In the absence of homologous DNA, the broken DNA is repaired by non homologous end joining (NHEJ), an error-prone method that often introduces disruptive insertions or deletions (indels). By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead


Pathology_Robbins

Pathology_Robbins. rror-prone method that often introduces disruptive insertions or deletions (indels). By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled with HDR include the repair of inherited genetic defects and the creation of pathogenic mutations.


Pathology_Robbins

Pathology_Robbins. of the system (and the excitement about its genetic engineering potential) comes from its impressive flexibility and specificity, which is substantially better than other previous editing systems. Applications include inserting specific mutations into the genomes of cells to model cancers and other diseases, and rapidly generating transgenic animals from edited embryonic stem cells. On the flip side, it now is feasible to selectively “correct” mutations that cause hereditable disease, or—perhaps more worrisome—to just eliminate less “desirable” traits. Predictably, the technology has inspired a vigorous debate regarding its application. The viability and normal activity of cells depend on a variety of fundamental housekeeping functions that all differentiated cells must perform. Many normal housekeeping functions are compartmentalized within membrane-bound intracellular organelles (


Pathology_Robbins

Pathology_Robbins. Many normal housekeeping functions are compartmentalized within membrane-bound intracellular organelles ( Fig. 1.6 ). By isolating certain cellular functions within distinct compartments, potentially injurious degradative enzymes or reactive metabolites can be concentrated or stored at high concentrations in specific organelles without risking damage to other cellular constituents. Moreover, compartmentalization allows for the creation of unique intracellular environments (e.g., low pH or high calcium) that are optimal for certain enzymes or metabolic pathways.


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Pathology_Robbins. New proteins destined for the plasma membrane or secretion are synthesized in the rough endoplasmic reticulum (RER) and physically assembled in the Golgi apparatus; proteins intended for the cytosol are synthesized on free ribosomes. Smooth endoplasmic reticulum (SER) may be abundant in certain cell types such as gonads and liver where it serves as the site of steroid hormone and lipoprotein synthesis, as well as the modification of hydrophobic compounds such as drugs into water-soluble molecules for export. Cells catabolize the wide variety of molecules that they endocytose, as well as their own repertoire of proteins and organelles—all of which are constantly being degraded and renewed. Breakdown of these constituents takes place at three different sites, ultimately serving different functions.


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Pathology_Robbins. Proteasomes are “disposal” complexes that degrade denatured or otherwise “tagged” cytosolic proteins and release short peptides. In some cases the peptides so generated are presented in the context of class I major histocompatibility molecules to help drive the adaptive immune response (Chapter 5). In other cases, proteasomal degradation of regulatory proteins or transcription factors can trigger or shut down cellular signaling pathways. Lysosomes are intracellular organelles that contain enzymes that digest a wide range of macromolecules, including proteins, polysaccharides, lipids, and nucleic acids. They are the organelle in which phagocytosed microbes and damaged or unwanted cellular organelles are degraded and eliminated. Peroxisomes are specialized cell organelles that contain catalase, peroxidase and other oxidative enzymes. They Relative volumes of intracellular organelles (hepatocyte) Compartment % total volume number/cell role in the cell


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Pathology_Robbins. Relative volumes of intracellular organelles (hepatocyte) Compartment % total volume number/cell role in the cell Cytosol 54% 1 metabolism, transport, protein translation Mitochondria 22% 1700 energy generation, apoptosis Rough ER 9% 1* synthesis of membrane and secreted proteins Smooth ER, Golgi 6% 1* protein modification, sorting, catabolism Nucleus 6% 1 cell regulation, proliferation, DNA transcription Endosomes 1% 200 intracellular transport and export, ingestion of extracellular substances Lysosomes 1% 300 cellular catabolism Peroxisomes 1% 400 very long-chain fatty acid metabolism


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Pathology_Robbins. Fig. 1.6 Basic subcellular constituents of cells. The table presents the number of various organelles within a typical hepatocyte, as well as their volume within the cell. The figure shows geographic relationships but is not intended to be accurate to scale. *Rough and smooth ER form a single compartment; the Golgi apparatus is organized as a set of discrete stacked cisternae interconnected by transport vesicles. (Adapted from Weibel ER, Stäubli W, Gnägi HR, et al: Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver, J Cell Biol 42:68, 1969.) play a specialized role in the breakdown of very long chain fatty acids, generating hydrogen peroxide in the process.


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Pathology_Robbins. The contents and position of cellular organelles also are subject to regulation. Endosomal vesicles shuttle internalized material to the appropriate intracellular sites or direct newly synthesized materials to the cell surface or targeted organelle. Movement of both organelles and proteins within the cell and of the cell in its environment is orchestrated by the cytoskeleton. These structural proteins also regulate cellular shape and intracellular organization, requisites for maintaining cell polarity. This is particularly critical in epithelia, in which the top of the cell (apical) and the bottom and side of the cell (basolateral) are often exposed to different environments and have distinct functions.


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Pathology_Robbins. Most of the adenosine triphosphate (ATP) that powers cells is made through oxidative phosphorylation in the mitochondria. However, mitochondria also serve as an important source of metabolic intermediates that are needed for anabolic metabolism. They also are sites of synthesis of certain macromolecules (e.g., heme), and contain important sensors of cell damage that can initiate and regulate the process of apoptotic cell death.


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Pathology_Robbins. Cell growth and maintenance require a constant supply of both energy and the building blocks that are needed for synthesis of macromolecules. In growing and dividing cells, all of these organelles have to be replicated (organellar biogenesis) and correctly apportioned in daughter cells following mitosis. Moreover, because the macromolecules and organelles have finite life spans (mitochondria, e.g., last only about 10 days), mechanisms also must exist that allow for the recognition and degradation of “worn out” cellular components. The final catabolism occurs in lysosomes. With this as a primer, we now move on to discuss cellular components and their function in greater detail. Plasma Membrane: Protection and Nutrient Acquisition


Pathology_Robbins

Pathology_Robbins. With this as a primer, we now move on to discuss cellular components and their function in greater detail. Plasma Membrane: Protection and Nutrient Acquisition Plasma membranes (and all other organellar membranes) are more than just static lipid sheaths. Rather, they are fluid bilayers of amphipathic phospholipids with hydrophilic head groups that face the aqueous environment and hydrophobic lipid tails that interact with each other to form a barrier to passive diffusion of large or charged molecules ( Fig. 1.7A ). The bilayer is composed of a heterogeneous collection of different phospholipids, which are distributed asymmetrically—for example, certain membrane lipids preferentially associate with extracellular or cytosolic faces. Asymmetric partitioning of phospholipids is important in several cellular processes:


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Pathology_Robbins. Phosphatidylinositol on the inner membrane leaflet can be phosphorylated, serving as an electrostatic scaffold for intracellular proteins; alternatively, polyphosphoinositides can be hydrolyzed by phospholipase C to generate intracellular second signals such as diacylglycerol and inositol trisphosphate. Phosphatidylserine is normally restricted to the inner face where it confers a negative charge and is involved in electrostatic interactions with proteins; however, when it flips to the extracellular face, which happens in cells undergoing apoptosis (programmed cell death), it becomes an “eat me” signal for phagocytes. In the special case of platelets, it serves as a cofactor in the clotting of blood.


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Pathology_Robbins. • Glycolipids and sphingomyelin are preferentially expressed on the extracellular face; glycolipids (and particularly gangliosides, with complex sugar linkages and terminal sialic acids that confer negative charges) are important in cell–cell and cell–matrix interactions, including inflammatory cell recruitment and sperm–egg interactions. Certain membrane components associate laterally with each other in the bilayer, leading to distinct domains called lipid rafts. Because inserted membrane proteins have different intrinsic solubilities in various lipid domains, they tend to accumulate in certain regions of the membrane (e.g., rafts) and to become depleted from others. Such nonrandom distributions of lipids and membrane proteins impact cell–cell and cell–matrix interactions, as well as intracellular signaling and the generation of specialized membrane regions involved in secretory or endocytic pathways.


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Pathology_Robbins. The plasma membrane is liberally studded with a variety of proteins and glycoproteins involved in (1) ion and metabolite transport, (2) fluid-phase and receptor-mediated uptake of macromolecules, and (3) cell–ligand, cell–matrix, and cell–cell interactions. Proteins interact with the lipid bilayer by one of four general arrangements ( Fig. 1.7B • Most proteins are transmembrane (integral) proteins, having one or more relatively hydrophobic α-helical


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Pathology_Robbins. 1.7 Plasma membrane organization and asymmetry. (A) The plasma membrane is a bilayer of phospholipids, cholesterol, and associated proteins. The phospholipid distribution within the membrane is asymmetric; phosphatidylcholine and sphingomyelin are overrepresented in the outer leaflet, and phosphatidylserine (negative charge) and phosphatidylethanolamine are predominantly found on the inner leaflet; glycolipids occur only on the outer face where they contribute to the extracellular glycocalyx. Non-random partitioning of certain membrane components such as cholesterol creates membrane domains known as lipid rafts. (B) Membrane-associated proteins may traverse the membrane (singly or multiply) via α-helical hydrophobic amino acid sequences; depending on the sequence and hydrophobicity of these domain, such proteins may be enriched or excluded from lipid rafts and other membrane domain. Proteins on the cytosolic face may associate with membranes through posttranslational modifications, fo


Pathology_Robbins

Pathology_Robbins. se domain, such proteins may be enriched or excluded from lipid rafts and other membrane domain. Proteins on the cytosolic face may associate with membranes through posttranslational modifications, for example, farnesylation or addition of palmitic acid. Proteins on the extracytoplasmic face may associate with the membrane via glycosyl phosphatidyl inositol linkages. Besides protein–protein interactions within the membrane, membrane proteins can also associate with extracellular and/or intracytoplasmic proteins to generate large, relatively stable complexes (e.g., the focal


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Pathology_Robbins. adhesion complex). Transmembrane proteins can translate mechanical forces (e.g., from the cytoskeleton or ECM) as well as chemical signals across the membrane. It is worth remembering that a similar organization of lipids and associated proteins also occurs within the various organellar membranes.


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Pathology_Robbins. segments that traverse the lipid bilayer. Integral membrane proteins typically contain positively charged amino acids in their cytoplasmic domains that anchor the proteins to the negatively charged head groups of membrane phospholipids. Proteins may be synthesized in the cytosol and posttranslationally attached to prenyl groups (e.g., farnesyl, related to cholesterol) or fatty acids (e.g., palmitic or myristic acid) that insert into the cytosolic side of the plasma membrane. Attachment to membranes can occur through glycosylphosphatidylinositol (GPI) anchors on the extracellular face of the membrane. Extracellular proteins can noncovalently associate with transmembrane proteins, which serve to anchor them to the cell.


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Pathology_Robbins. Many plasma membrane proteins function together as larger complexes; these may assemble under the control of chaperone molecules in the RER or by lateral diffusion in the plasma membrane. The latter mechanism is characteristic of many protein receptors (e.g., cytokine receptors) that dimerize or trimerize in the presence of ligand to form functional signaling units. Although lipid bilayers are fluid in the two-dimensional plane of the membrane, membrane components can nevertheless be constrained to discrete domains. This can occur by localization to lipid rafts (discussed earlier), or through intercellular protein–protein interactions (e.g., at tight junctions) that establish discrete boundaries; indeed, this strategy is used to maintain cell polarity (e.g., top/apical versus bottom/basolateral) in epithelial layers. Alternatively, unique domains can be formed through the interaction of membrane proteins with cytoskeletal molecules or an extracellular matrix (ECM).


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Pathology_Robbins. The extracellular face of the plasma membrane is diffusely studded with carbohydrates, not only as complex oligosaccharides on glycoproteins and glycolipids, but also as polysaccharide chains attached to integral membrane proteoglycans. This glycocalyx functions as a chemical and mechanical barrier, and is also involved in cell–cell and cell–matrix interactions.


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Pathology_Robbins. Small, nonpolar molecules such as O2 and CO2 readily dissolve in lipid bilayers and therefore rapidly diffuse across them, as do hydrophobic molecules (e.g., steroid-based molecules such as estradiol or vitamin D). Similarly, small polar molecules (<75 daltons in mass, such as water, ethanol, and urea) readily cross membranes. In contrast, the lipid bilayer is an effective barrier to the passage of larger polar molecules, even those only slightly greater than 75 daltons, such as glucose. Lipid bilayers also are impermeant to ions, no matter how small, because of their charge and high degree of hydration. We will next discuss specialized mechanisms that regulate traffic across plasma membranes.


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Pathology_Robbins. For each of the larger polar molecules that must cross membranes to support normal cellular functions (e.g., for nutrient uptake and waste disposal), unique plasma membrane protein complexes are typically required. For low-molecular-weight species (ions and small molecules up to approximately 1000 daltons), channel proteins and carrier proteins may be used (although this discussion focuses on plasma membranes, it should be noted that similar pores and channels are needed for transport across organellar membranes). Each transported molecule (e.g., ion, sugar, nucleotide) requires a transporter that is typically highly specific (e.g., glucose but not galactose): Channel proteins create hydrophilic pores that, when open, permit rapid movement of solutes (usually restricted by size and charge; Fig. 1.8 Carrier proteins bind their specific solute and undergo a series of conformational changes to transfer the ligand across the membrane; their transport is relatively slow.


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Pathology_Robbins. Fig. 1.8 Carrier proteins bind their specific solute and undergo a series of conformational changes to transfer the ligand across the membrane; their transport is relatively slow. In many cases, a concentration and/or electrical gradient between the inside and outside of the cell drives solute movement via passive transport (virtually all plasma membranes have an electrical potential difference across them, with the inside negative relative to the outside). In other cases, active transport of certain solutes against a concentration gradient is accomplished by carrier molecules (not channels) using energy released by ATP hydrolysis or a coupled ion gradient. Transporter ATPases include the notorious multidrug resistance (MDR) protein, which pumps polar compounds (e.g., chemotherapeutic drugs) out of cells and may render cancer cells resistant to treatment.


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Pathology_Robbins. Because membranes are freely permeable to water, it moves into and out of cells by osmosis, depending on relative solute concentrations. Thus, extracellular salt in excess of that in the cytosol (hypertonicity) causes a net movement of water out of cells, whereas hypotonicity causes a net movement of water into cells. The cytosol is rich in charged metabolites and protein species, which attract a large number of counterions that tend to increase the intracellular osmolarity. As a consequence, to prevent overhydration cells must constantly pump out small inorganic ions (e.g., Na+)—typically through the activity of membrane ion-exchanging ATPases. Loss of the ability to generate energy (e.g., in a cell injured by toxins or ischemia) therefore results in osmotic swelling and eventual rupture of cells. Similar transport mechanisms also regulate intracellular and intraorganellar pH; most cytosolic enzymes prefer to work at pH 7.4, whereas lysosomal enzymes function best at pH 5 or less.


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Pathology_Robbins. Uptake of fluids or macromolecules by the cell, called endocytosis, occurs by two fundamental mechanisms ( Fig. 1.8 ). Certain small molecules—including some vitamins—are taken up by invaginations of the plasma membrane called caveolae. For larger molecules, uptake occurs after binding to specific cell-surface receptors; internalization occurs through a membrane invagination process driven by an intracellular matrix of clathrin proteins. Clathrin is a hexamer of proteins that spontaneously assembles into a basketlike lattice to drive the invagination process. We shall come back to these later. The process by which large molecules are exported from cells is called exocytosis. In this process, proteins synthesized and packaged within the RER and Golgi apparatus http://ebooksmedicine.net


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Pathology_Robbins. are concentrated in secretory vesicles, which then fuse with the plasma membrane and expel their contents. Transcytosis is the movement of endocytosed vesicles between the apical and basolateral compartments of cells; this is a mechanism for transferring large amounts of intact proteins across epithelial barriers (e.g., ingested antibodies in maternal milk across intestinal epithelia) or for the rapid movement of large volumes of solute. In fact, increased transcytosis probably plays a role in the increased vascular permeability seen in tumors. We now return to the two forms of endocytosis mentioned earlier.


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Pathology_Robbins. We now return to the two forms of endocytosis mentioned earlier. • Caveolae-mediated endocytosis. Caveolae (“little caves”) are noncoated plasma membrane invaginations associated with GPI-linked molecules, cyclic adenosine monophosphate (cAMP)-binding proteins, SRC-family kinases, and the folate receptor. Caveolin is the major structural protein of caveolae. Internalization of caveolae with any bound molecules and associated extracellular fluid is denoted potocytosis—literally “cellular sipping.” Although caveolae likely participate in the transmembrane delivery of some molecules (e.g., folate), they also appear to contribute to the regulation of trans-membrane signaling and/or cellular adhesion via the internalization of receptors and integrins. Mutations in caveolin are associated with muscular dystrophy and electrical abnormalities in the heart.


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Pathology_Robbins. Fig. 1.8 ). Pinocytosis (“cellular drinking”) is a fluid-phase process. The plasma membrane invaginates and is pinched off to form a cytoplasmic vesicle; after delivering their cargo, endocytosed vesicles recycle back to the plasma membrane (exocytosis) for another round of ingestion. Endocytosis and exocytosis are tightly balanced and highly active, as a cell typically pinocytoses 10% to 20% of its own cell volume each hour, or about 1% to 2% of its plasma membrane each minute. Pinocytosis and receptor-mediated endocytosis begin with the formation of a clathrin-coated pit containing the ligand to be internalized (by itself or bound to the receptor), which rapidly http://ebooksmedicine.net invaginates and pinches off to form a clathrin-coated vesicle. Thus, trapped within the vesicle is a gulp of the extracellular milieu, as well as receptor-bound macromolecules as described below. The vesicles then rapidly uncoat and fuse with an acidic intracellular structure called the early


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Pathology_Robbins. is a gulp of the extracellular milieu, as well as receptor-bound macromolecules as described below. The vesicles then rapidly uncoat and fuse with an acidic intracellular structure called the early endosome, which progressively matures to late endosomes and ultimately fuses with lysosomes.


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Pathology_Robbins. Receptor-mediated endocytosis is the major uptake mechanism for certain macromolecules, as exemplified by transferrin and low-density lipoprotein (LDL). These macromolecules bind to receptors that localize to clathrin-coated pits. After binding to their specific receptors, LDL and transferrin are endocytosed in vesicles that mature into early and late endosomes. In the acidic environment of the endosome, LDL and transferrin release their bound ligands (cholesterol and iron, respectively), which then exit into the cytosol, and the LDL receptor and transferrin receptor subsequently recycle to the plasma membrane. Defects in receptor-mediated transport of LDL are responsible for familial hypercholesterolemia, as described in Chapter 7. The ability of cells to adopt a particular shape, maintain polarity, organize the intracellular organelles, and move about depends on the intracellular scaffolding of proteins called the cytoskeleton (


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Pathology_Robbins. Fig. 1.9 ). In eukaryotic cells, there are three major classes of cytoskeletal proteins: • Actin microfilaments are fibrils 5-to 9-nm in diameter formed from the globular protein actin (G-actin), the most abundant cytosolic protein in cells. G-actin monomers noncovalently polymerize into long filaments (F- Fig. 1.9 Cytoskeletal elements and cell–cell interactions. Interepithelial adhesion involves several different surface protein interactions, including tight junctions and desmosomes; adhesion to the ECM involves cellular integrins (and associated proteins) within hemidesmosomes. See text for details. actin) that intertwine to form double-stranded helices. In muscle cells, the filamentous protein myosin binds to actin and moves along it, driven by ATP hydrolysis (the basis of muscle contraction). In non-muscle cells, F-actin assembles via an assortment of actinbinding proteins into well-organized bundles and networks that control cell shape and movement.


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Pathology_Robbins. Intermediate filaments are fibrils 10-nm in diameter that comprise a large and heterogeneous family. Members include lamins A, B, and C, which contribute to the structure of nuclear lamina. Individual types of intermediate filaments have characteristic tissue-specific patterns of expression that are useful for identifying the cellular origin of poorly differentiated tumors. Vimentin: Mesenchymal cells (fibroblasts, endothelium) anchoring intracellular organelles Desmin: Muscle cells, forming the scaffold on which actin and myosin contract Neurofilaments: Axons of neurons, imparting strength and rigidity Glial fibrillary acidic protein: Glial cells that support neurons


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Pathology_Robbins. Neurofilaments: Axons of neurons, imparting strength and rigidity Glial fibrillary acidic protein: Glial cells that support neurons Cytokeratins: Epithelial cells express more than 30 distinct varieties with distinct patterns of expression in different lineages (e.g., lung versus gastrointestinal epithelia). These can serve as histochemical markers for various epithelia Intermediate filaments are found predominantly in a ropelike polymerized form and primarily serve to impart tensile strength and allow cells to bear mechanical stress. The nuclear membrane lamins are important not only for maintaining nuclear morphology but also for regulating nuclear gene transcription. The critical roles of lamins is emphasized by rare but fascinating disorders caused by lamin mutations, which range from certain forms of muscular dystrophy to progeria, a disease of premature aging. Intermediate filaments also form the major structural proteins of epidermis and hair.


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Pathology_Robbins. Microtubules are 25-nm-thick fibrils composed of noncovalently polymerized dimers of α-and β-tubulin arrayed in constantly elongating or shrinking hollow tubes with a defined polarity; the ends are designated “+” or “−.” The “−” end is typically embedded in a microtubule organizing center (MTOC or centrosome) near the nucleus where it is associated with paired centrioles, while the “+” end elongates or recedes in response to various stimuli by the addition or subtraction of tubulin dimers. Microtubules are involved in several important cellular functions: Support cables for “molecular motor” proteins that allow the movement of vesicles and organelles around cells. Kinesins are the motors for anterograde (− to +) transport, whereas dyneins move cargo in a retrograde direction (+ to −). The mechanical support for sister chromatid separation during mitosis The core of primary cilia, single nonmotile projections on nucleated cells that help regulate proliferation and differentiation


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Pathology_Robbins. The mechanical support for sister chromatid separation during mitosis The core of primary cilia, single nonmotile projections on nucleated cells that help regulate proliferation and differentiation The core of motile cilia (e.g., in bronchial epithelium) or flagella (in sperm) Cells interact and communicate with one another by forming junctions that provide mechanical links and enable surface receptors to recognize ligands on other cells. Cell junctions are organized into three basic types ( Fig. 1.9


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Pathology_Robbins. Fig. 1.9 Occluding junctions (tight junctions) seal adjacent cells together to create a continuous barrier that restricts the paracellular (between cells) movement of ions and other molecules. Viewed en face, occluding junctions form a tight meshlike network of macromolecular contacts between neighboring cells. The complexes that mediate these cell–cell interactions are composed of multiple proteins, including occludin and claudin. In addition to being a high-resistance barrier to solute movement, occluding junctions also maintain cellular polarity by forming the boundary between apical and basolateral domains of cells. Significantly, these junctions (as well as the desmosomes described later) are dynamic structures that can dissociate and reform as required to facilitate epithelial proliferation or inflammatory cell migration.


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Pathology_Robbins. Anchoring junctions (desmosomes) mechanically attach cells—and their intracellular cytoskeletons—to other cells or to the ECM. When the adhesion focus is between cells, and is small and rivetlike, it is designated a spot desmosome. When such a focus attaches the cell to the ECM, it is called a hemidesmosome. Similar adhesion domains can also occur as broad bands between cells, where they are denoted as belt desmosomes. Cell–cell desmosomal junctions are formed by the homotypic association of transmembrane glycoproteins called cadherins. In spot desmosomes, the cadherins are linked to intracellular intermediate filaments and allow extracellular forces to be mechanically communicated (and dissipated) over multiple cells. In belt desmosomes, the transmembrane adhesion molecules are associated with intracellular actin microfilaments, by which they can influence cell shape and/or motility.


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Pathology_Robbins. In belt desmosomes, the transmembrane adhesion molecules are associated with intracellular actin microfilaments, by which they can influence cell shape and/or motility. In hemidesmosomes, the transmembrane connector proteins are called integrins; like cadherins, these attach to intracellular intermediate filaments, and thus they functionally link the cytoskeleton to the ECM. Focal adhesion complexes are large macromolecular complexes that localize at hemidesmosomes, and include proteins that can generate intracellular signals when cells are subjected to increased shear stress, for example, endothelium in the bloodstream, or cardiac myocytes in a failing heart.


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Pathology_Robbins. Communicating junctions (gap junctions) mediate the passage of chemical or electrical signals from one cell to another. The junction consists of a dense planar array of 1.5to 2-nm pores (called connexons) formed by hexamers of transmembrane protein connexins. These pores permit the passage of ions, nucleotides, sugars, amino acids, vitamins, and other small molecules; the permeability of the junction is rapidly reduced by lowered intracellular pH or increased intracellular calcium. Gap junctions play a critical role in cell–cell communication; in cardiac myocytes, for example, cell-to-cell calcium fluxes through gap junctions allowing the myocardium to behave as a functional syncytium capable of coordinated waves of contraction—the beating of the heart. BiosyntheticMachinery:EndoplasmicReticulumandGolgiApparatus


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Pathology_Robbins. BiosyntheticMachinery:EndoplasmicReticulumandGolgiApparatus The structural proteins and enzymes of the cell are constantly renewed by a balance between ongoing synthesis and intracellular degradation. The endoplasmic reticulum (ER) is the site of synthesis of all transmembrane proteins and lipids needed for the assembly of plasma membrane and cellular organelles, including the ER itself. It is also the initial site of synthesis of all molecules destined for export out of the cell. The ER is organized into a mesh-like interconnected maze of branching tubes and flattened lamellae forming a continuous sheet around a single lumen that is topologically contiguous with the extracellular environment. The ER is composed of distinct domains that are distinguished by the presence or absence of ribosomes ( Fig. 1.6


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Pathology_Robbins. Rough ER (RER): Membrane-bound ribosomes on the cytosolic face of the RER translate mRNA into proteins that are extruded into the ER lumen or become integrated into the ER membrane. This process is directed by specific signal sequences on the N-termini of nascent proteins. Proteins insert into the ER fold and must fold properly in order to assume a functional conformation and assemble into higher order complexes. Proper folding of the extracellular domains of many proteins involves the formation of disulfide bonds. A number of inherited disorders, including many cases of familial hypercholesterolemia (Chapter 6), are cause by mutations that disrupt disulfide bond formation. In addition, N-linked oligosaccharides (sugar moieties attached to asparagine residues) are added in the ER. Chaperone molecules retain proteins in the ER until these modifications are complete and the proper conformation is achieved. If a protein fails to fold and assemble into complexes appropriately, it is


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Pathology_Robbins. molecules retain proteins in the ER until these modifications are complete and the proper conformation is achieved. If a protein fails to fold and assemble into complexes appropriately, it is retained and degraded within the ER. Moreover, excess accumulation of misfolded proteins—exceeding the capacity of the ER to edit and degrade them—leads to the ER stress response (also called the unfolded protein response or the UPR), which triggers cell death through apoptosis (Chapter 2).


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Pathology_Robbins. As an example of the importance of the ER editing function, the disease cystic fibrosis most commonly results from misfolding of the CFTR membrane transporter protein. In cystic fibrosis, the most common mutation in the CFTR gene results in the loss of a single amino acid residue (phenylalanine 508), leading in turn to misfolding, ER retention, and degradation of the CFTR protein. The loss of CFTR function leads to abnormal epithelial chloride transport, hyperviscous bronchial secretions and recurrent airway infections (Chapter 7).


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Pathology_Robbins. Golgi apparatus: From the RER, proteins and lipids destined for other organelles or for extracellular export are shuttled into the Golgi apparatus. This organelle consists of stacked cisternae that progressively modify proteins in an orderly fashion from cis (near the ER) to trans (near the plasma membrane); macromolecules are shuttled between the various cisternae within membrane-bound vesicles. As molecules move from cis to trans, the N-linked oligosaccharides originally added to proteins in the ER are pruned and further modified in a stepwise fashion; O-linked oligosaccharides (sugar moieties linked to serine or threonine) are also appended. Some of this glycosylation is important in directing molecules to lysosomes (via the mannose-6-phosphate receptor); other glycosylation adducts may be important for cell–cell or cell–matrix interactions, or for clearing senescent cells (e.g., platelets and red cells). In addition to the stepwise glycosylation of lipids and proteins, the cis


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Pathology_Robbins. may be important for cell–cell or cell–matrix interactions, or for clearing senescent cells (e.g., platelets and red cells). In addition to the stepwise glycosylation of lipids and proteins, the cis Golgi network is where proteins are recycled back to the ER, and the trans Golgi network is where proteins and lipids are dispatched to other organelles (including the plasma membrane), or to secretory vesicles destined for extracellular release. The Golgi complex is especially prominent in cells specialized for secretion, including goblet cells of the intestine, bronchial epithelium (secreting large amounts of polysaccharide-rich mucus), and plasma cells (secreting large quantities of antibodies).


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Pathology_Robbins. http://ebooksmedicine.net Smooth ER (SER): The SER in most cells is relatively sparse, forming the transition zone from RER to transport vesicles moving to the Golgi. However, in cells that synthesize steroid hormones (e.g., in the gonads or adrenals), or that catabolize lipid-soluble molecules (e.g., in the liver), the SER may be particularly conspicuous. Indeed, repeated exposure to compounds that are metabolized by the SER (e.g., phenobarbital, which is catabolized by the cytochrome P-450 system) leads to a reactive hyperplasia of the SER. The SER also is responsible for sequestering intracellular calcium; subsequent release from the SER into the cytosol can mediate a number of responses to extracellular signals. In addition, in muscle cells, a specialized SER called the sarcoplasmic reticulum is responsible for the cyclical release and sequestration of calcium ions that regulate muscle contraction and relaxation, respectively. WasteDisposal:LysosomesandProteasomes


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Pathology_Robbins. WasteDisposal:LysosomesandProteasomes As already mentioned briefly, cellular waste disposal depends on the activities of lysosomes and proteasomes ( Fig. 1.10 Lysosomes are membrane-bound organelles containing roughly 40 different acid hydrolases (i.e., enzymes that function best in acidic pH ≤5), including proteases, nucleases, lipases, glycosidases, phosphatases, and sulfatases. Lysosomal enzymes are initially synthesized in the ER lumen and then tagged with a mannose-6-phosphate (M6P) residue within the Golgi apparatus. Such M6P-modified proteins are subsequently delivered to lysosomes through trans-Golgi vesicles that express M6P receptors. The other macromolecules destined for catabolism in the lysosomes arrive by one of three other pathways ( Fig. 1.10


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Pathology_Robbins. Fig. 1.10 Material internalized by fluid-phase pinocytosis or receptor-mediated endocytosis passes from plasma membrane to early endosome to late endosome, and ultimately into the lysosome, becoming progressively more acidic in the process. The early endosome is the first acidic compartment encountered, whereas proteolytic enzymes only begin significant digestion in the late endosome; late endosomes mature into lysosomes.


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Pathology_Robbins. Senescent organelles and large protein complexes can be shuttled into lysosomes by a process called autophagy. Through poorly understood mechanisms, obsolete organelles are corralled by a double membrane derived from the ER; the membrane progressively expands to encircle a collection of structures and forms an autophagosome, which then fuses with lysosomes where the contents are catabolized. In addition to facilitating the turnover of aged and defunct cellular constituents, autophagy also is used to preserve cell viability during nutrient depletion. The significance of autophagy in cell biology was recognized by the award of the 2016 Nobel Prize to Yoshinori Ohsumi for his discoveries relating to the mechanism of autophagy. This topic is discussed in more detail in Chapter 2.


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Pathology_Robbins. Phagocytosis of microorganisms or large fragments of matrix or debris occur primarily in professional phagocytes (macrophages and neutrophils). The material is engulfed to form a phagosome that subsequently fuses with a lysosome.


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Pathology_Robbins. Fig. 1.10 ); these include denatured or misfolded proteins (akin to what occurs within the ER), as well as other proteins whose levels and half-life need to be tightly regulated (e.g., transcription factors). Many (but not all) proteins destined for proteasome destruction are targeted after covalent addition of a protein called ubiquitin. Polyubiquitinated molecules are progressively unfolded and funneled into the polymeric proteasome complex, a cylinder containing multiple different protease activities, each with its active site pointed at the hollow core. Proteasomes digest proteins into small (6–12 amino acids) fragments that can subsequently be further degraded to their constituent amino acids and recycled, or presented to immune cells in the context of major histocompatibility complex class I molecules, an important component of host immune surveillance.


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Pathology_Robbins. Mitochondria evolved from ancestral prokaryotes that were engulfed by primitive eukaryotes about 1.5 billion years ago. Their origin explains why mitochondria contain their own DNA genome (circularized, about 1% of the total cellular DNA), which encodes roughly 1% of the total cellular proteins and approximately 20% of the proteins involved in oxidative phosphorylation. Although their genomes are small, mitochondria can nevertheless perform all the steps of DNA replication, transcription, and translation. Interestingly, the mitochondrial machinery is similar to present-day bacteria; for example, mitochondria initiate protein synthesis with N-formylmethionine and are sensitive to anti-bacterial antibiotics. Mitochondria are dynamic, constantly undergoing fission and fusion with other mitochondria; in this way, http://ebooksmedicine.net


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Pathology_Robbins. Mitochondria are dynamic, constantly undergoing fission and fusion with other mitochondria; in this way, http://ebooksmedicine.net Fig. 1.10 Intracellular catabolism. (A) Lysosomal degradation. In heterophagy (right side), lysosomes fuse with endosomes or phagosomes to facilitate the degradation of their internalized contents (see


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Pathology_Robbins. Fig. 1.8 ). The end products may be released into the cytosol for nutrition or discharged into the extracellular space (exocytosis). In autophagy (left


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Pathology_Robbins. side), senescent organelles or denatured proteins are targeted for lysosome-driven degradation by encircling them with a double membrane derived from the ER and marked by LC3 proteins (microtubule-associated protein 1A/1B-light chain 3). Cell stressors such as nutrient depletion or certain intracellular infections can also activate the autophagocytic pathway. (B) Proteasome degradation. Cytosolic proteins destined for turnover (e.g., transcription factors or regulatory proteins), senescent proteins, or proteins that have become denatured because of extrinsic mechanical or chemical stresses can be tagged by multiple ubiquitin molecules (through the activity of E1, E2, and E3 ubiquitin ligases). This marks the proteins for degradation by proteasomes, cytosolic multisubunit complexes that degrade proteins to small peptide fragments. High levels of misfolded proteins within the ER trigger a protective unfolded protein response engendering a broad reduction in protein synthesis, but specif


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Pathology_Robbins. grade proteins to small peptide fragments. High levels of misfolded proteins within the ER trigger a protective unfolded protein response engendering a broad reduction in protein synthesis, but specific increases in chaperone proteins that can facilitate protein refolding. If this is inadequate to cope with the levels of misfolded proteins, apoptosis is induced.


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Pathology_Robbins. mitochondria can undergo regular renewal to stave off contributes the vast majority of cytoplasmic organelles to degenerative changes that might occur because of genetic the fertilized zygote, mitochondrial DNA is virtually disorders or oxygen free radical damage. Mitochondria entirely maternally inherited. However, because the protein turn over rapidly, with estimated half-lives ranging from 1 constituents of mitochondria derive from both nuclear and to 10 days, depending on the tissue, nutritional status, meta-mitochondrial genetic transcription, mitochondrial disorbolic demands, and intercurrent injury. Because the ovum ders may be X-linked, autosomal, or maternally inherited. (glucose, glutamine, ISCHEMIA, ATP Lipid, protein building blocks


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Pathology_Robbins. (glucose, glutamine, ISCHEMIA, ATP Lipid, protein building blocks Fig. 1.11 Roles of the mitochondria. Besides the efficient generation of ATP from carbohydrate and fatty acid substrates, mitochondria play an important role in intermediary metabolism, serving as the source of molecules used to synthesize lipids and proteins, and they also are centrally involved in cell life and-death decisions. Mitochondria provide the enzymatic machinery for oxidative phosphorylation (and thus the relatively efficient generation of energy from glucose and fatty acid substrates). They also have central roles in anabolic metabolism and the regulation programmed cell death, so-called “apoptosis” ( Fig. 1.11


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Pathology_Robbins. Fig. 1.11 Energy Generation. Each mitochondrion has two separate and specialized membranes. The inner membrane contains the enzymes of the respiratory chain folded into cristae. This encloses a core matrix space that harbors the bulk of certain metabolic enzymes, such as the enzymes of the citric acid cycle. Outside the inner membrane is the intermembrane space, site of ATP synthesis, which is, in turn, enclosed by the outer membrane; the latter is studded with porin proteins that form aqueous channels permeable to small (<5000 daltons) molecules. Larger molecules (and even some smaller polar species) require specific transporters.


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Pathology_Robbins. The major source of the energy needed to run all basic cellular functions derives from oxidative metabolism. Mitochondria oxidize substrates to CO2, and in the process transfer high-energy electrons from the original molecule (e.g., gluocse) to molecular oxygen to water. The oxidation of various metabolites drives hydrogen ion (proton) pumps that transfer H+ ions from the core matrix into the inter-membrane space. As these H+ ions flow back down their electrochemical gradient, the energy released is used to synthesize ATP.


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Pathology_Robbins. It should be noted that the electron transport chain need not necessarily be coupled to ATP generation. Thus, an inner membrane protein enriched in brown fat called thermogenin (or UCP-1 = uncoupling protein 1) is a proton transporter that can dissipate the proton gradient (uncouple it from oxidative phosphorylation) in the form of heat (nonshivering thermogenesis). As a natural (albeit usually low-level) byproduct of substrate oxidation and electron transport, mitochondria also are an important source of reactive oxygen species (e.g., oxygen free radicals, hydrogen peroxide); importantly, hypoxia, toxic injury, or even mitochondrial aging can lead to significantly increased levels of intracellular oxidative stress.


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Pathology_Robbins. Intermediate Metabolism. Pure oxidative phosphorylation produces abundant ATP, but also “burns” glucose to CO2 and H2O, leaving no carbon moieties for use as building blocks for lipids or proteins. For this reason, rapidly growing cells (both benign and malignant) increase glucose and glutamine uptake and decrease their production of ATP per glucose molecule—forming lactic acid in the presence of adequate oxygen—a phenomenon called the Warburg effect (or aerobic glycolysis). Both glucose and glutamine provide carbon moieties that prime the mitochondrial tricarboxylic acid (TCA) cycle, but instead of being used to make ATP, intermediates are “spun off” to make lipids, nucleic acids, and proteins. Thus, depending on the growth state of the cell, mitochondrial metabolism can be modulated to support either cellular maintenance or cellular growth. Ultimately, growth factors, nutrient supplies, and oxygen availability, as well as cellular signaling pathways and sensors that respond to these


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Pathology_Robbins. either cellular maintenance or cellular growth. Ultimately, growth factors, nutrient supplies, and oxygen availability, as well as cellular signaling pathways and sensors that respond to these exogenous factors, govern these metabolic decisions.


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Pathology_Robbins. Cell Death. Mitochondria are like the proverbial Dr. Jekyll and Mr. Hyde. On the one hand, they are factories of energy production in the form of ATP that allow the cells to survive; on the other hand, they participate in driving cell death when the cells are exposed to noxious stimuli that the cells cannot adapt to. The role of mitochondria in the two principle forms of cell death, necrosis and apoptosis, are discussed in Chapter 2. In addition to providing ATP and metabolites that enable the bulk of cellular activity, mitochondria also regulate the balance of cell survival and death.


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Pathology_Robbins. Cell communication is critical in multicellular organisms. At the most basic level, extracellular signals determine whether a cell lives or dies, whether it remains quiescent, or whether it is stimulated to perform a specific function. Intercellular signaling is important in the developing embryo, in maintaining tissue organization, and in ensuring that tissues respond in an adaptive and effective fashion to various threats, such as local tissue trauma or a systemic infection. Loss of cellular communication and the “social controls” that maintain normal relationships of cells can variously lead to unregulated growth (cancer) or an ineffective response to an extrinsic stress (as in shock).


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Pathology_Robbins. An individual cell is constantly exposed to a remarkable cacophony of signals, which must be “interpeted” and integrated into responses that benefit the organism as a whole. Some signals may induce a given cell type to differentiate, others may stimulate proliferation, and yet others may direct the cell to perform a specialized function. Multiple signals received in combination may trigger yet another totally unique response. Many cells require certain inputs just to continue living; in the absence of appropriate exogenous signals, they die by apoptosis. The sources of the signals that most cells respond to can be classified into several groups: Pathogens and damage to neighboring cells. Many cells have an innate capacity to sense and respond to damaged cells (danger signals), as well as foreign invaders such as microbes. The receptors that generate these danger signals are discussed in Chapters 3 and 5.


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Pathology_Robbins. Cell-cell contacts, mediated through adhesion molecules and/or gap junctions. As mentioned previously, gap junction signaling is accomplished between adjacent cells via hydrophilic connexons that permit the movement of small ions (e.g., calcium), various metabolites, and potential second messenger molecules such as cAMP. Cell-ECM contacts, mediated through integrins, which are discussed in Chapter 3 in the context of leukocyte attachment to other cells during inflammation. Secreted molecules. The most important secreted molecules include growth factors, discussed later; cytokines, a term reserved for mediators of inflammation and immune responses (also discussed in Chapters 3 and 5); and hormones, which are secreted by endocrine organs and act on different cell types (Chapter 20). Signaling pathways also can be classified into different types based on the spatial relationships between the sending and receiving cells:


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Pathology_Robbins. Signaling pathways also can be classified into different types based on the spatial relationships between the sending and receiving cells: Paracrine signaling. Cells in just the immediate vicinity are affected. Paracrine signaling may involve trans-membrane “sending” molecules that activate receptors on adjacent cells or secreted factors that diffuse for only short distances. In some instances, the latter is achieved by having secreted factors bind tightly to ECM. Autocrine signaling occurs when molecules secreted by a cell affect that same cell. This can serve as a means to entrain groups of cells undergoing synchronous differentiation during development, or it can be used to amplify (positive feedback) or dampen (negative feedback) a response. Synaptic signaling. Activated neurons secrete neurotransmitters at specialized cell junctions (synapses) onto target cells. Endocrine signaling. A hormone is released into the bloodstream and acts on target cells at a distance.


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Pathology_Robbins. Endocrine signaling. A hormone is released into the bloodstream and acts on target cells at a distance. Regardless of the nature of an extracellular stimulus (paracrine, synaptic, or endocrine), the signal it conveys is transmitted to the cell via a specific receptor protein. Signaling molecules (ligands) bind their respective receptors and initiate a cascade of intracellular events culminating in the desired cellular response. Ligands usually have high affinities for receptors and at physiologic concentrations bind receptors with exquisite specificity. Receptors may be present on the cell surface or located within the cell ( Fig. 1.12


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Pathology_Robbins. Fig. 1.12 Intracellular receptors include transcription factors that are activated by lipid-soluble ligands that easily transit plasma membranes. Examples include vitamin D and steroid hormones, which activate nuclear hormone receptors. In other settings, a small and/or nonpolar signaling ligand can diffuse into adjacent cells. Such is the case for nitric oxide (NO), through which endothelial cells regulate intravascular pressure. NO is generated by an activated endothelial cell and then diffuses into adjacent vascular smooth muscle cells; there it activates guanylyl cyclase to generate cyclic GMP, an intracellular second signal that causes smooth muscle relaxation.


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Pathology_Robbins. Cell-surface receptors are generally transmembrane proteins with extracellular domains that bind activating ligands. Depending on the receptor, ligand binding may (1) open ion channels (typically at the synapse between electrically excitable cells), (2) activate an associated GTP-binding regulatory protein (G protein), (3) activate an endogenous or associated enzyme, often a tyrosine kinase, or (4) trigger a proteolytic event or a change in protein binding or stability that activates a latent transcription factor. Activities (2) and (3) are associated with growth factor signaling pathways that drive cell proliferation, whereas activity (4) is a common feature of multiple pathways (e.g., Notch, Wnt, and Hedgehog) that regulate normal development. Understandably, signals transduced by cell surface receptors are often deranged in developmental disorders and in cancers.


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Pathology_Robbins. Binding of a ligand to a cell surface receptor mediates signaling by inducing clustering of the receptor (receptor http://ebooksmedicine.net Transcription of nuclear Transcription of Transcription A Nucleus hormone target gene notch target gene Inactivation by hydrolysis of GTP Activation of transcription


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Fig.

Pathology_Robbins. Fig.


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Pathology_Robbins. 1.12 Receptor-mediated signaling. (A) Categories of signaling receptors, including receptors that use a nonreceptor tyrosine kinase; a receptor tyrosine kinase; a nuclear receptor that binds its ligand and can then influence transcription; a seven-transmembrane receptor linked to heterotrimeric G proteins; Notch, which recognizes a ligand on a distinct cell and is cleaved yielding an intracellular fragment that can enter the nucleus and influence transcription of specific target genes; and the Wnt/Frizzled pathway where activation releases intracellular β-catenin from a protein complex that normally drives its constitutive degradation. The released β-catenin can then migrate to the nucleus and act as a transcription factor. Lrp5/Lrp6, low-density lipoprotein (LDL) receptor-related proteins 5 and 6, are highly homologous and act as coreceptors in Wnt/Frizzled signaling. (B) Signaling from a tyrosine kinase-based receptor. Binding of the growth factor (ligand) causes receptor dimerizati


Pathology_Robbins

Pathology_Robbins. s 5 and 6, are highly homologous and act as coreceptors in Wnt/Frizzled signaling. (B) Signaling from a tyrosine kinase-based receptor. Binding of the growth factor (ligand) causes receptor dimerization and autophosphorylation of tyrosine residues. Attachment of adapter (or bridging) proteins couples the receptor to inactive, GDP-bound RAS, allowing the GDP to be displaced in favor of GTP and yielding activated RAS. Activated RAS interacts with and activates RAF (also known as MAP


Pathology_Robbins

Pathology_Robbins. kinase kinase kinase). This kinase then phosphorylates mitogen-activated protein kinase (MAPK), and activated MAP kinase phosphorylates other cytoplasmic proteins and nuclear transcription factors, generating cellular responses. Activated RAS can also interact with other components, such as phosphatidyl 3-kinase (PI3 kinase), which activates other signaling systems. The cascade is turned off when the activated RAS eventually hydrolyzes GTP to GDP converting RAS to its inactive form. Mutations in RAS that lead to delayed GTP hydrolysis can thus lead to augmented proliferative signaling. GDP, Guanosine diphosphate; GTP, guanosine triphosphate; mTOR, mammalian target of rapamycin.


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Pathology_Robbins. Fig. 1.12 ). The common theme is that all of these perturbations cause a change in the physical state of the intracellular domain of the receptor, which then triggers additional biochemical events that lead to signal transduction. Cellular receptors are grouped into several types based on the signaling mechanisms they use and the intracellular biochemical pathways they activate ( Fig. 1.12 ). Receptor signaling most commonly leads to the formation or modification of biochemical intermediates and/or activation of enzymes, and ultimately to the generation of active transcription factors that enter the nucleus and alter gene expression:


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Pathology_Robbins. Receptors associated with kinase activity. Downstream phosphorylation is a common pathway of signal transduction. Changes in receptor geometry can stimulate intrinsic receptor protein kinase activity or promote the enzymatic activity of recruited intracellular kinases. These kinases add charged phosphate residues to target molecules. Tyrosine kinases phosphorylate specific tyrosine residues, whereas serine/threonine kinases add phosphates to distinct serine or threonine residues, and lipid kinases phosphorylate lipid substrates. For every phosphorylation event, there is also a potential counter-regulatory phosphatase, an enzyme that can remove the phosphate residue and thus modulate signaling; usually, phosphatases play an inhibitory role in signal transduction.


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Pathology_Robbins. Receptor tyrosine kinases (RTKs) are integral membrane proteins (e.g., receptors for insulin, epidermal growth factor, and platelet-derived growth factor [PDGF]); ligand-induced crosslinking activates intrinsic tyrosine kinase domains located in their cytoplasmic tails.


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Pathology_Robbins. Several kinds of receptors have no intrinsic catalytic activity (e.g., immune receptors, some cytokine receptors, and integrins). For these, a separate intracellular protein—known as a nonreceptor tyrosine kinase— interacts with receptors after ligand binding and phosphorylates specific motifs on the receptor or other proteins. The cellular homolog of the transforming protein of the Rous sarcoma virus, called SRC, is the prototype for an important family of such nonreceptor tyrosine kinases (Src-family kinases). SRC contains unique functional regions called Src-homology (SH) domains; SH2 domains typically bind to receptors phosphorylated by another kinase, allowing the aggregation of multiple enzymes, whereas SH3 domains mediate protein–protein interactions, often involving proline-rich domains.


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Pathology_Robbins. G-protein coupled receptors are polypeptides that characteristically traverse the plasma membrane seven times (hence their designation as seven-transmembrane or serpentine receptors); more than 1500 such receptors have been identified. After ligand binding, the receptor associates with an intracellular guanosine triphosphate (GTP)-binding protein (G protein). At baseline, these G proteins contain guanosine diphosphate (GDP); interaction with a receptor-ligand complex results in G protein activation through the exchange of GDP for GTP. Downstream signaling typically involves the generation of cAMP, and inositol-1,4,5,-triphosphate (IP3), the latter releasing calcium from the ER. Nuclear receptors. Lipid-soluble ligands can diffuse into cells where they interact with intracellular proteins to form a receptor-ligand complex that directly binds to nuclear DNA; the results can be either activation or repression of gene transcription.


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Pathology_Robbins. Other classes of receptors. Other receptors—originally recognized as important for embryonic development and cell fate determination—have since been shown to participate in the functions of mature cells, particularly within the immune system. These pathways rely on protein:protein interactions, rather than enzymatic activities, to transduce signals, which may serve to allow for very precise control. Receptor proteins of the Notch family: ligand binding to Notch receptors leads to proteolytic cleavage of the receptor and subsequent nuclear translocation of the cytoplasmic domain (intracellular Notch) to form a transcription complex.


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Pathology_Robbins. Wnt protein ligands act through a pathway involving transmembrane Frizzled family receptors, which regulate the intracellular levels of β-catenin. In the absence of Wnt, β-catenin is targeted for ubiquitindirected proteasome degradation. Wnt binding to Frizzled (and other coreceptors) recruits other proteins that disrupt the degradation-targeting complex. This stabilizes β-catenin, allowing it to translocate to the nucleus and form a transcription complex. ModularSignalingProteins,Hubs,andNodes


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Pathology_Robbins. ModularSignalingProteins,Hubs,andNodes The traditional linear view of signaling—that receptor activation triggers an orderly sequence of biochemical intermediates that ultimately leads to changes in gene expression and the desired biological response—is oversimplified. Instead, it is increasingly clear that any initial signal results in multiple primary and secondary effects, each of which contributes in varying degrees to the final outcome. This is particularly true of signaling pathways that rely on enzymatic activities, which typically modulate a web of polypeptides with complex interactions. For example, phosphorylation of any given protein can allow it to associate with a host of other molecules, resulting in multiple effects such as: Nuclear (or cytoplasmic) localization of transcription factors (see later) Activation of feedback inhibitory (or stimulatory) loops


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Pathology_Robbins. Nuclear (or cytoplasmic) localization of transcription factors (see later) Activation of feedback inhibitory (or stimulatory) loops Adaptor proteins play a key role in organizing intracellular signaling pathways. These proteins function as molecular connectors that physically link different enzymes and promote the assembly of complexes; adaptors can be integral membrane proteins or cytosolic proteins. A typical adaptor may contain a few specific domains (e.g., SH2 or SH3) that mediate protein–protein interactions. By influencing the proteins that are recruited to signaling complexes, adaptors can determine downstream signaling events. http://ebooksmedicine.net


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Pathology_Robbins. http://ebooksmedicine.net By analogy with computer networks, the protein– protein complexes can be considered nodes and the biochemical events feeding into or emanating from these nodes can be thought of as hubs. Signal transduction can therefore be visualized as a kind of networking phenomenon; understanding this higher-order complexity is the province of systems biology, involving a “marriage” of biology and computation.


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Pathology_Robbins. Most signal transduction pathways ultimately influence cellular function by modulating gene transcription through the activation and nuclear localization of transcription factors. Conformational changes of transcription factors (e.g., following phosphorylation) can allow their translocation into the nucleus or can expose specific DNA or protein-binding motifs. Transcription factors may drive the expression of a relatively limited set of genes or may have much more widespread effects on gene expression. Among the transcription factors that regulate the expression of genes that are needed for growth are MYC and JUN, whereas a transcription factor that triggers the expression of genes that lead to growth arrest is p53. Transcription factors have a modular design, often containing domains that bind DNA and others that interact with other proteins, such as components of the RNA polymerase complex required for transcription.


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Pathology_Robbins. • DNA-binding domains permit specific binding to short DNA sequences. Whereas some transcription factor binding sites are found in promoters, close to the site where transcription starts, it is now appreciated that most transcription factors bind widely throughout genomes, including to long-range regulatory elements such as enhancers. Enhancers function by looping back to gene promoters, and therefore are spatially located close to the genes that they regulate, even though it terms of genomic sequence they may appear to be far Table 1.1 GrowthFactorsInvolvedinRegenerationandRepair away. These insights highlight the importance of chromatin organization in regulating gene expression, both normal and pathologic.


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Pathology_Robbins. Table 1.1 GrowthFactorsInvolvedinRegenerationandRepair away. These insights highlight the importance of chromatin organization in regulating gene expression, both normal and pathologic. • For a transcription factor to induce transcription, it must also possess protein:protein interaction domains that directly or indirectly recruit histone-modifying enzymes, chromatin-remodeling complexes, and (most importantly) RNA polymerase—the large multiprotein enzymatic complex that is responsible for RNA synthesis. A major role of growth factors is to stimulate the activity of proteins that are required for cell survival, growth and division. Growth factor activity is mediated through binding to specific receptors, ultimately influencing the expression of genes that can: Promote entry of cells into the cell cycle Enhance biosynthesis of cellular components (nucleic acids, proteins, lipids, carbohydrates) required for a mother cell to give rise to two daughter cells


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Pathology_Robbins. Promote entry of cells into the cell cycle Enhance biosynthesis of cellular components (nucleic acids, proteins, lipids, carbohydrates) required for a mother cell to give rise to two daughter cells Although some growth factors are proteins that “just” stimulate cell proliferation and/or survival, it is important to remember that they also can drive a host of other activities, including migration, differentiation, and synthetic capacity. Some of the important growth factors relevant to tissue regeneration and repair are listed in Table 1.1 and described further in Chapter 3. Growth factors can be involved in the proliferation of cells at steady state as well as after injury, when irreversibly damaged cells must be replaced. Uncontrolled Hepatocytegrowthfactor(HGF)Fibroblasts,stromalcellsintheliver,Enhancesproliferationofhepatocytesandotherepithelialcells;(scatterfactor)endothelialcellsincreasescellmotility


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Pathology_Robbins. Keratinocytegrowthfactor(KGF)FibroblastsStimulateskeratinocytemigration,proliferation,anddifferentiation(i.e.,FGF-7) ECM,Extracellularmembrane. http://ebooksmedicine.net proliferation can result when the growth factor activity is dysregulated, or when growth factor signaling pathways are altered to become constitutively active. Thus, many growth factor pathway genes are proto-oncogenes, and gain-of-function mutations in these genes can convert them into oncogenes capable of driving unfettered cell proliferation and tumor formation. The following discussion summarizes selected growth factors that are involved in the important proliferative processes of tissue repair and regeneration; by virtue of their proliferative effects they can also drive tumorigenesis. Although the growth factors described here all involve receptors with intrinsic kinase activity, other growth factors may signal through each of the various pathways shown in Fig. 1.12


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Pathology_Robbins. Fig. 1.12 Epidermal growth factor and transforming growth factor-α. Both of these factors belong to the EGF family and bind to the same receptors, explaining their shared biologic activities. EGF and TGF-α are produced by macrophages and a variety of epithelial cells, and are mitogenic for hepatocytes, fibroblasts, and a host of epithelial cells. The “EGF receptor family” includes four membrane receptors with intrinsic tyrosine kinase activity; the best-characterized is EGFR1, also known as ERB-B1, or simply EGFR. EGFR1 mutations and/or amplification frequently occur in a number of cancers including those of the lung, head and neck, breast, and brain. The ERBB2 receptor (also known as HER2) is overexpressed in a subset of breast cancers. To treat malignancies, many of these receptors have been successfully targeted by antibodies and small molecule antagonists.


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Pathology_Robbins. Hepatocyte growth factor. Hepatocyte growth factor (HGF; also known as scatter factor) has mitogenic effects on hepatocytes and most epithelial cells. HGF acts as a morphogen during embryonic development (i.e., it influences the pattern of tissue differentiation), promotes cell migration (hence its designation as scatter factor), and enhances hepatocyte survival. HGF is produced by fibroblasts and most mesenchymal cells, as well as endothelium and nonhepatocyte liver cells. It is synthesized as an inactive precursor (pro-HGF) that is proteolytically activated by serine proteases released at sites of injury. The receptor for HGF is MET, which has intrinsic tyrosine kinase activity. It is frequently overexpressed or mutated in tumors, particularly renal and thyroid papillary carcinomas. Consequently, MET inhibitors are being evaluated as cancer therapies.


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Pathology_Robbins. Platelet-derived growth factor. PDGF is a family of several closely related proteins, each consisting of two chains (designated by pairs of letters). Three isoforms of PDGF (AA, AB, and BB) are constitutively active, while PDGF-CC and PDGF-DD must be activated by proteolytic cleavage. PDGF is stored in platelet granules and is released on platelet activation. Although originally isolated from platelets (hence the name), it also is produced by many other cells, including activated macrophages, endothelium, smooth muscle cells, and a variety of tumors. All PDGF isoforms exert their effects by binding to two cell surface receptors (PDGFR α and β), both having intrinsic tyrosine kinase activity. PDGF induces fibroblast, endothelial, and smooth muscle cell proliferation and matrix synthesis, and is chemotactic for these cells (and inflammatory cells), thus promoting recruitment of the cells into areas of inflammation and tissue injury.


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Pathology_Robbins. Vascular endothelial growth factor. Vascular endothelial growth factors (VEGFs)—VEGF-A, -B, -C, and -D, and PIGF (placental growth factor)—are a family of homodimeric proteins. VEGF-A is generally referred to simply as VEGF; it is the major factor responsible for angiogenesis, inducing blood vessel development, after injury and in tumors. In comparison, VEGF-B and PIGF are involved in embryonic vessel development, and VEGF-C and -D stimulate both angiogenesis and lymphatic development (lymphangiogenesis). VEGFs also are involved in the maintenance of endothelial cells lining mature vessels. Its expression is highest in epithelial cells adjacent to fenestrated endothelium (e.g., podocytes in the kidney, pigment epithelium in the retina, and choroid plexus in the brain). VEGF induces angiogenesis by promoting endothelial cell migration and proliferation (capillary sprouting), and the formation of the vascular lumina. VEGFs also induce vascular dilation and increase vascular


Pathology_Robbins

Pathology_Robbins. angiogenesis by promoting endothelial cell migration and proliferation (capillary sprouting), and the formation of the vascular lumina. VEGFs also induce vascular dilation and increase vascular permeability. As might be anticipated, hypoxia is the most important inducer of VEGF production through pathways that involve activation of the transcription factor hypoxia-inducible factor (HIF-1). Other VEGF inducers—produced at sites of inflammation or wound healing—include PDGF and TGF-α.


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Pathology_Robbins. VEGFs bind to a family of receptor tyrosine kinases (VEGFR-1, -2, and -3). VEGFR-2 is highly expressed in endothelium and is the most important for angiogenesis. Antibodies against VEGF are approved for the treatment of several tumors such as renal and colon cancers because cancers require angiogenesis for their spread and growth. Anti-VEGF antibodies also are used in the treatment of a number of ophthalmic diseases, including: “wet” age-related macular degeneration (AMD a disorder of inappropriate angiogenesis and vascular permeability that causes adult-onset blindness); the retinopathy of prematurity; and the leaky vessels that lead to diabetic macular edema. Finally, increased levels of soluble versions of VEGFR-1 (s-FLT-1) in pregnant women may contribute to preeclampsia (hypertension and proteinuria) by “sopping up” the free VEGF required for maintaining normal endothelium.


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Pathology_Robbins. Fibroblast growth factor. Fibroblast growth factor (FGF) is a family of growth factors with more than 20 members. Acidic FGF (aFGF, or FGF-1) and basic FGF (bFGF, or FGF-2) are the best characterized; FGF-7 is also referred to as keratinocyte growth factor (KGF). Released FGFs associate with heparan sulfate in the ECM, which serves as a reservoir for inactive factors that can be subsequently released by proteolysis (e.g., at sites of wound healing). FGFs transduce signals through four tyrosine kinase receptors (FGFR 1–4). FGFs contribute to wound healing responses, hematopoiesis, and development; bFGF has all the activities necessary for angiogenesis as well.


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Pathology_Robbins. Transforming growth factor-β. TGF-β has three isoforms (TGF-β1, TGF-β2, and TGF-β3) that belong to a family with about 30 members, including bone morphogenetic proteins (BMPs), activins, inhibins, and Müllerian inhibiting substance. TGF-β1 has the most widespread distribution, and it is more commonly referred to simply as http://ebooksmedicine.net


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Pathology_Robbins. TGF-β. It is a homodimeric protein produced by multiple cell types, including platelets, endothelium, and mononuclear inflammatory cells. TGF-β is secreted as a precursor that requires proteolysis to yield the biologically active protein. There are two TGF-β receptors, both with serine/threonine kinase activity that induces the phosphorylation of several downstream cytoplasmic transcription factors called Smads. Phosphorylated Smads form heterodimers with Smad4, allowing nuclear translocation and association with other DNA-binding proteins to activate or inhibit gene transcription. TGF-β produces multiple and often opposing effects depending on the tissue type and concurrent signals. Agents with such multiplicity of effects are called pleiotropic, and TGF-β is “pleiotropic with a vengeance.” Primarily, TGF-β drives scar formation by stimulating matrix synthesis through decreased matrix metalloproteinase (MMP) activity and increased activity of tissue inhibitors of proteinases (TIMPs).