Extracellular Matrix Molecules Fibronectin

Biochemical Properties. Fibronectins are glycoproteins consisting of two disulfide-linked subunits with a molecular weight of 220,000-250,000 kDa. Fibronectin was originally purified to homogeneity in 1970 by Mosesson and Umfleet from plasma cryoprecipitate and termed cold-insoluble globulin (CIg) (1). Later, fibronectin was detected as a cell surface protein on fibroblasts whose expression was lost upon transformation (2,3). In addition to existing as a disulfide-linked dimer, cell surface-associated fibro-nectin also exists as higher molecular weight disulfide-linked aggregates that are secreted by cells and accumu late as a major component of the basement membrane and ECM in vivo.

The primary structure of fibronectin consists of a series of three different types of modular units that combine to generate the functional domains of the protein. The two subunits of fibronectin are connected by a pair of interchain disulfide linkages at the carboxy-terminal end. The intact dimer appears as an extended, flexible molecule consisting of two linear arrays of globular domains. The identification and characterization of the individual globular domains has resulted largely from examination of proteo-lytic fragments of fibronectin, many of which retain biological function. The N-terminal domain of fibronectin binds fibrin (4-6), heparin (6-8), Staphylococcus aureus (8-11), thrombospondin (12), factor XIIIa transglutaminase (4,11), and IgG (13). Adjacent to the amino-terminal domain of fibronectin is a domain that binds gelatin and collagen (7,14). This interaction is important for localization of fibronectin to basement membranes and ECMs. The collagen-binding domain also binds to the C1q component of complement via the collagenous tail of C1q (15). Adjacent to the gelatin-binding domain of fibronectin is the cell-binding domain. Pierschbacher et al. (16) showed that the cell-binding activity of fibronectin resided within a 12-kDa proteolytic fragment that contains the tetrapeptide sequence Arg-Gly-Asp-Ser (RGDS) (17). Peptides containing soluble synthetic Arg-Gly-Asp (RGD) were shown to detach cultured cells and platelets from fibronectin substrates (18,19), implicating the RGDS sequence in cell binding.

In addition to binding collagen, fibronectin has an affinity for several other components of the ECM, including the glycosaminoglycans heparin, heparan sulfate and chon-droitin sulfate proteoglycan, and hyaluronic acid (6,2025). The interaction of fibronectin with these various components of the ECM is thought to play a role in the formation and maintenance of the ECM.

The high-affinity cell surface receptors for fibronectin are the integrins, which consist of heterodimeric glycopro-tein a- and /-subunits of approximately 140 and 120 kDa that bind to the RGD sequence of fibronectin (26). The extracellular domains mediate cell adhesion via binding to fibronectin and other ECM proteins. The cytoplasmic domains are responsible for transmitting signals to the cell interior following ligand engagement (outside-in signaling), and for modulating the affinity of the integrin for its ligand by interacting with intracellular signaling molecules (inside-out signaling). The cytoplasmic domains also interact with the actin-binding proteins filamin (27,28) and a-actinin (29), thereby connecting the cell membrane with the cytoskeleton. These interactions play a role in the formation of focal adhesions and in phagocytosis.

Fibronectin plays a central role in vertebrate development. Fibronectin matrices are utilized by migrating cells during embryogenesis (30-32). The migration of avian, amphibian, and mammalian neural crest cells is blocked by RGD-containing peptides and by antibodies to fibronec-tin or its cell surface receptor (33,34). Furthermore, inac-tivation of the fibronectin gene in mice leads to early embryonic lethality (35). Its role in development and in maintenance of the adult organism is due to its diverse influences on cell growth, adhesion, migration, and differentiation.

Role in Cell Adhesion. Fibronectin promotes the attachment of multiple cell types, including epithelial cells, fol-licular cells, ovary cells, neuronal cells, fibroblasts, kerati-nocytes, and adult liver cells. As previously mentioned, fibronectin is not solely an attachment factor for cells. In many cases, cell attachment to fibronectin is required for cell survival and promotion of cell growth. Fibronectin promotes cell division of numerous cell types, including follic-ular cells (36), primary chick fibroblasts (37), murine and human fibroblasts (38), neuroblastoma cells (39), kerati-nocytes (40), rat granulosa cells (41), and lymphocytes (42). Fibronectin also promotes DNA synthesis by serum-deprived quiescent normal hamster fibroblasts (43).

In addition to regulating cell division, fibronectin has been shown to modulate the differentiation of cells in culture. Fibronectin coating of culture surfaces inhibits morphological changes and biosynthesis of lipogenic enzymes associated with the differentiation of preadipocyte fibro-blasts (44,45). Fibronectin also attenuates hormone-induced expression of cytoskeletal proteins associated with granulosa cell differentiation (46). Additionally, exoge-nously added fibronectin inhibits both spontaneous and chemically induced differentiation of cultured normal human keratinocytes in vitro (47,48). Alternatively, fibronec-tin induces the differentiation of neural crest cells, neu-roepithelial embryonal carcinoma cells (F9), and neuroblastoma cells in vitro (49-51), consistent with observations showing that receptors for fibronectin are found on neural crest cell derivitives colocalized with fibronectin (52) in vivo. Fibronectin also promotes the differentiation of primary human hepatocytes, as measured by the expression of albumin and the liver-specific enzyme cyto-chrome P450 (53). It has been suggested that modulation of cell growth and differentiation by fibronectin is due in part to its role in regulating the cytoskeletal architecture of cells (36,50). The recent cloning of fibronectin receptors and identification of signaling pathways coupled to these receptors has provided evidence that specific signaling events play an important role in controlling cell growth and differentiation.

Collagen

Biochemical Properties. Collagen is the most abundant component of the ECM and has been used extensively as an attachment factor for cultured cells. Collagen is composed of three helical a-chains that are wrapped around each other in a triple helical arrangement. In some forms of collagen the three chains are identical, whereas in other forms the chains differ from one another. The a-chains are composed of a series of triplet Gly-X-Y residues, where X and Y may be any amino acid. Frequently, X is a proline and Y is a hydroxyproline. Alternatively, hydroxylysine may also occupy the X or Y position and contribute to interchain cross-linking of the collagen a-chains. Collagen is secreted from cells as procollagen, with each of the a-chains containing large amino- and carboxy-terminal non-helical domains. Although some collagens exist in vivo as intact procollagen, many forms of collagen undergo proteo-lytic removal of the amino- and carboxy-terminal nonhel-ical domains.

To date, more than 14 collagen types have been described, which are generally subgrouped into six classes defined by the structural forms in which they assemble within the ECM. Several forms of collagen display ordered fibrous structures. For example types I, II, III, V, and XI collagen form fibrillar structures that contribute to connective tissues such as cartilage, skin, bone, and tendon, whereas type IX and XII collagens are fibril associated and interact with type I and II collagen fibrils. Type IV collagen is a nonfibrillar collagen and is the principle collagen type found in basement membrane. Type IV collagen differs from the fibrillar collagens by its existence in tissues in an unprocessed procollagen form (54,55). Other collagen types include filamentous collagen (type VI), short-chain collagens (types VIII and X), and long-chain collagen (type VII).

Collagen matrices are associated with many cell attachment proteins in vivo, including fibronectin and laminin. Thus the role of collagen as an attachment factor may be divided into two types: (1) a direct mechanism whereby collagen serves as the substrate for cell attachment (VLA-2; collagen receptor); and (2) an indirect mechanism whereby a secondary attachment factor, through its interaction with collagen, serves as the substrate for cell attachment.

Role in Cell Adhesion. The principle collagens used as attachment factors in cell culture are the fibrillar collagens and the nonfibrillar type IV collagen. Fibrillar collagen isolated from connective tissues was the first collagen used in cell culture. A mouse tumor source of type IV collagen was subsequently identified, which produced large amounts of this collagen type (56,57). Collagen substrates serve as an attachment and growth-promoting factor for a variety of cells in culture. Collagen promotes the sustained growth of epithelial cells from various tissues including mammary epithelial cells (58-61), endometrial epithelial cells (62), vaginal epithelial cells (63), esophageal epithelial cells (64), and liver epithelial cells (65). Collagen substrates also promote the extended viability of primary chondrocytes

(66). Chondrocytes exhibit three-dimensional growth within collagen gels, deposit ECM, and incorporate or rearrange exogenous collagen into newly formed matrix, suggesting the formation of tissuelike structures.

In addition to providing attachment and growth-promoting activity, collagen also modulates the differentiation state of cells in culture. Collagen induces morphological and biochemical characteristics associated with the differentiated state of various epithelial cell types including human endometrial cells (62), trachial epithelial cells

(67), uterine epithelial cells (68), mammary epithelial cells (69), and intestinal epithelial cells (70). Collagen also promotes the differentiation of adrenocortical cells (71,72), osteoblasts (73), keratinocytes (74), hepatocytes (75-79), liver dendritic cell progenitors (80), thyroid follicle cells (81), smooth muscle cells (82), and Sertoli cells (83). Regulation of growth and differentiation, which is accompanied by alterations in gene expression, implicate collagen as an active signal-transducing molecule. Candidate receptors for collagen that may play a role in cell signaling include the integrins VLA-1 (84,85), VLA-2 (86-88), and VLA-3 (86-88).

Laminin

Biochemical Properties Laminin is the first extracellular protein expressed during development and plays a critical role in cellular development and tissue organization through modulation of cellular attachment, motility, growth, and differentiation. Laminin is a large glycoprotein that is a major component of basement membranes. The prototypical laminin molecule is composed of three subunits: a 440-kDa A-chain, a 200-kDa Bl-chain, and a 220-kDa B2-chain. These are joined together by disulfide linkages to form a cruciform-shaped structure possessing multiple globular and rodlike domains that are responsible for its cell-binding activity and its interaction with a variety of ECM-associated molecules. The terminal end of the long arm, which contains a large, multilobed globular domain and forms the base of the cruciform, is responsible for receptor-mediated cell attachment, promotion of neu-rite outgrowth, and heparin binding (89). Regions within the short B-chain arms provide a second cell attachment site and also play a role in cell signaling and binding of the glycoprotein nidogen/entactin. Similar to fibronectin, laminin also binds to bacteria (90) and glycosaminoglycans

Laminin promotes cell attachment through interaction with specific cell surface receptors. The most extensively studied laminin receptors are the integrins. Laminin binds to at least four VLA integrins: a1ß1 (92,93), a2ß1 (87,94), a3ß1 (95), and a6ß1 (92,96). Laminin also binds to the aVß3 integrin (97) and a6ß4 integrin (98,99). The interaction of laminin with integrins is believed to occur through a non-RGD-mediated mechanism, although RGD-contain-ing peptides can inhibit cell adhesion to the second cell adhesion site located within the central portion of the laminin cruciform (96). Other recently identified receptors for laminin include the 67-kDa laminin receptor, which has been implicated in tumor metastasis (100,101), and a 110-kDa laminin-binding protein from brain, which appears to be immunologically and biologically related to the ß-amyloid precursor protein (APP) family (102). The interaction of the 110-kDa laminin-binding protein with the Ile-Lys-Val-Ala-Val (IKVAV) residues of the laminin A-chain plays a role in neurite outgrowth (102). A distinct 110-kDa protein, termed nucleolin, was copurified with the APP-related 110-kDa protein and has also been identified as a laminin-binding protein (103) that may play a role in ECM signaling. Laminin also binds to the actin-binding cell surface protein connectin (104) and the 67-kDa elastin/ laminin receptor (105).

Role in Adhesion. Laminin plays an important role in the attachment, growth, and differentiation of many cell types in culture, including neural and epithelial cells. When added to the culture medium, laminin promotes Schwann cell attachment, growth, and the formation of a stellate, process-bearing morphology (106) and regulates glycolipid synthesis (107). Laminin promotes attachment and neurite outgrowth of sensory neurons (108) and induces the differentiation of neuroblastoma cells (51). Antibodies directed against the heparin-binding domain of laminin reduce neuronal viability and inhibit neurite outgrowth in vitro, indicating that the heparin-binding domain is responsible for the effects of laminin on neurite outgrowth and neuronal survival (109). Laminin also modulates the proliferation of cultured astrocytes (110) and oligodendrocytes (111). Laminin promotes the differentiation of cultured endothelial cells to form capillary-like structures (112). Laminin also induces the differentiation of various epithelial cells, as measured by /-casein production by cultured mammary epithelial cells (113,114), the formation of cordlike structures by Sertoli cells (115), and the expression of alkaline phosphatase and lactase activity by intestinal epithelial cells (116). Laminin promotes the attachment and survival of F9 embryonal carcinoma cells (117), while it potentiates the differentiation of PCC4uva embryonal carcinoma cells to neurons following treatment with retinoic acid and dibutyryl cyclic AMP (118). Laminin also promotes the differentiation of granulosa cells (119) and hepatocytes (120,121).

Vitronectin

Biochemical Properties. Vitronectin, or serum-spreading factor, is a glycoprotein that is present in the blood and in other tissues. Vitronectin was initially described as a cell-spreading and growth-promoting a1-glycoprotein enriched in a fraction of human serum isolated by glass bead chromatography (122). The spreading-promoting activity of this fraction was further purified from this fraction by Barnes et al. (58) and was shown to be biochemically and immunologically distinct from fibronectin and laminin (123,124). Vitronectin exists in two distinct forms in human serum: a 75-kDa single-chain form and a 65-kDa two-chain form (125,126). The 65-kDa form is the product of proteolytic processing and lacks the carboxy-terminal 10-kDa fragment (127). Sensitivity to proteolytic processing may result from allelic differences in the vitronectin gene (128). The gene encoding vitronectin was identified by expression cloning (127), and sequence analysis revealed a 1,545-bp open reading frame corresponding to the full-length plasma vitronectin. Sequence analysis of an independently isolated serum protein associated with complement, termed S-protein, revealed it to be identical to vitronectin (129).

The major source of circulating vitronectin is the liver (130), although it is also produced by various hematopoetic cell types including platelets, monocytes, and macrophages

(131.132). Vitronectin is also associated with tissues

(124.133). In situ hybridization analysis indicates that vi-tronectin mRNA is expressed early in mammalian development primarily in the liver and central nervous system (134), suggesting that it plays an important role in mammalian development. In the mouse, vitronectin mRNA is detected by day 10 in the liver and central nervous system. Expression of vitronectin mRNA in the central nervous system is first detected in the floor plate; later, vitronectin mRNA can be observed in the meninges of the cortex and the spinal cord. Vitronectin mRNA is associated with the vasculature of the central nervous system but not with blood vessels of peripheral tissues, suggesting that vitro-nectin may play a specific role in vascular function in the central nervous system. Surprisingly, mice deficient in vi-tronectin gene expression demonstrate normal development and survival, indicating that the functional role of vitronectin in development may be compensated for by other molecules (135).

The vitronectin gene is composed of eight exons and seven introns (136), which define the domain structure of the mature protein. The amino-terminal cysteine-rich 44-residue domain is identical to somatomedin B (137). The second domain contains an RGD sequence that, analogous to fibronectin, is responsible for cell attachment via a number of integrins (137,138) and also contributes to collagen binding (139,140). The second domain is also implicated in binding to plasminogen-activator inhibitor-1, and consistent with its localization to platelets, this interaction plays an important role in blood clotting. Vitronectin also binds to heparin (125), and a domain located toward the carboxy-terminal region of vitronectin is responsible for the heparin-binding activity of vitronectin (137,141), which is dependent on the conformational state of vitronectin. Although native plasma vitronectin binds heparin weakly, vitronectin denatured by 8 M urea or other denaturing agents has an increased affinity for heparin (125,141). De-naturation is hypothesized to expose a cryptic heparin-binding domain and suggests that the biological functions of vitronectin are dependent on its conformation. Recently a form of vitronectin distinct from plasma vitronectin has been identified within platelet a-granules, which is able to bind heparin (142) and may play a role in platelet function at sites of vascular injury. Additionally, similar to collagen and fibronectin, vitronectin binds to bacteria, including S. aureus, Escherichia coli, S. pneumoniae, and Neisseria gonorrhoeae (143-147), implicating vitronectin in the adherence and phagocytosis of bacteria by host immune defense cells.

Role in Adhesion. Based on the tissue distribution of vitronectin, it is not surprising that vitronectin promotes the attachment and growth of a wide variety of cell types in vitro, including endothelial cells, lymphoid cells,and neural cells (58). Human endothelial cells attach to vitro-nectin, adopt a flattened morphology, and exhibit increased viability (148). Vitronectin promotes the attachment and proliferation of IL-3-dependent mast cells (149). However, vitronectin is unable to replace IL-3 as a survival factor, indicating that vitronectin is not itself a survival factor for bone marrow-derived mast cells but is able to augment the IL-3-dependent signal in mast cells. Vitronectin promotes the attachment of myeloblast cells through the av-contain-ing integrins (150) and stimulates megakaryocyte propla-telet formation through interaction with av//3 integrin (151). Vitronectin also promotes the attachment and long-term survival of cultured glioma cells (152), and this interaction may be mediated by gangliosides (153). In addition, vitronectin mediates attachment and nerve growth factor-dependent neurite outgrowth of rat peochromoccy-toma cells via an RGD-dependent mechanism (154).

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