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PubMed
CAVEAT LECTOR
Shravan K. Chintala, Ph.D. and Jasti S. Rao, Ph.D.
Department of Neurosurgery, Box 064, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Received 9/28/96; Accepted 10/17/96; On-line 11/01/96
3. EXTRACELLULAR MATRIX COMPONENTS
Proteoglycans and glycosaminoglycans (GAG) are abundantly present in the brain parenchyma. Proteoglycans contain a core protein with one or more covalently bound glycosaminoglycan side chains. The major proteoglycans in the CNS are chondroitin sulfate (CS) and heparan sulfate. CS is a polymer consisting of alternating units of N-acetylgalactosamine and glucuronic acid. In the mature brain, CS, is located in the cytoplasm of some neurons (5-7) and astrocytes, and in myelinated and unmyelinated axon fibers, but not in oligodendrocytes and myelin (8). In contrast, CS proteoglycans are the major proteoglycans of the white and gray matters of the brain, located predominantly in the extracellular spaces of granular and molecular layers in the immature cerebellum, and they are believed to be associated with cell differentiation and migration in the central nervous system (9). Both heparan sulfate and chondroitin sulfate are present in the basement membrane (10). Heparan sulfate is found as membrane protein in synaptic vesicles and in the ECM of the neuromuscular junction (11, 12), and it is also present in the basement membranes of the Schwann's cells (13). Heparan sulfate proteoglycans have been found to induce cell motility (14).
Hyaluronic acid (HA or hyaluronan), a high molecular-weight proteoglycan found in the extracellular matrix, is the only proteoglycan that does not contain a core protein. A polysaccharide comprised of repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine, HA is found in most extracellular matrices and at the cell surface. Brain parenchyma contain several other proteoglycans, such as tenascin and cytotactin, in addition to glial hyaluronate binding protein (GHAP or hyaluronectin).
Glycosaminoglycans are found mainly within tumor tissue (15); their hyaluronic acid content has been shown to increase transiently during tumor cell migration and is usually found at the interface between tumor mass and host tissues (16). Apart from its major role in tumor cell invasion, HA has been implicated in many cell functions including neural crest migration (17). Elevated levels of HA have been correlated with tumor cell invasiveness (18). HA interacts with other extracellular matrix proteins via hyaluronan binding proteins and receptors such as cluster differentiation 44 (CD44). Recent studies have demonstrated that suppression of CD44 expression decreases migration and invasion of human glioma cells (19). Further, intracranial injection of CD44-suppressed cells led to relatively localized tumor growth and with a reduced malignant behavior, whereas control cells showed extensive invasion typical of highly malignant gliomas (17). Koochekpour et al. (20) demonstrated that HA-mediated cell detachment involved its high-affinity receptors, CD44. They also showed that HA induces cell detachment, stimulates migration, and promotes invasion through interaction with CD44. In 1994, Merzak et al. (21) showed for the first time that CD44 is involved in glioma cell invasion. Thiery et al. (22) had reported that antisense CD44 oligonucleotides inhibited the invasion of glioma cells in vitro, and that several components of basement membrane molecules, such as fibronectin, laminin, vitronectin, and collagen I, might be involved in CD44-mediated invasion of human glioma cells. The proposal is that once synthesized, HA becomes hydrated, opening up the extracellular space to invading tumor cells. This process is suggested to be facilitated through CD44-HA interaction. The role of HA in glioma cell invasion is complicated however, by the fact that astrocytes synthesize HA themselves (23), rather than tumor cells stimulating host fibroblasts to make HA (24). Our own results showed that invasion of glioblastoma cells was significantly increased in the presence of HA (25).
Gliomas also disseminate along the myelinated fiber tracts of white matter, which results in distant spread of tumor cells through the corpus callosum into the contralateral hemisphere (26-28). Caroni and Schwab (29) showed that C6 rat glioma cells attach to and spread on crude extracts of CNS myelin, and that the spreading of C6 glioma cells on myelin extracts depends on the expression of a membrane-bound metalloendoprotease (30). More recently, Giese et al. (31) found that established glioma cell lines as well as primary cells isolated from glioblastoma biopsy specimens, attach and migrate on crude myelin extracts. Although these findings indicated that glioma cells migrate and spread along existing anatomical structures such as myelin, the responsible component/molecule of the crude myelin extract has not been identified. As Giese et al. (31) suggested, the three-dimensional conformation of the associated proteins could be changed during the migrating process, and this in turn could influence the behavior of the glioma cells. It seems, however, that neither neural cell adhesion molecules nor integrins mediate the adhesion of glioma cells to crude myelin extracts.
Fibronectin is an Mr 500,000 glycoprotein found in most extracellular matrices as aggregates or fibrils. Fibronectin consists of two polypeptide chains of approximately Mr 250,000 linked by interchain disulfide bonds. Fibronectin has three homologous repeats type I (about 45 amino acids long with two disulfide bonds), type II (about 60 residues with two disulfide bonds), and a type III (about 90 residues with no disulfide bonds). The first major adhesive protein identified, fibronectin has many biological functions involving cell adhesion, migration, and invasion. Fibronectin mediates a variety of adhesive events by binding to fibrinogen/fibrin, collagen, heparan sulfate, and hyaluronic acid. Fibronectin is found at the gliomesenchymal junction of tumors and in tumor-associated blood vessels, and it is expressed by glioblastoma cell lines in vitro (32, 33). An arg-gly-asp (RGD) sequence in the third fibronectin repeat functions as an integrin receptor for most cells (34). Kochi et al. (35) showed that astrocytomas and glioblastomas do not express fibronectin and that fibronectin was confined to proliferating vessel walls and leptomeninges.
Vitronectin, originally known as S-protein and primarily found in the liver, is a multicellular serum protein that promotes cell adhesion, spreading, and migration of a variety of cell types (36). Vitronectin is found mainly in serum, skin, and wound tissue (37, 38). Vitronectin is absent in normal brain and early-stage glioblastoma but late-stage glioblastomas have recently been shown to express vitronectin (39). Vitronectin supports cell adhesion through integrins. Apart from modulating plasminogen (40) and plasminogen activator inhibitor (PAI-I) (40-43) vitronectin promotes cell adhesion mediated by integrins alphavß3, alphavß5, alphaIIß5 and alphavß1. To date, alphavß3 integrin appears to be specific for vitronectin, by the finding that low to high metastatic potential of human melanoma was correlated with an increased expression of vitronectin receptor alphavß3 (44). Gladson and Cheresh (39) recently showed that vitronectin may play a role during local invasion of glioblastoma. A recent study showed that, compared to fibronectin, laminin, and collagen IV, vitronectin was a poor adhesive and migratory protein for glioblastoma cells U-251 and SF-767 (45).
Tenascins are large disulfide-linked heterodimeric extracellular glycoproteins. Tenascin-C, the original member of a family of heterodimeric extracellular matrix proteins, is implicated in adhesion and migration of human glioma cells (46). However, the recent discovery that tenascin knock-out mice develop normally (47) and the tenascin's contradictory effects on adhesion and migration (48-50) raised a controversy concerning their basic role. To date, three members of the tenascin family have been identified, tenascin-C, tenascin-R, and tenascin-X. All known forms of tenascins consist of heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III repeats and a fibrinogen domain. Tenascin-C (also known as myotendinous antigen, glioma mesenchymal extracellular matrix, hexabrachion, J1-200/220, and cytotactin) is mainly found during normal embyrogenesis; it is prominent in the developing central nervous system and in developing connective tissues, and it is overexpressed in tumors (51, 52). Tenascin-C is expressed in malignant breast carcinomas (53), in wound healing (54, 55) and during newt limb regeneration (56). Tenascin-C is upregulated in most types of carcinomas including melanomas and gliomas (57-59). In the central nervous system, tenascin-C is found to be synthesized by glial and neural crest cells (60-62) and by satellite cells of the peripheral nervous system (63). Tenascin-R (also known as J1-160/180, januscin, and restrictin), so far identified in rat and chicken, seems to be specific to the central and peripheral nervous system (64-65). Tenascin-X mainly expressed in skeletal and heart muscles, was originally reported as a partial sequence encoded by gene X (66), but little is known about its function. Although tenascin was categorized as an antiadhesive protein (67-69), recent data show that tenascin supports cell adhesion involving the RGD sequence in the third fibronectin type III repeat that it seems to be mediated by V3 integrin (70, 71). A recent study showed that tenascin enhances U251.3 glioma cell migration by an RGD independent mechanism (46).
Laminins are a large group of adhesion glycoproteins found in all basement membranes and in hyperplastic blood vessels in gliomas, gliosarcomas, and menigiomas as an integral part of the glial limitans externa (72,74). Laminin is a structural glycoprotein found predominantly in basement membranes (75-77). It plays a role in migration, neurite outgrowth, proliferation, and differentiation (78, 79). The first identified laminin was purified from Engelbreth-Holm-Swarm sarcoma, and recent literature identifies at least six other forms. All the seven known laminins are composed of three subunits designated as alpha, ß, and gamma and association of disulfide-linked subunits give rise to different laminins (80). Laminin interacts with a variety of basement membrane components such as entactin/nidogen, type IV collagen, and heparan sulfate (80). The other laminin isoforms known are laminin-2 (merosin), laminin-3 (S-laminin), laminin-4 (S-merosin), laminin-5 (kalinin/nicein/epiligrin), laminin-6 (K-laminin), and laminin-7 (Ks-laminin) (78, 80).
Interstitial collagens are located in the leptomeninges and the fibromuscular layer of large blood vessels in the brain. Type IV collagen, mainly present in capillaries and large blood vessels, is the principal collagenous constituent of most basement membranes. The most common molecular form of type IV collagen is a heteropolymeric molecule, [a1(IV)]2 a2 (IV); other types of homopolymeric forms, [a1(IV)]3 and [a2(IV)]3, occur as well, and three additional type IV collagen chains are known to exist in kidney (81). Type IV collagen is secreted and assembled as a procollagen molecule, in which, each chain has an apparent molecular weight of 160,000-180,000. In rotary shadowed electron microscope preparations, type IV collagen appears as a rod with a knob like non-helical domain at the COOH- terminal end. Two to four monomers can be associated to form network like appearance in most basement membranes. Recent studies have shown that the glioblastoma cells are also capable of synthesizing type IV collagen in vitro (33). In an immunohistochemical study, Bjerkvig et al., (82) showed that type IV collagen was strongly expressed in tumor spheroids from rat glioma cell line BT4C but was negative in monolayers, and fibronectin was strongly expressed in BT4C and BT4Cn cell lines. In an immunofluorescence study, Bellon et al., (83) demonstrated that type IV collagen was localized to the subendothelial basement membrane of blood vessels in gliomas. Similar results were reported earlier by Rutka et al., (84).