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PubMed1995

A novel ligand for CD44 is serglycin, a hematopoietic cell lineage-specific proteoglycan. Possible involvement in lymphoid cell adherence and activation.

The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation.

Amino Acid Sequence↗
PubMed1995

Adhesive interactions between alternatively spliced CD44 isoforms.

Alternative splicing of a series of 10 contiguous exons present within the CD44 gene can generate a large number of differentially expressed CD44 isoforms that contain additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule proximal to the membrane spanning domain. Although distinct functions have been ascribed to certain of these isoforms, the effect of particular inserted domains on the ligand-binding specificity of the CD44 molecule remains unclear. In the present study, we demonstrate that while CD44H, the major CD44 isoform expressed on resting hemopoietic cells, and CD44R1, an alternatively spliced isoform present on transformed epithelial cells and certain activated and/or malignant hemopoietic cell types, can both bind avidly to hyaluronan, only CD44R1 can promote homotypic cellular aggregation when expressed in the CD44-negative murine lymphoma cell line TIL1. Experiments in which TIL1 cells transduced with different CD44 isoforms were tested for their ability to adhere to one another or to COS7 cells transfected with CD44R1, indicated that CD44R1 can recognize and bind a common determinant present on both CD44H and CD44R1. Monoclonal antibody blocking studies suggest further, that the determinant recognized by CD44R1 is located in a region of the CD44 molecule distinct from that involved in hyaluronan binding.

Alternative Splicing↗
PubMed1997

Widespread expression of chondroitin sulfate-type serglycins with CD44 binding ability in hematopoietic cells.

Serglycin is a family of small proteoglycans with Ser-Gly dipeptide repeats and is modified with various types of glycosaminoglycan side chains. We previously demonstrated that chondroitin sulfate-modified serglycin is a novel ligand for CD44 involved in the adherence and activation of lymphoid cells. In this study, we investigated the production and distribution of CD44 binding serglycins in various hematopoietic cells and characterized their carbohydrate side chains. Immunoprecipitation analysis using CD44-IgG and polyclonal antibody against the serglycin core peptide demonstrated that various serglycin species capable of binding CD44 are produced by a variety of hematopoietic cells including lymphoid cells, myeloid cells, and a few tumor cell lines. Glycosaminoglycans on these serglycins, which are essential for CD44 binding, are composed of chondroitin 4-sulfate or a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate, but no heparin or heparan sulfate side chain was detected. The serglycins are also secreted by normal splenocytes, lymph node lymphocytes, and bone marrow cells, whereas they are secreted in very small amounts by normal thymocytes. Secretion of serglycins is greatly enhanced by mitogenic stimulation with concanavalin A or lipopolysaccharide. Our results showed that serglycin, unlike hyaluronate, is produced and secreted in a functional (CD44 binding) form by many members of the hematopoietic system including various lymphocyte subsets. Our data suggest that serglycin may serve as a major ligand for CD44 in various events in the lymphohematopoietic system.

Animals↗
PubMed2000

Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.

The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.

Cell Differentiation↗
PubMed2000

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44.

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.

Animals↗
PubMed2001

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.

Animals↗
PubMed2005

Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.

Animals↗
PubMed2006

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Amino Acid Sequence↗
PubMed1997

Ultrastructural localisation of CD44 in the rat lung in experimental Goodpasture's syndrome.

Although CD44 is known to be involved in a wide array of cell to cell and cell to matrix interactions, its role in immune-mediated disease is not well understood. Therefore, using immunogold electron microscopy we have determined the precise localisation of CD44 in the rat lung in experimental Goodpasture's (GP) syndrome, a model of immune-mediated pulmonary disease. In normal rat lung CD44 was present on the surface of alveolar macrophages but was not detectable on endothelium. In GP syndrome there was strong CD44 expression on all infiltrating inflammatory leucocytes, both adherent to endothelium and within the alveolar spaces and interstitium. However the most striking finding was the progressively strong antibody staining for CD44 on pulmonary endothelium of alveolar capillaries and larger vessels over the 21 days of GP syndrome. In situ hybridisation confirmed that the endothelial CD44 staining was due to local protein synthesis. All epithelial cell surfaces, including bronchial epithelium and type I and II alveolar epithelial cells, were negative in normal rat lung and GP syndrome. De novo CD44 expression by endothelial cells during the progression of GP syndrome may contribute to leucocyte recruitment and cell-mediated lung injury.

Animals↗
PubMed1993

Regulated expression of exon v6 containing isoforms of CD44 in man: downregulation during malignant transformation of tumors of squamocellular origin.

CD44 is a family of glycoproteins involved in cell-cell and cell-matrix interactions. In addition to the major 90-kD form present on most hematopoietic cells, larger 140-230 kD forms are found on keratinocytes and carcinoma cell lines. These bigger isoforms of CD44 arise by alternative splicing that results in insertion of one or more of the "variant" exons into the extracellular part of the 90-kD constant form of the molecule. In rat, v6 (variant exon v6) containing form of CD44 confers metastatic potential to carcinoma cells, and therefore, it is of interest to study the distribution of this isoform in humans. We raised antibodies against a synthetic peptide containing a sequence encoded by the exon v6. A mAb thus obtained (designated Var3.1) strongly reacted with the plasma membranes of squamous cells in upper layers of skin and tonsil surface epithelia. Weaker staining was seen in germinal centers, vascular endothelia and enterocytes. Exon v6 containing forms of CD44 (CD44v6) were absent from tissue leukocytes and connective tissue components. In comparison, Hermes-3 epitope (on the constant part) containing forms of CD44 were preferentially localized in basal layers of epithelia, present on the surface on most leukocytes and connective tissue cells, and undetectable on the luminal surface of high endothelial venules. In benign neoplasms, epithelial cells stained with mAb Var3.1 like in normal tissues. In contrast, immunostaining of 30 squamous carcinoma specimens (both primary and metastatic lesions) revealed that malignant transformation resulted in downregulation or disappearance of Var3.1 epitope, but in majority of cases, not in diminished synthesis of the Hermes-3 epitope. Biochemical analyses showed that mAb Var3.1 recognized two major forms of CD44 (220 and 300 kD). In conclusion, epitopes on exon v6 and constant part of CD44 are differentially synthesized and regulated during normal and malignant growth of cells in man.

Adult↗
PubMed1994

Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function.

GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell-extracellular matrix interactions.

Amino Acid Sequence↗
PubMed1995

Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity.

CD44 splice variants have been shown to be involved in metastasis of carcinomas. In addition, the standard form of CD44 has been implicated in metastasis, particularly of melanomas and lymphomas. To investigate this, we have generated a CD44-negative mutant of the highly metastatic murine MDAY-D2 lymphosarcoma. The two CD44 alleles of this diploid cell line were sequentially disrupted by homologous recombination, using isogenic CD44 genomic constructs interrupted by a neomycin or hygromycin resistance-conferring gene. The resulting double knockout (DKO) cells had completely lost the capacity to bind to immobilized hyaluronic acid, but did not differ from MDAY-D2 cells in integrin expression or in vitro growth. Subcutaneous (s.c.) growth potential and metastatic capacity of MDAY-D2 and DKO cells were assessed by s.c. and i.v. injection of the lowest cell dose (10(3) or 10(4), respectively) that gave rise to tumor formation by MDAY-D2 cells in approximately 100% of the mice. Quite unexpectedly, we observed no difference at all in either s.c. growth rate or local invasion into surrounding tissues between MDAY-D2 cells and the CD44-negative DKO cells. Also hematogenous metastasis formation upon i.v. injection was similar: both parental and DKO cells metastasized extensively to the spleen, liver, and bone marrow. We conclude that, at least for these MDAY-D2 lymphosarcoma cells, the standard form of CD44 is dispensable for tumor growth and metastasis. Our results show that targeted disruption of genes in tumor cells is a feasible approach to study their role in tumorigenesis and metastasis.

Animals↗
PubMed1996

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

Apoptosis↗
PubMed2003

The liberation of CD44.

CD44 was once thought to simply be a transmembrane adhesion molecule that also played a role in the metabolism of its principal ligand hyaluronan. Investigations of CD44 over the past approximately 20 yr have established additional functions for CD44, including its capacity to mediate inflammatory cell function and tumor growth and metastasis. It has also become evident that intricate posttranslational modifications of CD44 regulate the affinity of the receptor for its ligands. In this review, we focus on emerging evidence that functional fragments of the cytoplasmic and ectodomain of CD44 can be liberated by enzymatic modification of cell surfaces as well as of cell-associated matrix. Based on the evidence discussed, we propose that CD44 exists in three phases, as a transmembrane receptor, as an integral component of the matrix, and as a soluble protein found in body fluids, each with biologically significant functions of which some are shared and some distinct. Thus, CD44 represents a model for understanding posttranslational processing and its emerging role as a general mechanism for regulating cell behavior.

Animals↗
PubMed1995

Glycosylation of CD44 negatively regulates its recognition of hyaluronan.

Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44.

Animals↗
PubMed2001

Feasibility of assessment of promoter methylation of the CD44 gene in serum of prostate cancer patients.

BACKGROUND AND PURPOSE: CD44 is an important metastasis-suppressor gene in prostate cancer. Downregulation of the CD44 gene is attributed to transcription repression by methylation of CpG islands in the promoter region. The feasibility of CD44 promoter methylation measurement as a diagnostic tool was assessed in the serum of patients with cancer of the prostate (CAP). MATERIALS AND METHODS: Seven serum samples of patients with CAP were investigated for CD44 promoter methylation by methylation-specific PCR. Three patients had proven metastatic disease, and four were free of metastases. Tissues from a variety of normal epithelia were assessed as well. RESULTS: Methylation of the CD44 promoter was readily detectable in all serum samples, although no distinction could be made between patients with and those without metastatic disease on the basis of the signal intensity of methylation-specific PCR products. Remarkably, tissue specimens from different normal epithelia, especially those of the colon and rectum, repeatedly showed aberrant methylation of the promoter region of CD44. CONCLUSIONS: In the serum of CAP patients, assessment of the methylation status of CpG islands in the promoter region of the CD44 gene is feasible using methylation-specific PCR. However, because of physiologic promoter methylation in normal tissues, including the colorectal mucosa, assessment of methylation of tumor-derived DNA in the serum of cancer patients lacks tissue specificity and seems not to be applicable in clinical settings.

CpG Islands↗
PubMed1995

Inhibition of HIV type 1 infection of mononuclear phagocytes by anti-CD44 antibodies.

Cellular CD4 is the primary membrane molecule that binds HIV-1 through interaction with viral gp120. Membrane glycolipids and cell adhesion molecules have also been noted to be involved in the interaction of HIV-1 with cells and in syncytium formation in infected cells. The purpose of this study was to determine the role of the cell adhesion molecule CD44 in HIV-1 infection of cells. Both normal blood monocytes and lymphocytes expressed CD44 as determined by flow cytometry using the anti-CD44 antibody A3D8. Anti-CD44 monoclonal antibodies A3D8, A1G3, and 5F12 [ascites, purified IgG, and F(ab')2] inhibited infection of monocytes and peritoneal macrophages with HIV-1-BaL and HIV-1-ADA, but had no effect on HIV-1-IIIB infection of mitogen-stimulated lymphocytes, or cells of a T lymphocyte line. CD44 monoclonal antibodies were not toxic for monocytes, and the observed inhibitory effect of CD44 monoclonal antibodies was not dependent on complement. These results suggest that CD44 may be a determinant of HIV-1 infection of mononuclear phagocytes in vitro.

Animals↗
PubMed1999

Functional hierarchy of simultaneously expressed adhesion receptors: integrin alpha2beta1 but not CD44 mediates MV3 melanoma cell migration and matrix reorganization within three-dimensional hyaluronan-containing collagen matrices.

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Antibodies, Monoclonal↗