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PubMed2002

Developmentally regulated interactions of human thymocytes with different laminin isoforms.

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.

CD4-Positive T-Lymphocytes↗

PubMed2003

Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line.

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an essential role in a wide array of cellular processes. The most well studied TGF-beta response in normal epithelial cells is growth inhibition. In some cell types, TGF-beta induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-beta, rendering NMuMG cells a good model system for studying these TGF-beta effects. METHOD: A National Institutes of Aging mouse 15,000 cDNA microarray was used to profile the gene expression of NMuMG cells treated with TGF-beta1 for 1, 6, or 24 hours. Data analyses were performed using GenePixPro and GeneSpring software. Selected microarray results were verified by northern analyses. RESULTS: Of the 15,000 genes examined by microarray, 939 were upregulated or downregulated by TGF-beta. This represents approximately 10% of the genes examined, minus redundancy. Seven genes previously not known to be regulated by TGF-beta at the transcriptional level (Akt and RhoB) or not at all (IQGAP1, mCalpain, actinin alpha3, Ikki, PP2A-PR53), were identified and their regulation by TGF-beta verified by northern blotting. Cell cycle pathway examination demonstrated downregulation of cyclin D2, c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. Examination of cell adhesion-related genes revealed upregulation of c-Jun, alpha-actinin, actin, myosin light chain, p120cas catenin (Catns), alpha-integrin, integrin beta5, fibronectin, IQGAP1, and mCalpain. CONCLUSION: Using a cDNA microarray to examine TGF-beta-regulated gene expression in NMuMG cells, we have shown regulation of multiple genes that play important roles in cell cycle control and EMT. In addition, we have identified several novel TGF-beta-regulated genes that may mediate previously unknown TGF-beta functions.

Animals↗

PubMed2005

Characterization of alphaX I-domain binding to Thy-1.

The beta2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alphaL subunit), CD11b (alphaM subunit), CD11c (alphaX subunit), or CD11d (alphaD subunit). alphaX-beta2 which binds several ligands including fibrinogen and iC3b is expressed in monocytes/macrophages and dendritic cells playing an important role in the host defense. Despite the unique characteristics on expression and regulation, alphaX-beta2 is less functionally characterized than other beta2 integrins. To understand the biological function of alphaX-beta2 more, we tested the possibility that alphaX-beta2 binds Thy-1, a membrane protein involved in cell adhesion and signaling regulation in neurons and T cells. Here we report that a ligand binding moiety of alphaX-beta2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K(D)) of alphaX I-domain binding to Thy-1 was 1.16muM and the affinity of the binding was roughly 2-fold higher than that of alphaM I-domain. Amino acid substitutions on the betaD-alpha5 of alphaX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha3-alpha4 and betaE-alpha6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a betaD-alpha5 loop, possibly alpha5 helix. Taken together, these results indicate that alphaX-beta2 specifically interacts with Thy-1. Additionally, kinetic analysis reveals a moderate affinity interaction in the presence of divalent cations. Given the reported role of Thy-1 in the regulation of T cell homeostasis and proliferation, it is tempting to speculate that alphaX-beta2 may be involved in Thy-1 function.

Animals↗

PubMed2000

Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)).

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.

Antibodies, Monoclonal↗

PubMed1998

Differential activities of secreted lymphotoxin-alpha3 and membrane lymphotoxin-alpha1beta2 in lymphotoxin-induced inflammation: critical role of TNF receptor 1 signaling.

Lymphotoxin (LT, LT alpha, TNF beta) is a member of the immediate TNF family that also includes TNF-alpha and lymphotoxin-beta (LT beta). LT is produced by activated lymphocytes and functions as either a secreted homotrimer or a membrane-associated heterotrimer that includes the transmembrane protein LT beta. Secreted LT alpha3 can bind to two cell surface receptors, TNFR1 and TNFR2, while the membrane-bound heterotrimer LT alpha1beta2 has been shown to interact with a distinct receptor, LT betaR. LT alpha induces inflammation at the sites of expression of a rat insulin promoter-driven lymphotoxin (RIPLT) transgene in the pancreas and kidney. To determine the role of the various ligands and their receptors in LT-induced inflammation, mice deficient in either TNFR1, TNFR2, or LT beta were crossed to RIPLT-transgenic mice. Our results indicate that LT alpha-induced inflammation is dependent on the interaction of LT alpha3 with TNFR1, and there is no obvious role for TNFR2, since in its absence, LT alpha-induced inflammation is quantitatively and qualitatively similar to that seen in the wild type. However, the absence of LT beta results in accentuated infiltration of the kidney with an increase in the proportion of memory cells in the infiltrate. These data show a crucial role for the secreted LT alpha3 signaling via TNFR1 in LT alpha-induced inflammation, and a separate and distinct role for the membrane LT alpha1beta2 form in this inflammatory process.

Animals↗

PubMed2006

Expression of laminin 5 by parental and c-Ha-ras-transformed HaCaT keratinocytes in organotypic cultures.

Tumor cells traverse the basement membrane zone and gain access to the underlying mesenchyme to eventually form metastases. Laminin 5 is a major component of the basement membrane and connects keratinocytes at the level of hemidesmosomes to the mesenchyme. Underneath invading tumor cells anti-laminin 5 staining is diminished, and laminin 5 degradation products can stimulate cell migration and epidermal growth factor (EGF) receptor signaling. To investigate laminin 5 expression in parental HaCaT and tumorigenic c-Ha-ras-transformed HaCaT II-4rt keratinocytes, the cells were cultivated under monolayer and organotypic culture conditions. In monolayer cultures, HaCaT and c-Ha-ras-transformed HaCaT II-4rt keratinocytes secreted comparable amounts of laminin 5. After 7 days of organotypic cultures, collagen IV, beta4-integrin, nidogen and laminin 5 were detected along the epithelial-mesenchymal interface of parental HaCaT keratinocytes, while staining for these proteins was patchy or absent in the organotypic cultures with c-Ha-ras-transformed HaCaT II-4rt cells. Immunoblotting analysis confirmed absence of laminin 5 deposition in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt while the protein was detected in organotypic cultures of HaCaT keratinocytes. Surprisingly, however, the alpha3 and gamma2 laminin chain transcripts were strongly induced in c-Ha-ras-transformed HaCaT II-4rt cells by organotypic culture conditions, indicating that invasive epidermal tumor cells retain high mRNA levels for laminin 5 chains and suggesting an autocrine/paracrine induction of the laminin chain mRNAs. Moreover, as laminin 5 was absent in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt cells, it suggests immediate degradation of the protein. Degradation products may further contribute to the malignant phenotype by enhancing cellular migration and EGF-receptor activation.

Cell Adhesion Molecules↗

PubMed2000

Mouse keratinocytes immortalized with large T antigen acquire alpha3beta1 integrin-dependent secretion of MMP-9/gelatinase B.

Remodeling of the extracellular matrix during tissue development, wound repair and tumor cell invasion depends on the coordinated regulation of cell adhesion receptors, matrix proteins and enzymes that proteolyse the extracellular matrix. Integrin alpha3beta1 is a major receptor on epidermal keratinocytes for laminin-5 in the cutaneous basement membrane and is required for normal basement membrane organization during skin development. alpha3beta1 is also expressed at high levels in the majority of adherent transformed cells and in most tumors, and it could have similar roles in extracellular matrix remodeling during tumorigenesis and cell invasion. In the present study, we show that alpha3beta1 expression is required in immortalized mouse keratinocytes (MK) for the production of the matrix metalloproteinase MMP-9/gelatinase B, an MMP that is coexpressed with alpha3beta1 in epithelial cell carcinomas and during wound healing, and contributes to the invasive potential of some tumor cells. MMP-9 was expressed in MK cells derived from wild-type mice, but not in MK cells derived from alpha3-null mice. Reconstitution of alpha3beta1 expression in alpha3-null MK cells through transfection with the alpha3 subunit restored MMP-9 secretion, indicating an alpha3beta1-dependent pathway for MMP-9 production. alpha3beta1-dependent expression of MMP-9 was associated with the immortalized phenotype, since nonimmortalized, primary keratinocytes required soluble growth factors, but not alpha3beta1, for efficient expression of MMP-9. Our results suggest that an alpha3beta1-independent pathway(s) for MMP-9 production is suppressed in keratinocytes immortalized with large T antigen, and that an alpha3beta1-dependent pathway is required for sustained production of MMP-9 in the absence of other pathways.

Actins↗

PubMed2001

Investigation into the mechanism of the loss of laminin 5 (alpha3beta3gamma2) expression in prostate cancer.

Laminin 5 is a pivotal hemidesmosomal protein involved in cell stability, migration, and anchoring filament formation. Protein and gene expression of the alpha3, beta3, and gamma2 chains of laminin 5 were investigated in normal and invasive prostate carcinoma using immunohistochemistry, Northern analysis, and in situ hybridization. Laser capture microdissection of normal and carcinomatous glands, in conjunction with RNA amplification and reverse Northern analysis, were used to confirm the gene expression data. Protein and mRNA expression of all three laminin 5 chains were detected in the basal cells of normal glands. In contrast, invasive prostate carcinoma showed a loss of beta3 and gamma2 protein expression with variable expression of alpha3 chains. Despite the loss of protein expression, there was retention of beta3 and gamma2 mRNA expression as detected by in situ hybridization, Northern and reverse Northern analysis. Our findings imply that an altered mechanism of translation of beta3 or gamma2 mRNAs into functional proteins contributes to failure of anchoring filaments and hemidesmosomal formation. The resultant hemidesmosome instability or loss would suggest a less stable epithelial-stromal junction, increased invasion and migration of malignant cells, and disruption of normal integrin signaling pathways.

Carcinoma↗

PubMed2003

Biological activities of homologous loop regions in the laminin alpha chain G domains.

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.

Actins↗

PubMed1995

Human melanoma integrins contribute to arrest and stabilization potential while flowing over extracellular matrix.

To form distant metastases, tumour cells must stabilize adhesive interactions that prevent detachment at secondary sites. Primary receptor-ligand interactions alone may not maintain prolonged adhesive contacts without secondary events that lead to adhesion stabilization. Computerized imaging methods enable us to examine various substrates for: (i) the wall shear adhesion threshold (WSAT), a measure of the dynamic adhesive potential of tumour cells; (ii) the number of tumour cells that adhered; and (iii) the adhesion stabilization lag time (ASLT) or length of time required for tumour cells to stabilize adhesive contacts capable of withstanding high wall shear force (up to 100 dynes/cm2). The relative WSAT ratios found were: wheat germ agglutinin (WGA) > laminin > fibronectin > vitronectin > collagen I > collagen IV > von Willebrand factor (vWF) (the greater the shear rate the higher the adhesive potential). The relative stabilization ratios found were as follows: laminin < fibronectin < vitronectin < collagen IV < collagen I < vWF < WGA (shorter times correlate with greater stabilization potential). Stabilization data using fibronectin as a substrate correlated the best with metastatic potential. Using three melanoma lines of different metastatic potential semiquantitative reverse transcriptase-polymerase chain reaction (PCR) showed a two- to four-fold increase in alpha1, alpha3, alpha4, alpha5, alpha6, and ICAM-1 in the highly metastatic 70W cells compared to the MeWo and non-metastatic 3S5 melanoma cells. There were no differences in alphav, beta1 and beta3 levels among the three melanoma lines, and PCR products for alphaIIb, alpha2, CD36, or ICAM-2 were not detected. The 70W cells also had higher levels of alphax and beta2 (CD11/CD18 and p150 leukocyte antigen) than either the MeWo or 3S5 cells. The data indicate that melanoma cells exhibit differences in the adhesion properties under fluid shear and differences in the expression of adhesion components that correlate with their metastatic potential.

Base Sequence↗

PubMed1997

Patterns of intermediate filaments, VLA integrins and HLA antigens in a new human biliary epithelial cell line sensitive to interferon-gamma.

BACKGROUND/AIMS: Intra-hepatic bile ducts are the primary site of damage in several immunologically mediated liver diseases. However, immunological processes underlying biliary epithelial cell recognition by T lymphocytes are poorly understood. Therefore, a convenient in vitro model that could mimic these immunologic disorders would be of great interest. METHODS: A human cell line (HuGB) was established from a metastasis of gallbladder adenocarcinoma in the liver. Intermediate filament expression was analysed by immunostaining, and gamma-glutamyl transpeptidase and albumin secretion were measured. VLA integrin expression pattern, expression of HLA class I and II antigens and ICAM-1 protein were analysed by flow cytometry and their modulation by interferon-gamma was quantitated using a QIFIKIT commercial kit. RESULTS: Histological analysis showed high similarity between the initial gallbladder adenocarcinoma and the established cell line. Cytokeratins 8 and 19 and vimentin showed strong positive staining in the established cell line. Gamma-glutamyl transpeptidase was secreted by these cells while albumin expression was negative. HuGB cells also expressed VLA-alpha2, VLA-alpha3, VLA-alpha6, VLA-beta1, but not VLA-alpha1, VLA-alpha4 and NCAM, a pattern of adhesion molecule expression compatible with the biliary epithelium. Also, similar to the biliary epithelium found in normal liver, HuGB cells expressed abundant HLA class I but few HLA class II antigens. We found that the expression of HLA antigens and ICAM-1 protein were increased during interferon-gamma treatment of HuGB cell line. CONCLUSIONS: Both phenotypic and morphological characteristics of HuGB cells suggested their biliary origin. Sensitivity of HuGB cells to interferon-gamma suggests that this new cell line could represent a suitable model to investigate the up-regulation of membrane antigens occurring in immune diseases involving biliary epithelial cells.

Adenocarcinoma↗

PubMed2004

Laminin-5 in epithelial tumour invasion.

Laminin-5 (LN-5), consisting of alpha3-, beta3-, and gamma2-chains, is a component of the cell adhesion complex containing hemidesmosomes and anchoring fibrils. This protein is a major constituent of the extracellular matrix and has recently proved to be an invasion marker for epithelial cells in many immunohistochemical surveys, indicating that it is frequently expressed in the invading edges of epithelial tumour cells. Additionally, intracellular accumulation of monomeric gamma2-chains has been widely observed in the invasive carcinoma cells, but its mechanism was not entirely understood. Epithelial carcinoma cells prefer to adhere onto the LN-5-rich basement membranes using the specific integrins as receptors. Induction of cell migration is an important function of LN-5 and the enhanced activity is observed in its truncated form after proteolytic shedding of the N-terminal fragments of gamma2-chains. This processing was demonstrated to be mediated mainly by several kinds of matrix metalloproteinases. The degraded fragments of gamma2-chains, released from invading carcinomas, can be immunodetected in biological fluids and potentially utilized in the clinical diagnosis of various epithelial cancers. Here, we summarize the previous clinical investigations of LN-5 in epithelial tumour progression, and also discuss what it can regulate in the cell physiological events.

Animals↗

PubMed2000

Differential expression of the LAMB3 and LAMC2 genes between small cell and non-small cell lung carcinomas.

To identify genes differentially expressed between small cell lung carcinoma (SCLC) cells and non-SCLC cells, mRNA differential display was applied to 3 SCLC cell lines and 6 non-SCLC cell lines. The LAMB3 gene was identified as being expressed only in non-SCLC cells and not in SCLC cells. The LAMB3 gene encodes the laminin beta3 chain, which is a unique component of laminin-5. Laminin-5 is a heterotrimer protein consisting of the alpha3, beta3, and gamma2 chains, and another unique component of laminin-5 is the gamma2 chain encoded by the LAMC2 gene. RT-PCR analysis of the LAMB3 and LAMC2 genes in 45 lung cancer cell lines revealed that both the LAMB3 and LAMC2 genes were co-expressed in 21 of 32 non-SCLC cell lines (66%) but only in one of 13 SCLC cell lines (8%). Coexpression of the LAMB3 and LAMC2 genes was also observed in all 4 cases of primary non-SCLC cells examined but not in the corresponding non-cancerous lung cells. Since alpha6beta4 integrin, the specific laminin-5 binding receptor, is known to be expressed only in non-SCLC cells and not in SCLC cells, it was indicated that laminin-5 is a critical microenvironmental factor for the growth of non-SCLC cells but not of SCLC cells. The differences in the expression of integrins and laminins would be critical factors to distinguish SCLC and non-SCLC cells, and such differences might be associated with the unique biological properties of SCLC cells, including metastatic potential and drug sensitivity.

Carcinoma, Non-Small-Cell Lung↗

PubMed2004

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation.

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.

Cell Adhesion↗

PubMed2005

Regulation of cell adhesion and type VII collagen binding by the beta3 chain short arm of laminin-5: effect of its proteolytic cleavage.

The basement membrane protein laminin-5 (Lm5), a heterotrimer of alpha3 (or alpha3A), beta3, and gamma2 chains, regulates cellular adhesion and motility. Here we examined the proteolysis and biological function of the laminin beta3 chain. First, we found that the beta3 chain of Lm5 is cleaved at its N-terminal, short arm by an endogenous proteinase(s) in normal human keratinocytes and some other cell lines. To examine the effect of beta3 chain cleavage, we expressed a wild-type Lm5 and two Lm5 mutants with partially deleted beta3 chains in HEK293 cells. Experiments with the purified Lm5 forms demonstrated that the deletion of the beta3 short arm or its N-terminal domain LN decreases the cell adhesion activity of Lm5, but does not significantly affect the motility activity. A recombinant beta3 short arm protein enhanced integrin-mediated cell adhesion to Lm5 by binding to an unidentified cell receptor. It was also found that the laminin EGF-like domain of the beta3 short arm is a binding site for type VII collagen. These results suggest that the beta3 short arm is involved not only in the matrix assembly of Lm5, but also in its cell adhesion activity. The proteolytic cleavage of the beta3 chain may modulate these functions of Lm5 in vivo.

Cell Adhesion↗

PubMed1999

Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow.

We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.

Animals↗

PubMed2007

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10).

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.

Blotting, Western↗

PubMed2003

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of alpha3 chain.

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.

Amino Acid Sequence↗