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PubMed2005

The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1 class.

ADAM Proteins↗
PubMed1998

Beta1 integrins participate in the hypertrophic response of rat ventricular myocytes.

Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that beta1 integrin function is involved in the alpha1-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti-beta1 integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of beta1 integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed beta1A integrin or the cardiac/skeletal muscle-specific beta1 isoform (beta1D) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free beta1 integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.

Adenoviridae↗
PubMed2005

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts.

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.

Calcium↗
PubMed1999

A distinct integrin-mediated phagocytic pathway for extracellular matrix remodeling by RPE cells.

PURPOSE: To characterize the phagocytosis of extracellular matrix components by retinal pigment epithelial cells and to determine which receptors and signal transduction pathways are involved. METHODS: Fluorescent latex beads were coated with fibronectin (FN), collagen type I or IV, or thrombospondin and incubated with human retinal pigment epithelial cells for 3 hours. Phagocytosis was quantified by flow cytometry. The effects of adhesion blocking antibodies to cell surface receptors (alpha1, alpha3, alpha5, beta1, alpha5beta1, alphavbeta3, alphavbeta5 integrins and CD36) and inhibitors of specific intracellular signaling pathways (tyrosine kinase phosphatidylinositol 3-kinase [PI3-kinase], protein kinase C [PKC], and mitogen-activated protein kinase) were determined using FN-coated beads. RESULTS: Phagocytosis of FN-coated beads was greater than phagocytosis of beads coated with collagen type I, collagen type IV, or thrombospondin or uncoated controls (P < 0.0005). Anti-alpha5, -beta1, and -alpha5beta1 antibodies markedly inhibited FN phagocytosis (P < 0.0005); the inhibitory effects of anti-alpha5 antibody were stronger in the initial stages (binding) than in the later stages (internalization) of phagocytosis. There was no significant effect on phagocytosis when anti-alpha1, -alpha3, -alphavbeta5, -alphavbeta3 or -CD36 antibodies were used. Fibronectin phagocytosis was decreased by inhibitors of tyrosine kinase (genistein, 100 microg/ml, P < 0.005) and PI3-kinase (wortmannin, 5 microM, P < 0.01), but these reagents did not affect the uncoated controls. The PKC inhibitor calphostin C (400 nM) nonspecifically increased the phagocytosis of FN-coated (P < 0.05) and uncoated beads (P < 0.01). CONCLUSIONS: Subconfluent retinal pigment epithelial cells preferentially phagocytose FN over other extracellular matrix components. Phagocytosis of FN utilizes the alpha5beta1 integrin, is mediated in part through tyrosine kinase and PI3-kinase signaling pathways, and is modulated by PKC. Phagocytosis of extracellular matrix by retinal pigment epithelial cells may represent a novel mechanism for remodeling of the provisional extracellular matrix during outer retinal wound healing.

CD36 Antigens↗
PubMed1997

Distribution of integrins and the extracellular matrix proteins in the baboon endometrium during the menstrual cycle and early pregnancy.

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.

Animals↗
PubMed2004

beta1-Integrins mediate enhancement of airway smooth muscle proliferation by collagen and fibronectin.

Airway smooth muscle (ASM) accumulation and enrichment of the extracellular matrix (ECM) with type I collagen and fibronectin are major pathologic features of airway remodeling in asthma. These ECM components confer enhanced ASM proliferation in vitro, but a requirement for specific integrin ECM receptors has not been examined. Here, we examined the mitogen platelet-derived growth factor (PDGF)-BB on beta1-integrin expression on human ASM cells cultured on these ECM substrates and defined the involvement of specific integrins in cell attachment and proliferation using integrin-neutralizing antibodies. PDGF-BB-dependent proliferation was enhanced two- to threefold by monomeric type I collagen or fibronectin and to a lesser extent by vitronectin; other interstitial ECM components (fibrillar type I and III collagen and tenascin-C) had no effect. Except for increased alpha3 expression induced by PDGF-BB and monomeric type I collagen or fibronectin, alpha1, alpha2, alpha4, alpha5, alphav, and alphavbeta3 integrins were unchanged compared with unstimulated cells on plastic. Blocking antibodies revealed alpha2beta1- and alphavbeta3-mediated attachment to monomeric type I collagen, whereas attachment to fibronectin required alpha5beta1. In contrast, enhancement of PDGF-BB-dependent proliferation by either monomeric type I collagen or fibronectin required alpha2beta1, alpha4beta1, and alpha5beta1 integrins. These data suggest multiple beta1-integrins regulate enhanced ASM proliferative responses.

Adult↗
PubMedDate not supplied

Cadherins and integrins in renal cell carcinoma: an immunohistochemical study.

AIMS AND BACKGROUND: The aim of this study was to determine the expression of cadherins and integrins in renal cell carcinoma (RCC) and their relationship with tumor morphology and TNM status. METHODS: Cadherin and integrin expression was investigated using an indirect immunoperoxidase technique, applying antibodies to E-, N-, P- and VE-cadherin and to alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alpha(v) integrin subunits. Correlation of semiquantitatively scored adhesion molecule levels with histopathological parameters (cytology, growth pattern, nuclear grade) and TNM status was performed for 24 RCCs (17 clear cell, 3 granular, 3 spindle cell and 1 chromophobe cell type according to the WHO classification). RESULTS: E-cadherin and N-cadherin were present in most cases (88% and 67%, respectively) and were usually coexpressed. T3 RCCs displayed higher E-cadherin and N-cadherin levels than T1/T2 tumors regardless of tumor grade, suggesting that impairment of their function might exist without actual loss from tumor cells. P-cadherin was found focally in two RCCs only, while VE-cadherin was present on stromal vessel endothelium in five tumors, showing no differences with regard to cell type, growth pattern, tumor grade or TNM status. All integrins were present in the studied RCCs (ranging from 12% for alpha5 to 79% for alpha3), including those that are normally absent from adult kidney tissue (alpha4 and alpha5). Tumors of higher grade showed increased alpha(v) and decreased alpha6 levels, while RCCs with metastases less often showed diffuse alpha3 presence and never expressed alpha5 integrin. CONCLUSIONS: Our results suggest that the level of expression of N-cadherin and some integrins (most notably alpha3, alpha6 and alpha5) is associated with the capacity of RCC for local and distant spread, regardless of tumor grade.

Antigens, CD↗
PubMed2000

Potential role of alphav and beta1 integrins as oocyte adhesion molecules during fertilization in pigs.

Integrin molecules are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction in rodents and humans. The objective of this study was to evaluate whether integrin molecules were present on the surface of pig oocytes, consistent with involvement in sperm-oocyte interaction in this species. Immunocytochemistry and confocal microscopy were used to evaluate the presence of beta1, and alpha1, alpha2, alpha3, alpha4, alpha5, alpha6 and alphav integrin subunits on the plasma membrane of pig oocytes. The beta1 and alphav integrin subunits were present consistently at the surface of pig oocytes; however, the remaining alpha integrin subunits evaluated were not routinely detected. The antibodies to the beta1 and alphav integrin subunits recognized appropriately sized protein bands on western blots of partially purified oocyte plasma membrane. These two antibodies also recognized oocyte plasma membrane protein isolated from a sperm plasma membrane affinity column. Sperm plasma membrane proteins of 137 and 93 kDa appeared to be the ligands for the beta1 integrin subunit as revealed by a western sandwich blot. Antibody to an extracellular domain of the beta1 integrin subunit reduced pig sperm-oocyte binding (P < 0.05), also indicating an assisting role for a beta1 oocyte integrin subunit in sperm-oocyte interaction in pigs. These results are consistent with an alphavbeta1 pig oocyte integrin interacting with a ligand on the sperm plasma membrane during fertilization.

Animals↗
PubMed2003

Integrins expressed with bladder extracellular matrix after stretch injury in vivo mediate bladder smooth muscle cell growth in vitro.

PURPOSE: It is unknown how bladder smooth muscle cells sense extrinsic mechanical stimuli. The integrins are a large versatile family of transmembrane mechanoreceptors that transduce extracellular matrix (ECM) alterations into the cell, thereby, regulating proliferation, differentiation and ECM synthesis. To our knowledge we provide the first evidence that the integrins may be involved in responses to whole bladder distention and bladder smooth muscle cell stretch. MATERIALS AND METHODS: Bladders from 100 to 120 gm. rats were stretched to 40 cm. H2O for 5 minutes. Five to 96 hours after distention whole bladder mRNAs were isolated for analysis of temporal expression of collagen and integrin genes. Separately quiescent primary culture bladder smooth muscle cells from 1-day-old Sprague-Dawley rats were stretched cyclically for 4 hours. Relative expression of select integrin subunit mRNAs was assessed by semiquantitative reverse transcriptase-polymerase chain reaction. Integrin blockade with asparagine-glycine-arginine peptides was used to determine the role of integrins in stretch induced proliferation and the cell cycle in bladder smooth muscle cells. RESULTS: Within 24 hours bladder distention stimulated collagen expression 2-fold (type I) and 5-fold (type III). Collagen levels beyond 24 hours were 8-fold (type I) and 2-fold (type III) greater than in controls, revealing an inverse temporal type I-to-III ratio beyond 24 hours. Coordinate alterations were observed in integrin and collagen expression. In vitro bladder smooth muscle cell integrin beta1, beta3 and alphav subunit expression was increased by mechanical stretch 2.5, 3.8 and 5-fold, respectively, while alpha1 expression decreased. Asparagine-glycine-arginine peptide inhibition of integrin function significantly inhibited stretch induced bladder smooth muscle cell proliferation and exit from the G2/M phase of the cell cycle. CONCLUSIONS: To our knowledge these results demonstrate for the first time that that bladder distention initiates dynamic alterations in ECM expression. The ability of integrin blockade to suppress stretch induced bladder smooth muscle cell proliferation and the coordinate changes in bladder ECM and integrin expression suggest that integrins mediate key responses to mechanical stimuli in the bladder. Furthermore, cell cycle analysis of resting and stretched bladder smooth muscle cells revealed novel avenues for the examination of integrin and stretch regulation of bladder smooth muscle cell growth.

Animals↗
PubMed1999

Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

Amino Acid Sequence↗
PubMed2003

Expression of RNAs encoding for alpha and beta integrin subunits in periodontitis and in cyclosporin A gingival overgrowth.

BACKGROUND: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling. AIM: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth. METHODS: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls. RESULTS: The RNA encoding for beta1, alpha2 and alpha5 integrin subunits were reduced in periodontitis gingiva. The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for alpha1 subunit was increased. The RNA encoding for alpha6 integrin was only reduced in cyclosporin A-treated gingiva. Immunohistochemistry showed that i) integrin alpha2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of alpha6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) beta1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva. CONCLUSION: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth.

Adult↗
PubMed2002

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions.

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.

Animals↗
PubMed2002

EGF and beta1 integrin convergently regulate migration of A431 carcinoma cell through MAP kinase activation.

We found that the convergently epidermal growth factor (EGF)-induced signal and the collagen-induced signal activate mitogen-activated protein kinase (MAPK), which induces migration. We examined the signaling mechanisms of EGF-induced cell migration on collagen using the A431 carcinoma cell. EGF (10 ng/ml) induced migration on collagen, but inhibited proliferation. Using a MAPK cascade inhibitor, PD98059, it was shown that EGF-induced migration on collagen was mediated by MAPK whereas EGF-induced migration on fibronectin and vitronectin was not. PD98059 also showed that activation of MAPK induced by EGF enhanced the adhesiveness of A431 cells to collagen. By Western blotting analysis, the kinetics of MAPK phosphorylation induced by EGF and collagen was examined separately, and convergently. First of all, EGF without collagen caused transient MAPK phosphorylation. Collagen without EGF caused MAPK to be immediately and transiently dephosphorylated, and rephosphorylated followed by sustained hyperphosphorylation. EGF together with collagen caused an immediate, and sustained, hyperphosphorylation. These facts suggest that the transient MAPK dephosphorylation induced by collagen is required for migration in order to maintain an appropriate level of sustained phosphorylation. Furthermore, we found that adhesion of A431 cells to collagen was blocked by the anti-beta1 integrin antibody or by the mixed antibodies composed of anti-alpha1, -alpha2, and -alpha3 antibodies, indicating that collagen-induced MAPK phosphorylation was mediated through alpha1beta1, alpha2beta1, and alpha3beta1 integrins.

Cell Movement↗
PubMed1997

Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.

The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.

Amino Acid Sequence↗
PubMed1999

Expression of beta1 and beta4 integrins in normal arachnoid membrane and meningiomas.

BACKGROUND: The aim of this work was to study the expression of alpha, beta1, and beta4 integrin subunits in meningiomas. METHODS: Seventeen atypical or anaplastic meningiomas were retrieved from the files of Hôpital de Bellevue, Saint-Etienne, France. They were compared with 17 benign meningiomas consecutively examined in 1997 and 6 schwannomas. The tumors were classified according to standard histologic criteria. Frozen sections were immunostained for alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1, and beta4 integrin subunits; collagen; laminin and fibronectin; cytokeratin; vimentin; neural cell adhesion molecule (NCAM); and MIB-1. RESULTS: The study included 7 fibrous meningiomas, 6 transitional meningiomas, 19 syncytial meningiomas, and 2 secretory meningiomas. The expression of alpha1, alpha3, alpha5, alpha6, and beta1 was constant. The expression of alpha1 was higher in fibrous meningiomas than in syncytial meningiomas. Only in transitional, syncytial, and secretory meningiomas was the expression of alpha2 detected. The expression of alpha2 and beta4 was associated with the expression of cytokeratin in the glandular structures of secretory meningiomas, whereas it was associated with NCAM expression in the whorls of meningothelial meningiomas. The expression of integrin receptors by tumor cells was strongly correlated with that of their respective ligands in the extracellular matrix. In invasive meningiomas, the expression of alpha3 and alpha6 by tumor cells was significantly lower. The higher the MIB-1 proliferation index, the lower the expression of alpha3. The 6 schwannomas expressed only alpha2, alpha3, alpha6, beta1, and beta4 integrins. CONCLUSIONS: Each histologic subtype of meningioma has a specific spectrum of integrin expression. The study of alpha3 and alpha6 may have prognostic value in the assessment of meningiomas. The study of the integrin profile is valuable for the differential diagnosis of fibrous meningiomas and schwannomas.

Antigens, Nuclear↗
PubMed2005

[Expression of keratinocyte basement membrane related genes during the process of re-epithelialization of burn wound in scalded rats].

OBJECTIVE: To study the expression of keratinocyte (KC) basement membrane (BM) related genes during the process of re-epithelialization of burn wound in scalded rats with cDNA microarray technique. METHODS: Twenty-four SD rats were inflicted with deep partial thickness scald with an area of 45 cm(2) on the back, and they were randomly divided into A [3 postscald day (PSD)], B (10 PSD), C (14 PSD) and D (re-epithelialization complete day) groups, with 6 rats in each group. Tissue samples were harvested from 1 cm of wound margin on 3, 10 and 14 PSD. On the re-epithelialization complete day, tissue samples were harvested from the center of the wound in D group and digested with enzyme into KC suspensions. Skin samples from the back of 6 uninjured rats were taken as normal control. The differential expression of KC BM related genes during different stages of re-epithelialization was assayed with cDNA microarray. RESULTS: The expression of laminin (LN) gamma 1 (2.068) and integrin beta8 (2.200) was up-regulated on 3 PSD compared with that in control. The expression of integrin beta1 and LN receptor 1 was up-regulated on 10 and 14 PSD, (2.472 and 2.658), while that of integrin beta2 and beta1 (0.419 and 0.462) down-regulated on 10 and 14 PSD, and the expression of type IV collagen alpha1 and alpha3 was up-regulated during re-epithelialization. CONCLUSION: The expression of integrin beta1, LN gamma 1, LN receptor 1, type IV collagen alpha1 and alpha3 genes were up-regulated during re-epithelialization, which might be beneficial to the construction of BM in new skin and the formation of stable conjunction between KC and BM.

Animals↗
PubMed2003

Alpha1beta1-integrin is an essential signal for neurite outgrowth induced by thrombospondin type 1 repeats of SCO-spondin.

In the central and peripheral nervous systems a heterogeneous group of proteins constituting the thrombospondin superfamily provides a cue for axonal pathfinding. They either contain or are devoid of the tripeptide RGD, and the sequence(s) and mechanism(s) which trigger in vitro their neurite-promoting activity have remained unclear. In this study, we reconsider the problem of whether sequences present in the thrombospondin type 1 repeats (TSRs), and independent of the well-known RGD-binding site, may activate integrins and account for their neurite-promoting activity. SCO-spondin is a newly identified member of the thrombospondin superfamily, which shows a multidomain organization with a great number of TSR motifs but no RGD sequence. Previous research has implicated oligopeptides derived from SCO-spondin TSRs in in-vitro development of various neuronal cell types. In this study, we investigate whether function-blocking antibodies directed against integrin subunits can block these effects in cell line B104, cloned from a neuroblastoma of the rat central nervous system. By two different approaches: flow cytometry revealing short-term effects and cell cultures revealing long-term effects, we show that: (a). activation of cell metabolism, (b). changes in cell size and structure, and (c). neurite-promoting activity induced by TSR oligopeptides are inhibited by function-blocking antibodies to beta1-subunit. Using a panel of function-blocking antibodies directed against various integrin alpha-subunits we show that the alpha1-subunit might be the partner of the beta1-subunit in B104 cells. Thus, we demonstrate that an original sequence within a TSR motif from SCO-spondin promotes neurite outgrowth through an intracellular signal driven by integrins, independently of an RGD-binding site.

Amino Acid Sequence↗
PubMed2001

Interactions between collagen IV and collagen-binding integrins in renal cell repair after sublethal injury.

Recent studies demonstrate that collagen IV selectively promotes the repair of physiological processes in sublethally injured renal proximal tubular cells (RPTC). We sought to further define the mechanisms of cell repair by measuring the effects of toxicant injury and stimulation of repair by L-ascorbic acid-2-phosphate (AscP), exogenous collagen IV, or function-stimulating integrin antibodies on the expression and subcellular localization of collagen-binding integrins (CBI) in RPTC. Expression of CBI subunits alpha1, alpha2, and beta1 in RPTC was not altered on day 1 after sublethal injury by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). On day 6, expression of alpha1 and beta1 subunits remained unchanged, whereas a 2.2-fold increase in alpha2 expression was evident in injured RPTC. CBI localization in control RPTC was limited exclusively to the basal membrane. On day 1 after injury, RPTC exhibited a marked inhibition of active Na(+) transport and a loss of cell polarity characterized by a decrease in basal CBI localization and the appearance of CBI on the apical membrane. On day 6 after injury, RPTC still exhibited marked inhibition of active Na(+) transport and localization of CBI to the apical membrane. However, DCVC-injured RPTC cultured in pharmacological concentrations of AscP (500 microM) or exogenous collagen IV (50 microg/ml) exhibited an increase in active Na(+) transport, relocalization of CBI to the basal membrane, and the disappearance of CBI from the apical membrane on day 6. Function-stimulating antibodies to CBI beta1 did not promote basal relocalization of CBI despite stimulating the repair of Na(+)/K(+)-ATPase activity on day 6 after injury. These data demonstrate that DCVC disrupts integrin localization and that physiological repair stimulated by AscP or collagen IV is associated with the basal relocalization of CBI in DCVC-injured RPTC. These data also suggest that CBI-mediated repair of physiological functions may occur independently of integrin relocalization.

Animals↗