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PubMed2006

Enhanced cell-substratum adhesion of E-cadherin-expressing cells is mediated by activation of the small GTPase protein, Rac1.

L cell transfectants stably expressing E-cadherin demonstrate a remarkable increase in adherence to extracellular matrix proteins, such as type IV collagen and fibronectin. This enhanced adhesion is mediated by integrin-type cell surface receptors, as assessed by inhibition with anti-receptor antibodies. Both the rate and efficiency of adhesion were enhanced 4- to 5-fold. In contrast, non-specific adhesion processes, such as cell attachment to polylysine-coated substrata, are unaffected by E-cadherin expression. Thus, integrin-mediated but not non-specific adhesion is modulated by E-cadherin expression. L cells expressing mutant E-cadherin molecules either lacking the cytoplasmic domain or bearing an amino acid substitution in the Ca2+-binding motif did not exhibit enhanced adhesion. The amount of collagen receptor, the alpha1 and beta1 integrin, did not change following expression of E-cadherin. Pull-down assays with the Cdc42/Rac interactive binding (CRIB) domain of the Rac effector, p21-activated kinase, revealed increased Rac-GTP levels in cells expressing wild-type E-cadherin. These results suggest that the activation of Rac is involved in the enhancement of integrin-mediated adhesion induced by E-cadherin expression.

Animals↗
PubMed2003

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1.

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.

Animals↗
PubMed2006

Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells.

Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.

Androstadienes↗
PubMed2005

Distinct expression of adhesion molecules on skin fibroblasts from patients with diffuse and limited systemic sclerosis. A pilot study.

OBJECTIVE: . Systemic sclerosis (SSc) is characterized by excessive production of collagen and other components of the extracellular matrix (ECM) by fibroblasts. The ECM receptors, integrins and CD44 (hyaluronan receptor), play a key role in the homeostasis of connective tissue and may also have a role in the pathogenesis of fibrosis. We investigated the expression of integrins and CD44 on skin fibroblasts from patients with limited and diffuse SSc. METHODS: We studied 13 patients with SSc, 8 with limited SSc, and 5 with diffuse SSc, and 8 control subjects. Fibroblasts were isolated and cultured from biopsies taken from the lesional skin of the second finger of the left hand. Cell-surface expression of beta1, beta3, alpha1-alpha6, alphav integrins, and CD44 was evaluated by immunofluorescence and flow cytometry analysis. RESULTS: Fibroblasts from limited SSc showed significantly decreased expression of alpha2, alpha3, alpha4 integrins, while diffuse SSc fibroblasts had significantly reduced expression of alpha5, alphav integrins, and CD44. Diffuse SSc also had significantly increased expression of alpha6 integrin on fibroblasts. In controls, the expression of alpha4 and alpha5 correlated positively, while in limited and diffuse SSc it did not. CONCLUSION: This is the first study evaluating separately the expression of adhesion molecules on skin fibroblasts from limited and diffuse subsets of SSc. We detected a distinct pattern of expression with decrease of collagen and fibronectin receptors in limited SSc, and downregulation of fibronectin and hyaluronan receptors in diffuse SSc. These results suggest that changes of fibroblasts/ECM interactions and mechanisms underlying the pathogenesis of fibrosis in SSc may differ in the single subset of the disease.

Cells, Cultured↗
PubMed2003

beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of beta-adrenergic receptor-stimulated apoptosis.

We have shown that the stimulation of beta-adrenergic receptors (beta-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of alphabeta-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases beta1 integrins expression in ARVMs via the stimulation of alpha1-AR, not beta-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited alpha1-AR-stimulated increases in beta1 integrins expression. Activation of beta1 integrins signaling pathway using laminin (LN) inhibited beta-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of beta1 integrins with anti-beta1 integrin antibodies prevented beta-AR-stimulated apoptosis. Treatment of cells using LN or anti-beta1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of alpha1-AR regulates beta1 integrins expression via the activation of ERK1/2, (2) beta1 integrins signaling protects ARVMs from beta-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of beta1-integrin signaling. These data suggest that beta1 integrin signaling protects ARVMs against beta-AR-stimulated apoptosis possibly via the involvement of ERK1/2.

Animals↗
PubMed1999

Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Characterization of the bovine placenta by cytoskeleton, integrin receptors, and extracellular matrix.

The cytoskeleton together with integrin receptors and proteins of the extracellular matrix provide sensitive indices of the development and organization of the bovine placenta. The bovine placenta is classified as synepitheliochorial because migrating trophoblast giant cells fuse with single uterine epithelial cells. This phenomenon may be interpreted as a restricted trophoblast invasion. Bovine placentomes from early placentation until term can be characterized by indirect immunohistochemical methods. In order to do so, placental tissues are snap-frozen in liquid nitrogen or perfusion-fixed in formalin and embedded in paraffin. Depending on the antibodies used, the different cell types within the cow placenta are identified either on frozen sections or on paraffin sections according to the expression of different cytoskeletal filaments (alpha smooth muscle actin, different cytokeratins, desmin and vimentin). The specific expression of integrin receptors (subunits alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha(v), beta1, beta3, and alpha4) as well as proteins of the extracellular matrix (collagens type I and IV, fibronectin, and laminin) in the different cell populations is also examined.

Animals↗
PubMed2006

Molecular phenotypes of notochordal cells purified from immature nucleus pulposus.

The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordal-like cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordal-like cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell-matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1beta via real-time quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of alpha6, alpha1 and beta1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or integrin-mediated cell-matrix interactions for these notochordal-like cells within the NP.

Animals↗
PubMed1998

Downstream events in mammary gland morphogenesis mediated by reexpression of the alpha2beta1 integrin: the role of the alpha6 and beta4 integrin subunits.

Our previous studies demonstrated that reexpression of the alpha2beta1 integrin by a poorly differentiated breast carcinoma cell line, Mm5MT, resulted in dramatic reversion of a malignant phenotype to a differentiated epithelial phenotype. We hypothesized that reexpression of the alpha2beta1 integrin may regulate expression of other genes, the expression of which contributed to the dramatic phenotypic change. We now show that reexpression of the alpha2beta1 integrin results in up-regulation of both the alpha6 and beta4 integrin subunits but no change in the alpha1, alpha3, alpha5, or beta1 integrin subunits or E-cadherin. To further investigate the role of the alpha6 and beta4 integrin subunits in mediating the phenotypic changes elicited by reexpression of the alpha2beta1 integrin, the alpha6 or beta4 integrin subunit was expressed in our Mm5MT model. Expression of either subunit increased adhesion to laminin-1. Although adhesion to collagen was unaltered, contraction of three-dimensional collagen gels was reduced. Expression of either the alpha6 or beta4 integrin subunit also restored some aspects of a less malignant phenotype, including the acquisition of contact inhibition and diminution of anchorage-dependent and anchorage-independent growth rates. The alpha6 and beta4 transfectants formed three-dimensional organized structures when grown in gels of reconstituted basement membrane but did not form the highly branched, duct-like structures formed by the alpha2 transfectants. In contrast to the reduced invasiveness of the alpha2 transfectants, the alpha6 and beta4 transfectants retained an invasive phenotype. These results suggest that expression of the alpha6beta4 integrin contributes to some but not all of the phenotypic changes elicited by reexpression of the alpha2 integrin subunit and modulates the function of other integrins on these cells. Using our Mm5MT model, we are defining the cascade of integrin expression required for maintenance of the differentiated mammary epithelial cell phenotype.

Animals↗
PubMed1999

Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB.

Pathologic remodeling of mesangial matrix after glomerular injury is the central biologic feature of glomerular scarring (sclerosis). Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF)-BB have been implicated in the development of glomerular scarring in rat and human glomerulonephritis. To clarify molecular and cellular mechanisms involved in abnormal mesangial remodeling, this study focused on the role of alpha1beta1 integrin, a collagen/laminin receptor, in rat mesangial cells, using collagen gel contraction as an experimental model of in vivo collagen matrix remodeling and scar formation. In addition, the influence of TGF-beta and PDGF-BB on mesangial cell (MC)-mediated collagen gel contraction in association with the alpha1beta1 integrin expression was evaluated. Integrin function blocking studies using anti-alpha1, beta1 subunit antibodies indicated that MC-alpha1beta1 integrin is essentially required not only for collagen-dependent adhesion/migration, but also for gel contraction. Protein synthesis and mRNA analysis experiments demonstrated that TGF-beta, but not PDGF-BB, increases the expression of alpha1beta1 integrin in mesangial cells cultured on plastic surface and in collagen gels. The upregulation of alpha1beta1 integrin expression by TGF-beta correlated with increases in gel contraction and collagen-dependent adhesion but not migration of mesangial cells. On the other hand, PDGF-BB enhanced MC-mediated gel contraction and migration without affecting cell adhesion to collagen I. Growth factor-induced collagen-dependent adhesion, migration, and gel contraction were significantly attenuated by incubation with anti-alpha1, beta1 subunit antibodies. Thus, these data indicate that alpha1beta1 integrin-mediated collagen matrix remodeling can be modulated by TGF-beta and PDGF-BB via different mechanisms. Alpha1 integrin-mediated mesangial matrix remodeling induced by TGF-beta or PDGF-BB may be a pathogenic mechanism leading to glomerular scarring.

Animals↗
PubMed1997

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Antibodies, Monoclonal↗
PubMed2003

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1).

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of beta 1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha 2 (63.8 11.3% positive cells), alpha 3 (93.3 7.0%), alpha 5 (50.4 12.0%) and alpha 6 (34.1 4.9%) integrins but not alpha1, alpha 4, alpha v or 4. Cells adhered well to laminin-1 (73.4 6.0%) and fibronectin (40.0 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha 2, alpha 3 and alpha 6 mediated laminin-1 adhesion, but neither alpha 3 nor alpha 5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 2.4% vs DMSO: 70.7 2.5%) while simultaneously reducing alpha 5 (24.2 19%) and alpha 6 (14.3 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha 3 and alpha 5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 2 cells vs DMSO: 64 6 cells), was blocked by an antibody against alpha 6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

Cell Adhesion↗
PubMed2003

Integrin alpha1beta1 and alpha2beta1 are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment.

As with many types of cancer, cell motility is an important factor in the progression and metastasis of hepatocellular carcinomas (HCC). HCC is associated with significant fibrosis in the liver. The fibrotic microenvironment in the liver is characterized by an altered composition of the extracellular matrix (ECM) and an abundance of growth factors that are likely conducive to migration of HCC cells. The purpose of this study was to delineate promigratory stimuli within the fibrotic microenvironment and to identify specific targets for prevention of HCC cell migration. We used a modified Boyden chamber system that allowed distinction between chemotactic (indirect stimulation) and haptotactic (direct stimulation) migration of two distinct HCC cell lines across the ECM-coated membrane. Fibrotic microenvironment-associated growth factors, such as transforming growth factor beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), induced chemotactic and haptotactic migration of HepG2 and Chang cells. Neutralizing antibodies to individual growth factors significantly decreased chemotactic and haptotactic migration. Haptotactic stimulation, but not chemotactic stimulation of HCC cell lines with TGF-beta1, bFGF, and EGF, induced production of matrix metalloproteinase (MMP) 2, a potential mediator of migration. Inhibition of MMPs significantly decreased haptotactic migration induced by individual growth factors but had an insignificant effect on chemotactic migration, suggesting an MMP-independent migration in this setting. Inhibition of cell-ECM interactions with blocking antibodies to alpha1 and alpha2 integrins were sufficient to inhibit both haptotactic and chemotactic migration induced by individual growth factors, strongly suggesting that targeting these integrins to abrogate pathogenic cell-ECM interactions might be a promising tool for inhibiting growth factor-induced invasion and metastasis of HCC.

Carcinoma, Hepatocellular↗
PubMed1997

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Animals↗
PubMed2000

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

Adrenal Medulla↗
PubMed1999

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

Amino Acid Sequence↗
PubMed2004

Integrin profile and in vivo homing of human smooth muscle progenitor cells.

BACKGROUND: Recently, we identified circulating smooth muscle progenitor cells (SPCs) in human peripheral blood. The integrin profile of such progenitors is currently unknown and may affect their in vivo homing characteristics. In this study, we determined the integrin profile of vascular progenitors and SPC adhesion to extracellular matrix (ECM) proteins in vitro and in vivo. METHODS AND RESULTS: SPCs and endothelial progenitor cells (EPCs) were isolated from peripheral blood of healthy human subjects, and expression of surface integrins and adhesion to several vascular ECM proteins were determined. Homing of SPCs in vivo to specific ECM protein was determined by intracoronary infusion of fluorescent SPCs into porcine coronary arteries containing a fibronectin-coated mesh stent. SPCs had high expression of beta1 integrin, moderate expression of alpha1, low levels of alpha(v)beta3, and did not express alpha(v)beta5, beta2, alpha2beta1, or alpha4beta1 integrins. In contrast, EPCs had high expression of alpha2beta1, alpha(v)beta3, alpha(v)beta5, beta1, and alpha1 and minimal expression of alpha4beta1. Moreover, SPCs showed increased adherence to fibronectin and collagen type I compared with vitronectin, consistent with their integrin profile, and demonstrated a similar degree of in vivo attachment to fibronectin-coated mesh. CONCLUSIONS: These data for the first time show a spectrum of integrin expression on vascular progenitors and suggest the potential importance of integrins in mediating adherence of SPCs to specific ECM both in vitro and in vivo.

Animals↗
PubMed2005

Invasive and noninvasive uveal melanomas have different adhesive properties.

AIMS: To establish if invasive and noninvasive uveal melanomas have differences in expression of adhesion molecules, and whether their adhesive interactions with the extracellular matrix (ECM) and endothelium vary. METHODS: Cells from an invasive and noninvasive uveal melanoma cell line and hepatic and dermal microvascular endothelial cells were assessed by flow cytometry for adhesion molecule expression. Tumour cell adhesion to ECM substrates (collagens I and IV, fibronectin, laminin, and vitronectin) and endothelial cells was also investigated using a commercially available assay or a fluorescence-based in vitro assay, respectively. The significance of results comparing cell lines was determined using a Student's t-test, whereby P-values of less than 0.05 were taken as significant. RESULTS: alpha1- and alpha4-integrins were not expressed by noninvasive cells, but were detected on invasive cells. The invasive cell line also expressed higher levels of other integrins than the noninvasive line. Correspondingly, invasive cells adhered in higher numbers to ECM substrates and endothelial cells, and for the latter, the difference was highly significant (P<0.001). No preference in adhesion of invasive cells for the hepatic endothelium was observed. CONCLUSIONS: Successful attachment to and migration through the ECM, basement membrane, and endothelium are vital processes involved in malignant progression. Differential expression of alpha1- and alpha4-integrins by invasive and noninvasive cells infers a role for these receptors in invasion, while the ability of invasive cells to adhere more efficiently to the endothelium suggests that this is a critical factor in uveal melanoma invasion.

Cell Adhesion↗