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PubMed2000

Phosphorylation of the beta1 integrin cytoplasmic domain: toward an understanding of function and mechanism.

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the beta1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of beta1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in beta1 phosphorylation and selective loss of beta1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with beta1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with beta1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with beta1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with beta1. A possible role for regulated beta1 phosphorylation in cell migration is discussed.

Animals↗

PubMed1996

beta1-Integrins dominate cell traffic of leukemic cells in human bone-marrow stroma.

Migration patterns of leukemic cells in bone marrow are largely regulated by cell contacts between leukemic cells and stromal cells or extra-cellular matrix. The mechanism of this interaction with bone-marrow stromal cells was studied in a human in vitro model. Migration behavior of erythroleukemia cell line K562, derived from a patient with chronic myeloid leukemia, was compared with that of the erythroleukemia cell line HEL92.1.7 and the promyelocytic leukemia cell line HL60 from acute leukemias. Interaction varied between low binding affinity (K562) to intensive cell interaction (HEL92.1.7) followed by invasion into the stromal cell monolayer. Some of the HL60 cells adhered to stromal cells, while the remainder migrated into the stromal cell monolayer. The role of adhesion molecules in these cell interactions was determined. Distinct expression of beta1-integrins ICAM-1, CD44 and VCAM-1 was detected on the different cell lines. Inhibition studies pointed to a dominant role of VLA-4- and VLA-5-mediated interactions. K562 lacked VLA-4 and a low binding affinity of the VLA-5 on these cells resulted in an absence of binding to the bone-marrow stroma. These results indicate the VLA-5/fibronectin, VLA-4/fibronectin and the VLA-4/VCAM-1 interaction pathways between leukemic cells and bone-marrow stroma.

Bone Marrow↗

PubMed2000

Lack of CD 29 (beta1 integrin) and CD 54 (ICAM-1) adhesion molecules in intravascular lymphomatosis.

Intravascular Lymphomatosis (IL) is a rare and usually aggressive form of non-Hodgkin's lymphoma characterized by the growth of neoplastic cells within vascular lumina that usually presents with skin or central nervous system (CNS) involvement. The mechanism(s) for the selective intravascular growth of this neoplasm remain(s) unexplained. We now report clinical and immunohistologic data on surgical material from 6 cases of IL; in 4 of 6 cases, autopsies were performed. Our IL cases shared the following features: (1) B-cell lineage; (2) lack of skin involvement at presentation; (3) aggressive behavior; and (4) lack of extravascular lymphomatous masses; in addition, 1 case had an associated gastric low-grade MALT lymphoma. We studied by immunohistochemistry formalin-fixed, paraffin-embedded sections with monoclonal antibodies to molecules known to be involved in lymphocyte and endothelial adhesion phenomena, that is, CD29 (beta1 integrin subunit), CD43 (leukosialin), CD44 (H-CAM), CD54 (ICAM-1), embryonal N-CAM (e-NCAM), and EMA (episialin). In all cases, the surfaces of IL aggregates reacted for CD44 but were consistently negative for CD29; also absent was CD54. Conversely, the integrity of the endothelial cells was underscored by their even reactivity for CD29, CD44, and CD54. Given that CD29 is currently regarded as critical for lymphocyte trafficking in general and for transvascular migration in particular, and CD54 is also involved in transvascular lymphocyte migration, we conclude that their consistent absence in IL may contribute to its intravascular and disseminated distribution pattern. The rather frequent association of IL with various conventional lymphomas is known; yet, one of our cases appears to be the first report of IL associated with a low-grade MALT lymphoma.

Aged↗

PubMed2006

Nano- and microscale holes modulate cell-substrate adhesion, cytoskeletal organization, and -beta1 integrin localization in SV40 human corneal epithelial cells.

Human corneal epithelial cells (HCECs) interface with a basement membrane in vivo that possesses complex nanoscale topographic features. We report that synthetic substrates patterned with nano- and microscale holes differentially modulate the proliferation, shape and adhesion of SV40 human corneal epithelial cells (SV40-HCECs) as a function of feature size: 1) Cell proliferation was inhibited on nanoscale features (features size less than 800 nm in pitch) compared to microscale features or planar substrates in identical culture conditions. 2) Cells on nanoscale holes had a stellate morphology compared to those on microscale features that were more evenly spread. 3) Cells adhered more to nanoscale features than to microscale features when exposed to shear stress in a laminar flow chamber. Transmission electron microscopy showed that cells cultured on the 400 nm pitch patterns had longer and more numerous filopodia and retraction fibers than cells cultured on the 1600 nm pitch patterns. Immunogold labeling of -beta1 integrins revealed that these receptors were localized at the cell periphery and in the aforementioned cytoskeletal elements. Our findings indicate that surface discontinuities and the activation of mechanochemical cell signaling mechanisms may contribute to the observed responses exhibited by SV40-HCECs cultured on nano- and microscale topography.

Cell Adhesion↗

PubMed2004

Cell-cell adhesion molecule CEACAM1 is expressed in normal breast and milk and associates with beta1 integrin in a 3D model of morphogenesis.

CEA cell adhesion molecule-1 (CEACAM1) is a cell-cell adhesion molecule that, paradoxically, is expressed in an apical location in normal breast epithelium. Strong lumenal membrane staining is observed in 100% of normal glands (11/11), low in atypical hyperplasia (2/6), high in cribiform ductal carcinoma in situ (DCIS) (8/8), but low in other types of DCIS (2/15). Although most invasive ductal carcinomas express CEACAM1 (21/26), the staining pattern tends to be weak and cytoplasmic in tumours with minimal lumena formation (grades 2-3), while there is membrane staining in well-differentiated tumours (grade 1). The 'normal' breast epithelial line MCF10F forms acini with lumena in Matrigel with apical membrane expression of CEACAM1. MCF7 cells that do not express CEACAM1 and fail to form lumena in Matrigel, revert to a lumen forming phenotype when transfected with the CEACAM1-4S but not the -4L isoform. CEACAM1 directly associates with and down-regulates the expression of beta1-integrin. Immuno-electron microscopy reveals numerous vesicles coated with CEACAM1 within the lumena, and as predicted by this finding, CEACAM1 is found in the lipid fraction of breast milk. Thus, CEACAM1 is a critical molecule in mammary morphogenesis and may play a role in the absorption of the lipid vesicles of milk in the infant intestinal tract.

Animals↗

PubMed1998

Beta1 integrin deficiency impairs migration and differentiation of mouse embryonic stem cell derived neurons.

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and beta1 integrin-deficient (G201) embryonic stem cell derived neurons. In D3 preparations complex networks of functionally coupled neurons with bi- and multipolar morphologies develop. In contrast, neuronal differentiation is retarded in G201 derived neurons, recognised by limited migration and restricted morphological differentiation. Furthermore, beta1 integrin deficiency causes a delay in expression of major neurotransmitters like GABA and glutamate as well as of synaptophysin. These findings indicate a prominent role of beta1 integrin for both morphological and chemical differentiation.

Animals↗

PubMed2006

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides.

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.

Adhesins, Bacterial↗

PubMed1998

Functional cooperation of beta1-integrins and members of the Ig superfamily in neurite outgrowth induction.

Neurite outgrowth is a central aspect of the ontogenetic formation of neural networks and is regulated by distinct groups of cell surface molecules. One protein involved in neurite elongation and fasciculation is the neural Ig superfamily member F11/contactin. We have shown previously that F11 promotes neurite extension of chick tectal neurons by interaction with the tectal receptor NrCAM, a member of the L1 subgroup of the Ig superfamily. By contrast, it does not induce outgrowth of retinal neurons despite the fact that these cells also express NrCAM, suggesting that in retinal cells the F11-NrCAM interaction alone is not sufficient to induce neurite extension. In this report we present a novel image analysis procedure to quantify neurite outgrowth and use it to demonstrate that F11 enhances the fibronectin-induced outgrowth response of embryonic retinal neurons. We reveal that NrCAM is the neuronal receptor mediating the enhanced outgrowth of retinal neurons, whereas the related F11-binding molecule NgCAM is not involved. Furthermore, we provide evidence that a beta1-integrin may represent the fibronectin-dependent receptor that cooperates indirectly with the F11-NrCAM pathway. Our results support the concept of a combinatorial labeling of cells in nervous system histogenesis by different classes of cell surface proteins, in particular by integrins and molecules of the Ig superfamily.

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↗

PubMed2004

Possible role of Malassezia furfur in psoriasis: modulation of TGF-beta1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis-affected patients.

BACKGROUND: Psoriasis is a disease characterized by an abnormal pattern of keratinocyte growth and differentiation. Malassezia furfur forms part of the normal human skin flora. It may also be involved in the pathogenesis of psoriasis. To define the role of M. furfur in the pathogenesis of psoriasis, we investigated how M. furfur regulates molecules involved in cell migration and proliferation. The experiments were performed using human keratinocytes and skin biopsies from M. furfur-positive and -negative psoriasis-affected patients. In addition, we examined the signal transduction mechanisms involved. MATERIALS AND METHODS: Western blot analysis was performed on human keratinocytes lysates treated or untreated with M. furfur and on biopsies from healthy and psoriasis patients. Signal transduction mechanisms involved were evaluated by electrophoretic mobility shift assay using the AP-1 inhibitor curcumin. RESULTS: We found that M. furfur up-regulates transforming growth factor-beta1 (TGF-beta1), integrin chain, and HSP70 expression in human keratinocytes via AP-1-dependent mechanism. In the biopsies of M. furfur-positive psoriasis-affected patients, an increase in TGF-beta1, integrin chains, and HSP70 expression was found. CONCLUSION: Our data suggest that M. furfur can induce the overproduction of molecules involved in cell migration and hyperproliferation, thereby favoring the exacerbation of psoriasis.

Blotting, Western↗

PubMed2004

Regulation of beta1 integrin expression by PKCepsilon in renal cancer cells.

Polarized cell movement represents an essential prerequisite for the progression and metastasis of malignant diseases. Protein kinase C (PKC) which physically associates with integrins has been implicated in the promotion of a migratory cell phenotype. In order to identify a direct link between PKC and integrins in renal cell carcinoma (RCC) the influence of PKC isoforms on integrin expression and possible consequences on proliferation and cell migration was analyzed in RCC cells. The constitutive expression of the PKC isoforms alpha, betaI, betaII, gamma, delta, epsilon, eta, theta, xi, lambda and micro was determined in the RCC cell line CCF-RC1. In addition, the influence of PKC inhibitors RO31-8220, GF109203X and GO6976 on the beta1, beta2 and beta3 integrin expression and cell proliferation of RCC cells was investigated by flow cytometry and by BrdU incorporation, respectively. Furthermore, the motility of CCF-RC1 cells was assessed through chamber chemotaxis analysis. All PKC isoforms tested were expressed in CCF-RC1 cells with the exception of PKClambda and theta. The PKC inhibitor RO31-8220 reduced beta1 integrin expression by 92% and inhibited proliferation by 42% of untreated cells, whereas cell migration remained uninfluenced by RO31-8220. GF109203X and GO6976 reduced beta1 integrin expression to approximately 50% of untreated cells. In contrast, beta2 and beta3 integrins were only weakly affected by RO31-8220, GF109203X and GO6976 treatment. The most significant influence on beta1 integrin expression was obtained by the PKC inhibitor RO31-8220. This leads to the assumption that PKCepsilon is involved in the regulation of beta1 integrin expression. Downregulation of beta1 integrins by RO31-8220 was associated with reduced proliferation, but did not influence migration. These findings provide a conceptual basis for treatment of renal cell carcinoma by interfering with tumor cell proliferation.

CD18 Antigens↗

PubMed1996

Regulation of plasminogen receptor expression on monocytoid cells by beta1-integrin-dependent cellular adherence to extracellular matrix proteins.

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

Carboxypeptidase B↗

PubMed2005

Nitric oxide inhibits T cell adhesion and migration by down-regulation of beta1-integrin expression in immunologically liver-injured mice.

Our previous study has reported that nitric oxide (NO) exerts a protective role in immunologically liver-injured mice induced by delayed-type hypersensitivity to picryl chloride. To explore the mechanism of the protection, we have now examined the effect of NO on T cell adhesion and migration. First, we isolated hepatocytes and nonparenchymal cells from the liver-injured mice and separated the nonparenchymal cells into Kupffer cell-enriched and lymphocyte-enriched populations. When these hepatocytes or the fractions of nonparenchymal cells were co-cultured with spleen T cells of the liver-injured mice in a Transwell system, the adhesive potential of the T cells was significantly inhibited in the presence of hepatocytes or the Kupffer cell-enriched population but not the lymphocyte-enriched population of nonparenchymal cells. This effect was dependent on NO production. The NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) could reverse this inhibition of cell adhesion and also decrease NO production. To confirm this effect of NO on T cells, we further examined the role of exogenous or endogenous NO on the adhesive activity of the Jurkat T cell line. As a result, the NO donor, S-nitroso-N-acetyl penicillamine (SNAP) caused a dose-dependent inhibition of the adhesion of Jurkat T cells. Furthermore, the binding ability of Jurkat T cells to collagen decreased gradually after co-incubation with macrophages stimulated by LPS+IFN-gamma, an effect which correlated well with the increasing NO level in the medium. Such opposite changes in cell adhesion and in NO production were also markedly reversed by L-NMMA. Moreover, treatment with SNAP reduced adhesion, transmigration, matrix metalloproteinase-9 production and beta1-integrin expression of spleen T cells of the liver-injured mice. Taken together, these findings suggest that NO can function as a down-regulator of T cell mobility, which might be one of the mechanisms by which NO exerts its protective effect in T cell-mediated liver injury.

Animals↗

PubMed2005

Suprabasal alpha 5 beta1 integrin expression stimulates formation of epidermal squamous cell carcinomas without disrupting TGFbeta signaling or inducing spindle cell tumors.

During epidermal chemical carcinogenesis benign papillomas convert to squamous cell carcinomas, some of which undergo epithelial-mesenchymal conversion to highly malignant spindle cell tumors. TGFbeta inhibits early stages of carcinogenesis but promotes the spindle cell phenotype in later stages. One hallmark of spindle cell tumors is upregulation of the alpha 5 beta 1 integrin fibronectin receptor. To examine the significance of altered alpha 5 beta1 integrin expression, we induced tumors in transgenic mice expressing alpha 5 beta1 in the suprabasal epidermal layers. Invalpha 5 beta1 mice developed threefold more papillomas and squamous cell carcinomas than wild-type (Wt) littermates; however, no spindle cell tumors or increased metastases were observed. Suprabasal expression of the alpha 6 beta 4 integrin increases squamous cell carcinoma formation and decreases TGFbeta sensitivity while alpha 3 beta1 may have the opposite effect. In contrast, nuclear phosphoSmad2 labeling in Invalpha 5 beta1 epidermis and tumors was indistinguishable from Wt, and suprabasal alpha 5 beta1 did not block TGFbeta-induced Smad2/3 translocation or growth inhibition in cultured keratinocytes. We conclude that upregulation of alpha 5 beta1 does not predispose the epidermis to undergo conversion to spindle cell tumors and that the mechanism by which alpha 5 beta1 influences susceptibility to carcinogenesis is independent of perturbed TGFbeta signaling.

Animals↗

PubMed1997

Oncogenic Ki-ras but not oncogenic Ha-ras blocks integrin beta1-chain maturation in colon epithelial cells.

Human colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-rasG12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-ras mutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-rasG12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-ras transfectant cells synthesized the mature, fully glycosylated forms of beta1 integrin. Instead of the mature integrin beta1-chain, a faster migrating beta1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered beta1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin beta1-chain and increased expression of the mature integrin beta1-chain. Altered glycosylation generated the new beta1 integrin form since integrin core beta1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on beta1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin beta1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin beta1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2005

Curcumin protects human chondrocytes from IL-l1beta-induced inhibition of collagen type II and beta1-integrin expression and activation of caspase-3: an immunomorphological study.

Interleukin 1beta (IL-1beta) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1beta activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1beta, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1beta-stimulated chondrocytes. Human articular chondrocytes were pre-treated with 10 ng/mI IL-1beta alone for 30 min before being co-treated with IL-1beta and 50 microM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor beta1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting. Transmission electron microscopy of chondrocytes stimulated with IL-1beta revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and beta1-integrin synthesis by IL-1beta was inhibited by curcumin. Additionally, curcumin antagonized IL-1beta-induced caspase-3 activation in a time-dependent manner. This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1beta-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.

Caspase 3↗

PubMed2002

Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1-integrin fibrillar adhesions.

The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL- cells and their wild-type VHL stably transfected counterparts. The levels of beta1 and alphav integrins were increased in VHL- cells when compared with VHL+ transfectants. Early after plating, both VHL+ and VHL- cells were capable of assembling classic "patch-like" alphav focal contacts. As the culture advanced and cells became confluent, alphav integrins partly relocated to the intercellular junctions in VHL+ transfectants, which then developed large beta1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL- cells were unable to assemble beta1 fibrillar adhesions, and alphav focal contacts remained unchanged at all stages of the culture. Exogenous activation of beta1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL- cells to assemble beta1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common beta1-integrin chain was delayed in VHL- cells when compared with VHL+ cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of beta1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL- renal cancer cells.

Cell Adhesion↗

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↗