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At least 523 records · Page 29Linked to original sources
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↗
PubMed2002

Integrin expression in the dermis during scar formation in humans.

To evaluate changes leading to human wound reorganization we examined by immunohistochemistry the expression of several extracellular matrix (ECM) receptors (alpha2 chain of VLA-2, alpha3 chain of VLA-3, alpha6 chain of VLA-6, alphav, and beta1/beta3 chains of integrins) in a series of biopsies of human skin wounds healing by primary intention. The first time point investigated in this study was day 6 after injury, i.e. when a fibrin clot has been almost completely replaced by the granulation tissue. Gradual changes in integrin expression in granulation tissue and in the dermal scar were observed from the first time point investigated and were characterized by an up-regulation of alpha2beta1 complex, alphav integrin subunit, and beta1 integrin subunit. At day 27, the expression of the alpha2 chain of VLA-2 in the scar decreased. The expression of alphav and beta1 integrin subunits decreased but was still detectable by day 35. Vitronectin expression from day 7 onwards was also increased and colocalized to the area of the wounded dermis, and decreased by day 27. Our data suggests that, during the remodelling of the provisional matrix of the wound, dermal fibroblasts express transiently mainly alpha2 and alphav subunits of integrins associated with up-regulation of the beta1 subunit. It seems that up-regulation of some chains of integrins may be involved in the control of deposition of ECM components associated with wound healing.

Cicatrix↗
PubMed1999

Laminin mediates tethering and spreading of colon cancer cells in physiological shear flow.

Under the physiological shear condition, cultured colon cancer cells bound to laminin (LM), but not to fibronectin or vitronectin. Most of the tethered cells did not roll, but arrested immediately and spread within 10-30 min on LM under the continuous presence of shear flow. The tethering of Colo201 was partially inhibited by monoclonal antibodies (mAbs) to alpha6 integrin and a combination of mAbs to beta1 and beta4 integrins, but not by mAb to 67KD laminin receptor. Some Colo201 cells still tethered at 4 degrees C. This suggests that alpha6beta1 and alpha6beta4 integrins participate in Colo201 tethering on LM, although other non-integrin molecules play roles. In contrast, the spread of Colo201 was effectively inhibited by the mAbs to integrin alpha2, alpha6 and beta1 chains. The effect of anti-alpha2 plus anti-alpha6 mAbs was almost equal to anti-beta1, suggesting that Colo201 cells mainly use alpha2beta1 and alpha6beta1 integrins for spreading on LM. When the cells were perfused on subconfluent endothelial cells (HUVEC) cultured on LM, they did not tether on HUVEC but did on coated LM exposed at intercellular gap area. Immunohistochemistry revealed that LM abundantly existed in the cytosol of human portal and hepatic vein endothelial cells. These data suggest that LM can mediate from tethering to spreading of colon cancer cells under the blood flow and plays an essential role in haematogeneous metastasis.

Antibodies, Monoclonal↗
PubMed1996

Cell adhesion and integrin binding to recombinant human fibrillin-1.

Fibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin alphaVbeta3. Integrins alphaIIbbeta3, alpha5beta1, alpha2beta1 and alpha1beta1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin alphaVbeta3.

Amino Acid Sequence↗
PubMed2001

Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.

Antibodies, Monoclonal↗
PubMed2006

Integrin-linked kinase functions as a downstream mediator of endothelin-1 to promote invasive behavior in ovarian carcinoma.

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ET(A)R) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by beta1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ET(A)R leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ET(A)R by ET-1 enhances the expression of alpha2beta1 and alpha3beta1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through beta1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3beta. The blockade of ET-1/ET(A)R-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase-dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3beta phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ET(A)R axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ET(A)R.

Animals↗
PubMed2003

Integrin receptors and ECM proteins involved in preferential adhesion of colon carcinoma cells to lung cells.

To assess the putative role of extracellular matrix (ECM) proteins on lung cells interacting with integrin receptors on colon carcinoma cells, an in vitro adhesion assay was used to investigate these factors. Tumor necrosis factor (TNF)-alpha treatment of fetal lung cell line MRC 9, upregulated expression of ECM proteins and also supported enhanced adhesion of PTC colon carcinoma cells. Antibodies to ECM proteins significantly blocked this enhanced adhesion of PTC cells. Similarly, antibody blocking of beta1 and beta2 integrin receptors on PTC cells revealed the integrin receptors involved in this enhanced adhesion. beta1 integrin receptors like alpha2beta1, alpha4beta1 and alpha5beta1 on PTC cells were found interacting with their ECM ligands like fibronectin and laminin on TNF-alpha stimulated MRC 9 cells. Interestingly, PTC cells were found to constitutively express alphaLbeta2, which is normally expressed by leukocytes. The data from the present study indicates that expression of multiple beta1 and beta2 integrins by colon carcinoma cells putatively allows these cells to successfully adhere to secondary sites like lungs.

Adenocarcinoma↗
PubMed1999

Expression of VLA-integrins and their related basement membrane ligands in gingiva from patients of various periodontitis categories.

Periodontitis is characterized by destruction of dento-gingival fibers and apical migration of the junctional epithelium. Tissue destruction may be associated with altered interactions between epithelium and connective tissue mediated by integrins localized in the basement membrane zone. We examined the expression of alpha2beta1, alpha3beta1, alpha4/alpha5/beta1, alpha6beta4 and their related extracellular matrix (ECM) ligands: laminin-1, laminin-5, and collagen type IV in untreated periodontitis sites of various categories. The expression and location of ECM proteins along the basement membrane were found to be similar between clinically healthy and periodontitis affected tissues. However, ECM proteins were more diffusely distributed in connective tissue (CT) of periodontitis tissues as streak-like/ fibrillar/granular stainings, particularly beneath the pocket epithelium (PE) and around the blood vessels. This may reflect an increase in inflammatory cell migration. The more widespread distribution of integrins alpha2beta1, alpha3beta1 in PE of periodontitis specimens may be related to disease activity and increased rate of keratinocyte proliferation and migration. Moreover, the weaker expression of alpha6beta4 in junctional epithelium (JE) of periodontitis affected tissues may be related to the epithelial detachment from the tooth surface. Clarification of expressions of integrins and their ligands in relation to known clinical disease susceptibility factors may provide information on the onset and progression mechanisms of periodontal disease destruction.

Adolescent↗
PubMed2001

Contraction-dependent apoptosis of normal dermal fibroblasts.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Antibodies↗
PubMed2002

Production and characterization of an in vitro engineered human oral mucosa.

The role of epithelial cells in oral pathologies is poorly understood. Until now, most studies have used normal or transformed epithelial cell monolayers, a system that largely bypasses oral mucosal complexity. To overcome these limitations, an engineered human oral mucosa (EHOM) model has been produced and characterized. Following histological and immunohistochemical analyses, EHOM showed well-organized and stratified tissues in which epithelial cells expressed proliferating keratins such as Ki-67, K14, and K19 and also differentiating keratin (K10). In this model, epithelial cells interacted with fibroblasts in the lamina propria by secreting basement membrane proteins (laminins) and by expressing integrins (beta1 and alpha2beta1). Cytokine analyses using cultured supernatants showed that cells in EHOM were able to secrete interleukins (IL) including IL-1beta and IL-8 and tumor necrosis factor alpha (TNF-alpha). Finally, cells in this engineered model were able to secrete different metalloproteinases such as gelatinase-A and gelatinase-B. In conclusion, using tissue engineering technology, we produced well-organized EHOM tissues. It is anticipated that this model will be useful for examining mechanisms involved in oral diseases under controlled conditions by modeling the interactions between mucosa and microorganisms in the oral cavity.

Animals↗
PubMed2000

Basement membrane laminin-5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for alpha3beta1 integrin.

Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors alpha2beta1, alpha3beta1 and alpha6beta4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immunoreactivity for chains of Ln-5 (alpha3-beta3-gamma2) and Ln-10 (alpha5-beta1-gamma1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln al chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for alpha2, alpha6, beta1 and beta4 Ints was found in all tumors and that for alpha3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the gamma2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to alpha3 and beta1 Int subunits, but not those to Int alpha2 or alpha6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln-5, produced by carcinoma cells, is a major BM component and the cells use the alpha3beta1 Int complex for adhesion to Ln-5.

Adenoma↗
PubMed1999

Osteoblast conditioned media contain TGF-beta1 and modulate the migration of prostate tumor cells and their interactions with extracellular matrix components.

Prostate cancers (PRCAs) frequently metastasize to bone. We show here that this process is facilitated by osteoblast-mediated tumor cell recruitment. Transforming growth factor-beta1 (TGF-beta1) is produced by osteoblasts in a latent form and is activated by proteases in a cell-dependent manner. This cytokine exhibits pleiotropic effects on cell-extracellular matrix (ECM) interactions and may influence tumor cell invasion and metastasis. Our purpose was to identify the potential molecular mechanisms involved in osteoblast-mediated cell recruitment and to characterize the effect of TGF-beta1 on adhesion, motility and invasiveness of a human prostate cancer cell line with high bone metastatic potential (PC3 cell line) in vitro. Conditioned media from osteoblast cultures (OB CM) enhanced PC3 cell chemotaxis and invasion of reconstituted basement membrane. These effects were blocked by a neutralizing TGF-beta1 polyclonal antibody but not by elution of the OB CM in agarose-heparin columns, suggesting that TGF-beta1, but not EGF-like proteins, contribute to PC3 cell recruitment. In addition, TGF-beta1 directly induced chemotaxis and invasion of PC3 cells in a dose-dependent manner. The TGF-beta1-mediated invasion and motility were accompanied by increased PC3 cell adhesion, spreading and alpha2beta1 and alpha3beta1 integrin expression. These events are involved in the cell adhesion to several components of basement membrane and ECM and in the selective invasion of metastatic tumor cells. Our results suggest that TGF-beta1 can influence cellular recognition of ECM components by prostatic cancer cells and can modulate cell adhesion and invasion leading to increased invasive potential. Given the widespread tissue distribution of TGF-beta1, and the high levels present in the bone, this cytokine may be an important autocrine-paracrine modulator of the bone invasive phenotype in vivo.

Animals↗
PubMed2006

The effects of the Maillard reaction on the physical properties and cell interactions of collagen.

The non-enzymic glycation of collagen occurs as its turnover decreases during maturation, with complex carbohydrates accumulating slowly and the end-products of these reactions being permanent. The nature of these advanced glycation end-reaction products (AGEs) can be categorised as: 1) cross-linking: intermolecular cross-linking may occur between two adjacent molecules and involve lysine to lysine or lysine to arginine residues. Several compounds have been characterised. They are believed to be located between the triple helical domains of adjacent molecules in the fibre resulting in major changes of the physical properties, primarily, fibre stiffness, thermal denaturation temperature and enzyme resistance, all of which increase slowly with age but the rate is accelerated in diabetes mellitus due to high glucose levels: 2) side-chain modifications: these changes alter the charge profile of the molecule affecting the interactions within the fibre and if they occur at specific sites can affect the cell-collagen interaction. Modification of arginine within the sites RGD and GFOGER recognised by the two specific integrins (alpha1beta2 and alpha2beta1) for collagen reduce cell interactions during turnover and for platelet interactions (alpha1beta2). These changes can ultimately affect repair of, for example, vascular damage and dermal wound healing in diabetes mellitus. Both types of modification are deleterious to the optimal properties of collagen as a supporting framework structure and as a controlling factor in cell matrix interactions. Glycation during ageing and diabetes is therefore responsible for malfunctioning of the diverse collagenous tissues throughout the body.

Aging↗
PubMed1998

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer.

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both alpha2beta1 and alpha3beta1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequence intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhesion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.

Adenocarcinoma, Scirrhous↗
PubMed1999

Regulation and function of the extracellular matrix protein tenascin-C in ovarian cancer cell lines.

The extracellular matrix glycoprotein tenascin-C (TN) is overexpressed in the stroma of malignant ovarian tumours particularly at the interface between epithelia and stroma leading to suggestions that it may be involved in the process of invasion (Wilson et al (1996) Br J Cancer 74: 999-1004). To define regulation of TN further and investigate its function in ovarian cancer, a range of cell line models were studied. Concentrations of secreted TN in media from cultures of ovarian fibroblast cell lines were at least 100-fold greater than from carcinoma cell lines. Evidence for paracrine regulation of TN secretion was obtained by co-culture of carcinoma cells with fibroblast cells wherein secretion into the media was greater than from fibroblasts alone. Transforming growth factor (TGF)-beta1, insulin-like growth factor (IGF)-II and progesterone all stimulated TN secretion while human choriogonadotropin (hCG), follicle-stimulating hormone (FSH) and gamma-interferon inhibited secretion. TGF-beta1 produced the greatest stimulation of TN in cultured fibroblasts and its co-expression with TN was examined in primary ovarian tumours. There was a significant association between the presence of moderate-strong expression of TN and TGF-beta1. Evidence for TN having a functional role in ovarian carcinoma was obtained from adhesion and migration assays. The PE01, PE04, SKOV-3 and 59M cell lines all demonstrated marked adhesion to plastic coated with TN relative to the control protein bovine serum albumin (BSA) and expressed alpha2beta1 and alpha3beta1 integrins. The SKOV-3 cell line migrated more rapidly through TN than through BSA indicating that TN can facilitate migration of ovarian carcinoma cells.

Animals↗
PubMed1999

Biologic roles of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins.

The biologic functions of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins (type I and IV collagens, fibronectin, and laminin) were investigated by using the G(D3)-deficient mutant clone (SK-MEL-28-N1) and the parent cell line SK-MEL28. SK-MEL-28-N1 (N1) (high G(M3) expression: G(M3), 97.3%; G(D3), 0%) was selected by treating SK-MEL-28 (high G(D3) but low G(M3): G(M3), 6.5%, G(D3), 93.5%) with an anti-G(D3) monoclonal antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells. The N1 clone showed significantly higher ability to adhere to type I and IV collagens and laminin than the parent clone SK-MEL-28. In the N1 clone, the expression of alpha2beta1 and alpha3beta1 integrin receptors was increased, whereas in SK-MEL-28, their expression was very low or undetectable. The treatment with monoclonal antibodies directed specifically to G(D3) expressed on SK-MEL-28 inhibited the cell attachment to type IV collagen (33% inhibition of control), fibronectin (59%), and laminin (71%). These findings suggest that gangliosides G(M3) (by influencing integrin receptor levels) and G(D3) (by interacting directly with matrix proteins) might play some functional roles in attachment to extracellular matrix proteins and thereby enhance the metastatic potency of melanoma cells.

Animals↗
PubMed1999

Early platelet activation by low density lipoprotein via p38MAP kinase.

Low Density Lipoprotein (LDL) is known to sensitize platelets for physiological agonists. To clarify the basis of this sensitization, we investigated the involvement of p38MAP Kinase (p38MAPK). As dual phosphorylation on Thr180 and Tyr182 of p38MAPK is the trigger for activation of the kinase, p38MAPK-activity was measured with an antibody that recognizes the dual-phosphorylated sequence. LDL induced a rapid and dose dependent activation of p38MAPK. The activation was not inhibited by a wide variety of inhibitors of platelet signalling, including TxA2-formation, Phospholipase C-activation, Ca2+-mobilization and ERK 1/2-activation. Only a slight reduction in p38MAPK-activation was observed when protein kinase C was inhibited. Activation of p38MAPK was strongly inhibited by a rise in cAMP. Thus, p38MAPK-activation was upstream of most signalling pathways and close to the LDL-receptor. A number of platelet receptors was screened with the use of antibodies. Integrins alphaIIbbeta3 and alpha2beta1, as well as the FcgammaRII-receptor, CD36 (platelet glycoprotein IV), CD68 (gp110) and Low Density Lipoprotein-receptor related protein (LRP) were not implicated in LDL-induced p38MAPK-activation. Inhibition of LDL binding by modification of apo B100 lysines reduced p38MAPK-activation by 80%. Activation of p38MAPK resulted in an increase in release of arachidonic acid, the precursor for thromboxane A2 synthesis. In conclusion, activation of p38MAPK might be the first step in platelet sensitization by LDL, leading to formation of arachidonate metabolites and increased aggregation and secretion responses to physiological agonists.

Humans↗
PubMed2001

A comparison of ocular melanocyte and uveal melanoma cell invasion and the implication of alpha1beta1, alpha4beta1 and alpha6beta1 integrins.

BACKGROUND/AIMS: Posterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The beta1 family of integrins, alphavbeta3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement in uveal melanoma we analysed the expression of the beta1 integrins, alphavbeta3, MMP-2, and MMP-9 in 10 primary posterior uveal melanomas, and correlated expression with invasive potential in vitro. Comparable studies were undertaken on cultures of melanocytes. METHODS: Expression of integrins was studied by immunohistochemistry, secretion of MMP-2 and MMP-9 by zymography, and the invasive potential was assessed using a transwell model. RESULTS: MMP-2 was secreted by all uveal melanomas and seven of 10 secreted MMP-9. Among uveal melanoma, invasion levels of 4-25% were observed and the major integrins expressed were alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1, and avbeta3. Melanocytes did not express alpha1beta1, alpha4beta1, and alpha6beta1. CONCLUSION: The laminin binding alpha6beta1 integrin was not expressed by either melanocytes or tumours with spindle morphology, which are considered to have a better prognosis. It is possible that expression of the alpha6beta1 integrin may prove useful as a prognostic indicator.

Adult↗