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PubMed1999

Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.

Animals↗

PubMed1995

The Ca2+ signaling capacity of the beta 2-integrin on HL60-granulocytic cells is abrogated following phosphorylation of its CD18-chain: relation to impaired protein tyrosine phosphorylation.

The phosphorylation state of the CD18-chain of beta 2-integrins have been shown not to mediate changes in the avidity of these receptors (i.e., inside-out signaling); however, no alternative functional significance has been proposed. Our study focused on how changes in the phosphorylation state of beta 2-integrin-receptors on HL60-granulocytic cells are related to its intracellular signal transduction properties (i.e., outside-in signaling). Engagement of beta 2-integrins on differentiated HL60 cells induced a transient increase in the cytosolic free Ca2+ concentration and an increased tyrosine phosphorylation of three major protein bands (70, 115, and 140 kDa). These signaling events occurred without any detectable phosphorylation of the CD18-chain. However, a strong phosphorylation of the CD18-chain by preexposure to phorbol myristate acetate (PMA) coincided with an abolishment of both the beta 2-integrin-induced Ca2+ signal and the protein tyrosine phosphorylations. By comparison, none of these effects were exhibited by 4-alpha-PMA, an analogue that does not activate protein kinase C. Thus, phosphorylation of the CD18-chain of beta 2-integrins is not required for outside-in signal transduction by these receptors, but it could constitute an effective mechanism by which the signaling properties of beta 2-integrins can be modulated by exogenous factors and possibly also by intracellular signals induced by other receptors. The fact that both the cytosolic free Ca2+ signal and protein tyrosine phosphorylations were abrogated by PMA suggests an intimate relationship between these two intracellular signals. To explore this possible relationship, we chelated the beta 2-integrin-induced Ca2+ signal with BAPTA. The beta 2-integrin-induced protein tyrosine phosphorylations were blocked by BAPTA but not by abolishment of the Ca2+ signal due to chelation with MAPT or by pretreatment with thapsigargin. These findings and the observation that pretreatment of cells with methyl-2,5-dihydroxycinnamate (a tyrosine kinase inhibitor) blocked the beta 2-integrin- but not the fMet-Leu-Phe-induced Ca2+ signal suggest that beta 2-integrin-induced tyrosine kinase activation occurs prior to and is a prerequisite for the subsequent Ca2+ signal.

CD18 Antigens↗

PubMed1996

Beta3 integrin expression in melanoma predicts subsequent metastasis.

Previous studies have suggested that differential expression and/or activation of integrins facilitates metastatic progression in murine and human melanoma. While recent data show that the integrin alphavbeta3 is involved in tumor angiogenesis and that tumor growth may be abrogated by alphavbeta3 inhibitors in vitro, the clinical significance of beta3 integrin expression in human malignant melanoma is not known. To assess the prognostic value of beta3 integrin expression, we examined primary cutaneous melanomas from 160 patients followed for a mean of 98 months or until death. We quantified the percentage of tumor area stained with beta3 integrin Ab CD-61 using an image analyzer. beta3 integrin expression was detected in 107/160 primary melanomas (69%). beta3-integrin-positive (beta3+) tumors were thicker (mean 2.98 +/- 0.3 mm) than beta3-integrin-negative (beta3-) melanomas (mean 1.64 +/- 0.2 mm) (P = 0.002). Patients with beta3+ melanomas were more likely to relapse (57/107, 53%) and to die from disease (45/107, 42%) than those with beta3- tumors (6/53, 11%; and 4/53, 8%, respectively) (P < 0.001). Overall survival was greater for beta3- than for beta3+ patients (mean 102 +/- 9 vs 69 +/- 6 months) (P = 0.001). These data show that beta3 integrin expression in primary cutaneous melanoma predicts subsequent metastatic progression. Further study of beta3 integrins in the development of melanoma metastases may yield new therapeutic strategies, as well as prognostic information, for the treatment of this cancer.

Antigens, CD↗

PubMed1999

Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.

Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding.

Animals↗

PubMed1992

Differential expression of cell surface integrins on human mast cells and human basophils.

Integrins are multifunctional recognition molecules and are expressed on various hematopoietic cells. In the present study expression of integrins on the cell surface of human mast cells and human basophils was investigated by using monoclonal antibodies (mAbs) and indirect immunofluorescence. Human mast cells were obtained from lung (n = 5), uterus (n = 5) and skin (n = 4). Human blood basophils were obtained from normal donors (n = 2). In addition, HMC-1 cells (human mast cell line) and KU-812 cells (a basophil cell line) were analyzed. Primary mast cells were found to react with mAbs directed against the common beta chain of beta 1 integrins (CD 29), the alpha chain of VLA-4 (CD 49 d) and VLA-5 (CD 49 e), the beta chain of beta 3 integrins (CD 61), and the alpha chain of the vitronectin receptor (VNR) (CD 51). Mast cells were not recognized by mAbs to beta 2 integrins (CD 18, CD 11 a, CD 11 b, CD 11 c), the alpha chain of VLA-2 (CD 49 b), and VLA-6 (CD 49 f). No differences in expression of integrins on human mast cells obtained from different organs were found. HMC-1 cells and primary mast cells expressed an almost identical pattern of integrins. Human basophils and KU-812 cells were found to react with mAbs directed against beta 1-integrins (CD 29, CD 49 b, CD 49 d, CD 49 e) and beta 2-integrins (CD 18, CD 11 a, CD 11 b, CD 11 c). Together, mast cells and blood basophils express a unique pattern of integrins. These cell surface structures may be involved in the distribution of basophils and tissue mast cells and their accumulation and function in inflammed tissues.

Antibodies, Monoclonal↗

PubMedDate not supplied

Hepatocyte growth factor upregulates alpha2beta1 integrin in Madin-Darby canine kidney cells: implications in tubulogenesis.

It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.

Animals↗

PubMed1990

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts.

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.

Animals↗

PubMed1995

Decreased integrin alpha 2, but normal response to TGF-beta in scleroderma fibroblasts.

The distribution and amount of integrin alpha2 were studied in cultured fibroblasts from normal subjects and scleroderma patients by immunofluorescence and immunoblotting using a monoclonal antibody against the human integrin alpha2 subunit. Integrin alpha2 was concentrated at the perinuclear regions in a dot-like pattern in normal fibroblasts on the glass coverslips until the 14th day after planting, and the staining pattern of integrin alpha2 was gradually changed to a dispersed dot-like pattern by the 19th day as examined by immunofluorescence microscopy by using the anti-integrin alpha2 antibody. No difference was observed in the distribution patterns between normal and scleroderma fibroblasts. By immunoblotting study, the amount of integrin alpha2 in scleroderma fibroblasts (n = 10) was less than that of normal fibroblasts (n = 10) (P < 0.01) in both cytosol and cytoskeleton-associated fractions. Furthermore, transforming growth factor beta (TGF-beta) increased the amount of integrin alpha2 in both normal fibroblasts and scleroderma cells by 33%. The total amount of integrin alpha2 in TGF-beta-stimulated scleroderma fibroblasts was less than that in TGF-beta-stimulated normal fibroblasts. These findings suggest that the amount of integrin alpha2, a collagen receptor, is reduced in scleroderma fibroblasts, but the integrin alpha2 production by TGF-beta stimulation is not impaired in scleroderma fibroblasts.

Adult↗

PubMed2004

Immunohistochemical localization of alphavbeta3 integrin receptor during experimental tooth movement.

During orthodontic treatment, multinucleated clast cells carry out the resorption of mineralized tissues. Adhesion of clast cells to the mineralized tissues is mediated by transmembrane cell-surface glycoproteins called integrins, specifically by the alphavbeta3 integrin, which plays an important role in the process of bone resorption. The role of the alphavbeta3 integrin in bone resorption leading to osteoporosis has been demonstrated, but its role in alveolar bone and root resorption during orthodontic tooth movement is unknown. This study examined the expression of the alphavbeta3 integrin during experimental tooth movement. Tooth movement was achieved in 16 male Sprague-Dawley rats (each weighing 120-200 g) with elastic bands between their maxillary first and second molars. The molar-bearing segments were dissected and processed for histologic and immunohistochemical examination. The expression of alphavbeta3 integrin was examined with 2 primary antibodies: a polyclonal anti-alphav integrin subunit antibody and a polyclonal anti-beta3 integrin subunit antibody. Negative controls were similarly processed but without incubation with primary antibodies. The alphavbeta3 integrin was expressed both by osteoclasts associated with alveolar bone resorption and by odontoclasts associated with root resorption during experimental tooth movement. Furthermore, the beta3 integrin subunit was expressed by the epithelial rests of Malassez in the periodontal ligament. Negative controls did not show immunolabeling. The alphavbeta3 integrin adhesion receptor is expressed during experimental tooth movement and might be involved in the process of mineralized tissue resorption and the functions of the epithelial rests of Malassez.

Alveolar Process↗

PubMed2005

Involvement of alpha4 integrins in maintenance of cardiac sympathetic axons.

Sympathetic neurons extend and maintain axons that innervate the myocardium, and proper innervation is important for cardiac function. However, the molecular basis for axon outgrowth and maintenance is not well understood. We have shown previously that the integrin alpha4beta1 is expressed on developing axons, and the alpha4 function is important for the development of innervation in vivo [Wingerd, K.L., Goodman, N.L., Tresser, J.W., Smail, M.M., Leu, S.T., Rohan, S.J., Pring, J.L., Jackson, D.Y., and Clegg, D.O., 2002. Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. J. Neurosci. 22,10772-10780]. Here we examine the function of alpha4beta1 integrins in the maintenance of cardiac sympathetic innervation in vitro and in vivo, and investigate integrin expression and function after myocardial infarction and in hypertensive rats. On substrates of vascular cell adhesion molecule-1 (VCAM-1), alpha4beta1 was required for both initial outgrowth and maintenance of neurites in vitro. On fibronectin substrates, initial outgrowth requires only alpha4 integrins, but maintenance requires both alpha4 integrins and RGD-dependent integrins. In vivo, in adult Long Evans rats, inhibition of alpha4 integrins resulted in decreased maintenance of sympathetic fibers innervating the apex of the heart. However, alpha4 integrins were not detected on most sympathetic axons that sprout after myocardial infarction, and alpha4 function was not required for sprouting. Spontaneously hypertensive rats (SHR) have increased numbers of cardiac sympathetic fibers compared to the parental Wistar strain, but many of these lack alpha4 expression, and alpha4 function is not required for maintenance of these fibers in the heart. These results suggest that developing sympathetic axons and sprouting sympathetic axons use different mechanisms of outgrowth, and that maintenance of cardiac sympathetic innervation involves alpha4 integrins in some rat strains.

Adrenergic Fibers↗

PubMed2006

Combination of integrin siRNA and irradiation for breast cancer therapy.

Up-regulation of integrin alpha(v)beta(3) has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin alpha(v)beta(3) in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin alpha(v) siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin alpha(v)beta(3)-positive). Integrin alpha(v)beta(3)-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin alpha(v)beta(3) expression in MDA-MB-435 cells and integrin alpha(v) siRNA can effectively reduce both alpha(v) and alpha(v)beta(3) integrin expression, leading to increased radiosensitivity. Integrin alpha(v) siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin alpha(v)beta(3)-positive cancer types.

Breast Neoplasms↗

PubMed2004

Alpha8beta1 integrin is upregulated in myofibroblasts of fibrotic and scarring myocardium.

Integrins mediate cell attachment to the extracellular matrix (ECM) regulating migration, proliferation, and differentiation. We previously reported the presence of alpha8beta1 integrin on cultured cardiac fibroblasts. Extending this information, we localized alpha8beta1 integrin in normal rat myocardial tissue, and investigated its expression pattern in rats chronically infused with angiotensin II (Ang II, 500 ng/kg/min), a well-recognized profibrotic factor. Alpha8beta1-integrin expression was analyzed by binding assay, western blotting, and immunohistochemistry. In normal myocardium, immunohistochemical staining for alpha8 was found in fibroblasts, as well as in the epicardium, endocardium, and valves. Vascular smooth muscle cells (VSMCs) of the media of cardiac arteries also stained positively. After 14-d-Ang II infusion, staining for fibronectin, as well as collagen staining by Sirius red, revealed extensive interstitial and perivascular fibrosis. Increased expression of alpha8 integrin in ventricular smooth muscle (SM) alpha-actin-positive fibroblasts (myofibroblasts) was also recorded. The upregulation of alpha8beta1 integrin was confirmed by binding assay and by western blotting. Microscopic scars, a characteristic of reparative fibrosis, were invaded by matrix proteins and by strongly alpha8- and SM alpha-actin-positive myofibroblasts. The results indicate that, in rat adult myocardium, alpha8beta1 integrin is expressed in fibroblasts and VSMC. In Ang II-infused animals, alpha8beta1-integrin expression was enhanced in the left ventricle and arteries. The coordinate regulation of alpha8beta1 integrin on fibroblasts and ECM proteins raises the possibility that this integrin is implicated in the deposition of matrix components leading to fibrosis.

Angiotensin II↗

PubMed1994

Expression of integrins and E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, and endometriosis.

OBJECTIVE: To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle. DESIGN: An immunohistochemical study. SETTING: Tertiary care university medical center. PATIENTS: Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles. INTERVENTIONS: A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead. MAIN OUTCOME MEASURES: The expression of cell adhesion molecules, including the integrin alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 and E-cadherin, as determined by immunohistochemistry on frozen sections. RESULTS: All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins alpha 2 beta 1 and alpha 3 beta 1 was found in epithelial cells in 13 of 16 cases. Integrin alpha 5 beta 1 was detected in 11 of 16 samples, and integrins alpha 4 beta 1 and alpha 6 beta 1 were detected in 5 of 16 samples. In PF, integrin alpha 3 beta 1 was found in epithelial cells in 12 of 16 samples, integrin alpha 5 beta 1 in 5 of 16, and integrins alpha 4 beta 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin. CONCLUSION: Integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Ascitic Fluid↗

PubMed2004

Granulocyte colony-stimulating factor receptor signals for beta1-integrin expression and adhesion in bladder cancer.

OBJECTIVES: To examine the association of granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) expression with increased beta1-integrin expression and determine the ability of autocrine G-CSFR signaling to promote bladder cancer cell adhesion by way of beta1-integrin. beta1-integrin is expressed at higher levels in more invasive bladder carcinoma cells and participates in the process of tissue invasion. Cancer cell invasion and metastasis in some ways mimic normal neutrophil behavior. In neutrophils, G-CSF acts through its receptor to enhance adhesion and migration. A significant fraction of bladder carcinoma has been reported to express both G-CSF and G-CSFR. METHODS: We examined bladder carcinoma tissue samples obtained from segmental or radical cystectomy specimens for expression of G-CSF, G-CSFR, and beta1-integrin using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical methods. We determined the G-CSFR-mediated beta1-integrin adhesion using a static adhesion assay and the bladder cancer cell line 5637. RESULTS: Eleven of 14 bladder cancer samples expressed G-CSFR. All 11 G-CSFR positive tumors also expressed G-CSF, and the G-CSF/G-CSFR positive tumors had elevated beta1-integrin protein levels. All but one G-CSFR negative tumor demonstrated low beta1-integrin protein levels. In four G-CSF/G-CSFR positive tumors for which distant urothelium was available for examination, G-CSF, G-CSFR, and beta1-integrin expression was also increased. In the 5637 cell line, we demonstrated G-CSFR-mediated upregulation of beta1-integrin-dependent adhesion to fibronectin and laminin. CONCLUSIONS: G-CSF/G-CSFR expression in some bladder cancers appears to be an early event during malignant transformation that increases beta1-integrin expression and adhesion and thereby may promote tissue invasion.

Autocrine Communication↗

PubMed1999

Depletion of alphaV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis.

Integrin heterodimers sharing the common alphaV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alphaV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alphaVbeta3 and alphaVbeta5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alphaV integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor alphaV integrin subunit was not affected. However, the maturation of alphaV integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of alphaV integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, alphaV integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation.

Alkaline Phosphatase↗

PubMed1998

Regulation of beta 1-integrin-mediated cell adhesion by the Cbl adaptor protein.

BACKGROUND: Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation. RESULTS: CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase. CONCLUSIONS: Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.

Amino Acid Substitution↗

PubMedDate not supplied

p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion.

We have previously shown that human breast cancer cells that overexpress erbB-2 are growth factor-independent. In order to test the contribution of erbB-2 to this and other transformed phenotypes without the genetic instability of cancer cells, erbB-2 was overexpressed in human mammary epithelial (HME) cells. ErbB-2-overexpressing HME cells exhibit several transformed phenotypes including cell surface alpha(4) integrin downregulation and invasiveness. We formulated a model for invasiveness that depends on a cell's ability to downregulate alpha(4) integrin. As small G-proteins play a role in cytoskeleton remodeling and as this is a likely route for alpha(4) integrin trafficking, we investigated the role of small G-proteins and their downstream signals in mediating alpha(4) integrin downregulation and invasiveness using Rac 1. Dominant-negative Rac 1 blocked erbB-2-mediated invasion and reversed erbB-2-mediated alpha(4) integrin downregulation. In addition, constitutively active Rac 1 induced alpha(4) integrin downregulation and invasiveness. In erbB-2-overexpressing and in constitutively active Rac 1-expressing cells, a p38MAP kinase (p38MAPK) inhibitor blocked invasiveness and reversed alpha(4) integrin downregulation. These data suggest a model in which erbB-2 signaling activates Rac 1, which, in turn, activates p38MAPK, leading to the downregulation of alpha(4) integrin. These data strengthen the model where loss of alpha(4) integrin at the cell surface, leading to reduced alpha(4) integrin binding to plasma fibronectin, plays a role in erbB-2-mediated invasiveness.

Actins↗

PubMed2003

Coordinated expression of integrin subunits, matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway?

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III-IV ovarian carcinomas were analyzed for the expression of alpha v and beta1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. Alpha v integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of beta1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of beta1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between alpha v integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of alpha v integrin subunit and labeling for IL-8 in carcinoma cells. The presence of beta1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of beta1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the beta1 (and possibly alpha v) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.

Adenocarcinoma, Mucinous↗