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PubMed2006

Regulation of prostate cell collagen receptors by malignant transformation.

Cell adhesion receptors, including the integrin-type collagen receptors (alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1) participate in cancer progression and invasion. Quantitative RT-PCR indicated that all 4 receptors are abundantly expressed in sarcoma-derived cell lines, whereas most carcinoma-derived cells express alpha1beta1 and alpha2beta1 only. This was surprising because alpha11beta1 has been connected previously to the progression of lung adenocarcinomas. To test the hypothesis that alpha11 expression may not persist in cultured cancer cells we analyzed fresh tissue samples of 104 total prostatectomies, keeping in mind that prostate cancer cell lines showed negligible alpha11 mRNA levels. In prostate alpha2 expression was significantly lower in poorly differentiated carcinomas when compared to benign lesions (p = 0.0331). In immunohistochemistry the protein levels of alpha2 integrin decreased significantly (p = 0.0001) and the protein levels of alpha11 subunit increased significantly (p = 0.029) with the increasing grade of carcinoma. Thus alpha11beta1 may replace alpha2beta1 during tumor progression. Our observations support the idea that alpha11beta1 may be expressed in tumors but the corresponding cell lines may lose the expression of this integrin. Previous studies have shown that in cell culture androgen receptor (AR) controls alpha2beta1 expression. We measured AR mRNA levels and the number of AR positive nuclei in the prostate samples and the results showed a significant correlation between alpha2beta1 and AR. Androgen receptors may control the mechanisms regulating integrin expression in prostate.

Carcinoma↗

PubMed2005

Maturational differences in superficial and deep zone articular chondrocytes.

To examine whether differences in chondrocytes from skeletally immature versus adult individuals are important in cartilage healing, repair, or tissue engineering, superficial zone chondrocytes (SZC, from within 100 microm of the articular surface) and deep zone chondrocytes (DZC, from 30%-45% of the deepest un-mineralized part of articular cartilage) were harvested from immature (1-4 months) and young adult (18-36 months) steers and compared. Cell size, matrix gene expression and protein levels, integrin levels, and chemotactic ability were measured in cells maintained in micromass culture for up to 7 days. Regardless of age, SZC were smaller, had a lower type II to type I collagen gene expression ratio, and higher gene expression of SZ proteins than their DZC counterparts. Regardless of zone, chondrocytes from immature steers had higher levels of Sox 9 and type II collagen gene expression. Over 7 days in culture, the SZC of immature steers had the highest rate of proliferation. Phenotypically, the SZC of immature and adult steers were more stable than their respective DZC. Cell surface alpha5 and alpha2 integrin subunit levels were higher in the SZC of immature than of adult steers, whereas beta1 integrin subunit levels were similar. Both immature and adult SZC were capable of chemotaxis in response to fetal bovine serum or basic fibroblast growth factor. Our data indicate that articular chondrocytes vary in the different zones of cartilage and with the age of the donor. These differences may be important for cartilage growth, tissue engineering, and/or repair.

Age Factors↗

PubMed1997

Localization of extracellular matrix molecules, integrins and their regulators, TGF betas, is correlated with axon pathfinding in the spinal cord of normal and Danforth's short tail mice.

To clarify the roles of extracellular matrix molecules (ECMs) and their receptors, integrins, in axonal pathfinding, their spatiotemporal localization was studied immunohistochemically in the developing mouse spinal cord. Immunoreactivities for collagen type IV (Col IV), laminin, integrins (alpha2, alpha3 and beta1 subunits), and transforming growth factor (TGF) betas (TGF beta2 and TGF beta3), putative regulators of gene expression of these molecules, were localized on the midline of the ventral spinal cord, or the 'floor plate', in normal ICR mice. These immunoreactions appeared at embryonic day 9 (E9), when commissural axons began to cross the floor plate, and peaked at E13-E14 when more axons crossed, turned rostrally, and projected longitudinally. At this stage, TAG-1-immunopositive commissural axons passed through the floor plate region. Longitudinal axons, on the other hand, projected ipsilaterally parallel to the midline. Immunoreactions for ECMs, integrins and TGF betas were weakly positive at the midline until postnatal day 0 (P0), after which they decreased markedly. In Danforth's short tail (Sd) heterozygous mutants with discontinuous floor plates, immunoreactions for ECMs, integrins, and TGF betas were not detected at P0. Few crossing axons were seen at this stage, and longitudinally ascending axons crossed via aberrant paths in these regions. These results suggest that the restricted localization of ECMs, integrins, and TGF betas at the floor plate is involved in the pathfinding of commissural and longitudinal axons.

Animals↗

PubMed2004

Modulation of anti-adhesion molecule MUC-1 is associated with arctiin-induced growth inhibition in PC-3 cells.

BACKGROUND: Lignans have been reported to possess anti-tumor activity in various cancer cells. However, their anticancer effects in human prostate cancer have not been well established. Here, we examine the effect of arctiin, a lignan compound, on growth regulation in prostate cancer PC-3 cells. We postulated that arctiin modulates the attachment/detachment of PC-3 cells and we investigated the role of arctiin on MUC-1 expression. METHODS: The effect of arctiin on PC-3 cell growth was examined using an MTT assay method and cell number was calculated by means of a standard regression line. The expressions of MUC-1 and integrins alpha2, alpha5, and beta1 were detected using FACScan flow cytometric analysis. Levels of MUC-1 mRNA were determined using reverse transcriptase PCR (RT-PCR). RESULTS: Treatment of PC-3 cells with arctiin decreased the cell number in a concentration- and time-dependent manner in serum-containing condition. Arctiin preferentially induced cell detachment, but did not have anti-proliferation or cytotoxic effects in PC-3 cells. The arctiin-induced effect was inhibited by cycloheximide, indicating that protein synthesis was required. FACScan flow cytometric analysis demonstrated that arctiin increased the expression of the anti-adhesion mucin MUC-1, but did not affect integrin expression in PC-3 cells. The arctiin-induced increase in MUC-1 protein expression was due to up-regulation of mRNA, as revealed by RT-PCR analysis. CONCLUSIONS: Arctiin significantly induces cell detachment and decreases the cell numbers via the up-regulation of MUC-1 mRNA and protein in PC-3 cells.

Cell Adhesion↗

PubMed2001

The role of the extracellular matrix in articular chondrocyte regulation.

The in vivo role of the extracellular matrix and the manner in which it interfaces with soluble regulators remains largely unknown. The current study reports the extracellular Type II collagen modulation of transforming growth factor-beta 1-stimulated proliferation, proteoglycan synthesis, messenger ribonucleic acid expression for transforming growth factor-beta 1, and integrin messenger ribonucleic acid expression in articular chondrocytes from adults. This study shows that this cytokine modulation occurs through a mechanism initiated by the attachment of Type II collagen to the beta1-integrin. Transforming growth factor-beta 1 stimulated deoxyribonucleic acid and proteoglycan synthesis in a bimodal fashion. Extracellular Type II collagen increased transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis, aggrecan gene expression as much as 400%, and alpha1(II) procollagen gene expression as much as 180% in a dose-dependent fashion. Heat inactivation of the Type II collagen abrogated the observed effects on deoxyribonucleic acid and proteoglycan synthesis. In contrast to Type II collagen, heat-denatured collagen and bovine serum albumin showed none of the observed effects. The presence of Type II collagen in the alginate bead cultures was found to diminish the messenger ribonucleic acid expression for alpha2 integrin and alter the cellular distribution pattern of the beta1 integrin receptors. Blocking of the beta1-integrin with cyclic-peptides containing the Arg-Gly-Asp sequences and antibodies reduced chondrocyte attachment to Type II collagen by 93%. The physiologic effects shown by the chondrocyte as a result of blocking this attachment to Type II collagen were a significant reduction in transforming growth factor-beta 1-stimulated deoxyribonucleic acid and proteoglycan synthesis. The conclusions elucidate the role played by the extracellular matrix in cytokine-specific regulation of the articular chondrocyte. The authors have shown that extracellular Type II collagen acts through a beta1-integrin mediated mechanism to modulate the chondrocyte response to transforming growth factor-beta 1.

Aggrecans↗

PubMed2006

Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptor-mediated pathway.

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Genetic factors underlying differential blood platelet sensitivity to inhibitors.

Blood platelets are not only the primary defence mechanism involved in physiological hemostasis, but also their disorders constitute a crucial risk factor in arterial thrombosis. As arterial thrombi are composed of predominantly platelets formed under conditions of elevated shear stress at sites of atherosclerotic vascular injury and disturbed blood flow, the prevention of arterial thrombosis has been for years the main target for antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. Some clinical studies have indicated that platelet glycoprotein polymorphisms are genetic factors contributing to arterial thrombosis. In spite of some discrepancies between different studies, there is substantial evidence that the integrin beta3 P1(A2) allele, the variants GPIbalpha Met145 and GPIbalpha (-5C) haplotype or the integrin alpha2 haplotype 1 (807T) each contribute to the risk for and morbidity of thrombotic disease. In this article, we reviewed a role of the aforementioned polymorphisms in modulating platelet function and platelet response to inhibitors. The paper focuses on the association between Pl(A1/A2) polymorphism and sensitivity (or resistance) to aspirin and the inhibitory efficacy of GPIIb-IIIa antagonists. Additionally, a potential role of 807C/T polymorphism (GPIa), polymorphisms of GPIb and platelet purinoreceptor P2Y12 in affecting platelet sensitivity to blocking agents is discussed.

Blood Platelets↗

PubMed1999

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Adolescent↗

PubMed2003

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

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

Carcinoma, Hepatocellular↗

PubMed2002

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

Animals↗

PubMed1996

Differential expression of integrin alpha subunits supports distinct roles during lung branching morphogenesis.

Epithelial branching morphogenesis is a process by which a continuous epithelium, embedded in mesenchyme, forms tubules that extend and branch into the surrounding mesenchyme. The morphogenetic process is responsible for the architecture of many organs including the lung. Proper expression and function of extracellular matrix (ECM) molecules, such as collagens and laminins, are necessary for branching to occur normally. However, little is known about the role of epithelial cell surface molecules that mediate epithelial-matrix interactions during this process. We have studied the expression patterns of cell surface collagen and laminin integrin receptor alpha subunits, alpha 1, alpha 2, alpha 3, and alpha 6, in relation to that of collagen and laminin during lung branching morphogenesis. The alpha 1 integrin subunit was present on endothelia and smooth muscles around airways and large blood vessels. The mesenchyme expressed high levels of alpha 2 and alpha 6 but not alpha 3, whereas the epithelium expressed all three integrin subunits. In contrast to the widespread epithelial expression of alpha 3 and alpha 6, the epithelial expression of alpha 2 was restricted to branch tips. By performing in situ hybridization and immunofluorescence on serial sections, we found that alpha 2 protein expression on the epithelium correlated spatially and temporally with high level expression of collagen IV and laminin-1 mRNAs, suggesting that the alpha 2-expressing epithelial cells were in the process of producing and assembling their collagen and laminin matrices. While the expression of alpha 3 and alpha 6 on all lung epithelia suggests that these integrins may be important to lung epithelial development, the unique expression pattern of the alpha 2 subunit suggests that the alpha 2 beta 1 integrin may be important at branch tips either in the process of collagen/laminin synthesis and assembly or extension of the epithelial tubules into the mesenchyme.

Animals↗

PubMed2000

Inhibition of distal lung morphogenesis in Nkx2.1(-/-) embryos.

In vitro and in vivo results are consistent with a critical role for NKX2.1, an epithelial homeodomain transcription factor in lung morphogenesis. Nkx2.1 null mutant embryos die at birth due to respiratory insufficiency caused by profoundly abnormal lungs. However, the precise role of NKX2.1 in the multistep process of lung structural morphogenesis and differentiation of various pulmonary cell types remains unknown. In the current study, we tested the hypothesis that the mutant lungs do not undergo branching morphogenesis beyond the formation of the mainstem bronchi and therefore consist solely of dilated tracheobronchial structures. To test this hypothesis, we determined the spatial and temporal expression pattern of a number of extracellular matrix (ECM) proteins and their cellular receptors, including alpha-integrins, laminin, and collagen type IV. Although laminin is expressed in the mutant Nkx2.1(-/-) lungs, expression of alpha-integrins and collagen type IV is significantly reduced or absent. In addition, examination of regionally specific expression of differentially spliced Vegf (vascular endothelial growth factor) transcripts, clearly indicates that the epithelial phenotype of the Nkx2.1(-/-) lungs is similar to the tracheobronchial epithelium. In contrast to wild-type lungs in which both Vegf1 and Vegf3 are developmentally expressed, Nkx2.1(-/-) lungs are characterized by predominant expression of Vegf1 and reduced or absent Vegf3. A similar pattern of Vegf expression is also observed in isolated tracheo-bronchial tissue. The sum of these findings suggest that at least two separate pathways may exist in embryonic lung morphogenesis: proximal lung morphogenesis is Nkx2.1 independent, while distal lung morphogenesis appears to be strictly dependent on the wild-type activity of Nkx2.1.

Actins↗

PubMed1996

Role of integrins in regulation of collagen phagocytosis by human fibroblasts.

Phagocytosis of collagen fibrils by fibroblasts is an important pathway for degradation of extracellular matrix in mature connective tissues. To study regulatory mechanisms in phagocytosis, 2-microns fluorescent beads coated with either collagen (COL) or bovine serum albumin (BSA) were incubated with human gingival fibroblasts in vitro. For these studies single cell suspensions were prepared by trypsinization, and bead internalization and collagen receptor expression were assessed by flow cytometry. After 3-h incubations, up to 8-fold more cells internalized COL beads than BSA-coated beads. Increased collagen coating concentration was associated with elevated proportions of cells that internalized COL beads, and was observed also in the presence of competing fibronectin-coated beads. The number of beads per cell and the percent of phagocytic cells increased proportionally with higher bead loadings. At > 4 beads per cell a maximum of approximately 80% of cells were phagocytic. Cells reacted with mAbs against the alpha 1, alpha 2, and alpha 3 integrin subunits were, respectively, 5%, 98% and 93% positively stained above background controls. All cells that internalized COL beads exhibited alpha 2 staining but there were large proportions of phagocytic cells that were not stained for alpha 1. In unfixed cells, bead internalization caused an immediate reduction of surface staining of membrane-bound alpha 2 by approximately 55% which returned to control levels within 3 h, indicating that cell-surface alpha 2 was internalized by phagocytosis. Preincubation of cells with up to 8 COL beads per cell reduced the proportion of phagocytic cells and the number of internalized beads after a second COL bead incubation 4 h later. To assess the relationship between the percent of phagocytic cells and alpha 2 integrin levels, serum starvation and cycloheximide experiments were conducted. Compared to controls, serum starvation for 24 h induced a 3.2-fold increase of cells internalizing COL beads but did not alter alpha 2 staining levels. In contrast, 3 h cycloheximide treatment reduced alpha 2 staining to 60% of control levels and this treatment also inhibited COL bead internalization. GRGDTP peptide as well as mAbs against the alpha 1 and alpha 2 subunits significantly reduced internalization of COL beads by 1.8 to 2.6-fold, whereas GRGESP peptide and alpha 3 mAb exerted no effect. Internalization of BSA beads was not affected by any of these treatments. Collectively, these data indicate that the alpha 2 integrin, along with other, as yet unidentified components, is likely involved in COL bead internalization. The alpha 2 integrin subunit is rapidly recycled or synthesized following a phagocytic load. In contrast, the alpha 1 integrin is not directly required for phagocytosis but may regulate the internalization step.

Amino Acid Sequence↗

PubMed1995

Appearance and distribution of laminin A chain isoforms and integrin alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits in the developing human small intestinal mucosa.

BACKGROUND: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. METHODS: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4, four potential receptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. RESULTS: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins alpha 6 beta 1 and alpha 6 beta 4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the alpha 2 beta 1 and alpha 3 beta 1 integrins was found time- and site-restricted. The alpha 2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the alpha 3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19-20-week-old crypts. CONCLUSIONS: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development.

Antigens, CD↗

PubMed2002

Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo.

Epithelial cell survival is dependent on extracellular signals provided by both soluble factors and by adhesion. In the mammary gland, extensive apoptosis of epithelial cells occurs rapidly when lactation ceases, but the mechanism of apoptosis induction is not known. In tissue culture, mammary epithelial cells require laminin as a survival ligand and specific beta1 integrins are necessary to suppress apoptosis. To explore the possibility that dynamic changes in cell-matrix interactions contribute to the onset of apoptosis during mammary involution in vivo, a detailed immunohistochemical analysis of the expression of integrin subunits and their extracellular matrix ligands during mouse mammary gland development has been performed. The kinetics of apoptosis were determined by using tissue samples obtained from virgin, pregnant, lactating, and involuting gland. The maximal elevation of apoptosis occurred within 24 hr of weaning as determined by histologic analysis and caspase-3 staining. A wide variety of laminin subunits, together with nidogen-1 and -2, and perlecan were identified within the basement membrane region of epithelial ducts, lobules, and alveoli in both human and mouse mammary gland. However, no change in the distribution of any of the basement membrane proteins or their cognate integrin receptors was observed during the transition from lactation to apoptosis. Instead, we discovered that altered ligand-binding conformation of the beta1 integrin to a nonbinding state coincided with the immediate onset of mammary apoptosis. This finding may provide a novel dynamic mechanism for inhibiting the transduction of extracellular matrix survival signals, thereby contributing to the onset of apoptosis in a developmental context in vivo.

Amino Acid Sequence↗

PubMed1997

Integrin alpha chain cytoplasmic tails regulate "antibody-redirected" cell adhesion, independently of ligand binding.

Here we describe a novel "antibody-redirected cell adhesion" (ARCA) assay. This assay measures heterotypic cell-cell adhesion, resulting from antibody bridging between Fc gamma receptors type II (CD32) on leukocytes, and clustered integrins on adherent cell monolayers. This ARCA activity, facilitated by integrins alpha3 beta1 or alpha4 beta1, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the alpha4 cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although alpha4 tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM-1 to intact alpha4-transfected K562 cells. In conclusion, the integrin alpha chain tail can positively regulate integrin-dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand-binding considerations.

Actin Cytoskeleton↗

PubMed2004

Expression profile of genes associated with antimetastatic gene: nm23-mediated metastasis inhibition in breast carcinoma cells.

Metastases of various malignancies have been shown to be inversely related to the abundance of nm23 protein expression. However, the downstream pathways involved in nm23-mediated suppression of metastasis have not been elucidated. In the present investigation, we used cDNA microarrays to identify novel genes and functional pathways in nm23-mediated spontaneous breast metastasis. Microarray experiments were performed in a pair of cell lines, namely, C-100 (only vector transfected; highly metastatic) and H1-177 (nm23 transfected; low metastatic), derived from human mammary carcinoma cell line MDA-MB-435. The cDNA microarray analysis using GeneSpring software revealed significant as well as consistent alterations in the expression (up- and downregulation) of 2158 genes in a total of 18889 genes between high and low metastatic cells. Some of these genes were grouped into 6 functional categories, namely, invasion and metastasis, apoptosis and senescence, signal transduction molecules and transcription factors, cell cycle and repair, adhesion, and angiogenesis to extrapolate an association between these genes and different functional pathways involved in nm23-regulated metastasis. The results suggest that nm23 gene plays a major role in metastasis and its mechanism of action of metastasis suppression may involve downregulation of genes associated with cell adhesion, motility (integrins alpha2, -8, -9, -L and -V, collagen type VIII alpha1, fibronectin 1, catenin, TGF-beta2, FGF7, MMP14 and 16, ErbB2) and possibly certain tumor/metastasis suppressors (2 members of SWI/SNF-related matrix-associated proteins 2 and 5 and PTEN).

Blotting, Western↗

PubMed2001

Differential regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression in co-cultures of prostate cancer and stromal cells.

Tumor-stromal interactions have been suggested to be a critical factor in both tumor invasion and tumor metastasis. Here, we examined the role of tumor-stromal interactions using co-cultures of prostate cancer (PC) cells derived from primary and metastatic tumors with primary or immortalized stromal (fibroblast and smooth muscle) cells and their effect on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression. Co-cultures of PC and stromal cells showed enhanced levels of pro-MMP-9 and reduced levels of TIMP-1 and TIMP-2. Whereas enhanced expression of pro-MMP-9 occurred in PC cells, the TIMPs were down-regulated in stromal cells. Induction of pro-MMP-9 and reduction of TIMP expression did not require cell-cell contact and were mediated by a soluble factor(s) present in the conditioned medium of the effector cell. Collagen I is a potent inducer of pro-MMP-9 in PC cells. Consistently, preliminary characterization of the soluble factor in the fibroblast conditioned medium revealed m.w. of approximately 100 to 250 kDa, and its effect on pro-MMP-9 expression was partly inhibited by an anti-alpha2 integrin antibody, a major collagen I receptor. Expression of pro-MMP-9 protein and mRNA was also induced in metastatic PC-3 cells grown in human fetal bone implants in SCID mice. Together, these findings demonstrate the importance of tumor-stromal interactions in the regulation of MMP and TIMP expression and their potential role in PC progression.

Animals↗