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At least 595 records · Page 33Linked to original sources
PubMed2006

Suprabasal overexpression of beta-1 integrin is induced by bovine papillomavirus type 1.

In a normal stratified squamous epithelium, beta1-integrin is expressed in basal epithelial cells. In BPV-induced fibropapillomas beta1-integrin is overexpressed and aberrantly localized, with uniform expression in the lower spinous layer, and sporadic expression within the mid-spinous region that co-localizes with expression of the viral E5 and E7 oncoproteins. In situ hybridization of fibropapillomas for beta1-integrin RNA revealed sporadic hybridization in the spinous layer, indicating transcriptional induction. Beta1-integrin expression in cultured keratinocytes requires exogenous EGF in the media, but this requirement is lost if E7 is expressed, and E7 was able to abrogate the EGF-requirement of normal keratinocytes for the activation of ERK and DNA synthesis. Within fibropapillomas, suprabasal expression of E5 and E7 correlated with suprabasal expression of beta1-integrin and PCNA, indicating that vegetative viral replication in the spinous layer correlated with the expression of E7 and beta1 integrin. The ability of BPV-1 E7 to support beta1-integrin expression and EGF independent DNA synthesis and the activation of ERK are the first biochemical correlates of its expression in keratinocytes.

Animals↗
PubMed2003

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro.

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.

Alkaloids↗
PubMed2003

Transcriptional regulation of the beta1C integrin splice variant in human prostate adenocarcinoma.

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behaviour, including motility and invasiveness, cell growth and cell survival. beta1C integrin, an alternatively spliced variant of the human beta1 integrin, has been shown to inhibit cell proliferation. We have previously demonstrated that beta1C integrin mRNA and protein are present in normal prostate and are down-regulated in prostate adenocarcinoma. To explore some of the molecular mechanisms regulating beta1C integrin gene expression, we have analysed the transcriptional activity of the beta1 integrin gene in neoplastic and normal human prostate tissue. Run-on analysis demonstrates that the transcription rate of the beta1 integrin gene is significantly reduced in prostate cancer specimens compared to normal prostate, thus accounting for the reduction in mRNA levels of the beta1 integrin variants. Moreover, the decrease in transcriptional activity of the beta1 integrin gene directly correlates to the reduction of beta1C integrin steady-state mRNA levels (r=0.78).

Aged↗
PubMed2006

Intracerebral peripheral blood stem cell (CD34+) implantation induces neuroplasticity by enhancing beta1 integrin-mediated angiogenesis in chronic stroke rats.

Although stem cell-based treatments for stroke and other neurodegenerative diseases have advanced rapidly, there are still few clinical treatments available. In this study, rats receiving intracerebral peripheral blood hematopoietic stem cell (CD34+) (PBSC) transplantation showed much more improvement in neurological function after chronic cerebral ischemia in comparison with vehicle-treated control rats. Using laser-scanning confocal microscopy, implanted PBSCs were seen to differentiate into glial cells [GFAP+ (glial fibrillary acidic protein-positive)], neurons [Nestin+, MAP-2+ (microtubule-associated protein 2-positive), Neu-N+ (neuronal nuclear antigen-positive)], and vascular endothelial cells [vWF+ (von Willebrand factor-positive)], thereby enhancing neuroplastic effects in the ischemic brain. Cortical neuronal activity, as evaluated by 1H-MRS (proton magnetic resonance spectroscopy), also increased considerably in PBSC-treated rats compared with a vehicle-treated control group. In addition, PBSC implantation promoted the formation of new vessels, thereby increasing the local cortical blood flow in the ischemic hemisphere. These observations may be explained by the involvement of stem cell-derived macrophage/microglial cells, and beta1 integrin expression, which might enhance this angiogenic architecture over the ischemic brain. Furthermore, quantitative reverse transcription-PCR analysis showed significantly increased modulation of neurotrophic factor expression in the ischemic hemisphere of the PBSC-transplanted rats compared with vehicle-treated control rats. Thus, intracerebral PBSC transplantation might have potential as a therapeutic strategy for treating cerebrovascular diseases.

Animals↗
PubMed2003

Novel role for a complement regulatory protein (CD46) in retinal pigment epithelial adhesion.

PURPOSE: There is increasing evidence that the complement system may play a significant role in one of the leading diseases causing blindness in the elderly population, age-related macular degeneration. In this study, a novel role in the retina for a regulatory protein in the complement system, CD46, is proposed. METHODS: The retinal pigment epithelium (RPE) was obtained from human donor eyes as well as human immortalized RPE cell lines (ARPE19). Immunohistochemistry and confocal microscopy were used to immunolocalize CD46 and beta1 integrin. Immunoprecipitation experiments with antibodies to either CD46 or beta1 integrin were performed on RPE cell lysates. A cell adhesion assay was used to determine the proportion of RPE cells that adhere to Bruch's membrane explants from donor eyes. RESULTS: Immunohistochemistry and confocal microscopy demonstrated that CD46 was polarized to the basal surface of the RPE along with beta1 integrin, shown previously to be involved in RPE adhesion. Immunoprecipitation experiments demonstrated that CD46 and beta1 integrin coprecipitated from RPE cell lysates when either protein was used as the precipitating antibody. The adhesion assay showed that antibodies to either CD46 or beta1 integrin reduced RPE adhesion to the surface of Bruch's membrane compared with the control. CONCLUSIONS: These findings suggest that this complement regulatory protein, which protects host cells from autologous complement attack, may have a functional interaction with beta1 integrin in the eye that is related to RPE adhesion to its basement membrane and Bruch's membrane.

Aged↗
PubMed2001

beta1 Integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation.

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 microg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1beta > RANTES MIP-1alpha. In contrast, using filters coated with fibronectin (20 microg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic: thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 microg/mL). To determine the involvement of beta1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to alpha2beta1; however, complete inhibition required a combination of mAbs to alpha1beta1 and alpha2beta1. In comparison, migration on fibronectin was inhibited by mAb to alpha3beta1 and alpha5beta1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1alpha, MIP-1beta, and RANTES), as well as the nature and concentration of the ECM substrate involved.

Cell Line↗
PubMed2001

A novel orthotopic implantation model of human esophageal carcinoma in nude rats: CD44H mediates cancer cell invasion in vitro and in vivo.

A new orthotopic esophageal cancer model was developed by implanting fragments of xenografts of T.T human esophageal squamous carcinoma cells into the cervical esophagus of athymic rats. The rats had symptoms analogous to the human clinical course such as respiratory distress, dysphagia, vomiting of blood, or Horner syndrome, followed by death resulting from suffocation. Microscopic metastases of lymph node were observed around the tumor in 3 of 18 rats. A new cell line (T.T-1) was established from these metastases. Flow cytometry showed that T.T-1 and T.T parental cells had nearly the same surface levels of beta1-integrin, alpha2-integrin, alpha3-integrin and E-cadherin, and no expression of CD44v3, CD44v6 and alpha5-integrin. T.T-1 cells had a higher level of CD44H, however, and a greater binding efficiency to the extracellular matrix components; laminin, type IV collagen, hyaluronic acid, and fibronectin than T.T cells. Anti-CD44H antibody significantly decreased the binding efficiency of T.T-1 cells. T.T-1 cells were also significantly more invasive than T.T cells through all the extracellular matrix components except hyaluronic acid. After orthotopic implantation histological examination showed that T.T-1 tumors invaded beyond the esophageal mucosa and tracheal muscle layer and obstructed the esophagus and trachea. No invasion was observed with T.T tumors. Rats with T.T-1 or T.T tumors survived an average of 32.0 and 50.7 days, respectively (p < 0.01). In addition T.T-1 tumors expressed higher levels of CD44H mRNA than T.T tumors. In summary, our newly developed orthotopic implantation model is a valid model of esophageal cancer because it followed the same clinical course experienced by humans. Moreover, using cells derived from this model, we were able to demonstrate that CD44H is involved in esophageal cancer cell invasion.

Animals↗
PubMed1996

Fibronectin enhances the migration rate of human neutrophils in vitro.

Efficient polymorphonuclear neutrophil (PMN) migration depends on specific interactions between PMNs, endothelial cells, and extracellular matrix (ECM) proteins. We investigated the relationship between PMN migration and the ECM molecule fibronectin (FN). We used an in vitro migration assay system to show that human PMNs migrated across an FN-coated filter barrier toward a formyl-Met-Leu-Phe (fMLP) chemoattractant gradient in greater numbers than across (uncoated) bare fitters. In 1 h of fMLP stimulation, 69 +/- 6% of the PMNs had migrated across the FN-coated filters, whereas 46 +/- 5% of PMNs migrated across bare filters. This effect was specific to FN; coating the filters with the ECM protein vitronectin did not enhance migration. Monoclonal antibodies against FN or against the alpha5 or beta1 integrin subunits of the FN receptor inhibited the enhanced PMN migration response across FN-coated filters. These findings indicate that the extracellular matrix protein FN enhances PMN migration and that this response is mediated by the alpha5beta1 FN receptor.

Antibodies↗
PubMed1997

Transendothelial migration induces rapid expression on neutrophils of granule-release VLA6 used for tissue infiltration.

Little is known of the mechanisms allowing neutrophils to infiltrate tissue after transendothelial migration. We postulated that VLA6 might be involved in neutrophil infiltration because it revealed as the most expressed beta1 integrins among VLA5, VLA4, and VLA3, which also appeared to define subsets within the blood neutrophil population. Transendothelial migration up-regulated by threefold (5,000 to 15,000 receptors) VLA6 expression on neutrophils. VLA6 up-regulation was transient, peaking in 6 min and returning to baseline in 1 h when tested in response to N-formyl-methionyl-leucyl-phenylalanine. Neutrophil degranulation experiments revealed a steady correlation between expression of VLA6 and granule content release, notably beta-glucuronidase, indicating that VLA6 molecules were preformed and stored mostly in azurophilic granules. Migration across fibroblast monolayers of neutrophils preactivated for VLA6 up-regulation was blocked when they were preincubated with anti-VLA6. However, anti-VLA6 had no effect on transfibroblast migration of non-preactivated neutrophils. These results indicate that VLA6 was functional only on activated neutrophils that used their up-regulated VLA6 to cross fibroblasts. Activation of neutrophils by transendothelial migration induces rapid expression of granule release VLA6 that appears to be a key adhesion mechanism used by a subset of neutrophils to infiltrate tissue.

Cell Degranulation↗
PubMed1999

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation.

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.

Amino Acid Sequence↗
PubMed1996

Interleukin-1beta expression is induced by adherence and is enhanced by Fc-receptor binding to immune complex in THP-1 cells.

Adherence of monocytes to endothelial cells and subsequently to basement membrane represents the initial steps in monocyte migration from the vasculature to the interstitium. We investigated the role of adhesion to endothelial cells and basement membrane in the induction of the cytokine IL-1beta. We demonstrated that mRNA for IL-1beta is induced in adherent THP-1 cells, but not in a matrix-specific manner. Adherence to fibrinogen, however, causes an increase in mRNA for IL-1beta. A background level of IL-1beta mRNA induction was observed in cells adherent to all matrices, including the non-specific human serum albumin substrate, as compared to non-adherent cells cultured in teflon troughs. In addition, antibodies to CD11a, CD11b, beta1 integrin, VLA4, (alpha)v(beta)3 (VNR), and ICAM-1 did not induce significant IL-1beta mRNA when THP-1 cells were adherent to those immunoglobulins. THP-1 cells adherent to immune complexes of anti-CD11a, anti-CD11b, anti-VLA4, anti-VNR, and anti-ICAM-1 showed greater mRNA induction than cells adherent to primary antibodies alone. THP-1 cells adherent to non-specific immune complexes gave the highest level of mRNA induction. Secretion of IL-1beta protein, measured by ELISA at 24 h, was greatest when cells were adherent to immobilized immune complexes or to fibrinogen. Our results demonstrate that a general adherence-induced increase in IL-1beta gene expression is greatly enhanced by the presence of immune complex.

Antigen-Antibody Complex↗
PubMed2005

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment.

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.

Alkylating Agents↗
PubMed2004

Glycosphingolipids govern gene expression.

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.

Animals↗
PubMedDate not supplied

Bromelain reversibly inhibits invasive properties of glioma cells.

Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that alpha3 and beta1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a transactivating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation.

Animals↗
PubMed2000

Disaccharides generated from heparan sulphate or heparin modulate chemokine-induced T-cell adhesion to extracellular matrix.

We have found previously that disaccharides (DS) enzymatically generated from heparin or heparan sulphate can modulate tumour necrosis factor-alpha (TNF-alpha) secretion from immune cells in vitro and cell-mediated immune reactions in vivo. Here, we show that such DS can modulate the adhesion and migration of human T cells. We found that certain heparin- and heparan sulphate-derived DS induced, in a dose-dependent manner, the adhesion of human T cells to both extracellular matrix (ECM) and immobilized fibronectin (FN); maximal T-cell adhesion occurred with 1 ng/ml of DS. The levels of T-cell adhesion to ECM that were induced by the tested DS molecules resembled those induced by the prototypic chemokine, macrophage inflammatory protein 1beta (MIP-1beta). However, the kinetics of DS-induced T-cell adhesion to FN resembled that induced by phorbol myristate acetate (PMA), but not that induced by MIP-1beta. This adhesion appeared to involve beta1 integrin recognition and activation, and was associated with specific intracellular activation pathways. Although a first exposure of T cells to certain DS molecules appeared to result in cell adhesion, a subsequent exposure of T cells to pro-adhesive chemokines, such as MIP-1beta or RANTES, but not to other pro-adhesive stimuli, for example interleukin-2 or CD3 cross-linking, resulted in inhibition of T-cell adhesion to and chemotactic migration through FN. Hence, we propose that the breakdown products of tissues generated by inflammatory enzymes are part of an intrinsic functional programme, and not necessarily molecular waste. Moreover, because the DS molecules exert their modulatory functions within a limited time, it appears that the historical encounters of the tissue-invading cells with the constituents of inflamed loci may dictate the cells' behaviour upon subsequent exposure to proinflammatory mediators.

Antibodies, Monoclonal↗
PubMed2005

Prostaglandin E2 stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase Calpha, and c-Src pathway in primary cultured rat osteoblasts.

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

Animals↗
PubMed2005

Disabled-2 (Dab2) is required for transforming growth factor beta-induced epithelial to mesenchymal transition (EMT).

Transforming growth factor beta (TGFbeta) induces an epithelial to mesenchymal transition (EMT) during both physiological and pathological processes; however, the mechanism underlying this transition is not fully elucidated. Here, we have demonstrated that TGFbeta induces the expression of the adaptor molecule disabled-2 (Dab2) concomitant with the promotion of EMT. We show that TGFbeta induces a transient accumulation of Dab2 to the membrane and increases Dab2 binding to beta1 integrin. Furthermore, small interfering RNA (siRNA)-mediated silencing of Dab2 expression in mouse mammary gland epithelial cells results in inhibition of integrin activation, shown by a decrease of both TGFbeta-induced focal adhesion kinase phosphorylation and cellular adherence, leading to apoptosis and inhibition of EMT. Forced re-expression of human Dab2, not targeted by the mouse siRNA sequence, rescues cells from apoptosis and restores TGFbeta-mediated integrin activation and EMT. These results are confirmed in the F9 teratocarcinoma cell line, a model for retinoic acid-induced visceral endoderm differentiation in which we demonstrate that ablation of retinoic acid-induced Dab2 expression levels, by stable siRNA silencing of Dab2, blocks visceral endoderm differentiation. Our findings indicate that Dab2 plays an important regulatory role during cellular differentiation and that induction of differentiation in the absence of Dab2 expression commits the cell to apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells.

The expression level and functional activity of estrogen receptor alpha is an important determinant of breast physiology and breast cancer treatment. However, it has been difficult to identify the signals that regulate estrogen receptor because cultured mammary epithelial cells generally do not respond to estrogenic signals. Here, we use a combination of two- and three-dimensional culture systems to dissect the extracellular signals that control endogenous estrogen receptor alpha. Its expression was greatly reduced when primary mammary epithelial cells were placed on tissue culture plastic; however, the presence of a reconstituted basement membrane in combination with lactogenic hormones partially prevented this decrease. Estrogen receptor alpha expression in primary mammary fibroblasts was not altered by these culture conditions, indicating that its regulation is cell type specific. Moreover, estrogen receptor-dependent reporter gene expression, as well as estrogen receptor alpha levels, were increased threefold in a functionally normal mammary epithelial cell line when reconstituted basement membrane was added to the medium. This regulatory effect of reconstituted basement membrane was reproduced by two of its components, collagen-IV and laminin-1, and it was blocked by antibodies against alpha2, alpha6 and beta1 integrin subunits. Our results indicate that integrin-mediated response to specific basement membrane components, rather than cell rounding or cell growth arrest induced by reconstituted basement membrane, is critical in the regulation of estrogen receptor alpha expression and function in mammary epithelial cells.

Animals↗