Expression Of Several Integrins Research Articles

Merocytophagy contributes to dissemination of Francisella tularensis and is enhanced by SYK signaling in macrophages

Abstract Merocytophagy is a phagocytic process characterized by the transfer of cytosolic content between macrophages and the formation of a double-membraned vesical within recipient cells. Studies conducted in our laboratory have demonstrated that proteins and intracellular pathogens can be transferred between cells by this contact-dependent mechanism. In particular, Francisella tularensis, a highly infectious pathogen that spreads from cell to cell by hijacking the phagocytic pathway in macrophages, has been shown to replicate within cells that receive cargo via merocytophagy. While these observations may point to an alternative route of F. tularensis dissemination in vivo, it is also possible that this mechanism of bacterial spread could amplify immune responses. Ongoing studies aim to characterize molecular and cellular players in merocytophagy to gain a better understanding of this phenomenon and to develop strategies to assess merocytophagy in vivo in the future. In cell-to-cell contact studies, inhibition of spleen tyrosine kinase (SYK) expression and activity showed decreased cargo transfer by merocytophagy. Pharmacologic inhibition of SYK was also associated with decreased surface expression of several integrins, giving evidence that cell-to-cell contact is stabilized by integrins during merocytophagic transfer in macrophages. Supported by grants from NIH and NIGMS (R56 AI139476 and T32 GM008336).

Understanding the role of integrins in breast cancer invasion, metastasis, angiogenesis, and drug resistance

Integrins are cell adhesion receptors, which are typically transmembrane glycoproteins that connect to the extracellular matrix (ECM). The function of integrins regulated by biochemical events within the cells. Understanding the mechanisms of cell growth by integrins is important in elucidating their effects on tumor progression. One of the major events in integrin signaling is integrin binding to extracellular ligands. Another event is distant signaling that gathers chemical signals from outside of the cell and transmit the signals upon cell adhesion to the inside of the cell. In normal breast tissue, integrins function as checkpoints to monitor effects on cell proliferation, while in cancer tissue these functions altered. The combination of tumor microenvironment and its associated components determines the cell fate. Hypoxia can increase the expression of several integrins. The exosomal integrins promote the growth of metastatic cells. Expression of certain integrins is associated with increased metastasis and decreased prognosis in cancers. In addition, integrin-binding proteins promote invasion and metastasis in breast cancer. Targeting specific integrins and integrin-binding proteins may provide new therapeutic approaches for breast cancer therapies. This review will examine the current knowledge of integrins' role in breast cancer.

Regulation of integrin α6A by lactogenic hormones in rat pancreatic β-cells: Implications for the physiological adaptation to pregnancy.

During pregnancy, the maternal β-cell mass is increased in order to adapt to the physiological changes in insulin demand. Lactogenic hormones stimulate rodent β-cell attachment and proliferation in vitro. The aim of this study was to identify adhesion molecules involved in expansion of the β-cell mass during pregnancy in the rat. Quantitative RT-PCR was used to evaluate the expression of several integrins and laminins in isolated neonatal rat islets in response to growth hormone (GH) and prolactin (PRL) treatment. Double-immunofluorescence staining of rat pancreas was used to localize the expression of integrin α6β1. β-cell proliferation was evaluated by incorporation of bromodeoxyuridine (BrdU). The role of STAT5 phosphorylation was tested by addition of STAT5 mutants. We found that the mRNA level of integrin-α6A, was upregulated 2.5-fold by PRL or GH. During pregnancy, a biphasic 3.4-4.5-fold increase of integrin-α6A and B mRNA levels was detected. A disintegrin peptide (DP) reduced the hormone-stimulated mitotic activity in neonatal rat β-cells from 2.9±0.4-fold to 1.3±0.3-fold. The hormone-induced expression of α6β1 integrin was shown to be mediated via STAT5 as a dominant negative (DN) mutant prevented and a constitutive active (CA) mutant augmented the hGH-stimulated expression. The DP was found to inhibit hGH-induced transactivation of the PRL receptor promoter 1A and reduce the hGH-induced phosphorylation of STAT5. These results show that integrin-α6 in β-cells is upregulated by lactogenic hormones and is required but not sufficient for the expansion of the β-cell mass in pregnancy in the rat, which may have implications for the understanding and treatment of gestational diabetes mellitus.

Abstract 4476: Role of MAP-Kinase suppressor in IBD

Abstract BACKGROUND & AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood. MATERIALS & METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells. RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p<0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p<0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes. CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD. Citation Format: Sharad Khare, Qiong Zhang. Role of MAP-Kinase suppressor in IBD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4476.

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells

ABSTRACTVascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

Overexpressions of Vimentin and Integrins in Human Metastatic Spine Tumors.

ObjectiveTo comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue.MethodsDegenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins αv, β1, and β3 was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR).ResultsImmunohistochemical staining showed that vimentin and integrin αv was broadly expressed in all tissues examined. By contrast, integrin β1 was weakly expressed only in 38.4% (5/13) of tissues. Integrin β3 was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin αv in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin β1 was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin β3 was variable.ConclusionSpinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin αv and β1 in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.

The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway

The aryl hydrocarbon receptor (AhR) is commonly described as a transcription factor, which regulates xenobiotic-metabolizing enzymes. Recent studies have suggested that the binding of ligands to the AhR also activates the Src kinase. In this manuscript, we show that the AhR, through the activation of Src, activates focal adhesion kinase (FAK) and promotes integrin clustering. These effects contribute to cell migration. Further, we show that the activation of the AhR increases the interaction of FAK with the metastatic marker, HEF1/NEDD9/CAS-L, and the expression of several integrins. Xenobiotic exposure, thus, may contribute to novel cell-migratory programs.

Integrin expression during reverse remodeling in the myocardium of heart failure patients

The main anchoring proteins of myocardial cells with each other and with the extracellular matrix are integrins present in the membranes of myocardial cells. These integrins are important for maintaining the architecture of the myocardial tissue and the mechanotransduction in the heart. Heart failure leads to various alterations in the myocardium, such as changes in morphology, and in expression of mRNAs, miRNAs, and proteins. Left ventricular assist device (LVAD) support in heart failure patients has been described to induce reverse remodeling of the myocardium and thus to (some degree of) reversal of the aforementioned alterations. In this study, we evaluated whether changes in expression of integrins α-1, -3, -5, -6, -7, -10, -11 and β-1, -3, -5 and -6 play a role during reverse remodeling. Three-step immunoperoxidase staining procedures were applied on frozen heart tissue sections to locate the various integrins tested. Integrin mRNA expression was established by standard Q-PCR procedures. It was shown that mRNA expression of several integrins changes significantly during LVAD support, however without subsequent changes in immunohistochemical detectable quantities. Various integrins showed different locations within the myocardium. LVAD-induced reversed remodeling did not result in significant integrin protein expression, although changes in integrin mRNA expression suggested an adaptation to unloading.

Insights into the Biology of Mobilized Cells through Innovative Treatment Schedules of the CXCR4 Antagonist AMD3100.

The CXCR4 antagonist AMD3100 mobilizes hematopoietic stem/progenitor cells (HSPC), but most available data are on combination with G-CSF, rather than its use as a single modality for HSPC mobilization and transplantation. Because of low efficacy of the standard single-dose regimen, we additionally tested in mice the effect of pulsed or continuous AMD3100-infusion: Repeat injections as little as 4 hours apart for up to 6 days did not result in CFU-C accumulation or curtail the size of AMD3100-responsive HSPC pools. In contrast to the 3-fold increase in circulating CFU-C after bolus or repeat injections, after 5 days of continuous AMD3100-infusion circulating CFU-C were increased 30-fold over baseline, from 301±41/mL to 10,687±757/mL compared to 1,088±75/mL after a bolus. These numbers were sustained at least until day 8 and, of interest, were not further augmented by injection of an AMD3100-bolus, suggesting satiating doses of AMD3100-infusion. Furthermore, at least equally efficient mobilization by AMD3100-bolus or -infusion was documented in splenectomized hosts, indicating that the contribution of spleen to mobilization by AMD3100 is negligible. Long-term repopulating cells in AMD3100-infusion-mobilized blood, assessed by limiting dilution transplantation, were also increased, to 273(205–362) CRU/mL, vs. 77(56–106) CRU/mL after bolus. Of interest, the number of HSPC mobilized by AMD3100-infusion was much higher than by standard twice-daily dosing of G-CSF, and AMD3100 effectively mobilized two G-CSF resistant/refractory mouse models (G-CSFR−/−, Plasminogen−/−), while not surprisingly, mice treated with the Gi protein inhibitor Pertussis toxin were resistant to AMD3100-induced mobilization. Subsequent studies addressed the phenotype and the in vitro and in vivo functional behavior of AMD3100-mobilized HSPC. The immunophenotype of AMD3100-mobilized c-kit+ cells differed significantly from that of steady-state bone marrow (BM) c-kit+ cells. Thus expression of several integrins was lower, while CD26 expression was significantly higher than on BM c-kit+ cells. AMD3100-infusion-mobilized blood and BM CFU-C migrated equally efficiently towards SDF-1 in vitro. In vivo marrow homing of AMD3100-infusion-mobilized blood CFU-C was >50% increased over BM CFU-C, similarly to what we recently showed for G-CSF-infusion-mobilized blood. These properties differ from those of in vitro AMD3100-incubated BM-HSPC, in which migration was blocked, and homing was normal or reduced. Kinetics of hematopoietic recovery after transplantation of 100 μl AMD3100- or G-CSF-infusion-mobilized blood to lethally irradiated hosts were similar except for delayed platelet recovery in AMD3100-mobilized blood recipients. Partial BrdU labeling experiments for up to 3 days before mobilization (resulting in 20–40% BrdU labeling of c-kit+ cells in BM) documented that labeled cells were consistently underrepresented (<5%) among AMD3100-mobilized c-kit+ cells. These data defend the notion that only non-cycling HSPC are found in mobilized blood (>80% of AMD3100-mobilized c-kit+ cells are in G0), but extend it to conclude that such a status precedes their exit from BM, rather than being a consequence of modification in the blood environment. In summary, we are presenting a novel efficacious mobilization scheme which fully supports transplantation demands in normal and G CSF-refractory donors. Furthermore, our data expand previous knowledge about size and turn-over of AMD3100-mobilizable HSPC pools.

Down-regulation of CD9 in Human Ovarian Carcinoma Cell Might Contribute to Peritoneal Dissemination: Morphologic Alteration and Reduced Expression of β1 Integrin Subsets

Peritoneal dissemination is one of the main causes of death in cancer patients. Pathophysiology of metastasis has been well investigated, but the mechanism of diffuse spread of tumor colonies in the peritoneal cavity is not fully understood. CD9 is a member of tetraspanin and its down-regulation is known to be involved in poor prognosis. To investigate the significance of the down-regulation of CD9, HTOA, an ovarian carcinoma cell line that highly expressed CD9, was transiently transfected with small interfering RNA (siRNA) against CD9, and CD9-negative cells (HTOA(CD9-)) were purified. HTOA(CD9-) showed altered adhesion patterns on Matrigel, collagen, fibronectin, and laminin compared with those of control siRNA-transfected HTOA (control-HTOA). Flow cytometry and fluorescence cytostainings revealed that the expression levels of integrins beta1, alpha2, alpha3beta1, alpha5, and alpha6 were lower in HTOA(CD9-) than those of control-HTOA. HTOA(CD9-) showed altered expression of junctional and cytoskeletal molecules. By time-lapse video microscopy, control-HTOA showed solid adhesion to extracellular matrix and formed cobblestone pattern, whereas HTOA(CD9-) showed weaker adhesion and were distributed as diffuse spots. To examine whether the expression level of CD9 change during tumor dissemination, HTOA-P, a highly disseminative subclone of HTOA, was established. HTOA-P showed distinctive down-regulation of CD9 at mRNA and protein levels, and showed similar morphologic alteration as HTOA(CD9-) did. These findings indicate that the down-regulation of CD9 may be an acquired event in the process of tumor dissemination. Down-regulated CD9 may attenuate the expression of several integrins and rearrange junctional and cytoskeletal molecules that might contribute to dissemination of ovarian carcinomas.

Characterization of Retinoic Acid Receptor-deficient Keratinocytes

Retinoids are essential for normal epidermal growth and differentiation and show potential for the prevention or treatment of various epithelial neoplasms. The retinoic acid receptors (RARalpha, -beta, and -gamma) are transducers of the retinoid signal. The epidermis expresses RARgamma and RARalpha, both of which are potential mediators of the effects of retinoids in the epidermis. To further investigate the role(s) of these receptors, we derived transformed keratinocyte lines from wild-type, RARalpha, RARgamma, and RARalphagamma null mice and investigated their response to retinoids, including growth inhibition, markers of growth and differentiation, and AP-1 activity. Our results indicate that RARgamma is the principle receptor contributing to all-trans-retinoic acid (RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs, the synthetic retinoid N-(4-hydroxyphenyl)-retinamide inhibited growth; this was not observed with RA, 9-cis RA, or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic acid. Finally, both RARalpha and RARgamma differently affected the expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.

The Highly Reducing Sugar 2-Deoxy-d-Ribose Induces Apoptosis in Human Fibroblasts by Reduced Glutathione Depletion and Cytoskeletal Disruption

2-deoxy-D-Ribose (dRib), the most reducing sugar, induces apoptosis in normal human fibroblasts, as judged by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation and mitochondrial depolarization. This effect is independent from culture conditions, such as cell density and the presence or absence of serum in the culture milieu, suggesting that dRib-induced apoptosis is cell cycle-independent. dRib was found also to provoke disruption of the actin filament network and detachment from the substratum, while at the same time, interestingly, it increases the expression of several integrins and cell adhesion molecules. Furthermore, dRib was found to reduce the intracellular levels of reduced glutathione (GSH). The apoptotic process was not affected by the macromolecular-synthesis inhibitors cycloheximide and actinomycin D. On the contrary, the antioxidant N-acetyl-L-cysteine (NAC) fully blocks the dRib-induced apoptosis by preventing GSH depletion, while it also inhibits actin-filament-network disruption and mitochondrial depolarization. The above indicate that dRib induces apoptosis in human fibroblasts by a mechanism involving glutathione metabolism and oxidative stress, as well as disturbance of cytoskeletal integrity and cell adhesion.

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β4subunit is present in association with α6chain on both normal and transformed epithelial cells. Recently α6β4heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β4subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α6β4has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β1(TGF-β1) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α6β4expression is modulated by TGF-β1, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β1cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β4subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β1.

Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats.

Recently, we showed that magnesium deficiency induces lesions in knee joint cartilage from 5-week-old rats that are very similar to ofloxacin-induced cartilage defects. We concluded that quinolone-induced arthropathy is probably due to chelation of magnesium and thus a deficit in functionally available magnesium in joint cartilage (Stahlmann et al. 1995). As magnesium deficiency in joint cartilage could impair chondrocyte-matrix interaction which is mediated by cation-dependent integrin receptors of the beta 1-subfamily, we investigated integrin expression in joint cartilage from untreated, ofloxacin-treated and magnesium-deficient Wistar rats. With immunohistochemical methods using monoclonal and polyclonal antibodies, we showed that the integrin pattern in joint cartilage from rats corresponded largely to integrin expression described for human cartilage tissue: beta 1, alpha 1, alpha 3 and alpha v subunits and the alpha 5 beta 1 and alpha v beta 3 heterodimers were consistently expressed. Joint cartilage lesions were detected in ofloxacin-treated and magnesium-deficient rats. Lesions were more pronounced in the quinolone-treated group. Expression of several integrins was reduced in the vicinity of lesions after oral treatment with 2 x 600 mg ofloxacin/kg for 1 day. Gross-structural lesions (e.g., cleft formation, unmasked collagen fibres) in magnesium-deficient rats were very similar but changes in integrin expression were less pronounced. On the other hand, changes in cartilage matrix composition showed similar alterations in ofloxacin-treated and magnesium-deficient rats: fibronectin deposition in the cartilage matrix increased in both groups while glycosaminoglycan content decreased. In summary, similar defects occur in ofloxacin-treated and magnesium-deficient rats and with immunohistochemical methods subtle differences are demonstrable.

Regulation of integrin gene expression by substrate adherence.

Under substrate adherent conditions, integrin gene expression can be regulated by transforming growth factor-beta, interleukin-1 beta, and prostaglandins. This report demonstrates a new mechanism that can differentially control the expression of several integrins. When MG-63 osteosarcoma cells are maintained in suspension, up-regulation of several integrin alpha-subunits takes place. Within as little as 4 h, the mRNA levels for both the alpha 2- and alpha 4-subunits are increased 4- and 6-fold, respectively. It was found that mRNA levels for the alpha 2-, alpha 4-, and alpha v-subunits were markedly increased in several differentiated cell lines under nonadherent conditions; however, cells that did not express a given integrin under substrate adherent conditions also did not express this integrin when maintained in suspension. The alpha 5-subunit did not upregulate during suspension growth. By immunocytochemistry, changes in integrin mRNA levels were confirmed at the protein level. Both cytochalasin B and a phorbol ester were found to induce the expression of the alpha 2-subunit, but not the alpha 4- and alpha 5-subunits, in a dose-dependent fashion. Many investigators have documented changes in gene expression that result from changes in "cell shape." These phenomena may result from up-regulation of integrin gene expression induced by the lack of substrate adherence.