SEARCH · PubMed Health

Results for “Integrin alpha2”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources

PubMed2004

Structure of the integrin alpha2beta1-binding collagen peptide.

We have determined the 1.8A crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)(2)-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)(3). The central GFOGER hexapeptide is recognised specifically by the integrins alpha2beta1, alpha1beta1, alpha10beta1 and alpha11beta1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin alpha2-I domain (IBP(c)) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP(c) main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin-collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP(c) complex crystal structure.

Amino Acid Motifs↗

PubMed2006

Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts.

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

Actins↗

PubMed2001

Arg-gingipain is responsible for the degradation of cell adhesion molecules of human gingival fibroblasts and their death induced by Porphyromonas gingivalis.

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are two major cysteine proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis, which has been shown to act as major pathogen in the development and progression of periodontal diseases. These enzymes are also important for this organism to proliferate and survive in periodontal pockets. Here we show that Rgp is responsible for the disruption of fibronectin-integrin interactions in human gingival fibroblasts by P. gingivalis. Fibroblasts incubated with the culture supernatant of P. gingivalis showed a time-dependent loss of the adhesion activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fibronectin and integrin subunits alpha2, beta1 and beta3 in the fibroblast culture largely disappeared with the treatment. The detached cells became committed to death by disruption of contacts between adhesion molecules. In contrast, the culture supernatants from the Rgp-deficient mutants produced no significant changes in either cell adhesion or viability. Prior treatment of the culture supernatant of P. gingivalis with an Rgp inhibitor, but not a Kgp inhibitor, strongly inhibited the detachment of fibroblasts followed by cell death. These results suggest that Rgp disrupts the integrin-fibronectin interactions in fibroblasts, thereby contributing to the damage of periodontal tissues in periodontal diseases caused by P. gingivalis.

Adhesins, Bacterial↗

PubMed2006

Mixture of sugar and povidone--iodine stimulates wound healing by activating keratinocytes and fibroblast functions.

The topical application of a mixture of sugar and povidone--iodine (PI) has been reported to accelerate the healing of cutaneous wounds and ulcers by promoting re-epithelialization and granulation tissue formation as well as having an anti-microbial effect. To clarify the mechanisms accounting for the efficacy of a 70% sugar and 3% PI paste (U-PASTAtrade mark) (SP), various keratinocytes and fibroblasts functions, including proliferation, collagen synthesis, integrin expression, and cytokine and proteinase secretions in the presence of SP were investigated. Cultured human keratinocytes and fibroblasts were treated with various concentrations of SP, SU and PI. The secretion of urokinase-type plasminogen activator (u-PA), transforming growth factor (TGF)-alpha and interleukin-1alpha from keratinocytes, was detected by ELISA. Collagen synthesis of fibroblasts was examined by means of detecting proline uptake. Furthermore, integrin expressions of these cells were analyzed using a flow cytometer. SP and PI increased intra-cellular u-PA of keratinocytes and stimulated the secretion of u-PA and TGF-alpha. Sugar accelerated the extra-cellular u-PA level only. Both SP and sugar increased the collagen synthesis of fibroblasts. SP and PI also remarkably induced the expressions of extra-cellular matrix receptor integrins, alpha1, alpha2, alpha3, alpha4, alpha5 and beta1, on the surface of keratinocytes and fibroblasts. SP, the mixture of sugar and PI, is likely to act on wounds not only as an antibiotic agent, but also as a modulator for keratinocytes and fibroblasts.

Anti-Infective Agents↗

PubMed2006

E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion.

Much remains to be learned about how cell-cell and cell-matrix interactions are coordinated to influence the earliest development of neoplasia. We used novel 3D human tissue reconstructs that mimic premalignant disease in normal epidermis, to directly investigate how loss of E-cadherin function directs conversion to malignant disease. We used a genetically tagged variant of Ha-Ras-transformed human keratinocytes (II-4) expressing dominant-interfering E-cadherin fusion protein (H-2k(d)-Ecad). These cells were admixed with normal human keratinocytes and tumor cell fate was monitored in 3D reconstructed epidermis upon transplantation to immunodeficient mice. Tumor initiation was suppressed in tissues harboring control- and mock-infected II-4 cells that lost contact with the stromal interface. By contrast, H-2k(d)-Ecad-expressing cells persisted at this interface, thus enabling incipient tumor cell invasion upon in vivo transplantation. Loss of intercellular adhesion was linked to elevated cell surface expression of alpha2, alpha3 and beta1 integrins and increased adhesion to laminin-1 and Types I and IV collagen that was blocked with beta1-integrin antibodies, suggesting that invasion was linked to initial II-4 cell attachment at the stromal interface. Collectively, these results outline a novel aspect to loss of E-cadherin function that is linked to the mutually interdependent regulation of cell-cell and cell-matrix adhesion and has significant consequences for the conversion of premalignancy to cancer.

Animals↗

PubMed2005

Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination: morphologic alteration and reduced expression of beta1 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.

Adenocarcinoma↗

PubMed2005

Collagen phagocytosis by fibroblasts is regulated by decorin.

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.

Animals↗

PubMed1997

Expression of cell adhesion molecules in normal nerves, chronic axonal neuropathies and Schwann cell tumors.

Cell adhesion molecules (CAMs) play a role in the normal development and regeneration of tissues as well as in the biological behaviour of tumors. We studied the immunohistochemical expression of various CAMs, such as neural cell adhesion molecule (NCAM), its polysialylated isoform (PSA-NCAM), epithelial (E-) cadherin, and beta1 integrins (alpha2beta1, alpha5beta1, alpha6beta1) in a series of frozen specimens of 10 normal nerves, 5 axonal neuropathies, 26 benign Schwannomas and 2 malignant peripheral nerve sheath tumors (MNST). NCAM was expressed by non-myelinating Schwann cells from normal nerves and overexpressed by Schwann cells from patients with chronic axonal neuropathies and Schwannomas. The expression was lower in MNST. Expression of PSA-NCAM was heterogeneously displayed by Schwann cells from the various tissues studied. Anti E-cadherin immunoreactivity was present in myelin sheath in normal nerves and axonopathies. It was expressed in some Schwannomas especially in vestibular Schwannomas. Integrins VLA alpha2 and VLA alpha6 were widely expressed by Schwann cells from normal nerves, axonal neuropathies and Schwannomas but their expression was low in MNST. VLA alpha5 was not expressed by Schwann cells from normal nerve and Schwannomas but present in chronic axonal neuropathies and MNST. In addition VLA alpha6 was strongly expressed by perineurial cells. These data show that CAMs have a characteristic pattern of expression in normal nerve. Also, some CAMs are always expressed by Schwann cells but the expression of others differs in normal nerves versus axonopathies or tumors, suggesting a role of the microcellular environment in the regulation of CAM expression. Schwannomas have different pattern of expression than MNST.

Axons↗

PubMedDate not supplied

Characterization and functionality of cell surface molecules on human mesenchymal stem cells.

We have characterized adhesion molecules on the surface of multipotential human mesenchymal stem cells (hMSCs) and identified molecules whose ligands are present on mature hematopoietic cells. Flow cytometric analysis of hMSCs identified the expression of integrins: alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta4, in addition to ICAM-1, ICAM-2, VCAM-1, CD72, and LFA-3. Exposure of hMSCs to IL-1alpha, TNFalpha or IFNgamma up-modulated ICAM-1 surface expression, whereas only IFNgamma increased both HLA-class I and -class II molecules on the cell surface. Whole cell-binding assays between the hMSCs and hematopoietic cell lines showed that T lymphocytic lines bound hMSCs with higher affinity than lines of either B lymphocytes or those of myeloid lineage. Experiments using autologous T lymphocytes isolated from peripheral blood mononuclear cells showed that hMSCs exhibited increased affinity for activated T-lymphocytes compared to resting T cells by quantitative whole cell binding and rosetting assays. Flow cytometric analysis of rosetted cells demonstrated that both CD4+ and CD8+ cells bound to hMSCs. To determine the functional significance of these findings, we tested the ability of hMSCs to present antigen to T lymphocytes. hMSCs pulsed with tetanus toxoid stimulated proliferation and cytokine production (IL-4, IL-10, and IFNgamma) in a tetanus-toxoid-specific T cell line. Maximal cytokine production correlated with maximal antigen-dependent proliferation. These data demonstrate physiological outcome as a consequence of interactions between hMSCs and human hematopoietic lineage cells, suggesting a role for hMSCs in vivo to influence both hematopoietic and immune function(s).

Adult↗

PubMed1998

A population of CD62Llow Nk1.1- CD4+ T cells that resembles NK1.1+ CD4+ T cells.

In MHC class II-/- C57BL/6 (II-/-) mouse spleen, a small population of CD4+ T cells is present of which NK1.1+ CD4+ (NK) T cells comprise 40 to 45%. We report here that many of the NK1.1- CD4+ T cells derived from II-/- mice are also NK T cells. They produce large amounts of IL-4 in response to anti-CD3 ligation and do so without any requirement for the presence of IL-4 in the priming culture, a property characteristic of NK T cells. Their IFN-gamma production is large and is enhanced by IL-12. In addition, II-/- NK1.1- CD4+ T cells produce IL-4 as a result of culture with L cells expressing murine CD1 (L-CD1). We report that CD49b, a component of integrin VLA-2, is expressed on the majority of both NK1.1+ and NK1.1- NK T cells. NK1.1- NK T cells also exist in wild-type C57BL/6 mice. Evidence supporting this is that Vbeta8 usage by CD62Llow NK1.1- CD4+ T cells was approximately 5% higher than that by CD62Lhigh CD4+ T cells in wild-type mice in keeping with the estimated proportion of NK1.1- NK T cells in the CD62Llow population. CD62Llow CD4+ T cells from beta2-m(-/-) mice, which lack NK T cells, showed no increase in Vbeta8 usage. When activated by anti-CD3 or L-CD1, CD62Llow NK1.1- CD4+ T cells from conventional but not beta2-m(-/-) and CD1-/- mice produce IL-4 in a manner indistinguishable from II-/- NK1.1- CD4+ T cells. NK1.1- NK T cells in normal mouse spleens are approximately as numerous as NK1.1+ NK T cells.

Animals↗

PubMed2002

HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human epidermis and in hyperproliferation conditions, including cancers, in adult epidermis. Published 2002 Wiley-Liss, Inc.

Adult↗

PubMed2004

Surface receptors identify mouse NK1.1+ T cell subsets distinguished by function and T cell receptor type.

Natural killer T (NKT) lymphocytes rapidly produce several cytokines, including IL-4 and IFN-gamma, upon activation, and act as regulatory cells at an early interphase of innate and adaptive immune responses. They have been implicated as important elements in diverse immune responses including the regulation of autoimmune disease, the immune response to infections, and the prevention of tumor metastasis. The broad spectrum of their activities suggested that functionally different subsets of NKT cells may exist. We demonstrate two functionally distinct splenic NKT populations identified by the expression of CD49b and CD69, respectively. Each NKT subset was represented by the amplified transgenic NKT cell population in a distinct transgenic mouse line expressing a CD1d-restricted TCR. CD49bhigh CD69- NKT cells, termed NKT1 cells by us, were high producers of IFN-gamma after stimulation, but essentially devoid of IL-4-synthesizing cells. Most NKT1 cells used diverse (non-Valpha14-canonical) TCR. The CD69+ CD49(-/low) NKT cell population, which we term NKT2, produced large quantities of IL-4 and substantial amounts of IFN-gamma upon activation and were dominated by cells using the canonical Valpha14-Jalpha18 T cell receptor. Knowledge of the unique roles of the different NKT cell subsets in specific situations will be essential for our understanding of NKT cell biology.

Animals↗

PubMed2005

Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.

Carcinoma, Adenoid Cystic↗

PubMed2007

Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells.

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.

Animals↗

PubMed2003

Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins.

We performed a proteomic analysis of monocytes primed by lipopolysaccharide (LPS) in vitro, using two-dimensional gels stained with Coomassie blue. We found 16 proteins of approximately 500 detected that either increased or decreased in abundance as a result of priming by LPS (14 with P </= 0.05). The proteins were identified by comparing the masses of their tryptic peptides with those of all known proteins, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the SWISS-PROT database. Identities were confirmed by matching the sequence of several tryptic peptides, using liquid chromatography electrospray-ionization quadrupole ion trap mass spectrometry. There were increases in the protective enzymes superoxide dismutase and catalase, in four calgranulins, in the cytokine pre-B cell enhancing factor, and in annexin 2, macrophage capping protein, transketolase, pyruvate kinase, and serine/threonine protein kinase 10. Proteins that decreased in abundance were integrin alpha-IIB, protein disulfide isomerase, and platelet-activating factor acetylhydrolase. Many of these altered proteins have interesting functions in inflammation.

Amino Acid Sequence↗

PubMed1996

The regulated expression of a TATA-less, platelet-specific gene, alphaIIb.

The megakaryocyte (MK)-specific integrin, alphaIIb, is the alpha-subunit of the alphaIIb/beta3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alphaIIb gene is specifically expressed in MKs, this gene was studied as a potential model for MK-specific gene expression. Previous studies have defined some of the important regulatory elements in 912 bp of the immediate 5'-flanking region of this gene. These studies defined several important elements including two GATA-binding elements and an Ets-binding element. Using a primary rat marrow expression system, we demonstrated that one of the GATA-binding elements, -454 bp upstream of the transcriptional start site (GATA454), is critical for expression of the alphaIIb gene. A potential negative regulatory element was found between -100 and -200 bp upstream of both the rat and human alphaIIb genes. The biological basis by which this negative regulatory region effects expression is not well understood. Recent studies have focused on the issue of the molecular basis by which this TATA-less gene is properly transcribed. We found that a GA-rich region approximately 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site that interacts with an Ets-consensus site approximately 20 bp further upstream. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alphaIIb gene. In studies looking at more distal regulatory elements, we have found, using the primary rat marrow expression system, that 2.9 kb of 5'-flanking alphaIIb sequence has as high a level of expression as the 912 bp construct. Whether either of these lengths of 5'-flanking region can result in tissue-specific expression in transgenic models is presently being investigated. In addition, while a published report suggests that the two genes alphaIIb and beta3 are physically linked within a 250 kb region of genomic DNA, analysis of yeast artificial chromosome clones and genomic pulsed field gel electrophoresis analysis are consistent with these two genes not being tightly linked and being >1 mb apart, suggesting that these two genes do not form a single, tissue-specific locus.

Animals↗

PubMed1995

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro.

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Actins↗

PubMed2001

Three-dimensional collagen regulates collagen gene expression by a mechanism that requires serine/threonine kinases and is independent of mechanical contraction.

Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗