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PubMed2006

CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins.

The mechanisms leading to prostate cancer metastasis are not understood completely. Although there is evidence that the CXC chemokine receptor (CXCR) 4 and its ligand CXCL12 may regulate tumor dissemination, their role in prostate cancer is controversial. We examined CXCR4 expression and functionality, and explored CXCL12-triggered adhesion of prostate tumor cells to human endothelium or to extracellular matrix proteins laminin, collagen, and fibronectin. Although little CXCR4 was expressed on LNCaP and DU-145 prostate tumor cells, CXCR4 was still active, enabling the cells to migrate toward a CXCL12 gradient. CXCL12 induced elevated adhesion to the endothelial cell monolayer and to immobilized fibronectin, laminin, and collagen. Anti-CXCR4 antibodies or CXCR4 knock out significantly impaired CXCL12-triggered tumor cell binding. The effects observed did not depend on CXCR4 surface expression level. Rather, CXCR4-mediated adhesion was established by alpha5 and beta3 integrin subunits and took place in the presence of reduced p38 and p38 phosphorylation. These data show that chemoattractive mechanisms are involved in adhesion processes of prostate cancer cells, and that binding of CXCL12 to its receptor leads to enhanced expression of alpha5 and beta3 integrins. The findings provide a link between chemokine receptor expression and integrin-triggered tumor dissemination.

Cell Line, Tumor↗
PubMed2004

Tenascin-C deposition requires beta3 integrin and Src.

In this study we now show that deposition of the mesenchymal matrix marker, tenascin-C (TN-C), is mediated through beta3 expression and activation of Src. There was a striking upregulation of TN-C matrix organization in cell lines expressing beta3 and activated Src when compared to cell lines with neither of these attributes. When beta3 function was suppressed so was the deposition of TN-C. The same was true for function and activation of Src. When Src was inactive, the deposition of TN-C was low. We also determined that one of the downstream effectors of Src, MAPK, was also required to promote TN-C deposition. When MAPK activation was inhibited, TN-C deposition was also decreased. MMP activation is also implicated in TN-C deposition. The broad spectrum MMP inhibitor, GM6001, suppressed TN-C organization. These results indicate that beta3 integrin ligand binding and the activation of the Src/MAPK/MMP pathway modulate deposition of TN-C.

Animals↗
PubMed1996

Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21-L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Antigens, CD↗
PubMed2006

Molecular mechanisms of prothrombotic risk due to genetic variations in platelet genes: Enhanced outside-in signaling through the Pro33 variant of integrin beta3.

In recent years inherited variations in platelet proteins have emerged as potential risk factors that could predispose individuals to arterial thrombosis. Although many studies have examined the association of platelet gene polymorphisms with particular disease states, the underlying mechanisms by which most of these polymorphisms contribute to the pathophysiology of thrombosis have remained largely unexplored. This review will focus on the cellular and molecular features by which these genetic changes affect platelet physiology. Although many genes have been investigated in this regard, only the genes encoding integrins beta3 and alpha2, and the platelet Fc receptor, Fc(gamma)RIIA, have been studied in any depth. In some cases (such as integrin alpha2), evidence supports a quantitative trait locus. For other genes, nonsynonymous nucleotide substitutions lead to structural and functional consequences. A large portion of this review will focus on the widely studied Leu33Pro (Pl(A)) polymorphism of integrin beta3, and will consider the potential mechanisms by which the Pro33 polymorphism could induce a prothrombotic risk. A detailed understanding of how polymorphisms modulate platelet physiology will be important for understanding individual differences in response to antiplatelet therapy.

Animals↗
PubMed1997

Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain.

The amino-terminal domain of each integrin beta subunit is hypothesized to contain an ion binding site that is key to cell adhesion. A new hypothesis regarding the structure of this site is suggested by the crystallization of the I domains of the integrin alphaL and alphaM subunits (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281). In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin beta3 and beta5 subunits. Here, we examined the putative metal binding site within beta3 and beta5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin beta subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either beta subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the beta subunits is considerably different than the I domains. Thus, the integrin beta subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.

Amino Acid Sequence↗
PubMed2001

Antibodies to alpha5beta1 and alpha(v)beta3 integrins react with Candida albicans alcohol dehydrogenase.

It has been hypothesized that Candida albicans possesses integrin-like receptors on its cell surface. This is because C. albicans binds numerous fluid-phase extracellular matrix (ECM) proteins on its cell surface and adheres to the same ECM proteins when immobilized. In addition, numerous antibodies to human integrins (receptors for ECM proteins) bind to the fungal cell surface and in so doing inhibit the binding of the respective proteins. To demonstrate the presence of such a cell surface integrin, a cDNA library of C. albicans yeast cells was screened with polyclonal antiserum to the human fibronectin receptor (alpha5beta1 integrin). Clones isolated by this screening technique also reacted specifically to antiserum against the human vitronectin receptor (alpha(v)beta3 integrin). DNA sequence analysis of the cloned insert predicted a 350 aa protein (37 kDa). This predicted protein showed 75% homology at the nucleotide sequence level to alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae. In vitro transcription/translation of the cloned inserts yielded a 37 kDa protein that was immunoprecipitated with antibodies to the alpha5beta1 and alpha(v)beta3 integrins and an antibody to a C. albicans fibronectin receptor. These antibodies and an mAb to the human vitronectin receptor demonstrated an antigen of -37 kDa present in the cell-wall preparations of C. albicans and in spent growth medium. All four antibodies reacted with authentic ADH. The possible significance of these results in relation to C. albicans adherence is discussed.

Alcohol Dehydrogenase↗
PubMed2002

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors.

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.

Animals↗
PubMed1998

Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events.

Amino Acid Sequence↗
PubMed2005

Synthesis of 2,5-thiazole butanoic acids as potent and selective alpha(v)beta3 integrin receptor antagonists with improved oral pharmacokinetic properties.

We describe a series of 2,5 thiazole containing compounds, which are potent antagonists of the integrin alpha(v)beta3 and show selectivity relative to the other integrins, such as alpha(IIb)beta3 and alpha(v)beta6. These analogs were demonstrated to have high bioavailability relative to other relative heterocyclic analogs.

Administration, Oral↗
PubMed1998

Phagocytosis of Escherichia coli by insect hemocytes requires both activation of the Ras/mitogen-activated protein kinase signal transduction pathway for attachment and beta3 integrin for internalization.

Insect hemocytes in response to lipopolysaccharide (LPS) of Gram-negative bacteria facilitate binding and internalization of either cell-associated or cell-free LPS (Charalambidis, N. D., Foukas L. C., and Marmaras V. J. (1996) Eur. J. Biochem. 236, 200-206). An early event in LPS signaling in hemocytes involves protein tyrosine phosphorylation (Charalambidis N. D., Zervas C. G., Lambropoulou M., Katsoris P. G., and Marmaras V. J.(1995) Eur. J. Cell Biol. 67, 32-41). Here we report further data of LPS-mediated signal transduction responsible for Escherichia coli phagocytosis. We demonstrate that both adhesion of hemocytes to substrata and LPS stimulation can cause activation of p44(MAPK) in Ceratitis capitata hemocytes but with distinct kinetics indicating different functions. In addition, we showed that Drk, a homolog protein to the mammalian GRB2, is implicated in the transmission of LPS signaling, indicating that the Ras/mitogen-activated protein kinase pathway is involved. Either the cell-free or the cell-associated LPS appears to attach to the hemocyte surface by the same mechanism that is based on the cross-linking of LPS to membrane-associated p47 via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase. By contrast, the cell-free LPS internalization into the hemocytes differs from the cell-associated LPS internalization. For E. coli internalization integrin receptors as well as cytoskeletal rearrangements are required, as judged by inhibition of E. coli internalization in the presence of the RGD peptide, beta3-integrin antibodies, and cytochalasin D.

Animals↗
PubMedDate not supplied

The beta3 integrin antagonist m7E3 reduces matrix metalloproteinase activity and smooth muscle cell migration.

Treatment with c7E3 (abciximab, ReoPro) has been associated with a reduction in coronary events and the need for revascularization. Some of these beneficial effects may be due to blockade of the alphavbeta3 integrin receptor on smooth muscle cells (SMCs), however very little is known about the mechanisms involved. The current studies were designed to test the hypothesis that beta3 integrin antagonists inhibit the arterial response to injury by reducing matrix metalloproteinase (MMP) activity in the vessel wall. Male Sprague-Dawley rats were treated with daily intraperitoneal injections of the monoclonal antibody m7E3 at a dose of 6 mg/kg/day. MMP-9 activity was reduced by 73%, and MMP-2 activity by 75%, in the injured carotids of the m7E3-treated rats compared to saline-treated controls. By contrast, tissue inhibitor metalloproteinase (TIMP) activity was not changed. SMC migration assayed at 4 days after injury was reduced from 56.7 +/- 14 cells/mm(2) intimal surface area in controls to 17.5 +/- 5 cells/mm(2) in m7E3-treated rats (p = 0.02). Medial cell replication measured at 4 days and intimal cell replication measured at 7 days were not affected. Intimal cross-sectional area, measured 14 days after injury was reduced by 28% after m7E3 treatment (p = 0.05). Intimal smooth muscle cell number and the ratio of intima/media cross-section area were also reduced. By contrast, intimal SMC density was not affected by m7E3 treatment, indicating no effect on matrix accumulation. We conclude that treatment with m7E3 reduced SMC migration following vascular injury, possibly via an inhibitory effect on MMP activity, and this resulted in a decrease in intimal size at 14 days after injury.

Abciximab↗
PubMed2005

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells.

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.

Actin Cytoskeleton↗
PubMed2006

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family.

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.

Amino Acid Sequence↗
PubMed2004

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Actins↗
PubMed2003

Disruption of the beta3 663-687 disulfide bridge confers constitutive activity to beta3 integrins.

The platelet fibrinogen receptor, integrin alphaIIbbeta3, is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, trans-membrane, and cytoplasmic regions of the glycoprotein beta3 in the formation of functional complexes with alpha subunits. Progressive carboxy-terminal deletions of beta3 revealed that surface exposure of alphaIIbbeta3 or alphavbeta3 could not occur in the absence of the transmembrane domain of beta3. In contrast, internal deletions 616 to 690 of the carboxy-terminal regions of the beta3 ectodomain led to surface exposure of constitu tive active receptors in CHO cells, as indicated by the enhanced rate of cell adhesion to immobilized ligands and spontaneous binding to soluble fibrinogen or activation-dependent antibody PAC-1. The functional analysis of cysteine mutations within the 616 to 690 region of beta3 or chimeric beta3-beta7 subunits revealed that disruption of the C663-C687 disulfide bridge endows constitutive activity to the alphaIIbbeta3 receptor. It is concluded that the carboxy-terminal tail of the beta3 ectodomain, so-called beta tail domain (betaTD), is not essential for cell surface expression of beta3 receptors. However, a basal, nonactivated, low ligand-affinity state of the beta3 integrins demands a normal conformation of this domain.

Animals↗
PubMed1996

The ligand recognition specificity of beta3 integrins.

AlphaIIbbeta3 (platelet membrane glycoprotein IIb-IIIa) and alphavbeta3 are members of the beta3 subfamily of integrin adhesion receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD), originally described by Scarborough et al. (Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol. Chem. 268, 1066-1073) has been employed as a high affinity ligand for alphaIIbbeta3 to examine the specificity of the beta3 integrins. cHarGD interacted with high affinity with purified alphaIIbbeta3 (Kd = 10 nM) or with platelets (Kd = 120 nM). While cHarGD was specific for alphaIIbbeta3 in the presence of Ca2+, it bound to both beta3 integrins in the presence of Mn2+. Barbourin, a snake venom disintegrin containing a reactive KGD sequence, remained alphaIIbbeta3-specific, even in the presence of Mn2+. cHarGD became cross-linked to a site in beta3 of alphaIIb beta3, which is distinct from that of RGD peptides. These results allow identification of at least four classes of beta3 ligands: Class I, represented by RGD peptides and vitronectin, react similarly with alphaIIbbeta3 and alphavbeta3; Class II, represented by cHarGD, gamma-chain peptides and fibrinogen, react with both receptors in the presence of Mn2+ but only with alphaIIbbeta3 in the presence of Ca2+; Class III, represented by barbourin, are alphaIIbbeta3-specific under all cation conditions; Class IV, represented by osteopontin, bind primarily to alphavbeta3.

Amino Acid Sequence↗
PubMed1999

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway.

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.

Cell Adhesion Molecules↗
PubMed1998

Human integrin beta3 gene expression: evidence for a megakaryocytic cell-specific cis-acting element.

The human integrin beta3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for beta3 expression, a number of upstream regulatory regions in the beta3 gene were identified. (1) The three regions from -1159 to -584, -290 to -146, and -126 to -115 demonstrated positive, negative, and negative activity, respectively. (2) The region from -115 to +29 of the beta3 gene was sufficient for cell-specific activity. Deletion of the sequence from -115 to -89 produced a 6- to 40-fold reduction in reporter gene activity in beta3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in beta3-expressing endothelial and melanoma cell lines, and 1.3- and 2. 8-fold reduction, respectively, in non-beta3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions -113 to -108) is a megakaryocytic cell line-specific cis-acting element. (3) The region from -89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position -70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5' region of the beta3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment beta3 gene expression.

Animals↗