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At least 37 records · Page 2Linked to original sources
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↗
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↗
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↗
PubMed1996

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Animals↗
PubMed1997

Expression of alpha(v)beta3 integrin is less frequent in ovarian epithelial tumors of low malignant potential in contrast to ovarian carcinomas.

Ovarian tumors of low malignant potential (LMPs) histologically lack invasion and have excellent prognosis in contrast to invasive carcinomas. Integrins are heterodimeric adhesion molecules thought to play a role in cell migration and tumor progression. The alpha(v)beta3 integrin in particular mediates melanoma invasion in vitro and promotes neoplastic angiogenesis, facilitating breast cancer metastasis. In addition, alpha(v)beta3 expression has been detected in ovarian cancer cell lines and in a limited number of human ovarian cancer samples. The distribution pattern of this integrin in LMPs is not known. We examined tissue sections from 19 LMPs and 31 ovarian carcinomas for alpha(v)beta3, in addition to alpha5beta1 and alpha2beta1 integrins, which have been shown to be expressed in ovarian cancer cell lines. Variable immunoreactivities of alpha5beta1 and alpha2beta1 were detected in both LMPs and ovarian carcinomas. Most (74.2%) ovarian carcinomas were alpha(v)beta3 positive, whereas only 36.8% of LMPs were positive, which is statistically significant (P = .009). These results establish the distribution pattern of alpha2beta1, alpha5beta1, and alpha(v)beta3 integrins in LMPs. The biological significance of the less frequent expression of alpha(v)beta3 in LMPs compared with carcinomas of the ovary needs further elucidation.

Adult↗
PubMed1997

Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions.

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

Antibodies, Monoclonal↗
PubMed1997

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

Amino Acids↗
PubMed1996

Up-regulated expression of the beta3 integrin and the 92-kDa gelatinase in human HT-144 melanoma cell tumors grown in nude mice.

In situ changes in the repertoire of integrins and proteolytic enzymes have been demonstrated during melanoma metastasis. To investigate whether established human melanoma cell lines, injected into nude mice, could undergo phenotypic changes similar to those observed in in situ lesions, we studied 3 melanoma cell lines of distinct metastatic origin, adherent HT-144 and SK-MEL-2 cells, and non-adherent SK-MEL-1 cells for integrin expression, proteolytic enzyme repertoire and invasive potential after in vitro culture. Heterogeneity in integrin expression, such as elevated levels in alpha(v)beta3 in SK-MEL-1 and SK-MEL-2 cells and low expression in HT-144 cells, correlated with their in vitro invasiveness, since only the adherent HT-144 and SK-MEL-2 cells were able to invade Matrigel, and in addition, secreted a 72-kDa gelatinase. In contrast, no similar correlation could be established in nude mice, as all 3 cell lines, including the non-adherent SK-MEL-1 cells, were tumorigenic when injected s.c., while only HT-144 consistently produced experimental lung metastasis. Immunochemical analysis of the integrin profile in s.c. xenografts revealed over-expression of alpha(v), beta1 and beta3 integrins exclusively in HT-144 cells, as well as increased expression of beta3 in HT-144 cell lung metastases, as confirmed by PCR analysis using species-specific primers, while zymography and Western-blot analysis demonstrated de novo expression of the 92-kDa gelatinase MMP-9 in HT-144 xenografts. Our results highlight a positive correlation between up-regulated beta3 integrin and MMP-9 expression in human HT-144 melanoma cell tumors grown in nude mice.

Animals↗
PubMed1997

Inhibition of angiogenesis in vitro and in vivo: comparison of the relative activities of triflavin, an Arg-Gly-Asp-containing peptide and anti-alpha(v)beta3 integrin monoclonal antibody.

Disintegrin which contains the amino acid sequence Arg-Gly-Asp (RGD), has been implicated as a recognition site in interactions between extracellular matrix (ECM) and cell membrane receptors. Triflavin, a 7.5 kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of disintegrins. Integrin alpha(v)beta3 has recently been identified as a marker of angiogenic blood vessels and therefore anti-alpha(v)beta3 mAb may significantly inhibit angiogenesis. Therefore, this study was designed to compare the relative activity of triflavin and anti-alpha(v)beta3 mAb in human umbilical vein endothelial cell (HUVEC) adhesion and migration in vitro, and on angiogenesis induced by TNF(alpha) in chicken chorioallantoic membrane (CAM). In this study, it was shown that triflavin (0.1 to 0.4 microM) dose-dependently inhibited the adhesion of HUVECs to ECMs (i.e., vitronectin, fibronectin, laminin and collagen type IV). At a concentration of 10 microM, anti-alpha(v)beta3 mAb almost completely inhibited the adhesion of cells to vitronectin, had a moderate inhibitory effect on fibronectin and laminin, but only a slight inhibitory effect on collagen type IV. On the other hand, vitronectin and fibronectin promote a significantly greater extent of cell adhesion and migration than laminin or collagen type IV over a wide range of concentrations (5 to 15 microg/ml). In cell migration studies, triflavin (0.4 microM) inhibited more markedly vitronectin- and fibronectin-mediated migration than that mediated by laminin- and collagen type IV. Comparison of the relative effectiveness of triflavin with anti-alpha(v)beta3 mAb, showed that triflavin was at least twenty to thirty times more potent than anti-alpha(v)beta3 mAb at inhibiting cell adhesion and migration. Furthermore, we used TNF(alpha) as an inducer of angiogenesis in the CAM assay. Close examination of the effects of triflavin and anti-alpha(v)beta3 mAb on TNF(alpha)-induced angiogenesis revealed the presence of discontinuous and disrupted blood vessels. However, anti-alpha(v)beta3 mAb showed a significant effect only at a higher concentration (10 microM). These results suggest that the inhibition of angiogenesis may have been due to interference with the adhesion and migration of endothelial cells to ECMs. The results also indicate that triflavin has a more powerful inhibitory effect than anti-alpha(v)beta3 mAb on angiogenesis, suggesting that triflavin could theoretically be used as a reasonable therapeutic adjuvant for therapy or prevention of angiogenesis-induced diseases.

Allantois↗
PubMed1997

Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site.

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to alpha(v) beta3 and alphaIIb beta3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with alphaIIb beta3 and alpha(v) beta3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to alphaIIb beta3 and decreased binding to alpha(v) beta3. This substitution impaired the ability of echistatin to induce LIBS in alpha(v) beta3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind alphaIIb beta3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with alphaIIb beta3 and alpha(v) beta3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized alpha(v) beta3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified alpha(v) beta3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of alphaIIb beta3 and alpha(v) beta3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

Animals↗
PubMed1997

Identification of a region in the integrin beta3 subunit that confers ligand binding specificity.

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin beta subunit involved in determining ligand binding specificity. Chimeras of beta3 and beta5, the most homologous integrin beta subunits, were expressed with alphav on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for alphavbeta3. The results of the study show that when exons C and D of the beta3 subunit (residues 95-233) are substituted into beta5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type alphavbeta3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, beta3 residues 164-202, into the backbone of beta5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin beta subunits, suggesting that it may play a role in determining ligand selection by all integrins.

Amino Acid Sequence↗
PubMed1997

The human integrin beta3 gene is 63 kb and contains a 5'-UTR sequence regulating expression.

The human blood platelet fibrinogen receptor, integrin alphaIIbbeta3 (glycoprotein IIb-IIIa) is an archetypal member of the integrin family of adhesive molecules and is the only integrin encoded by genes physically linked in the genome. Because studies on the normal and abnormal expression of any gene require a thorough understanding of its organization, the initial goals of the current study were to determine the size and complete the genomic organization for the beta3 gene. We now report the isolation of the entire beta3 gene in a single P1 plasmid and for the first time have linked the first and second exons on a contiguous fragment of DNA. Using pulsed-field gel analysis, we determined the full size of the beta3 gene to be 63 kb and show a large (16.7 kb) first intron; based on this information, we propose a uniform numbering system for the beta3 exons. We have completed the 5' genomic structure and generated a long-range restriction map. The promoter and the 5' end of the first intron were found to have approximately 50% sequence identity with a region of the avian beta3 gene known to possess functional transcriptional activity. Analysis of three different homologous regions led to the identification of a sequence in the 5'-UTR of the human gene, CCGCGGGAGG, which shares 90% identity with the avian gene and which bound nuclear proteins in DNaseI and electrophoretic mobility shift assay studies. Mutating this sequence caused a 2.6-fold reduction in reporter gene activity. In these studies we have (1) determined the full length and 5' organization of the beta3 gene, (2) identified a large region of homology between the 5' regions of the avian and human genes, and (3) identified a sequence in the 5'-UTR that augments gene expression. Knowing the genomic structure of beta3 has permitted the uncovering of new mechanisms of mutagenesis causing Glanzmann thrombasthenia (Jin et al, J Clin Invest 98:1745, 1996), and our findings will be valuable for such genetic analyses as well as for studies on the transcriptional regulation of beta3 and other integrin genes.

Antigens, CD↗
PubMed1996

Antibody to beta3 integrin inhibits osteoclast-mediated bone resorption in the thyroparathyroidectomized rat.

The cell surface integrin, alphaVbeta3, is important for the attachment of osteoclasts to bone matrix and the subsequent resorption of bone. The present study was designed to determine the effects of F11, a monoclonal antibody to the rat beta3 subunit, on calcium mobilization in a rat model of bone resorption. Male Sprague Dawley rats became hypocalcemic within 18 h after thyroparathyroidectomy. Synthetic PTH-related protein (PTHrP(1-34)) administered to control rats caused serum calcium to return to normal. Anti-beta3 treatment of rats after thyroparathyroidectomy inhibited the calcemic response to PTHrP by 65%. Circulating F11 was biologically active as demonstrated by osteoclast retraction and by the inhibition of adenosine diphosphate-induced platelet aggregation via inhibition of the platelet integrin alphaIIbbeta3 in ex vivo assays. F11 antibody was localized by immunohistological staining to osteoclasts in long bones, suggesting that the mechanism of action of the antibody was via a direct effect upon osteoclasts. Echistatin and calcitonin also inhibited calcemic responses to PTHrP in this in vivo model, whereas an isotype-matched, control antibody was ineffective. These studies provide the first direct evidence in vivo that osteoclast-mediated bone resorption is regulated via beta3 integrin.

Animals↗
PubMed1997

The selective engulfment of apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and requires intracellular and extracellular calcium.

Dendritic cells derived in vitro from monocytes are known to be poor phagocytes. Here we show that, unlike macrophages, monocyte-derived dendritic cells indeed fail to take up opsonized particles or necrotic cells; however, apoptotic bodies are efficiently engulfed by dendritic cells. The temperature dependence and the sensitivity to cytochalasin D indicate that the apoptotic body engulfment is representative of early stages of phagocytosis. Inhibition studies with ligands for surface molecules involved in recognition of apoptotic bodies, such as vitronectin receptor, CD36 and phosphatidylserine receptor, revealed that apoptotic body engulfment by dendritic cells is mediated preferentially by the vitronectin receptor alpha(v)beta3, while all the receptors, with different efficiency, are engaged in phagocytosis of apoptotic bodies by macrophages. The interaction between apoptotic bodies and dendritic cells elicits a rise in intracellular free calcium concentration ([Ca2+]i) which is essential for the process of engulfment. Either intra- or extracellular Ca2+ buffering inhibits apoptotic body engulfment by dendritic cells and [Ca2+]i increases, indicating the involvement of both intra- and extracellular Ca2+. In contrast, Ca2+ mobilization is dispensable for macrophage phagocytosis of apoptotic bodies. The different requirements of Ca2+ in macrophages and dendritic cells is possibly due to the differential usage of phagocytic receptors (CD36 vs. alpha(v)beta3) and might reflect different fates of apoptotic bodies in the two cell types.

Apoptosis↗
PubMed1996

Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3.

Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin-inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co-immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells.

Amino Acid Sequence↗
PubMed1997

Ectopic expression of platelet integrin alphaIIb beta3 in tumor cells from various species and histological origin.

The integrin alphaIIb beta3 is a membrane receptor which was considered to be expressed only in cells of megakaryocytic lineage. We have shown that alphaIIb beta3 is expressed in mouse melanoma B16a cells, and in human prostate adenocarcinoma cells. The purpose of this study was to determine whether the megakaryocytic product alphaIIb beta3 was functionally expressed in other non-megakaryocyte lineage tumor cells. By using the reverse transcription polymerase chain reaction (RT-PCR), we have obtained data demonstrating that alphaIIb beta3 is expressed in a variety of tumor cell lines (17) derived from different species (human, rat and mouse) and of different histological origins (skin, blood, lung, liver, kidney, cervix, colon, bladder, breast and prostate). Immunostaining of tumor cells with a monoclonal antibody (MAb) to alphaIIb beta3 demonstrates that alphaIIb beta3 protein is also expressed in tumor cells. A protein kinase C activator PMA stimulates adhesion of tumor cells to fibronectin and fibrinogen, and this stimulated adhesion is blocked by a function-blocking MAb directed to alphaIIb beta3. Our results indicate that the megakaryocytic gene product alphaIIb beta3 integrin is widely expressed among tumor cells of non-megakaryocytic lineage, suggesting that ectopic expression of this integrin may play an important role in tumor progression.

Adenocarcinoma↗
PubMed1996

Competition for a unique response element mediates retinoic acid inhibition of vitamin D3-stimulated transcription.

We have identified a novel steroid hormone response element in the avian beta3 integrin promoter. This sequence, comprising three hexameric direct repeat half-sites separated by nine and three nucleotides binds vitamin D receptor (VDR)-retinoid X receptor (RXR) and retinoic acid receptor (RAR)-RXR heterodimers. VDR-RXR binds direct repeats separated by three base pairs, and RAR-RXR recognizes half-sites separated by nine bases, whereas the central half-site interacts with both heterodimers. Retinoic acid and 1, 25-dihydroxyvitamin D3 activate both a genomic fragment including the transcriptional start site and an oligonucleotide containing the three repeats, linked to a heterologous promoter. Co-addition of the steroids produces neither synergy nor an additive effect; rather the result equals that for retinoic acid alone. Scatchard analysis demonstrates that RAR-RXR has greater affinity than VDR-RXR for the composite element. Based on these findings we propose a model in which there is specific, polarity-defined binding of VDR-RXR and RAR-RXR to three half-sites, which form two overlapping steroid response elements, with the central half-site common to both. Our results identify a novel mechanism by which one steroid hormone can modulate the activity of a second, by competing for a shared half-site in a composite response element.

Animals↗