Biomedical subjects

Ulhas P Naik

Publications and source records attributed to Ulhas P Naik.

11 recordsLinked to original sources

PubMed2007

Cytoplasmic binding partners of the platelet integrin alphaIIb beta3.

Platelets function physiologically in mediating hemostasis, but are also associated with many pathological conditions, such as thrombosis, which can lead to myocardial infarction and/or stroke. Therefore, the study of platelet regulation and signaling has been of great interest and is necessary for generating effective anti-platelet therapeutics. One platelet signaling molecule of particular interest is the integrin alphaIIb beta3, which binds Fg and mediates platelet cross-linking. The integrin itself as well as cytoplasmic proteins that interact with alphaIIb beta3 have become potential targets for anti-platelet therapies. One such protein that has been shown to directly regulate alphaIIb beta3 function is calcium- and integrin-binding protein 1 (CIB1). CIB1 has been implicated in alphaIIb beta3 activation and outside-in signaling through the integrin. By increasing our understanding of CIB1 and other proteins that like it, associate with integrin alphaIIb beta3, and the signaling events that result from those interactions, we may bring ourselves closer to more effective therapies. In the present work, we explore known cytoplasmic binding partners of the integrin alphaIIb beta3 and their effect on alphaIIb beta3, focusing on CIB1.

Blood Platelets↗

PubMed2006

Deletion of JAM-A causes morphological defects in the corneal epithelium.

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

Animals↗

PubMed2006

Fibroblast growth factor-2 failed to induce angiogenesis in junctional adhesion molecule-A-deficient mice.

OBJECTIVE: We have previously shown that JAM-A regulates fibroblast growth factor-2 (FGF-2)-induced endothelial cell morphology, proliferation, and migration. Whether JAM-A is involved in FGF-2-induced angiogenesis in vivo is not known. We used JAM-A null mice to conclusively determine the role of JAM-A in FGF-2-induced neovascularization. METHODS AND RESULTS: We generated JAM-A null (JAM-A(-/-)) mice using gene trap technology. These mice, although viable and fertile, exhibited distorted Mendelian and sex ratios, suggesting partial embryonic lethality. Retinal fluorescein angiogram did not reveal any significant morphological differences in the vasculature of JAM-A(-/-) mice compared with wild-type (JAMA-A(+/+)) littermates. To evaluate the role of JAM-A in angiogenesis, we performed an aortic ring assay. FGF-2-induced microvessel growth was evident in aortic rings from JAM-A(+/+) mice, but FGF-2 failed to induce microvessel sproutings in aortic rings from JAM-A(-/-) mice. In a Matrigel plug assay, a known in vivo model for angiogenesis, we found that FGF-2 induced a robust vessel growth in JAM-A(+/+) mice, whereas FGF-2 failed to induce blood vessel formation in plugs from JAM-A(-/-) mice. CONCLUSIONS: Our results using JAM-A(-/-) mice presented here conclusively establish an essential role for JAM-A in FGF-2-induced angiogenesis.

Animals↗

PubMed2006

Junctional adhesion molecule-A-induced endothelial cell migration on vitronectin is integrin alpha v beta 3 specific.

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin superfamily, and is mainly expressed in the tight junctions of both epithelial and endothelial cells. We have recently shown that JAM-A is involved in basic fibroblast growth factor (bFGF)-induced angiogenesis. Here, we show that, when ectopically expressed in human umbilical vein endothelial cells (HUVECs), JAM-A induced enhanced cell migration on vitronectin, but had no effect on fibronectin. Use of antibodies that block integrin function indicated that the migration on vitronectin is specific to integrin alpha(v)beta(3) and not to integrin alpha(v)beta(5). JAM-A-induced migration was inhibited by anti-JAM-A antibody. Additionally, overexpression of a JAM-A cytoplasmic domain deletion mutant failed to induce HUVEC migration. Addition of phosphoinositide 3-kinase and protein kinase C inhibitors blocked JAM-A-induced migration, suggesting that these kinases act downstream of JAM-A. Immunoprecipitation analysis showed that JAM-A interacts with integrin alpha(v)beta(3), and this association was increased by engagement of the ligand-binding site of the integrin by Arg-Gly-Asp-Ser (RGDS) peptide. Furthermore, activation of both focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) on vitronectin was enhanced by JAM-A overexpression but not by its cytoplasmic domain deletion mutant. Taken together, these results suggest that signaling through JAM-A is necessary for alpha(v)beta(3)-dependent HUVEC migration and implicate JAM-A in the regulation of vascular function.

Cell Adhesion Molecules↗

PubMed2005

JAM-A expression during embryonic development.

Cell adhesion molecules of the immunoglobulin superfamily play an important role in embryonic development. We have shown recently that JAM-A, a member of this family expressed at endothelial and epithelial tight junctions, is involved in platelet activation, leukocyte transmigration, and angiogenesis. Here, we determine the expression pattern of the JAM-A gene during embryogenesis using transgenic mice expressing lacZ under the control of the endogenous JAM-A promoter. Histochemical staining for beta-galactosidase in heterozygous mouse embryos was first seen in the inner cell mass and trophectoderm of the blastocyst. By 8.5 days post coitum (dpc), JAM-A gene activity was detected in the endoderm and part of the surface ectoderm. At 9.5 dpc, JAM-A expression began to localize to certain organ systems, most notably the developing inner ear and early vasculature. Localization of JAM-A to embryonic vasculature was confirmed by double-staining with antibodies against JAM-A and platelet endothelial cell adhesion molecule-1, a known endothelial cell marker. As organogenesis progressed, high levels of JAM-A expression continued in the epithelial component of the inner ear as well as the epithelium of the developing skin, olfactory system, lungs, and kidneys. In addition, JAM-A gene activity was found in the developing liver, choroid plexuses, and gut tubes. Immunofluorescent staining with a JAM-A antibody was performed to confirm that expression of the JAM-A-beta-galactosidase fusion protein accurately represented endogenous JAM-A protein. Thus, JAM-A is prominently expressed in embryonic vasculature and the epithelial components of several organ systems and may have an important role in their development.

Animals↗

PubMed2005

The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions.

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.

Amino Acid Sequence↗

PubMed2004

OptiRNAi, an RNAi design tool.

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Animals↗

PubMed2003

Essential role of junctional adhesion molecule-1 in basic fibroblast growth factor-induced endothelial cell migration.

OBJECTIVE: Recently, we have shown that blocking of junctional adhesion molecule-1/A (JAM-1/A) inhibits basic fibroblast growth factor (bFGF)-induced angiogenesis. Because the process of endothelial cell proliferation is a key initial step of neovascularization, we studied the effect of functional knockdown of JAM-1 on human umbilical vein endothelial cell (HUVEC) adhesion and migration induced by bFGF. METHODS AND RESULTS: We introduced small interfering RNAs specific to JAM-1 in HUVECs, stimulated them with bFGF, and studied the resultant adhesion and migration of these cells on vitronectin and fibronectin. We show that depletion of JAM-1 inhibits bFGF-induced HUVEC migration specifically on vitronectin. This inhibition is not attributable to the failure of junctional organization, because expression and distribution of other junctional proteins remained unaffected. This inhibition was in fact attributed to an inability of JAM-1-depleted HUVECs to adhere and spread on vitronectin. Furthermore, we find that JAM-1-depleted HUVECs failed to activate extracellular signal-related kinase (ERK) in response to bFGF treatment. CONCLUSIONS: Our results show that JAM-1 is required for the bFGF-induced ERK activation that leads to endothelial cell migration on vitronectin. These data thus implicate JAM-1 as an integral part of both bFGF and ERK signaling pathways in endothelial cells.

Cell Adhesion↗

PubMed2003

Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen.

Platelet spreading on the subendothelium in response to vascular injury is fundamental to the regulation of physiologic hemostasis. Previously, we have shown that, when bound to glycoprotein IIb (GPIIb), calcium- and integrin-binding protein (CIB) regulates platelet spreading on immobilized fibrinogen (Fg). In this study, we investigated the signaling events that occur downstream of CIB in the absence of signaling that occurs as a result of granular secretion. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate that CIB induces cell migration. Immunofluorescence analysis of CIB localization indicates that endogenous CIB accumulates in areas of focal adhesions, and its overexpression up-regulates the formation of focal adhesion complexes compared with control cells. Immunoprecipitation analysis indicates that CIB associates with focal adhesion kinase (FAK), a key regulator in focal complex formation, and up-regulates its activity. Overexpression of dominant-negative FAK, FRNK, along with CIB in CHO cells completely inhibits CIB-induced cell migration. Further, confirmation of these data in the platelet system indicates that CIB and FAK associate throughout all stages of platelet spreading but only on Fg binding to GPIIb/IIIa. Taken together, our results suggest that CIB regulates platelet spreading through the regulation of FAK activation.

Amino Acid Sequence↗

PubMed2003

Signaling through JAM-1 and alphavbeta3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and alphavbeta3 complex.

Growth factor-induced neovascularization has received a great deal of attention because it is fundamental to the growth and metastasis of solid tumors. This multistep process requires extensive signaling through growth factor receptors and integrins. Among the integrins involved in this process, integrin alphavbeta3 is specific to basic fibroblast growth factor (bFGF)-induced angiogenesis. Here we show that junctional adhesion molecule 1/A (JAM-1/A) and alphavbeta3 form a complex in the absence of bFGF. JAM-1, which is normally localized at the cell-cell junctions of quiescent endothelial cells, redistributes to the cell surface on bFGF treatment. Blockage of the extracellular domain of JAM-1 inhibits bFGF-induced endothelial cell morphology, proliferation, and angiogenesis. Additionally, mutation in the JAM-1 cytoplasmic domain blocks bFGF-induced mitogen-activated protein (MAP) kinase activation and ablates its ability to induce endothelial cell tube formation, suggesting that signaling through JAM-1 is key to bFGF-induced signaling. Immunoprecipitation analysis suggests that bFGF signaling dissociates the JAM-1/ alphavbeta3 complex, allowing for signaling through JAM-1 and alphavbeta3. In addition, blockage of either JAM-1 or alphavbeta3 inhibits bFGF-induced MAP kinase activation. Thus, our results suggest that signaling through JAM-1 and alphavbeta3 is necessary for bFGF-induced angiogenesis.

Allantois↗

PubMed2003

Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen.

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets. Immunoprecipitation analyses indicate that CIB and glycoprotein IIb/IIIa (GPIIb/IIIa) interact with each other as platelets adhere to immobilized Fg, and together they associate with the platelet cytoskeleton. Introduction of anti-CIB antibody or GPIIb cytoplasmic peptide into platelets blocks lamellipodia but not filopodia formation. GPIIb peptide-induced inhibition of platelet spreading is recovered by the incorporation of recombinant CIB protein, suggesting that interaction between CIB and GPIIb/IIIa is required for progression from filopodial to spread morphologies. Further, anti-CIB- or GPIIb peptide-induced inhibition of platelet spreading can be overcome by the addition of exogenous adenosine diphosphate (ADP). These data suggest that formation of the CIB-GPIIb/IIIa complex may be necessary for initiation of downstream signaling events, such as ADP secretion, that lead to platelet spreading.

Adenosine Diphosphate↗