Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.     

Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells

Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the alpha 2 beta 1 integrin, beta 2 chain integrins, and vitronectin receptor (alpha v beta 3) were not involved in cellular attachment to invasin.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.     

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of Fc epsilon R II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the alpha or the beta form of Fc epsilon R II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both alpha and beta forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either alpha or beta, were unaffected after incubation with anti-sense oligonucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural localization of integrin subunits alpha3 and alpha6 in capillarized sinusoids of the human cirrhotic liver

Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.     

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.     

Reelin binds alpha3beta1 integrin and inhibits neuronal migration

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.     

Synthesis of highly potent and selective hetaryl ureas as integrin alpha(V)beta3-receptor antagonists

Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Triplex formation with alpha anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides.

Nuclease-resistant alpha anomers of pyrimidine-rich CT- and purine-rich GA- and GT-containing oligonucleotides were investigated for their triplex-forming potential and compared with their corresponding nuclease-sensitive beta anomers. Both 23mer CT-alpha and 23mer CT-beta had quite similar triplex binding affinities. Synthetic 23mer GT-alpha oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-beta oligonucleotides. The orientation of third strand GT-alpha binding was parallel to the purine strand of the duplex DNA target, whereas the orientation of third strand GT-beta binding was found to be antiparallel. Triplex formation with both GT oligonucleotides showed the typical dependence on magnesium and temperature. In contrast, 23mer GA-alpha oligonucleotides did not support triplex formation in either orientation under a variety of experimental conditions, whereas the corresponding 23mer GA-beta oligonucleotides demonstrated strong triplex formation in the antiparallel orientation. GA-alpha oligonucleotides covalently conjugated to acridine were similarly unable to demonstrate triplex formation. GA-alpha oligonucleotides, in contrast to GT-alpha oligonucleotides, were capable of self-association, detectable by gel retardation and UV spectroscopy, but competing self-association could not fully account for the lack of triplex formation. Thus for in vivo triplex gene regulation strategies using GT oligonucleotides the non-natural alpha anomer may be a feasible alternative to the natural beta anomer, allowing for a comparable degree of triplex formation without rapid cellular degradation. However, alpha anomeric inversion does not appear to be a feasible alternative in applications involving GA oligonucleotides.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Protein phosphatase-1 inhibitor-3 is an in vivo target of caspase-3 and participates in the apoptotic response

Inh3 (inhibitor-3) is a potent inhibitor of protein phosphatase-1 that selectively associates with PP1gamma1 and PP1alpha but not the PP1beta isoform. We demonstrate that Inh3 is a novel substrate for caspase-3 and is degraded in vivo during apoptosis induced by actinomycin D. Inh3 was not degraded in apoptotic MCF-7 cells, which lack caspase-3. These experiments establish that Inh3 is a novel physiological substrate of caspase-3. Electroporation of the caspase-3-resistant Inh3-D49A mutant into HL-60 cells resulted in a significant attenuation of apoptosis induced by actinomycin D. These results show that Inh3 degradation contributes to the apoptotic process. Immunofluorescence based examination of the subcellular localizations of Inh3 and PP1gamma1 revealed a major relocalization of the cellular pool of PP1gamma1 from the nucleolus to the nucleus and then to the cytoplasm during actinomycin D-induced apoptosis. A similar redistribution of PP1alpha from the nucleus to the cytoplasm occurred. These results are consistent with an unexpected discovery that significant fractions of the cellular pools of PP1gamma1 and PP1alpha are associated with Inh3 in HL-60 cells. Thus, Inh3 is a major factor in the cellular economy of PP1gamma1 and PP1alpha subunits. The unscheduled relocalization of this large a pool of PP1 subunits and their release from a potent inhibitor could deregulate a diverse range of essential cellular processes and signaling pathways. We discuss the significance of these findings in relation to working hypotheses whereby Inh3 destruction could contribute to the apoptotic process.     

Differential gene regulation by specific gain-of-function JNK1 proteins expressed in Swiss 3T3 fibroblasts

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1alpha or p46JNK1beta. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1alpha or MKK7-JNK1beta were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1alpha and MKK7-JNK1beta proteins, whereas 13 genes were selectively increased by MKK7-JNK1alpha and 13 genes were selectively increased by MKK7-JNK1beta. The set of genes selectively induced by MKK7-JNK1beta included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1beta exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.