Abstract: Dendritic spines are the major sites of excitatory synaptic input, and their morphological changes have been linked to learning and memory processes. Here, we report that growing microtubule plus ends decorated by the microtubule tip-tracking protein EB3 enter spines and can modulate spine morphology. We describe p140Cap/SNIP, a regulator of Src tyrosine kinase, as an EB3 interacting partner that is predominantly localized to spines and enriched in the postsynaptic density. Inhibition of microtubule dynamics, or knockdown of either EB3 or p140Cap, modulates spine shape via regulation of the actin cytoskeleton. Fluorescence recovery after photobleaching revealed that EB3-binding is required for p140Cap accumulation within spines. In addition, we found that p140Cap interacts with Src substrate and F-actin-binding protein cortactin. We propose that EB3-labeled growing microtubule ends regulate the localization of p140Cap, control cortactin function, and modulate actin dynamics within dendritic spines, thus linking dynamic microtubules to spine changes and synaptic plasticity.
Abstract: The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient "per se" to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1-increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway.
Abstract: p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.
Abstract: Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium pervanadate to boost the abundance of phosphorylation of the EGF receptor. Nine phosphorylation sites (pT669, pS967, pS1002, pY845, pY974, pY1045, pY1086, pY1148, and pY1173) of EGF receptor were quantified from EGF-stimulated cells in suspension and adherent conditions. Our data sets revealed that EGF stimulation of adherent cells induced higher levels of tyrosine phosphorylation relative to EGF stimulation of suspended cells. In contrast, EGF stimulation of adherent cells induced lower levels of serine and threonine phosphorylation relative to EGF stimulation of suspended cells. These findings are consistent with the hypothesis that cellular adhesion modulates phosphorylation of plasma membrane receptor tyrosine kinases relevant for EGF-induced signal transduction processes. Furthermore, our results suggest that strong phosphatase inhibitors should be used to generate reference datasets in comparative phosphoproteomics experiments.
Abstract: We recently identified p140Cap as a novel adaptor protein, expressed in epithelial-rich tissues and phosphorylated upon cell matrix adhesion and growth factor treatment. Here, we characterise p140Cap as a novel Src-binding protein, which regulates Src activation via C-terminal Src kinase (Csk). p140Cap silencing increases cell spreading, migration rate and Src kinase activity. Accordingly, increased expression of p140Cap activates Csk, leading to inhibition of Src and downstream signalling as well as of cell motility and invasion. Moreover, cell proliferation and "in vivo" breast cancer cell growth are strongly impaired by high levels of p140Cap, providing the first evidence that p140Cap is a novel negative regulator of tumour growth.
Abstract: The Cas family of multiadaptor and scaffold molecules has an essential role in intracellular signaling events. Although these proteins do not have enzymatic or transcriptional activity, they spatially and temporally control signaling events through their ability to undergo changes in phosphorylation and to associate with effectors proteins in multimolecular complexes. The involvement of p130Cas in cell motility as a component of the integrin signaling machinery is well established. Here, we discuss recent developments that highlight a fundamental role in cell transformation and microbial pathogenesis and the implications of these developments on p130Cas function under normal and pathological conditions.
Abstract: To investigate the mechanisms through which p130Cas adaptor protein is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone. Mammary epithelial cells isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. Consistently with the double-transgenic mice model, p130Cas is overexpressed in a significant subset of human breast cancers and high levels of p130Cas in association with HER2 expression correlate with elevated proliferation. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a putative target for clinical therapy.
Abstract: We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.
Abstract: The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.
Abstract: Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.
Abstract: As vascular endothelial growth factor (VEGF), interleukin-3 (IL-3), released into the tumor microenvironment stimulates motogenic and mitogenic activity of normal and transformed cells. In the present study, we investigate the effects of IL-3 and VEGF on neoplastic vascular growth. Engagement of IL-3 receptor beta common (IL-3R beta c) contributes to both IL-3- and VEGF-induced Rac1 activation, cell migration and in vitro tube-like structure formation as shown by the expression of the dominant-negative IL-3R beta c construct (Delta455). In normal and transformed endothelial cells (EC) as well as in HEK 293 cells expressing KDR and IL-3R, VEGF and IL-3 treatment induces the formation of a KDR/IL-3R beta c complex. Moreover, as shown by the IL-3R Delta455 mutant or by the kinase dead KDR, functional receptors are required for this interaction. Consistent with the contribution of IL-3R beta c in both IL-3- and VEGF-mediated angiogenic signal, a reduced number of vessels inside tumors are found in mice injected with cells expressing the IL-3R Delta455 mutant. Thus, these findings provide a novel mechanism through which IL-3 and VEGF support cell survival and tumor neovascularization.
Abstract: Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.
Abstract: Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)-ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry-based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor-bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALK(K210R) or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.
Abstract: Steroid hormones bind to their receptors and trans-activate target genes. Rapid non-genomic action of steroid hormones has been proposed in addition to the one at the genomic level. Estrogen has been described to activate c-Src kinase and this activation has been shown to be responsible for estrogen-dependent mitogenicity. A major substrate of c-Src kinase activity is the cytoskeletal protein p130Cas, originally identified in v-Src-transformed cells. We show that in the human breast carcinoma T47D cells, upon estrogen treatment, p130Cas rapidly and transiently associates with the estrogen receptor alpha in a multi-molecular complex containing the c-Src kinase and the p85 subunit of PI 3-kinase. Association of p130Cas with the estrogen receptor alpha occurs within 3 minutes of estrogen treatment and is dependent on c-Src kinase activation. Transient overexpression of p130Cas in T47D cells increases estrogen-dependent Src kinase and Erk1/2 MAPKs activities and accelerates their kinetics of stimulation. A similar effect was detected on estrogen-dependent cyclin D1 expression, suggesting a role for p130Cas in regulating estrogen-dependent cell cycle progression. Double-stranded small RNA interference (siRNA) by silencing endogenous p130Cas protein, was sufficient to inhibit estrogen-dependent Erk1/2 MAPKs activity and cyclin D1 induction, demonstrating the requirement of p130Cas in such events. Therefore, our data show that the adaptor protein p130Cas associates with the estrogen receptor transducing complex, regulating estrogen-dependent activation of c-Src kinase and downstream signaling pathways.
Abstract: Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.
Abstract: Integrin signalling co-ordinates with signalling originating from growth factor receptors in the co-operative control of cell proliferation, survival and migration. Increasing evidence suggests that integrins form physical complexes at the cell membrane with growth factor receptors, giving rise to signalling platforms at the adhesive sites. It is probable that at these sites integrins regulate adhesion and at the same time physically constrain and direct the response to soluble growth factors towards proliferation or survival stimuli. These co-operative effects might depend on integrin ability to activate growth factor receptors. In the present paper, we summarize our recent study showing that integrin-dependent adhesion triggers ligand-independent EGFR (epidermal growth factor receptor) activation to transduce downstream signalling. In addition, we also show that integrin-induced signalling pathways are necessary for EGF-dependent transcriptional response, demonstrating the requirement of the co-operation between cell-matrix adhesion and EGFR to achieve full biological responses.
Abstract: Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.
Abstract: Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.
Abstract: Advanced glycation end products (AGEs) have been implicated in the accelerated vascular injury occurring in diabetes. We recently reported that LDL prepared from type 2 diabetic patients (dm-LDL), but not normal LDL (n-LDL) triggered signal transducers and activators of transcription STAT5 activation and p21(waf) expression in endothelial cells (ECs). The aims of the present study were to investigate the role of LDL glycation in dm-LDL- mediated signals and to analyze the molecular mechanisms leading to STAT5 activation. We found that glycated LDL (gly-LDL) triggered STAT5 activation, the formation of a prolactin inducible element (PIE)-binding complex containing STAT5, and increased p21(waf) expression through the activation of the receptor for AGE (RAGE). We also demonstrated that dm-LDL and gly-LDL, but not n-LDL treatment induced the formation of a stable complex containing the activated STAT5 and RAGE. Moreover, gly-LDL triggered src but not JAK2 kinase activity. Pretreatment with the src kinase inhibitor PP1 abrogated both STAT5 activation and the expression of p21(waf) induced by gly-LDL. Consistently, gly-LDL failed to activate STAT5 in src(-/-) fibroblasts. Collectively, our results provide evidence for the role of glycation in dm-LDL-mediated effects and for a specific role of src kinase in STAT5-dependent p21(waf) expression.
Abstract: Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-Ã -go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.
Abstract: The Dbl oncogene is a putative exchange factor for the small GTPases RhoA and Cdc42, which are involved in actin polymerization into stress fibers and filopodia, respectively. We report here that, upon adhesion to fibronectin, Dbl-transformed NIH3T3 cells display a contracted, polygonal shape with a high number of short stress fibers. In contrast, untransformed NIH3T3 cells acquire the characteristic fibroblast morphology and organize a regular mesh of long stress fibers. We show that in Dbl-transformed and in untransformed NIH3T3 cells the different shape and actin cytoskeleton organization observed in the early steps of adhesion involves activation of distinct GTPases. Upon adhesion to fibronectin, cell morphology of Dbl-transformed NIH3T3 cells depends on activation of RhoA and not of Cdc42. In contrast Cdc42 activation is necessary to untransfected NIH3T3 cells to acquire their fibroblast shape. In both Dbl-transformed and in untransformed NIH3T3 cells a basal Rac activation is necessary to support stress fiber organization, while constitutive Rac activation promotes ruffles and lamellipodia formation. As a consequence of RhoA activation, Dbl-transformed cells show high activity of ROCK-alpha and CRIK kinases, two known RhoA effectors. In addition Dbl-transformed and NIH3T3 cells expressing the constitutive active form of RhoA are less motile on fibronectin than cells expressing constitutive active Cdc42. We conclude that in NIH3T3 cells in response to fibronectin the expression of the Dbl oncogene leads to a predominant activation of RhoA which both supports the peculiar cell shape and actin cytoskeleton organization in stress fibers and regulates cell motility.
Abstract: Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.
Abstract: Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.
Abstract: Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to beta1- or alphav-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell-matrix interaction.
Abstract: Cell matrix adhesion regulates actin cytoskeleton organization through distinct steps, from formation of filopodia and lamellipodia in the early phases of cell adhesion to organization of focal adhesions and stress fibers in fully adherent cells. In this review, we follow the events induced by integrin-mediated adhesion, such as activation of GTPases Cdc42 and Rac and their effectors and their role in actin polymerization leading to formation of lamellipodia and filopodia and cell spreading. We also show that actin stress fiber and focal adhesion formation following adhesion requires cooperation between integrin-mediated signaling and additional stimuli, including activation of PKC, Rho GTPases, and PTKs such as p125Fak and Src.
Abstract: Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, beta1 or alphav integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5x10(3) receptors per cell). These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.
Abstract: The Dbl oncogene is the putative exchange factor for two small GTP-binding proteins, RhoA and CDC42 which are involved in the polymerization of actin to produce stress fibers and filopodia, respectively. We report here that Dbl oncogene-transformed NIH3T3 cells show actin stress fibers only when cells are plated on fibronectin. Plating of cells on collagen I and IV as well as on poly-D-lysine and gelatin induces polymerization of actin to form filopodia, lamellipodia and membrane ruffles but not stress fibers. The putative collagen receptors, alpha1/beta1 and alpha2/beta1 integrins are expressed at reduced level in Dbl-transformed cells compared to untransformed NIH3T3 fibroblasts. Nevertheless, adhesion to collagens is not altered. Inhibitory monoclonal antibody to mouse integrin beta1 subunit blocked adhesion of both Dbl-transformed and untransformed NIH3T3 cells, demonstrating that adhesion to collagen I and IV is mediated by the beta1 family of integrins. Dbl product rapidly induces the depolymerization of actin stress fibers, rounding up of the cells, and formation of filopodia and lamellipodia when microinjected in NIH3T3 cells plated on gelatin. Thus, Dbl may exert its effect on actin cytoskeleton organization in response to extracellular proteins by altering integrin-mediated signalling pathways.
Abstract: BHK-21 c13 monolayer and suspension cells were investigated with regard to some phenotypic features which could bear on the quality of foot-and-mouth disease virus (FMDV) antigen produced in them. Despite good viability, suspension cells differed from monolayer cells in fundamental features of susceptibility to FMDV. Most important, FMD virus particles grown in suspension cells at high passage levels were shown to be largely degraded following inactivation with an aziridine compound. Suspension cells were characterised by a downregulation in the surface expression of both alpha 5 and alpha V integrin chains. According to the results of binding assays, both integrins could act as FMDV receptors on BHK-21 c13 cells. Reduced surface expression of integrins was correlated with disappearance of actin stress fibres, which would play a role in regular encapsidation of viral RNA and hence in stability of virus particles. With regard to FMD vaccine production, practical suggestions are put forward to evaluate the quality of BHK-21 c13 cells and FMDV Ag, which must prove stable during downstream processing.
Abstract: A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.
Abstract: Platelet-activating factor (PAF) is a potent activator of angiogenesis and controls the motility and the shape of vascular endothelium. The mechanism(s) whereby PAF exerts its action are in part known. Here we report that the biological active (R)PAF enantiomer administrated to cultured endothelial cells induces the early phosphorylation in tyrosine residues of focal adhesion kinase (p125FAX) and paxillin, two molecules involved in the early signaling and cytoskeleton assembly in cells that undergo integrin-mediated adhesion or are challenged by neuropeptides or lysophosphatidic acid. The phenomenon is rapidly turned on, lasts for a few minutes and is adhesion-independent indicating that the chain of events induced by (R)PAF, including p125FAK activation, precedes adhesion. The inhibitory effect of WEB2086, a PAF receptor antagonist, and the lack of activity exerted by the (S)PAF enantiomer, indicate that (R)PAF-mediated p125FAK activation, is PAF receptor-dependent. Calphostin C, an inhibitor of protein kinase C blocks the effect of (R)PAF on p125FAK phosphorylation suggesting that protein kinase C activation is up-stream the activation of this tyrosine kinase. When endothelial cells are exposed to a substratum that allows adhesion and spreading. (R)PAF-stimulated cells, change their adhesive phenotype and start migrating. Inhibitors of tyrosine kinases, like 3-(1,4,-dihydroxytetralyl) methylen-2-oxindole and herbimycin A, reduce the cells migration, the transendothelial flux of albumin and the enhancement of p125FAK activity induced by (R)PAF. The observation that increased tyrosine phosphorylation of p125FAK and its ensuring association with focal adhesion occurs rapidly upon (R)PAF challenge indicates that this signaling molecule has a primary and independent role also in the signaling cascade initiated by (R)PAF.
Abstract: Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.
Abstract: This study describes the adhesion of human osteoblasts, cultured in vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77-100%, in 2 h and at 55 nM substrata concentration, and it was accompanied by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the beta 1 integrin and antibodies to the alpha 5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against alpha 2, alpha 3, alpha 5, alpha v, beta 1 and beta 3 integrins we detected synthesis of alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and an alpha v beta 1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus alpha 4, alpha 1 and beta 5 subunits. In cells adhering in the presence of serum we showed organization of beta 3 and alpha v integrins in focal contacts. In cells adhering to fibronectin alpha 5 and beta 1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.
Abstract: p125FAK is a major tyrosine kinase phosphorylated in response to integrin-dependent adhesion. In this study we use vanadate and phenylarsine oxide (PAO), known inhibitors of phosphotyrosine phosphatases (PTPases), as a tool to artificially modulate p125FAK phosphorylation in human endothelial and in Chinese hamster ovary cells. Vanadate treatment strongly upregulates in a dose-dependent manner the level of tyrosine phosphorylation of several proteins in adherent cells. PAO induces a more restricted profile of tyrosine-phosphorylated proteins, increasing primarily a broad band of 120-140 kDa. Maximal stimulation of p125FAK tyrosine phosphorylation is reached at 10 microM PAO. In contrast, in vanadate-treated cells the p125FAK tyrosine phosphorylation shows a biphasic curve, being increased at high doses of vanadate (100 microM) and downregulated at low doses (25 microM). Immunofluorescence analysis of cells treated with PTPase inhibitors showed a direct correlation between the level of p125FAK tyrosine phosphorylation and the assembly of focal adhesions and actin stress fibers. Downregulation of p125FAK tyrosine phosphorylation is observed by treating cells with cytochalasin D (CD), a drug known to rapidly disrupt the actin cytoskeleton. When PTPase inhibitors are added in combination to CD, the level of tyrosine phosphorylation of p125FAK remains high and focal adhesions and actin stress fibers are preserved from the CD-mediated disruption. Based on these data we suggest that assembly of actin cytoskeleton plays an important role in inhibiting PTPases involved in p125FAK tyrosine phosphorylation.
Abstract: Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either beta 1, alpha 3, alpha 5, alpha 6 or beta 3 integrin subunits. Increased phosphorylation of the 100-130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100-130 kDa complex was identified as the focal adhesion tyrosine kinase p125FAK. The phosphorylation of the p125FAK was also observed by inducing beta 1 integrin clustering in non adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.
Abstract: Adhesion of human neuroblastoma cells (SK-N-SH clone SY5Y) to laminin or collagen type IV promotes tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a protein of 180 kDa. The same pattern of tyrosine phosphorylation was observed when SY5Y cells were allowed to adhere to culture dishes coated with monoclonal antibodies directed to the integrin subunits expressed in the cells, alpha 1, alpha 3, and beta 1, indicating that these receptors are responsible for this signaling mechanism. Using specific antibodies we identified the focal adhesion kinase p125FAK as a component of the 100- to 130-kDa phosphoproteins. Treatment with genistein or herbimycin A, two specific tyrosine kinase inhibitors, greatly reduced the tyrosine phosphorylation of the 100- to 130- and the 180-kDa proteins in response to laminin or collagen IV. Concomitantly, neurite outgrowth on the matrix proteins was strongly inhibited. This effect was observed in two distinct neuroblastoma cell lines, SY5Y and SK-N-BE. Genistein and herbimycin A treatment did not affect cell viability nor cause retraction of preformed neurites. These data suggest that matrix-induced tyrosine phosphorylation events are involved in neurite extension.
Abstract: Monoclonal antibodies (mAbs) against cultured human ovary carcinoma cells were produced. We obtained 7 mAbs which reacted diffusely with carcinoma of the ovary but only weakly with vessels and the surface epithelial layer of normal ovary. Biochemical characterization of these mAbs indicated that 3 out of 7 were specific for the alpha 3 chain of the Vla-3 integrin, a receptor for fibronectin, collagen and laminin. Using one of these mAbs, we have studied, by immunohistochemical methods, the distribution of alpha 3 beta 1 integrin in mucinous, serous and endometrioid cystoadenocarcinoma of the ovary and in their normal equivalent: endocervical, tubal and endometrial epithelia. The results show that alpha 3 beta 1 is present in cell-cell contact areas and more abundantly at the junction between epithelial cells and basement membrane in endocervical, tubal epithelia, in epithelium lining the cavity of the uterus and in surface epithelium of the ovary. However, endometrial glands showed only weak and fragmented positivity at the basal pole of the cells. 26 out of 31 ovarian cancers studied, expressed the alpha 3 beta 1 integrin. However, basal localization, typical of normal epithelia, is not prominent or disappears in tumors and is replaced by a more diffuse reaction with variable immunohistochemical staining of the neoplastic cells. Furthermore, a comparative analysis of the expression of alpha 3 beta 1 and its ligands, laminin (LM), fibronectin (FN) and collagen IV (Coll IV), demonstrated that basal polarization of Vla-3 was always correlated with the presence of laminin and Coll IV, intrinsic components of the basement membranes.
Abstract: Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.
Abstract: In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.
Abstract: In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.
Abstract: Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.
Abstract: The interaction with matrix components of the basal membrane is an important factor in the control of vascular endothelial cell function in both normal and pathological conditions. In the present work we define integrin receptors in vascular endothelial cells and analyze whether their expression is affected by endothelial cell activators. Using in vitro adhesion tests we show that human endothelial cells (HEC) can attach and spread on substrates coated with several matrix components including fibronectin, laminin, collagen type IV, fibrinogen and vitronectin. Using specific antibodies we detect integrin receptor complexes of the beta 1 and beta 3 families that can support adhesion of HEC to the above matrix proteins. These are the alpha 2/beta 1 collagen receptor, the alpha 3/beta 1 receptor for fibronectin, collagens and laminin, the alpha 5/beta 1 fibronectin receptor, the alpha 6/beta 1 laminin receptor and the alpha V/beta 3 receptor for vitronectin and fibrinogen. When HEC are exposed to a combination of tumor necrosis factor alpha (TNF alpha) and immune interferon (IFN-gamma) the amount of the alpha V/beta 3 vitronectin receptor at the cell surface was decreased by a factor of 50-70%, while the beta 1 integrin complexes were not affected. HEC cells thus express receptors for several matrix components and inflammatory mediators such as TNF alpha and IFN-gamma can selectively alter the expression of some of them.
Abstract: Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) belong to a family of glycoprotidic growth factors required for the survival, growth and differentiation of haematopoietic precursors and which affect the function of circulating mature cells. They are produced by resting or stimulated stromal cells of the haematopoietic microenvironment (fibroblasts and endothelium) and by immunocompetent cells (T cells and monocytes/macrophages). The action of these CSF molecules was thought to be restricted to cells of haematopoietic origin. Here, we report that G-CSF and GM-CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.
Abstract: A 26-kDa protein, originally described in human fibroblasts superinduced for interferon beta (IFN-beta) production, and termed IFN-beta 2 by other investigators, is induced by cycloheximide and by a 22-kDa, interleukin 1 (IL-1)-related factor. Although the structure and sequence of the corresponding gene show nonhomology with the IFN-beta gene, the gene is identical to that of B-cell stimulatory factor 2, a human interleukin, and displays a very potent growth and differentiation factor activity for B lymphocytes. In this work we show that IL-1 beta and tumor necrosis factor (TNF) strongly induce the 26-kDa protein in FS-4 fibroblasts and in some transformed cell lines. Addition of cycloheximide to recombinant (r)IL-1 beta and rTNF further enhances the level of 26-kDa-protein mRNA. We determined the kinetics of induction and the amounts of rTNF and rIL-1 beta required for optimal induction of this mRNA in FS-4 cells and in HeLa H21 cells and found that rIL-1 beta is a more efficient inducer of 26-kDa protein mRNA than is TNF. By analyzing the inducibility of the 26-kDa protein gene by rTNF and rIL-1 beta in a series of transformed cell lines that differ in their sensitivity to the cytotoxic action of TNF, we report a direct correlation between the 26-kDa protein mRNA expression and the resistance of these cells to the cytotoxic effect of TNF.
Abstract: Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.
Abstract: 2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.
Abstract: Among the various proteins which are induced when human cells are treated with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological function and its possible contribution to the various antiviral and non-antiviral actions exerted by interferons, we cloned a full-length cDNA copy of the 1.8-1.9 X 10(3)-base 56-kDa-protein mRNA and determined its sequence. The cDNA contains 1662 nucleotides derived from the 56-kDa-protein mRNA, including a poly(A) tail of 20 residues. Primer extension experiments indicate that the 5' end of this cDNA clone is probably located only 13 nucleotides downstream of the actual cap site of the 56-kDa-protein mRNA. It consists of a 64-nucleotide-long 5'-non-coding segment, a coding segment of 1434 nucleotides terminated by a TAG triplet and a 141-nucleotide 3'-non-coding segment. The encoded protein of 478 amino acid residues has a molecular mass of 55335 Da and a single potential site for N-glycosylation. The protein contains an excess of basic amino acids and most of them are localized in the carboxy-terminal half of the molecule. A single [35S]methionine-labeled 56-kDa protein was obtained using an SP64 construction to allow the cell-free transcription and translation of the cloned cDNA. Microinjection of this labeled protein in Xenopus oocytes indicates that the 56-kDa protein is cytoplasmic.
