A proteomic approach to the investigation of early events involved in vascular smooth muscle cell activation

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive feature is the basis of the VSMC migration and proliferation involved in cardiovascular diseases. We have used a proteomic approach to the molecular changes that promote the switch of VSMC from having a quiescent to activated-proliferating phenotype. In particular, we have focused on modulations occurring during tyrosine-phosphorylation following cell activation by serum or single growth factors, such as insulin-like growth factor 1 or platelet-derived growth factor. A comparison of two-dimensional polyacrylamide gel profiles from quiescent or activated-proliferating VSMC has allowed us to recognize a number of differences in protein expression. Several differentially expressed proteins have been identified by mass spectrometry, and their time-course changes during tyrosine-phosphorylation have been documented from time zero till 48 h after stimulation. We have documented a general decrease of the tyrosine-phosphorylation level within the first few minutes after stimulation followed by a recovery that is quick and dramatic for some chaperones and redox enzymes but not so significant for enzymes of glucose metabolism. With regard to cytoskeleton components, no remarkable fluctuations have been detected at the earliest time points, except for those relative to α-actin, which displays an impressive decrease. A comparison of the early stages of cell stimulation after the administration of serum or single growth factors has brought to light important differences in the phosphorylation of chaperones, thereby suggesting their crucial role in VSMC activation. This work was partially supported by two FIRB 2001 project grants to Dr. G. Rainaldi and to Prof. G. Camici.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

The regulation of caveolin expression and localization by serum and heparin in vascular smooth muscle cells

Caveolae have been implicated in growth factor receptor and G-protein coupled receptor signaling in vascular cells. It has been postulated that caveolin, the structural protein of caveolae, may act as a general tyrosine kinase inhibitor by binding and inhibiting signaling molecules involved in the activation of the MAP kinase proliferation cascade. Using an in vitro model of VSMC proliferation, we found that serum stimulation caused a dose dependent decrease in both caveolin-1 and caveolin-2 protein levels in human coronary artery smooth muscle cells. Heparin, an inhibitor of VSMC proliferation, inhibited the serum-induced loss of caveolin-1 and caveolin-2. In addition, heparin caused an increase in both caveolin-1 and caveolin-2 localization to caveolae-enriched sucrose gradient membrane fractions when compared to serum alone. Taken together, caveolin may play an important role in the regulation of VSMC proliferation and heparin and serum have opposing effects on caveolin expression and localization in VSMC.     

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.     

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.     

Analysis of the growth factor requirements for stimulation of WI-38 cells after extended periods of density-dependent growth arrest

When cultures of WI-38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI-38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long-term quiescent WI-38 cells (stimulated 26 days after plating) and short-term quiescent WI-38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short- and long-term quiescent WI-38 cells, in that the same defined medium (supplemented with epidermal growth factor [EGF], recombinant human insulin-like growth factor 1 [IGF-1], and dexamethasone [DEX]) stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long-term quiescent WI-38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long-term density-arrested WI-38 cells.     

The growth-regulated gene 1B6 is identified as the heavy chain of calpactin I

The expression of 1B6, a growth-regulated sequence isolated from a Syrian hamster fibroblast cDNA library, was studied in BALB/c 3T3 cells. The level of cytoplasmic 1B6 mRNA (1600 bases) was low in quiescent cells and plateaued in mid/late G1 after the cells were stimulated with 15% fetal calf serum (FCS). Protein synthesis was not required for the induction of 1B6 mRNA; therefore, the expression of 1B6 is a primary response to serum stimulation. The induction of 1B6 mRNA was also observed after stimulation with insulin, epidermal growth factor, and fibroblast growth factor but not with platelet-derived growth factor. When quiescent cells were serum-stimulated, the percentage of cells that became committed to enter DNA synthesis was proportional to the length of their incubation with serum. To determine if 1B6 expression was also correlated with the time of exposure to serum, quiescent cells were stimulated with a pulse of 15% FCS and the abundance level of 1B6 induced by that pulse was determined. The amount of 1B6 mRNA increased with increasing time of exposure to serum and paralleled the increase in the percentage of nuclei that were induced into DNA synthesis by the serum pulse. Comparison of the nucleotide sequence of the p1B6 cDNA to the GenBank database revealed a striking identity of 1B6 to the 3' end of p36, the heavy chain of calpactin I. The previous characterization of p36 as a substrate for tyrosine kinases suggests a possible role for 1B6/p36 in cell proliferation.     

STAT-3-dependent cytosolic phospholipase A2 expression is required for thrombin-induced vascular smooth muscle cell motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).     

The role of signal-transducing events in the proliferative response of cells to a mitogenic viral K-ras protein

Activated oncogenic ras proteins are powerful mitogenic agents which by themselves can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how ras proteins induce proliferation we examined the early events in the G0 to G1 transition caused by the activation of a thermolabile K-ras protein in quiescent, serum-starved tsKSV-transformed NRK cells. We show that ras reactivation, in the absence of exogenous growth factors, triggered a rapid surge in free cytosolic Ca2+ and diacylglycerol production, which led to a transient increase in membrane-associated protein kinase C (PKC) activity which was necessary for G1 transit. Unlike TPA-stimulated PKC activity, the ras-induced increase in PKC was readily extracted from membranes by EGTA. These signal transducing events occurred despite the fact that ras activation did not induce the tyrosine phosphorylation of any known surface receptor. The results indicate that the K-ras protein triggers the G0 to G1 transition by an intracellular mechanism and not indirectly via autocrine stimulation.     

G‐CSF‐dependent neutrophil differentiation requires downregulation of MAPK activities through the Gab2 signaling pathway

Granulocyte colony‐stimulating factor (G‐CSF) stimulation of myeloid cells induced tyrosine‐phosphorylation of cellular proteins. One of the tyrosine‐phosphorylated proteins was found to be a scaffold protein, Grb2‐associated binding protein 2 (Gab2). Another member of Gab family protein, Gab3, was exogenously overexpressed in neutrophil progenitor cells to make the Gab3 protein to compete with the endogenous Gab2 for the G‐CSF‐dependent signaling. In Gab3‐overexpressed cells, the level of tyrosine phosphorylation of endogenous Gab2 by G‐CSF stimulation was markedly downregulated, while the phosphorylation of Gab3 was significantly enhanced. The Gab3‐overexpressed cells continuously proliferated in the medium containing G‐CSF and lost the ability to differentiate to the mature neutrophil, characterized by the lobulated nucleus. The G‐CSF stimulation‐dependent tyrosine phosphorylation of Gab3, the association of SHP2 to Gab3 and the following mitogen‐activated protein kinase (MAPK) activation were prolonged in the Gab3‐overexpressed cells, compared to the parental cells, where the binding of SHP2 to Gab2 protein and thereby the activation of MAPK were not sustained after G‐CSF stimulation. Inhibition of MAPK by pharmaceutical inhibitor restored the Gab3‐overexpressed cells to the ability to differentiate to mature neutrophil. Therefore, G‐CSF‐dependent Gab2 phosphorylation and following its downregulation led the short‐term MAPK activation. The downregulation of MAPK after transient Gab2 phosphorylation was necessary for the consequent neutrophil differentiation induced by G‐CSF stimulation.     

Disruption of the Rb--Raf-1 interaction inhibits tumor growth and angiogenesis

The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G(1) phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.     

RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation

Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.     

Insulin-Like Growth Factor (IGF) Binding Protein 2 Functions Coordinately with Receptor Protein Tyrosine Phosphatase β and the IGF-I Receptor To Regulate IGF-I-Stimulated Signaling

Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development and progression of atherosclerosis. IGF binding proteins (IGFBPs) modify IGF-I actions independently of IGF binding, but a receptor-based mechanism by which they function has not been elucidated. We investigated the role of IGFBP-2 and receptor protein tyrosine phosphatase β (RPTPβ) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPβ, which led to its dimerization and inactivation. This enhanced PTEN tyrosine phosphorylation and inhibited PTEN activity. Utilization of substrate trapping and phosphatase-dead mutants showed that RPTPβ bound specifically to PTEN and dephosphorylated it. IGFBP-2 knockdown led to decreased PTEN tyrosine phosphorylation and decreased AKT Ser473 activation. IGFBP-2 enhanced IGF-I-stimulated VSMC migration and proliferation. Analysis of aortas obtained from IGFBP-2^−/− mice showed that RPTPβ was activated, and this was associated with inhibition of IGF-I stimulated AKT Ser473 phosphorylation and VSMC proliferation. These changes were rescued following administration of IGFBP-2. These findings present a novel mechanism for coordinate regulation of IGFBP-2 and IGF-I signaling functions that lead to stimulation of VSMC proliferation. The results have important implications for understanding how IGFBPs modulate the cellular response to IGF-I.     

Rapid phosphorylation of microtubule-associated proteins through distinct mitogenic pathways

Mitogenic stimulation of sparse quiescent Swiss 3T3 cells with serum induces a transient reorganization of microtubules which may be necessary for generation or transduction of the mitogenic signal(s). Recently, several studies have shown that microtubule-associated proteins (MAPs) modulate microtubule-mediated functions in vitro and in vivo. We have analyzed, by two-dimensional electrophoresis, the molecular changes in MAPs associated with microtubules in situ following cell activation. By as early as 15 min after addition of serum, several of the MAPs present in quiescent cells are lost from the assembled microtubule fraction while one additional MAP becomes evident. This new MAP is a phosphoprotein whose appearance is independent of protein synthesis. Four additional MAPs also become phosphorylated, and this phosphorylation is accompanied by a partial redistribution of MAPs into the unassembled soluble fraction. Stimulation of cells with purified platelet-derived growth factor or phorbol tumor promoter, a direct activator of protein kinase C, also induces phosphorylation of the same MAPs and DNA synthesis. These results demonstrate that activation of the protein kinase C pathway is sufficient to promote the phosphorylation of MAPs and mitogenesis. However, epidermal growth factor, which does not activate protein kinase C, also stimulates phosphorylation of MAPs and DNA replication. Furthermore, down-regulation of the protein kinase C pathway does not prevent these responses. We conclude that phosphorylation of MAPs and mitogenesis can proceed through protein kinase C-dependent and -independent pathways in 3T3 cells.     

p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.

Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidly and transiently activated in quiescent cells after addition of various agonists, including insulin, epidermal growth factor, platelet-derived growth factor, and phorbol esters. We show here that addition of the growth factors thrombin or basic fibroblast growth factor to CCL39 fibroblasts rapidly induces tyrosine phosphorylation of the p42 MAP kinase protein and concomitantly stimulates MAP kinase enzymatic activity. To elucidate the signaling pathways utilized in this activation, we took advantage of the sensitivity of CCL39 cells to the toxin of bordetella pertussis, which ADP-ribosylates two Gi proteins in this cell system. We show that pretreatment of cells with the toxin inhibited thrombin stimulation of MAP kinase by greater than 75% but had no detectable effect on the stimulation induced by basic fibroblast growth factor. We also demonstrate that these two growth factors that synergize for mitogenicity are able to cooperate in activation of MAP kinase and that this synergism is partially sensitive to pertussis toxin. Finally, we describe a 44-kDa protein, the tyrosine phosphorylation of which appears to be coregulated with p42 MAP kinase. We conclude that p42 MAP kinase (and the pp44 protein) are at or are downstream from a point of convergence of two different receptor-induced signaling pathways and might well play a key role in integrating those signals.     

几种扩血管多肽对bFGF促血管平滑肌细胞增殖作用的影响

目的和方法：研究肾上腺髓质素（Ａｄｍ）、降钙素基因相关肽（ＣＧＲＰ）及Ｃ－型心房利太（ＣＮＰ）对碱性成纤维细胞生长因子（ｂＦＧＦ）促血管平滑细胞（ＶＳＭＣ）增殖作用的影响及其机制。结果：孵育２４ｈ后,ｂＦＧＦ刺激ＶＳＭＣ增殖较对照组增加２．１倍（Ｐ〈０．０１）,细胞内蛋白磷酸化程度增加１．４倍（Ｐ〈０．０１）,ＰＫＣ及ＭＡＰＫ活性分别增加１．５和２．５倍（Ｐ〈０．０１０;Ａｄｍ．ＣＧＲＰｔ ＣＮＰ     

Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin

The sodium/proton exchanger type 1 (NHE-1) plays an important role in the proliferation of vascular smooth muscle cells (VSMC). We have examined the regulation of NHE-1 by two potent mitogens, serotonin (5-HT, 5-hydroxytryptamine) and angiotensin II (Ang II), in cultured VSMC derived from rat aorta. 5-HT and Ang II rapidly activated NHE-1 via their G protein-coupled receptors (5-HT(2A) and AT(1)) as assessed by proton microphysiometry of quiescent cells and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Activation of NHE-1 was blocked by inhibitors of phospholipase C, CaM, and Jak2 but not by pertussis toxin or inhibitors of protein kinase C. Immunoprecipitation/immunoblot studies showed that 5-HT and Ang II induce phosphorylation of Jak2 and induce the formation of signal transduction complexes that included Jak2, CaM, and NHE-1. The cell-permeable Ca(2+) chelator BAPTA-AM blocked activation of Jak2, complex formation between Jak2 and CaM, and tyrosine phosphorylation of CaM, demonstrating that elevated intracellular Ca(2+) is essential for those events. Thus, mitogen-induced activation of NHE-1 in VSMC is dependent upon elevated intracellular Ca(2+) and is mediated by the Jak2-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE-1, similar to the pathway previously described for the bradykinin B(2) receptor in inner medullary collecting duct cells of the kidney [Mukhin, Y. V., et al. (2001) J. Biol. Chem. 276, 17339-17346]. We propose that this pathway represents a fundamental mechanism for the rapid regulation of NHE-1 by G(q/11) protein-coupled receptors in multiple cell types.