p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle

Angiotensin II (ANG II) is a multifunctional hormone that exerts potent vasoconstrictor and hypertrophic effects on vascular smooth muscle. Here, we demonstrate that the p38 mitogen-activated protein (MAP) kinase pathway is involved in ANG II-induced vascular contraction. Addition of ANG II to rat aortic smooth muscle cells (SMC) caused a rapid and transient increase of p38 activity through activation of the AT(1) receptor subtype. This response to ANG II was strongly attenuated by pretreating cells with antioxidants and diphenylene iodonium and was mimicked by exposure of cells to H(2)O(2). Stimulation of p38 by ANG II resulted in the enzymatic activation of MAP kinase-activated protein (MAPKAP) kinase-2 and the phosphorylation of heat shock protein 27 (HSP27) in aortic SMC. Pretreatment of cells with the specific p38 MAP kinase inhibitor SB-203580 completely blocked the ANG II-dependent activation of MAPKAP kinase-2 and phosphorylation of HSP27. ANG II also caused a robust activation of MAPKAP kinase-2 in the intact rat aorta. Incubation with SB-203580 significantly decreased the potency of ANG II to induce contraction of rat aortic rings and depressed the maximal hormone response. These results suggest that the p38 MAP kinase pathway selectively modulates the vasoconstrictor action of ANG II in vascular smooth muscle.     

p38 MAP kinase negatively regulates angiotensin II-mediated effects on cell cycle molecules in human coronary smooth muscle cells

Many of the signaling events in VSMC stimulated by angiotensin II (AngII) are mediated by members of the mitogen-activated protein kinase (MAPK) family, including p38 MAPK. The role of p38 MAPK in AngII-mediated cell cycle regulation is poorly understood. Therefore, we examined the involvement of p38 MAPK signaling in AngII-stimulated DNA synthesis, phosphorylation of the retinoblastoma protein (Rb), and expression of the G1-phase cyclin D1 in human coronary artery smooth muscle cells (CASMC). AngII (1 microM) stimulated p38 MAPK and ERK1/2 activation. Pretreatment with the p38 MAPK inhibitors SB203580 (10 microM) (SB) or SKF-86002 (10 microM) (SKF) potently inhibited AngII-induced p38 MAPK activation, but enhanced AngII-mediated ERK1/2 activation. AngII-induced-phosphorylation of Rb (Ser 795 and Ser 807/811), -cyclin D1 expression, and -DNA synthesis was also markedly enhanced by pharmacological inhibition of the p38 MAPK pathway. The present study demonstrates that p38 MAPK negatively regulates AngII-induced ERK1/2 activity, Rb phosphorylation, cyclin D1 expression, and DNA-synthesis in human CASMC. These findings support an important role for p38 MAPK in modulating AngII-mediated VSMC hyperplasia.     

Hypoxia activates beta(1)-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells

The progress in the use of HAART for the treatment of HIV-infected individuals has been limited by the development of viral resistance and the maintenance of viral latency. New therapeutic strategies geared toward improvement in the host's immune response are now being considered. We found that IFN-gamma induces CIITA through the JAK-STAT pathway and inhibits HIV-1 replication in latently infected cells. Its effect appears to be mediated through the reciprocal action of Tat and CIITA. With this beneficial effect, IFN-gamma and its inducers can be considered as an adjunct to the currently available therapy. We also addressed the safety of using simvastatin, an HMG-CoA reductase inhibitor, to treat dyslipidemia often associated with the use of protease inhibitors. Simvastatin did not show any unfavorable effects on HIV replication, thus could be used safely unless there are any drug interactions when administered.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells

Although it is known that transforming growth factor (TGF)-beta induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF-beta stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The VEGF synthesis induced by TGF-beta was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF-beta in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF-beta-stimulated synthesis of VEGF (each in a dose-dependent manner). PD169316 or SB203580 attenuated the TGF-beta-induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF-beta stimulates the synthesis of VEGF in aortic smooth muscle cells.     

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.     

IL-1beta stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-kappa B

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1beta and that mitogen-activated protein (MAP) kinase signaling and NF-kappaB activation are involved in IL-1beta-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1-2 ng/ml) of IL-1beta in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-kappaB binding site. Treatment of myotubes with IL-1beta resulted in a dose- and time-dependent increase of IL-6 production accompanied by an approximately 25-fold increase in IL-6 mRNA levels. IL-1beta stimulated NF-kappaB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1beta. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1beta regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-kappaB.     

IL-1beta, BK, and TGF-beta1 attenuate PGI2-mediated cAMP formation in human pulmonary artery smooth muscle cells by multiple mechanisms involving p38 MAP kinase and PKA

We have previously shown that interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1beta, BK, and TGF-beta1 reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Galphai protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Galphas protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1beta, BK, and TGF-beta1. These studies suggest that IL-1beta, BK, and TGF-beta1 impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.     

Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.     

白介素-1 β通过JNK/p38信号-转导通路调控肾系膜细胞表达α-平滑肌肌动蛋白

为探讨细胞内丝裂素原活化蛋白激酶（MAPK）家族各亚类信号转导通路在炎症性细胞因子白介素－1β（IL－1β）对大鼠肾系膜细胞（rMC)表型标志物α-平滑肌肌动蛋白（α－SMA）表达及其分布中的调控作用,以IL－1β（10ng/ml)刺激体外培养的rMC,用电穿孔基因转染及免疫杂交法观察IL－1β对α－SMA基因启动子活性及蛋白表达的作用,并用共聚焦荧光显微镜及透射电镜观察IL－1β刺激前后细胞内α-SMA及微丝的分布变化。通过应用PD98059和SB203580特异阻断ERK和p38通路、共转染显性失活JNKK基因特异阻断JNK通路,观察阻断对IL－1β刺激所致α-SMA表达或启动子活性的影响。结果显示,IL－1β刺激6h可明显上调α－SMA启动子活性,在1－2d内显著促进其蛋白合成;IL－1β刺激24h后,细胞内α-SMA及微丝在细胞核周的分布增加。阻断ERK通路对IL－1β诱导的α-SMA表达无明显影响;阻断JNK及p38通路均可使IL－1β诱导的α－SMA表达明显受抑;阻断p38通路的作用比阻断JNK通路更强,而且对基础状态的α-SMA表达也有抑制作用。上述结果提示,IL－1β可刺激rMC发生表型转化,其表型标志物α－SMA可通过基因转录增强而增加蛋白表达,在细胞内的分布向核周转位积聚。JNK及p38通路是介导IL－1β刺激rMC α-SMA表达的主要信号转导途径,而ERK通路不影响IL－1β的这一作用。     

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.     

Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading

We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal (F(o)). Each strip was then contracted isotonically (at 0.32 F(o)) with ACh (maintained at 10(-4) M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 microM SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Stiffness changes in cultured airway smooth muscle cells

Airwaysmooth muscle (ASM) cells in culture stiffen when exposed tocontractile agonists. Such cell stiffening may reflect activation ofthe contractile apparatus as well as polymerization of cytoskeletalbiopolymers. Here we have assessed the relative contribution of thesemechanisms in cultured ASM cells stimulated with serotonin(5-hydroxytryptamine; 5-HT) in the presence or absence of drugs thatinhibit either myosin-based contraction or polymerization offilamentous (F) actin. Magnetic twisting cytometry was used to measurecell stiffness, and associated changes in structural organization ofactin cytoskeleton were evaluated by confocal microscopy. We found that5-HT increased cell stiffness in a dose-dependent fashion and alsoelicited rapid formation of F-actin as marked by increased intensity ofFITC-phalloidin staining in these cells. A calmodulin antagonist (W-7),a myosin light chain kinase inhibitor (ML-7) and a myosin ATPaseinhibitor (BDM) each ablated the stiffening response but not theF-actin polymerization induced by 5-HT. Agents that inhibited theformation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, andY-27632) attenuated both baseline stiffness and the extent of cellstiffening in response to 5-HT. Together, these data suggest thatagonist-evoked stiffening of cultured ASM cells requires actinpolymerization as well as myosin activation and that neitheractin polymerization nor myosin activation by itself is sufficient toaccount for the cell stiffening response.