PKC-delta-dependent pathways contribute to PDGF-stimulated ERK1/2 activation in vascular smooth muscle

Platelet-derived growth factor (PDGF) is an important regulator of vascular smooth muscle (VSM) cell growth and migration and has been identified as a key mediator of neointima formation resulting from vascular injury. PDGF exerts its effects, in part, through activation of ERK1/2. Previously, we reported that PKC-delta, specifically compared with PKC-alpha, mediated phorbol ester- and ATP-dependent activation of ERK1/2 in VSM cells. The purpose of this study was to determine whether PKC-delta was involved in PDGF-dependent activation of ERK1/2 in VSM cells. The addition of PDGF resulted in the activation, and Src family kinase-dependent tyrosine phosphorylation, of PKC-delta. Treatment with rottlerin (0.1-10 microM), a selective PKC-delta inhibitor, or adenoviral overexpression of kinase-negative PKC-delta significantly attenuated PDGF-induced activation of ERK1/2. The effects of the PKC-delta inhibitors decreased with increasing concentrations of activator PDGF. Interestingly, treatment with Go6976 (0.1-3 microM), a selective inhibitor of cPKCs, or adenoviral overexpression of kinase-negative PKC-alpha also inhibited PDGF-stimulated ERK1/2. Furthermore, inhibition of cPKC activity with Go6976 or overexpression of kinase-negative PKC-alpha attenuated PKC-delta activation and tyrosine phosphorylation in response to PDGF. These studies indicate involvement of both PKC-delta and PKC-alpha isozymes in PDGF-stimulated signaling in VSM and suggest an unexpected role for PKC-alpha in the regulation of PKC-delta activity.     

CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle

In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca²⁺. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca²⁺/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa²⁺-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibitsCa²⁺-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca²⁺/calmodulin-dependent activation of PYK2.

     

PKC-delta and CaMKII-delta 2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle

ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-₂, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-Ras^N17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-₂ coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- ₂; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus     

ERK activation and mitogenesis in human airway smooth muscle cells

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.     

Angiotensin II induces tyrosine nitration and activation of ERK1/2 in vascular smooth muscle cells

Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.     

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

Rho activation in excitatory agonist-stimulated vascular smooth muscle

Small GTPase Rho and its downstream effector, Rho kinase, havebeen implicated in agonist-stimulated Ca²⁺ sensitization of20-kDa myosin light chain (MLC₂₀) phosphorylation andcontraction in smooth muscle. In the present study we demonstrated forthe first time that excitatory receptor agonists induce increases inamounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aorticsmooth muscle. Using a pull-down assay with a recombinant RhoA-bindingprotein, Rhotekin, we found that a thromboxane A₂ mimetic,U-46619, which induced a sustained contractile response, induced asustained rise in the amount of GTP-RhoA in a dose-dependent mannerwith an EC₅₀ value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxaneA₂ receptor-mediated and reversible. Other agonistsincluding norepinephrine, serotonin, histamine, and endothelin-1 (ET-1)also stimulated RhoA, albeit to lesser extents than U-46619. Incontrast, ANG II and phorbol 12,13-dibutyrate failed to increaseGTP-RhoA. The tyrosine kinase inhibitor genistein substantiallyinhibited RhoA activation by these agonists, except for ET-1. Thusexcitatory agonists induce Rho activation in an agonist-specificmanner, which is thought to contribute to stimulation ofMLC₂₀ phosphorylation Ca²⁺ sensitivity.

     

Myosin-linked calcium regulation in vascular smooth muscle.

Rat liver microsomal membranes have been shown to contain a biosynthetic pathway of UDP-glucose. In addition, they are able to bind UDP-glucose in a reversible manner, As UDP-glucose is also metabolized in these membranes, the study of the binding has been performed with a microsomal Triton X 100 extract. This reversible binding depends on pH (maximum at pH 8.1) and manganous ions, and disappears at pH 6.5. It exhibits a high affinity (K-diss equals 3 mu M), and a narrow specifity for UDP-glucose. Proteolytic digestion inhibits the binding up to 90%, showing that the UDP-glucose receptor has a proteic nature. These binding characteristics have been also found in the membranes themselves, indicating that the detergent solubilization does not destroy the protein binding capacity.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60min) with insulin or A-II increased phosphorylation of ERK1/2 at 15min and ERK5 at 5min. Chronic treatment (< or = 8h) with insulin increased ERK1/2 phosphorylation by 4h and ERK5 by 8h. A-II-stimulated phosphorylation of ERK1/2 by 8h and ERK5 by 4h. The EC(50) for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1nM, whereas the EC(50) for A-II was 2nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.     

Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells

Mechanical stress contributes to vascular disease related to hypertension. Activation of ERK is key to mediating cellular proliferation and vascular remodeling in response to stretch stress. However, the mechanism by which stretch mediates ERK activation in the vascular tissue is still unclear. Caveolin, a major component of a flasklike invaginated caveolae, acts as an adaptor protein for an integrin-mediated signaling pathway. We found that cyclic stretch transiently induced translocation of caveolin from caveolae to noncaveolar membrane sites in vascular smooth muscle cells (VSMCs). This translocation of caveolin was determined by detergent solubility, sucrose gradient fractionation, and immunocytochemistry. Cyclic stretch induced ERK activation; the activity peaked at 5 min (the early phase), decreased gradually, but persisted up to 120 min (the late phase). Disruption of caveolae by methyl-beta-cyclodextrin, decreasing the caveolar caveolin and accumulating the noncaveolar caveolin, enhanced ERK activation in both the early and late phases. When endogenous caveolins were downregulated, however, the late-phase ERK activation was subsided completely. Caveolin, which was translocated to noncaveolar sites in response to stretch, is associated with beta1-integrins as well as with Fyn and Shc, components required for ERK activation. Taken together, caveolin in caveolae may keep ERK inactive, but when caveolin is translocated to noncaveolar sites in response to stretch stress, caveolin mediates stretch-induced ERK activation through an association with beta1-integrins/Fyn/Shc. We suggest that stretch-induced translocation of caveolin to noncaveolar sites plays an important role in mediating stretch-induced ERK activation in VSMCs.     

Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation

We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.     

Metabolic activation of AMP kinase in vascular smooth muscle.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.