Signaling In Vascular Smooth Muscle Research Articles

Nicotine Impairs Smooth Muscle cAMP Signaling and Vascular Reactivity.

This study aimed to determine nicotine's impact on receptor-mediated cyclic adenosine monophosphate (cAMP) synthesis in vascular smooth muscle (VSM). We hypothesize that nicotine impairs β adrenergic-mediated cAMP signaling in VSM, leading to altered vascular reactivity. The effects of nicotine on cAMP signaling and vascular function were systematically tested in aortic VSM cells and acutely isolated aortas from mice expressing the cAMP sensor TEpacVV (Camper), specifically in VSM (e.g., CamperSM). Isoproterenol (ISO)-induced β-adrenergic production of cAMP in VSM was significantly reduced in cells from second-hand smoke (SHS)-exposed mice and cultured wild-type VSM treated with nicotine. The decrease in cAMP synthesis caused by nicotine was verified in freshly isolated arteries from a mouse that had cAMP sensor expression in VSM (e.g., CamperSM mouse). Functionally, the changes in cAMP signaling in response to nicotine hindered ISO-induced vasodilation, but this was reversed by immediate PDE3 inhibition. These results imply that nicotine alters VSM β adrenergic-mediated cAMP signaling and vasodilation, which may contribute to the dysregulation of vascular reactivity and the development of vascular complications for nicotine-containing product users.

Abstract 063: Role of Ribosomal S6 Kinase 2 in Vascular Myogenic Tone and Blood Pressure Regulation

Smooth muscle (SM) contractile force is generated through phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase. However, emerging evidence also suggests that additional Ser/Thr kinases may contribute to the pathways that regulate SM contractility. Recently, we published that the 90 kDa ribosomal S6 kinase (RSK), extensively studied in cancers of epithelial origin, is a major new player in the regulation of SM contractility. RSKs are unusual kinases having two catalytic domains separated by a linker having multiple phosphorylation sites and are activated by a cascade of ERK phosphorylations and by PDK, which phosphorylates RSK at Ser227 resulting in transition to a fully active enzyme. The thromboxane agonist, U46619 leads to activation of ERK1/2 and PDK1 in SM. We find that 4 th order mesenteric arteries are significantly more dilated at a pressure of 80 mm Hg in Rsk2 -/- mice compared to wild-type (WT) (2.0±0.8 vs 12.8±4.5 %, p <0.05, respectively). The contractile response to dose-dependent phenylephrine stimulation is significantly smaller in the absence of RSK2, p <0.0001 two-way ANOVA, while EC50 for phenylephrine contractions did not differ. Using isolated arcades of mesenteric vessels, phosphorylation of myosin RLC20 and myosin phosphatase regulatory subunit and RSK Ser227 (PDK site) and Thr 577 (ERK site) increased with in 30secs following an increase in intraluminal pressure to 80 mm Hg to induce myogenic tone development. Mean baseline systolic blood pressure (SBP, mmHg) was significantly lower in Rsk2 -/- animals (WT=107.4±1.6 and RSK2 -/- =95.8±2.2, n=23, p< 0.0002). In view of the lower SBP in the Rsk2 -/- animals, we examined the potential role for RSK signaling in the kidney, since RSK2 is known to phosphorylate NHE3. By immunohistochemistry, we show for the first time the expression of RSK2 in human kidney tubules. Lithium excretion, moles/kg/day, was increased in Rsk2-/- mice compared to WT (21.7±3.7 vs 48.6±7.9, respectively), indicative of defective renal Na + reabsorption. Taken together, RSK2 signaling in vascular SM and the kidney is a novel mechanism for the regulation of SM contractility and of BP. RSK2 may be a potential selective target in hypertension.

Abstract 420: G3BP1 Activation of NFATc Signaling is Inhibited by LRP6 in Aortic Vascular Smooth Muscle Cells

When fed a high fat diet, male LDL receptor -deficient (LDLR-/-) mice develop type 2 diabetes (T2D) with concomitant arteriosclerotic calcification. We recently identified that vascular smooth muscle (VSM) LRP6 expression inhibits pro-sclerotic noncanonical Wnt signaling, mediated in part by protein arginine (Arg) methylation relays that converge on NFAT- dependent transcriptional programs. To better understand the mechanisms whereby Arg methylation mediates LRP6 actions,immunoaffinity mass spectrometry analysis was performed on aortic VSM extracts from SM22-LRP6(fl/fl);LDLR-/- and LRP6(fl/fl);LDLR-/- control mice. Applying a 2.5- fold cutoff threshold, alterations were noted in 499 monomethylated (MMA), 109 symmetric dimethylated (SDMA), and 65 asymmetric dimethylated (ADMA) protein cognates. One protein, G3BP1 (GTPase activating-protein binding-protein 1), demonstrated increases in MMA on residues 433, 435 & 458 -- with concomitant reductions SDMA on residue 458 —with LRP6 deficiency. G3BP1 exhibited both nuclear and non-nuclear localization, consistent with NTF2 and RRM structural motifs. Transient transfection and MMA western blot analysis confirmed that protein Arg methyltransferase 1 recognized G3BP1 as a substrate. This modification was inhibited by co-expression of LRP6. G3BP1activated osteopontin (OPN)- and NFATc3- dependent transcriptional responses, with NFAT transcription enhanced by co-expression of either Fzd10 or constitutively active cdc42. LRP6 inhibited G3BP1+NFAT activation of artificial and native (OPN, TNF) NFATc3-targeted promoter - reporter constructs. Moreover, NFATc3 protein levels were increased in nuclear extracts of LRP6-deficient VSM and reduced by G3BP1-directed siRNA, with concomitant reduction in genomic targets. Structure-function analysis mapped transcriptional activation of NFAT signaling by G3BP1 to the 32 amino-acid C-terminal domain encompassing Arg methylation cognates. Thus, G3BP1 is a cellular target of Arg methylation that is negatively regulated by LRP6 and mediates inflammatory osteogenic signaling in VSM. Strategies targeting G3BP1 and/or its Arg-methylation may help limit arteriosclerotic calcification and vascular disease in the setting of type 2 diabetes.

CaM kinase IIδ2-dependent regulation of vascular smooth muscle cell polarization and migration

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

PKC-δ-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.