Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.     

The RGS proteins add to the diversity of soybean heterotrimeric G-protein signaling

Regulator of G-protein signaling (RGS) proteins are a family of highly diverse, multifunctional proteins that function primarily as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of GTP hydrolysis by Gα proteins and essentially regulate the duration of active signaling. Recently, we have identified two chimeric RGS proteins from soybean and reported their distinct GAP activities on individual Gα proteins. A single amino acid substitution (Alanine 357 to Valine) of RGS2 is responsible for differential GAP activity. Surprisingly, most monocot plant genomes do not encode for a RGS protein homolog. Here we discuss the soybean RGS proteins in the context of their evolution in plants, their relatedness to non-plant RGS protein homologs and the effect they might have on the heterotrimeric G-protein signaling mechanisms. We also provide experimental evidence to show that the interaction interface between plant RGS and Gα proteins is different from what is predicted based on mammalian models.     

Characterization of vascular smooth muscle cell phenotype in long-term culture

Summary Studies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavoir, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concommitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression. This work was supported by grants from from National Institutes of Health, Bethesda, MD, to DMW (HL35684), JW (HL36412), and JM and RL (SCOR HL 14212).     

Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis

In a previous study, we have demonstrated that overexpression of the tissue inhibitors of metalloproteinases-4 (TIMP-4) can inhibit the neointima formation in the rat carotid model. To define the functions of tissue inhibitor of metalloproteinases-4 (TIMP-4) in SMCs, we transduced human TIMP-4 cDNA into rat aortic SMCs by using adenoviral vector. Overexpression of TIMP-4 blocked the conversion of pro-MMP-2 to the active form and inhibited basic fibroblast growth factor-induced migration by 56.7% (p < 0.01). Overexpression of TIMP-4 markedly increased apoptotic cell death without changing their proliferation. Importantly, overexpression of human TIMP-4 in the wall of balloon-injured rat carotid artery also increased SMC apoptosis. The percentages of TUNEL-positive cells of total cells increased significantly in AdTIMP-4 infected group compared with AdGFP infected group. These findings demonstrate that TIMP-4 can inhibit SMCs migration and induce apoptosis in vitro and in vivo, which may generate new targets for prevention and treatment of vascular diseases.     

Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been partially identified. In this study, we demonstrate that Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), is expressed on VSMCs isolated from the intima of atherosclerotic plaques of carotid arteries. Stimulation with HGF led to activation of Met as well as to activation of PI3-K, PKB/Akt, MEK, and the MAP kinases Erk1 and -2. Moreover, HGF induced lamellipodia formation, a characteristic feature of motile cells, and promoted VSMC migration across fibronectin-coated filters. The HGF-induced cell migration was mediated by beta1 integrins and required PI3-K activation. Our results suggest a role for the HGF-Met signaling pathway in the pathogenesis of atherosclerosis and restenosis.     

DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α_1A-adrenergic receptor-stimulated intracellular Ca²⁺ mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability.

Structured summary

MINT-8049215: Rgs4 (uniprotkb:P49799) physically interacts (MI:0915) with DHHC3 (uniprotkb:Q8R173) by anti-tag coimmunoprecipitation (MI:0007)     

Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Background

The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.

Results

Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [³H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.

Conclusions

Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.     

Cell-cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of β-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that β-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.     

影响主动脉平滑肌细胞迁移的因素

平滑肌细胞(vascular smooth muscle cell,VSMC)的迁移对血管发育、动脉粥样硬化和术后再狭窄等起到关键性的作用。主要从激发VSMC迁移的关键炎性细胞因子、细胞间相互作用的核心成员、microRNA、细胞骨架和上述各因素的迁移信号通路这几方面来综述VSMC的迁移。     

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.     

Epidermal growth factor up-regulates the expression of nestin through the Ras-Raf-ERK signaling axis in rat vascular smooth muscle cells

The contractile-synthetic phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event during atherosclerosis progression. Although many studies have reported possible cytokines and growth factors implicated to this process, the critical factors affecting the VSMC phenotype remain unclear due to the lack of early de-differentiation marker identifications. In this study, we showed that nestin, an intermediate filament protein, is expressed in primary cultures of rat VSMCs representing the synthetic phenotype and its expression is diminished as these cells re-differentiate after serum deprivation. However, the regulation of nestin expression was never reported despite its common usage as an early differentiation marker. Herein, we showed that nestin expression is regulated by epidermal growth factor (EGF) via de novo RNA and protein synthesis. Furthermore, signaling analyses revealed that the EGF-induced nestin re-expression is mediated through the activation of the Ras-Raf-ERK signaling axis. This is the first report to show that nestin expression is regulated by an extracellular signaling molecule.     

EBP50 is involved in the regulation of vascular smooth muscle cell migration and cytokinesis

Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several elements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Neuromedin B modulates phosphate-induced vascular calcification

Vascular calcification is the heterotopic accumulation of calcium phosphate salts in the vascular tissue and is highly correlated with increased cardiovascular morbidity and mortality. In this study, we found that the expression of neuromedin B (NMB) and NMB receptor is upregulated in phosphate-induced calcification of vascular smooth muscle cells (VSMCs). Silencing of NMB or treatment with NMB receptor antagonist, PD168368, inhibited the phosphate-induced osteogenic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling and VSMC apoptosis. PD168368 also attenuated the arterial calcification in cultured aortic rings and in a rat model of chronic kidney disease. The results of this study suggest that NMB–NMB receptor axis may have potential therapeutic value in the diagnosis and treatment of vascular calcification.     

Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells

Accumulation of transglutaminase 2 (TG2) is often associated with mineral deposits in vasculature. Here, we demonstrate that purified TG2 stimulated a 3-fold increase in matrix mineralization and up-regulation of osteoblastic markers in cultured primary vascular smooth muscle cells (VSMCs). Extracellular TG2 interacts with the low density lipoprotein related-protein 5 receptor and activates beta-catenin signaling in VSMCs. These results suggest that TG2 may promote vascular calcification by activating the beta-catenin signaling pathway.