Sebastien Taurin

Phosphorylation of β-Catenin by Cyclic AMP-dependent Protein Kinase

β-Catenin is a signaling molecule that promotes cell proliferation by the induction of gene transcription through the activation of T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors. The canonical mechanism of the regulation of β-catenin involves its phosphorylation by casein kinase 1 at the Ser-45 site and by glycogen synthase kinase 3 (GSK3) at the Thr-41, Ser-37, and Ser-33 sites. This phosphorylation targets β-catenin to ubiquitination and degradation by the proteasome system. Mitogenic factors promote β-catenin signaling through the inhibition of GSK3, resulting in reduced β-catenin phosphorylation, its stabilization, and subsequent accumulation in the nucleus, where it stimulates TCF/LEF-dependent gene transcription. In the present study, we have shown that (i) β-catenin can be phosphorylated by protein kinase A (PKA) in vitro and in intact cells at two novel sites, Ser-552 and Ser-675; (ii) phosphorylation by PKA promotes the transcriptional activity (TCF/LEF transactivation) ofβ-catenin; (iii) mutation of Ser-675 attenuates the promoting effect of PKA; (iv) phosphorylation by PKA does not affect the GSK3-dependent phosphorylation ofβ-catenin, its stability, or intracellular localization; and (v) phosphorylation at the Ser-675 site promotes the binding of β-catenin to its transcriptional coactivator, CREB-binding protein. In conclusion, this study identifies a novel, noncanonical mechanism of modulation of β-catenin signaling through direct phosphorylation of β-catenin by PKA, promoting its interaction with CREB-binding protein.

Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na+]i/[K+]i Ratio in Cultured Vascular Smooth Muscle Cells

Abstract— Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na+]i/[K+]i-responsive signaling pathway that may play an important role in the regulation of programmed cell death in VSMCs.

Critical Role of Serum Response Factor in Pulmonary Myofibroblast Differentiation Induced by TGF-β

Transforming growth factor-beta (TGF-beta) is a cytokine implicated in wound healing and in the pathogenesis of pulmonary fibrosis. TGF-beta stimulates myofibroblast differentiation characterized by expression of contractile smooth muscle (SM)-specific proteins such as SM-alpha-actin. In the present study, we examined the role of serum response factor (SRF) in the mechanism of TGF-beta-induced pulmonary myofibroblast differentiation of human lung fibroblasts (HLF). TGF-beta stimulated SM-alpha-actin expression in HLF, which paralleled with a profound induction of SRF expression and activity. Inhibition of SRF by the pharmacologic SRF inhibitor (CCG-1423), or via adenovirus-mediated transduction of SRF short hairpin RNA (shSRF), blocked the expression of both SRF and SM-alpha-actin in response to TGF-beta without affecting Smad-mediated signaling of TGF-beta. However, forced expression of SRF on its own did not promote SM-alpha-actin expression, whereas expression of the constitutively transactivated SRF fusion protein (SRF-VP16) was sufficient to induce SM-alpha-actin expression, suggesting that both expression and transactivation of SRF are important. Activation of protein kinase A (PKA) by forskolin or iloprost resulted in a significant inhibition of SM-alpha-actin expression induced by TGF-beta, and this was associated with inhibition of both SRF expression and activity, but not of Smad-mediated gene transcription. In summary, this is the first direct demonstration that TGF-beta-induced pulmonary myofibroblast differentiation is mediated by SRF, and that inhibition of myofibroblast differentiation by PKA occurs through down-regulation of SRF expression levels and SRF activity, independent of Smad signaling.

Inhibition of Na+, K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i[K+]i ratio

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.

Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i.

Recent studies have demonstrated the tissue-specific effect of Na+/K+ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na+/K+ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na+/K+ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na+/K+ pump inhibition in K+-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by 3H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K+ and Na+, whereas dissipation of the Na+ gradient abolished the antiapoptotic action of K+-free medium. Comparative analysis of these results and modulation of intracellular Na+ and K+ content by the above-listed stimuli showed that interaction of ouabain with Na+/K+-ATPase triggered necrosis independently of inhibition of Na+/K+ pump-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio, whereas protection against apoptosis under Na+/K+ pump inhibition in K+-depleted medium was mediated by [Na+]i elevation. The role of Na+/K+ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.

Phosphorylation of β-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells

Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.

c-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium- mediated, calcium-independent mechanism

In this study, we examined the effect of Na+‐K+ pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain‐treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c‐Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c‐Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca2+ response element binding protein (CREB)‐1 and c‐Myc was not altered. Markedly augmented c‐Fos expression was also observed under Na+‐K+ pump inhibition in potassium‐depleted medium. Na+‐K+ pump inhibition triggered c‐Fos expression via elevation of the [Na+]i/[K+]i ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c‐Fos expression in high‐potassium‐low‐sodium medium and from the comparison of dose responses of Na+‐K+ pump activity, [Na+]i and [K+]i content and c‐Fos expression to ouabain. A fourfold increment of c‐Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na+]i but did not affect [K+]i. Augmented c‐Fos expression was also observed under VSMC depolarization in high‐potassium medium. Increments in both c‐Fos expression and 45Ca uptake in depolarized VSMC were abolished under inhibition of L‐type Ca2+ channels with 0.1 μM nicardipine. Ouabain did not affect the free [Ca2+]i or the content of exchangeable [Ca2+]i. Ouabain‐induced c‐Fos expression was also insensitive to the presence of nicardipine and [Ca2+]o, as well as chelators of [Ca2+]o (EGTA) and [Ca2+]i (BAPTA). The effect of ouabain and serum on c‐Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk‐1, serum response factor, CREB and activator protein‐1 transcription factors identified within the c‐Fos promoter. These results suggest that Na+‐K+ pump inhibition triggers c‐Fos expression via [Na+]i‐sensitive [Ca2+]i‐independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.

Inhibition of Na+,k+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [na+]i/[k+]i ratio: possible implication in vascular remodelling

Objectives Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. Methods DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. Results Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. Conclusions Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.

Gβγ-mediated Prostacyclin Production and cAMP-dependent Protein Kinase Activation by Endothelin-1 Promotes Vascular Smooth Muscle Cell Hypertrophy through Inhibition of Glycogen Synthase Kinase-3

Endothelin-1 (ET1) is a vasoactive peptide that stimulates hypertrophy of vascular smooth muscle cells (VSMC) through diverse signaling pathways mediated by Gq/Gi/G13 heterotrimeric G proteins. We have found that ET1 stimulates the activity of cAMP-dependent protein kinase (PKA) in VSMC as profoundly as the Gs-linked β-adrenergic agonist, isoproterenol (ISO), but in a transient manner. PKA activation by ET1 was mediated by type-A ET1 receptors (ETA) and recruited an autocrine signaling mechanism distinct from that of ISO, involving Gi-coupled βγ subunits of heterotrimeric G proteins, extracellular signal-regulated kinases ERK1/2, cyclooxygenase COX-1 (but not COX-2) and prostacyclin receptors. In the functional studies, inhibition of PKA or COX-1 attenuated ET1-induced VSMC hypertrophy, suggesting the positive role of PKA in this response to ET1. Furthermore, we found that ET1 stimulates a Gβγ-mediated, PKA-dependent phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3), an enzyme that regulates cell growth. Together, this study describes that (i) PKA can be transiently activated by Gi-coupled agonists such as ET1 by an autocrine mechanism involving Gβγ/calcium/ERK/COX-1/prostacyclin signaling, and (ii) this PKA activation promotes VSMC hypertrophy, at least in part, through PKA-dependent phosphorylation and inhibition of GSK3.

Myocardin-dependent Activation of the CArG Box-rich Smooth Muscle γ-Actin Gene: PREFERENTIAL UTILIZATION OF A SINGLE CArG ELEMENT THROUGH FUNCTIONAL ASSOCIATION WITH THE NKX3.1 HOMEODOMAIN PROTEIN*

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds a 10-bp element known as the CArG box, located in the proximal regulatory region of hundreds of target genes. SRF activates target genes in a cell- and context-dependent manner by assembling unique combinations of cofactors over CArG elements. One particularly strong SRF cofactor, myocardin (MYOCD), acts as a component of a molecular switch for smooth muscle cell (SMC) differentiation by activating cytoskeletal and contractile genes harboring SRF-binding CArG elements. Here we report that the human ACTG2 promoter, containing four conserved CArG elements, displays SMC-specific basal activity and is highly induced in the presence of MYOCD. Stable transfection of a non-SMC cell type with Myocd elicits elevations in endogenous Actg2 mRNA. Gel shift and luciferase assays reveal a strong bias for MYOCD-dependent transactivation through CArG2 of the human ACTG2 promoter. Substitution of CArG2 with other CArGs, including a consensus CArG element, fails to reconstitute full MYOCD-dependent ACTG2 promoter stimulation. Mutation of an adjacent binding site for NKX3.1 reduces MYOCD-dependent transactivation of the ACTG2 promoter. Co-immunoprecipitation, glutathione S-transferase pulldown, and luciferase assays show a physical and functional association between MYOCD and NKX3.1; no such functional relationship is evident with the related NKX2.5 transcription factor despite its interaction with MYOCD. These results demonstrate the ability of MYOCD to discriminate among several juxtaposed CArG elements, presumably through its novel partnership with NKX3.1, to optimally transactivate the human ACTG2 promoter.Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds a 10-bp element known as the CArG box, located in the proximal regulatory region of hundreds of target genes. SRF activates target genes in a cell- and context-dependent manner by assembling unique combinations of cofactors over CArG elements. One particularly strong SRF cofactor, myocardin (MYOCD), acts as a component of a molecular switch for smooth muscle cell (SMC) differentiation by activating cytoskeletal and contractile genes harboring SRF-binding CArG elements. Here we report that the human ACTG2 promoter, containing four conserved CArG elements, displays SMC-specific basal activity and is highly induced in the presence of MYOCD. Stable transfection of a non-SMC cell type with Myocd elicits elevations in endogenous Actg2 mRNA. Gel shift and luciferase assays reveal a strong bias for MYOCD-dependent transactivation through CArG2 of the human ACTG2 promoter. Substitution of CArG2 with other CArGs, including a consensus CArG element, fails to reconstitute full MYOCD-dependent ACTG2 promoter stimulation. Mutation of an adjacent binding site for NKX3.1 reduces MYOCD-dependent transactivation of the ACTG2 promoter. Co-immunoprecipitation, glutathione S-transferase pulldown, and luciferase assays show a physical and functional association between MYOCD and NKX3.1; no such functional relationship is evident with the related NKX2.5 transcription factor despite its interaction with MYOCD. These results demonstrate the ability of MYOCD to discriminate among several juxtaposed CArG elements, presumably through its novel partnership with NKX3.1, to optimally transactivate the human ACTG2 promoter.