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Nickolai O Dulin

Publications and source records attributed to Nickolai O Dulin.

14 recordsLinked to original sources
PubMed2006

Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase.

Beta-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 beta-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 beta-catenin to ubiquitination and degradation by the proteasome system. Mitogenic factors promote beta-catenin signaling through the inhibition of GSK3, resulting in reduced beta-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) beta-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) of beta-catenin; (iii) mutation of Ser-675 attenuates the promoting effect of PKA; (iv) phosphorylation by PKA does not affect the GSK3-dependent phosphorylation of beta-catenin, its stability, or intracellular localization; and (v) phosphorylation at the Ser-675 site promotes the binding of beta-catenin to its transcriptional coactivator, CREB-binding protein. In conclusion, this study identifies a novel, noncanonical mechanism of modulation of beta-catenin signaling through direct phosphorylation of beta-catenin by PKA, promoting its interaction with CREB-binding protein.

Animals↗
PubMed2005

Cl- secretion in ATP-treated renal epithelial C7-MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A2 and protein kinase A.

This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

Adenosine Triphosphate↗
PubMed2004

Regulation of serum response factor-dependent gene expression by proteasome inhibitors.

Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.

Animals↗
PubMed2004

Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP.

Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM-alpha-actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity.

Adenosine Triphosphate↗
PubMed2004

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl- cotransport, intracellular Cl- and L-type Ca2+ channels.

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

Animals↗
PubMed2003

Serum response factor function and dysfunction in smooth muscle.

Tight control of smooth muscle cell (SM) proliferation, differentiation, and apoptosis requires a balance between signaling and transcriptional events. Recent developments in vascular research revealed that serum response factor (SRF) function is important for the regulation of each of these processes. The cloning and characterization of several SM specific genes and the discovery that SRF is central for their expression fueled studies aimed at understanding the role of molecular partners including co-activators and co-repressors. Perturbations of pathways involving SRF are associated with abnormalities in the myogenic program and aberrant phenotypic consequences. Surprisingly, studies on airway SM have remained an underrepresented area of investigation. Our laboratory described a novel regulatory mechanism of SRF function in airway myocytes by modulation of its subcellular localization. This review summarizes current knowledge on the structure and function of this essential transcription factor as well different modes of regulating SRF expression and activity that are becoming key players in directing SM function in health and disease.

Animals↗
PubMed2003

Fractalkine (CX3CL1) stimulated by nuclear factor kappaB (NF-kappaB)-dependent inflammatory signals induces aortic smooth muscle cell proliferation through an autocrine pathway.

Fractalkine (also known as CX3CL1), a CX3C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX3C receptor 1 (CX3CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX3CR1. TNF-alpha activated nuclear factor kappaB (NF-kappaB) and induced fractalkine and CX3CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory kappaB (IkappaB)-alpha, dnIkappaB-beta, dnIkappaB kinase (IKK)-gamma, kinase-dead (kd) NF-kappaB-inducing kinase (NIK) and kdIKK-beta, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt ('PDTC') or MG-132, significantly attenuated TNF-alpha-induced fractalkine and CX3CR1 expression. Furthermore, expression of dn TNF-alpha-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-alpha signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX3CR1 antibodies attenuated TNF-alpha-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-alpha-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide 3-kinase (PI 3-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-kappaB activation, and induced SMC cell-cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-alpha induces the expression of fractalkine and CX3CR1 in rat aortic SMCs and that this induction is mediated by NF-kappaB activation. We also show that fractalkine induces its own expression, which is mediated by the PI 3-kinase/PDK1/Akt/NIK/IKK/NF-kappaB signalling pathway. More importantly, fractalkine increased cell-cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.

Androstadienes↗
PubMed2003

Activation of cGMP-dependent protein kinase by protein kinase C.

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.

Cyclic GMP-Dependent Protein Kinase Type I↗
PubMed2003

Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA.

Endothelin-1 (ET1) and ATP stimulate contraction and hypertrophy of vascular smooth muscle cells (VSMC) by activating diverse signalling pathways. In this study, we show that in VSMC, ET1 and ATP stimulate transient and sustained activation of protein kinase A (PKA), respectively. Using a dominant negative PKA mutant (PKA-DN), we examined the functional significance of PKA activation in the signalling of ET1 and ATP. Overexpression of PKA-DN did not alter the ET1- or ATP-induced phosphorylation of the extracellular signal-regulated protein kinase, Erk2. ATP stimulated a profound, PKA-dependent activation of cAMP-response element (CRE), whereas the effect of ET1 was negligible. Both ET1 and ATP stimulated serum response factor (SRF)-dependent gene expression. Overexpression of PKA-DN potentiated the effects of ET1 and ATP on SRF activity, whereas stimulation of PKA by isoproterenol, forskolin or by overexpression of the PKA catalytic subunit decreased SRF activity. These data demonstrate that (i) PKA negatively regulates SRF activity and (ii) ET1 and ATP stimulate opposing pathways, whose balance determines the net activity of SRF.

Adenosine Triphosphate↗
PubMed2003

What evidence implicates airway smooth muscle in the cause of BHR?

Bronchial hyperresponsiveness (BHR), the occurrence of excessive bronchoconstriction in response to relatively small constrictor stimuli, is a cardinal feature of asthma. Here, we consider the role that airway smooth muscle might play in the generation of BHR. The weight of evidence suggests that smooth muscle isolated from asthmatic tissues exhibits normal sensitivity to constrictor agonists when studied during isometric contraction, but the increased muscle mass within asthmatic airways might generate more total force than the lesser amount of muscle found in normal bronchi. Another salient difference between asthmatic and normal individuals lies in the effect of deep inhalation (DI) on bronchoconstriction. DI often substantially reverses induced bronchoconstriction in normals, while it often has much less effect on spontaneous or induced bronchoconstriction in asthmatics. It has been proposed that abnormal dynamic aspects of airway smooth muscle contraction velocity of contraction or plasticity- elasticity balance might underlie the abnormal DI response in asthma. We suggest a speculative model in which abnormally long actin filaments might account for abnormally increased elasticity of contracted airway smooth muscle.

Actins↗
PubMed2002

Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.

It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

Animals↗
PubMed2002

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/Ca(2+) 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 (45)Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca(2+) channels with 0.1 microM nicardipine. Ouabain did not affect the free [Ca(2+)](i) or the content of exchangeable [Ca(2+)](i). Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca(2+)](o), as well as chelators of [Ca(2+)](o) (EGTA) and [Ca(2+)](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 [Ca(2+)](i)-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.

Animals↗
PubMed2002

RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain.

RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the G alpha subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser(264) was then identified as the 14-3-3-binding site of RGS3. The S(264)A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind G alpha(q). Signalling studies showed that the S(264)A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3.

14-3-3 Proteins↗