scholarly journals Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf

1998 ◽  
Vol 335 (2) ◽  
pp. 241-246 ◽  
Author(s):  
Stephen J. FULLER ◽  
Stephen G. FINN ◽  
Julian DOWNWARD ◽  
Peter H. SUGDEN
Keyword(s):  
Cell Size ◽  
Reporter Gene ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Guanine Nucleotide ◽  
Phosphatidylinositol 3 Kinase ◽  
Transcriptional Responses ◽  
Response Element ◽  
Hypertrophic Response ◽  
Phosphatidylinositol 3

Treatment of cultured neonatal ventricular myocytes with oncogenic Ras increases their size and stimulates the re-expression of genes which are normally restricted to the fetal stage of ventricular development, including atrial natriuretic factor (ANF) and skeletal muscle (SkM)-α-actin. To determine which signalling pathways mediate these responses, myocytes were transfected with oncogenic (V12) Ras mutants which interact selectively with different effectors and their effects on luciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas), itself, activated ANF–LUX 9.6-fold, whereas mutants of V12HRas, which selectively stimulate Ral guanine nucleotide dissociation stimulator (Ral.GDS) (E37G), c-Raf (D38E) and phosphatidylinositol 3-kinase (PI-3-K; Y40C) enhanced ANF–LUX expression 3.0-, 3.7- and 1.7-fold respectively. The full response of ANF–LUX to V12HRas was restored by using a combination of the individual effector domain mutants. Likewise, SkM-α-actin–LUX expression was activated 12.0-, 3.5-, 4.5- and 3.0-fold by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern of activation was also observed using a c-fosserum-response element–LUX reporter gene. Cell size was also increased by each of the mutants, but simultaneous expression of all three mutant constructs was needed to reconstitute the full effect of V12HRas on cell size (50% increase). Transfection with a constitutively active mutant of PI-3-K (p110K227E) stimulated ANF–LUX, SkM-α-actin–LUX, c-fos-serum-response element–LUX and Rous sarcoma virus–LUX by 3.1-, 3.2-, 2.1- and 2.9-fold respectively, but the co-transfected cytomegalovirus-β-galactosidase reporter gene was activated to a similar extent (1.9-fold). These results suggest that Raf, Ral.GDS and PI-3-K can all transduce transcriptional responses to V12HRas, but that the specific induction of genes associated with the hypertrophic response is not mediated through PI-3-K.

scholarly journals The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element

2000 ◽  
Vol 278 (6) ◽  
pp. H1769-H1774 ◽  
Author(s):  
Michael R. Morissette ◽  
Valerie P. Sah ◽  
Christopher C. Glembotski ◽  
Joan Heller Brown
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Common Element ◽  
Luciferase Reporter ◽  
Serum Response Element ◽  
Transcriptional Responses ◽  
Response Element ◽  
Serum Response

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c- fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

Signals from type 1 sphingosine 1-phosphate receptors enhance adult mouse cardiac myocyte survival during hypoxia

2007 ◽  
Vol 293 (5) ◽  
pp. H3150-H3158 ◽  
Author(s):  
Jianqing Zhang ◽  
Norman Honbo ◽  
Edward J. Goetzl ◽  
Kanu Chatterjee ◽  
Joel S. Karliner ◽  
...  
Keyword(s):  
Cardiac Myocyte ◽  
Adult Mouse ◽  
Ventricular Myocytes ◽  
Mouse Cell ◽  
Biologically Active ◽  
Phosphatidylinositol 3 Kinase ◽  
Cytochrome C Release ◽  
Pharmacological Agents ◽  
Sphingosine 1 Phosphate ◽  
Phosphatidylinositol 3

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S1P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S1P to Gi protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S1P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S1P1 receptors were observed to transduce signals from exogenous S1P, an S1P1 receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW2871. S1P1 receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S1P-S1P1 receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S1P1 receptor function was closely associated with activation of Akt, inactivation of GSK-3β, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S1P1 receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

scholarly journals Beta-3 Adrenoreceptors protect from hypertrophic remodelling through AMP-Activated Protein Kinase and Autophagy

2020 ◽  
Author(s):  
Emilie Dubois-Deruy ◽  
Roselle Gelinas ◽  
Christophe Beauloye ◽  
Hrag Esfahani ◽  
Chantal Dessy ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Recombinant Adenovirus ◽  
Neonatal Rat ◽  
Specific Expression ◽  
Hypertrophic Response ◽  
Neonatal Rat Ventricular Myocytes ◽  
Amp Activated Protein Kinase

ABSTRACTAimsThe abundance of beta3-adrenergic receptors (β3-ARs) is upregulated in diseased human myocardium. We previously showed that cardiac-specific expression of β3-AR inhibits the hypertrophic response to neurohormonal stimulation. Here, we further analyzed signalling pathways involved in the anti-hypertrophic effect of β3-AR.MethodsIn vitro hypertrophic responses to phenylephrine (PE) were analyzed in neonatal rat ventricular myocytes (NRVM) infected with a recombinant adenovirus expressing the human β3-AR (AdVhβ3). We confirmed results in mice with cardiomyocyte-specific moderate expression of human β3-AR (β3-TG) and WT littermates submitted to thoracic transverse aortic constriction (TAC) for 9 weeks.ResultsWe observed a colocalization of β3-AR with the AMP-activated protein kinase (AMPK) both in neonatal rat andin adult mouse cardiomyocytes. Treatment of NRVM with PE induced hypertrophy and a decrease in phosphorylation of Thr172-AMPK (/2, p=0.0487) and phosphorylation of Ser79- acetyl-CoA carboxylase (ACC) (/2.6, p=0.0317), inducing an increase in phosphorylated Ser235/236 S6 protein (x2.5, p=0.0367) known to be involved in protein synthesis. These effects were reproduced by TAC in WT mice, but restored to basal levels in β3-AR expressing cells/mice. siRNA targeting of AMPK partly abrogated the anti-hypertrophic effect of β3-AR in response to PE in NRVM (x1.3, p<0.0001). Concomitant with hypertrophy, autophagy measured by microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio and p62 abundance was decreased by PE in NRVM (/2.6, p=0.0010 and x3, p=0.0016, respectively) or TAC in WT mice (/5.4, p=0.0159); and preserved in human β3-AR expressing cells and mice, together with reduced hypertrophy.ConclusionsCardiac-specific moderate expression of β3-AR inhibits the hypertrophic response in part through AMPK activation followed byinhibition of protein synthesis and preservation of autophagy. Activation of the cardiac β3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodelling.

scholarly journals Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response

1996 ◽  
Vol 317 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Angela CLERK ◽  
Judith GILLESPIE-BROWN ◽  
Stephen J. FULLER ◽  
Peter H. SUGDEN
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Hypertrophic Response ◽  
Selective Antagonist ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Stimulation Of

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1–8) [(Des-Arg9)BK] stimulated PtdInsP2 hydrolysis by 3–4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 μM [BK(1–8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-Δ and nPKC-ϵ from the soluble to the particulate fraction. EC50 values for nPKC-Δ translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-ϵ translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-Δ and nPKC-ϵ by BK(1–8) were more than 5 μM. The classical PKC, cPKC-α, and the atypical PKC, aPKC-ζ, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3–5 min, 30–35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1–8) were more than 10 μM. The order of potency [BK≈kallidin ≫ BK(1–8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-Δ and nPKC-ϵ translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2 BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1–8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-Δ and nPKC-ϵ, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

scholarly journals Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

2015 ◽  
Vol 26 (6) ◽  
pp. 1119-1128 ◽  
Author(s):  
Bjorn D. M. Bean ◽  
Michael Davey ◽  
Jamie Snider ◽  
Matthew Jessulat ◽  
Viktor Deineko ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
New Yeast ◽  
Phosphatidylinositol 3

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.

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