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Astrid E. Alewijnse - One of the best experts on this subject based on the ideXlab platform.
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LPS differentially affects vasoconstrictor responses: a potential role for RGS16?
Journal of Physiology and Biochemistry, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Astrid E. Alewijnse, M. Tjon-atsoi, S. L. M. PetersAbstract:El lipopolisacárido bacteriano LPS está implicado en la mayor parte de las alteraciones cardiovasculares propias del shock séptico. Se investiga en este trabajo sobre los efectos de la exposición al LPS en la contractilidad vascular en general y sobre el papel de las proteínas reguladoras de la señalización de proteínas G (RGS) en las alteraciones vasculares inducidas por el LPS. La exposición (22 h) de anillos aórticos de rata a diversas concentraciones de LPS (3, 10, 30 μg/ml) afecta de forma diferencial las respuestas contráctiles inducidas por la activación de 4 diferentes receptores acoplados a proteínas G (α1-adrenorreceptores, de angiotensina II, de serotonina y ET-1 de endotelina). Asi, el pretratamiento con LPS no afecta la contracción inducida por endotelina, mientras que reduce la de fenilefrina y angiotensina II e incrementa la de serotonina. Además, el tratamiento con LPS aumenta la expresión de RNAm de RGS16 tanto en anillos aórticos como en células VSMC, pero no de RNAm de RGS2, RGS3, RGS4 y RGS5. Los cambios de RNAm de RGS16 podrían contribuir a la regulación diferencial de las respuestas contráctiles a los vasoconstrictores en presencia de LPS. The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to α_1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (α_1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.
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Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells
European Journal of Pharmacology, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P1/S1P3–5 agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was Gi/o-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.
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LPS differentially affects vasoconstrictor responses: a potential role for RGS16?
Journal of physiology and biochemistry, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Astrid E. Alewijnse, M. Tjon-atsoi, Stephan L. M. PetersAbstract:The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to α1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (α1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.
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S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells.
European Journal of Pharmacology, 2008Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P 1 receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P 2 and S1P 3 receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P 1 or S1P 3 receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P 1 receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P 1 receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P 1 receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.
John H Kehrl - One of the best experts on this subject based on the ideXlab platform.
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the aorta and heart differentially express rgs regulators of g protein signalling proteins that selectively regulate sphingosine 1 phosphate angiotensin ii and endothelin 1 signalling
Biochemical Journal, 2003Co-Authors: Hyeseon Cho, Kathleen Harrison, Owen Schwartz, John H KehrlAbstract:Normal cardiovascular development and physiology depend in part upon signalling through G-protein-coupled receptors (GPCRs), such as the angiotensin II type 1 (AT(1)) receptor, sphingosine 1-phosphate (S1P) receptors and endothelin-1 (ET-1) receptor. Since regulator of G-protein signalling (RGS) proteins function as GTPase-activating proteins for the G alpha subunit of heterotrimeric G-proteins, these proteins undoubtedly have functional roles in the cardiovascular system. In the present paper, we show that human aorta and heart differentially express RGS1, RGS2, RGS3S (short-form), RGS3L (long-form), PDZ-RGS3 (PDZ domain-containing) and RGS4. The aorta prominently expresses mRNAs for all these RGS proteins except PDZ-RGS3. Various stimuli that are critical for both cardiovascular development and function regulate dynamically the mRNA levels of several of these RGS proteins in primary human aortic smooth muscle cells. Both RGS1 and RGS3 inhibit signalling through the S1P(1) (formerly known as EDG-1), S1P(2) (formerly known as EDG-5) and S1P(3) (formerly known as EDG-3) receptors, whereas RGS2 and RGS4 selectively attenuate S1P(2)-and S1P(3)-receptor signalling respectively. All of the tested RGS proteins inhibit AT(1)-receptor signalling, whereas only RGS3 and, to a lesser extent, RGS4 inhibit ET(A)-receptor signalling. The conspicuous expression of RGS proteins in the cardiovascular system and their selective effects on relevant GPCR-signalling pathways provide additional evidence that they have functional roles in cardiovascular development and physiology.
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regulator of g protein signaling 3 RGS3 inhibits gβ1γ2 induced inositol phosphate production mitogen activated protein kinase activation and akt activation
Journal of Biological Chemistry, 2001Co-Authors: Srikumar Sinnarajah, Carmen W. Dessauer, Sue Goo Rhee, John H KehrlAbstract:Abstract Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which Gαi and Gαq hydrolyze GTP to GDP, thereby limiting the duration in which GTP-Gαi and GTP-Gαq can activate effectors. Since GDP-Gα subunits rapidly combine with free Gβγ subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of Gβγ subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind Gβ5 in the absence of a Gγ subunit, RGS proteins are not known to directly influence Gβγ signaling. Here we show that RGS3 binds Gβ1γ2 subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with Gβ1γ2 inhibits Gβ1γ2-induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of Gβ1γ2 signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect Gβ1γ2 signaling in these assays. Consistent with the in vivo results, RGS3 inhibits Gβγ-mediated activation of phospholipase Cβ in vitro. Thus, RGS3 may limit Gβγ signaling not only by virtue of its GTPase-activating protein activity for Gα subunits, but also by directly interfering with the activation of effectors.
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RGS3 Is a GTPase-Activating Protein for Giα and Gqα and a Potent Inhibitor of Signaling by GTPase-Deficient Forms of Gqα and G11α
Molecular pharmacology, 2000Co-Authors: Astrid Scheschonka, Srikumar Sinnarajah, Carmen W. Dessauer, Peter Chidiac, Chong-shan Shi, John H KehrlAbstract:Many Regulators of G proteinSignaling (RGS) proteins accelerate the intrinsic GTPase activity of Giα and Gqα-subunits [i.e., behave as GTPase-activating proteins (GAPs)] and several act as Gqα-effector antagonists. RGS3, a structurally distinct RGS member with a unique N-terminal domain and a C-terminal RGS domain, and an N-terminally truncated version of RGS3 (RGS3CT) both stimulated the GTPase activity of Giα (except Gzα) and Gqα but not that of Gsα or G12α. RGS3 and RGS3CT had Gqα GAP activity similar to that of RGS4. RGS3 impaired signaling through Gq-linked receptors, although RGS3CT invariably inhibited better than did full-length RGS3. RGS3 potently inhibited GqαQ209L- and G11αQ209l-mediated activation of a cAMP-response element-binding protein reporter gene and GqαQ209L induced inositol phosphate production, suggesting that RGS3 efficiently blocks Gqα from activating its downstream effector phospholipase C-β. Whereas RGS2 and to a lesser extent RGS10 also inhibited signaling by these GTPase-deficient G proteins, other RGS proteins including RGS4 did not. Mutation of residues in RGS3 similar to those required for RGS4 Giα GAP activity, as well as several residues N terminal to its RGS domain impaired RGS3 function. A greater percentage of RGS3CT localized at the cell membrane than the full-length version, potentially explaining why RGS3CT blocked signaling better than did full-length RGS3. Thus, RGS3 can impair Gi- (but not Gz-) and Gq-mediated signaling in hematopoietic and other cell types by acting as a GAP for Giα and Gqα subfamily members and as a potent Gqα subfamily effector antagonist.
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regulation of chemotactic and proadhesive responses to chemoattractant receptors by rgs regulator of g protein signaling family members
Journal of Biological Chemistry, 1998Co-Authors: Edward P Bowman, John H Kehrl, Kirk M Druey, James J Campbell, Astrid Scheschonka, Eugene C ButcherAbstract:Serpentine Galphai-linked receptors support rapid adhesion and directed migration of leukocytes and other cell types. The intracellular mechanisms mediating and regulating chemoattractant-directed adhesion and locomotion are only now beginning to be explored. RGS (for regulator of G-protein signaling) proteins are a recently described family that regulate Galphai-stimulated pathways by acting as GTPase-activating proteins. Little is known about the GTPase activity of the Galphai proteins involved in adhesion and chemotaxis, or the significance of their regulation to these responses. Using transiently transfected lymphoid cells as a model system, we show that expression of RGS1, RGS3, and RGS4 inhibits chemoattractant-induced migration. In contrast, RGS2, a regulator of Galphaq activity, had no effect on cell migration to any chemoattractant. RGS1, RGS3, and RGS4 also reduced rapid chemoattractant-triggered adhesion, although the proadhesive response appears quantitatively less sensitive to RGS action than chemotaxis. The results suggest that the duration of the Galphai signal may be a particularly important parameter in the chemotactic responses of leukocytes, and demonstrate the potential for RGS family members to regulate cellular adhesive and migratory behaviors.
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Potential Role for a Regulator of G Protein Signaling (RGS3) in Gonadotropin-Releasing Hormone (GnRH) Stimulated Desensitization
Endocrinology, 1997Co-Authors: Jimmy D. Neill, Kirk M Druey, Astrid Scheschonka, L. Wayne Duck, Jeffrey C. Sellers, Lois C. Musgrove, John H KehrlAbstract:The cellular and molecular mechanisms of gonadotrope desensitization are unknown but transduction of the GnRH signal is known to involve sequentially the GnRH receptor, Gqα protein, phospholipase C β-1, inositol-1,4,5-trisphosphate (IP3), and intracellular Ca+2 release. Here, we report the results of studies of a new family of proteins known as regulators of G protein signaling (RGS) that recently have been implicated in desensitization of several ligand induced processes. Using DNA-mediated transfection, we co-expressed the GnRH receptor and RGS1, 2, 3, or 4 in COS-1 cells. Control cells and those expressing RGS1, 2, and 4 produced five fold increases in IP3 levels during the 30 sec after treatment with GnRH. In contrast, RGS3 expression suppressed by 75% the GnRH-induced IP3 responses. RGS3 was shown to bind Gqα protein in a model in vitro system: recombinant RGS3-glutathione-S-transferase (GST) fusion protein bound five-fold more 35S-met labeled Gqα protein than did with GST alone, suggesting that the ...
Mariëlle C. Hendriks-balk - One of the best experts on this subject based on the ideXlab platform.
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LPS differentially affects vasoconstrictor responses: a potential role for RGS16?
Journal of Physiology and Biochemistry, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Astrid E. Alewijnse, M. Tjon-atsoi, S. L. M. PetersAbstract:El lipopolisacárido bacteriano LPS está implicado en la mayor parte de las alteraciones cardiovasculares propias del shock séptico. Se investiga en este trabajo sobre los efectos de la exposición al LPS en la contractilidad vascular en general y sobre el papel de las proteínas reguladoras de la señalización de proteínas G (RGS) en las alteraciones vasculares inducidas por el LPS. La exposición (22 h) de anillos aórticos de rata a diversas concentraciones de LPS (3, 10, 30 μg/ml) afecta de forma diferencial las respuestas contráctiles inducidas por la activación de 4 diferentes receptores acoplados a proteínas G (α1-adrenorreceptores, de angiotensina II, de serotonina y ET-1 de endotelina). Asi, el pretratamiento con LPS no afecta la contracción inducida por endotelina, mientras que reduce la de fenilefrina y angiotensina II e incrementa la de serotonina. Además, el tratamiento con LPS aumenta la expresión de RNAm de RGS16 tanto en anillos aórticos como en células VSMC, pero no de RNAm de RGS2, RGS3, RGS4 y RGS5. Los cambios de RNAm de RGS16 podrían contribuir a la regulación diferencial de las respuestas contráctiles a los vasoconstrictores en presencia de LPS. The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to α_1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (α_1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.
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Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells
European Journal of Pharmacology, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P1/S1P3–5 agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was Gi/o-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.
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LPS differentially affects vasoconstrictor responses: a potential role for RGS16?
Journal of physiology and biochemistry, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Astrid E. Alewijnse, M. Tjon-atsoi, Stephan L. M. PetersAbstract:The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to α1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (α1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.
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S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells.
European Journal of Pharmacology, 2008Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P 1 receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P 2 and S1P 3 receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P 1 or S1P 3 receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P 1 receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P 1 receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P 1 receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.
Ulrike Mende - One of the best experts on this subject based on the ideXlab platform.
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Gq/11-Mediated Signaling and Hypertrophy in Mice with Cardiac-Specific Transgenic Expression of Regulator of G-Protein Signaling 2
PloS one, 2012Co-Authors: Cindy Park-windhol, Peng Zhang, Ming Zhu, Leonard Chaves, Angel E. Maldonado, Michelle E. King, Lisa M. Rickey, Darragh Cullen, Ulrike MendeAbstract:Cardiac hypertrophy is a well-established risk factor for cardiovascular morbidity and mortality. Activation of Gq/11-mediated signaling is required for pressure overload-induced cardiomyocyte (CM) hypertrophy to develop. We previously showed that among Regulators of G protein Signaling, RGS2 selectively inhibits Gq/11 signaling and its hypertrophic effects in isolated CM. In this study, we generated transgenic mice with CM-specific, conditional RGS2 expression (dTG) to investigate whether RGS2 overexpression can be used to attenuate Gq/11-mediated signaling and hypertrophy in vivo. Transverse aortic constriction (TAC) induced a comparable rise in ventricular mass and ANF expression and corresponding hemodynamic changes in dTG compared to wild types (WT), regardless of the TAC duration (1-8 wks) and timing of RGS2 expression (from birth or adulthood). Inhibition of endothelin-1-induced Gq/11-mediated phospholipase C β activity in ventricles and atrial appendages indicated functionality of transgenic RGS2. However, the inhibitory effect of transgenic RGS2 on Gq/11-mediated PLCβ activation differed between ventricles and atria: (i) in sham-operated dTG mice the magnitude of the inhibitory effect was less pronounced in ventricles than in atria, and (ii) after TAC, negative regulation of Gq/11 signaling was absent in ventricles but fully preserved in atria. Neither difference could be explained by differences in expression levels, including marked RGS2 downregulation after TAC in left ventricle and atrium. Counter-regulatory changes in other Gq/11-regulating RGS proteins (RGS4, RGS5, RGS6) and random insertion were also excluded as potential causes. Taken together, despite ample evidence for a role of RGS2 in negatively regulating Gq/11 signaling and hypertrophy in CM, CM-specific RGS2 overexpression in transgenic mice in vivo did not lead to attenuate ventricular Gq/11-mediated signaling and hypertrophy in response to pressure overload. Furthermore, our study suggests chamber-specific differences in the regulation of RGS2 functionality and potential future utility of the new transgenic model in mitigating Gq/11 signaling in the atria in vivo.
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RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro.
Journal of biochemistry and molecular biology, 2007Co-Authors: Thomas Anger, Ulrike Mende, Nils Klintworth, Christian Stumpf, Werner G. Daniel, Christoph D. GarlichsAbstract:Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate G(q)and G(i/o)-induced ERK and Akt phosphorylation. To isolate G(q)- and G(i/o)-mediated effects, we exclusively expressed muscarinic M(2) or M(3) receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in M(2)-/M(3)- expressing cells through carbachol stimulation. In co-expressions, M(3)/G(q)-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. M(2)/G(i/o) induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on M(2)/G(i/o)-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of G(q)- and G(i/o)-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards G(q) or G(i/o) proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.
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Regulation of cardiomyocyte signaling by RGS proteins: Differential selectivity towards G proteins and susceptibility to regulation
Journal of molecular and cellular cardiology, 2006Co-Authors: Jianming Hao, Christina Michalek, Wei Zhang, Ming Zhu, Ulrike MendeAbstract:Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2-RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited Gq/11-mediated phospholipase C beta stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited Gq/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both Gq/11 and Gi/o signaling (carbachol-induced cAMP inhibition). Gs signaling was unaffected, and, contrary to reports in other cell lines, RGS2-RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced Gq/11 signaling (alpha1-adrenergic stimulation or Galphaq* overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of Gq/11 signaling (RGS3-RGS5) were unchanged. Endogenous RGS2 (but not RGS3-RGS5) expression was also up-regulated in cells with enhanced AC signaling (beta-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of Gq/11 signaling that could potentially become a promising target for ameliorating Gq/11-mediated signaling and growth.
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selective loss of fine tuning of gq 11 signaling by rgs2 protein exacerbates cardiomyocyte hypertrophy
Journal of Biological Chemistry, 2006Co-Authors: Wei Zhang, Thomas Anger, Jialin Su, Agnieszka Gach, Xiaomei Xu, Ronglih Liao, Ulrike MendeAbstract:Abstract Alterations in cardiac G protein-mediated signaling, most prominently Gq/11 signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced Gq/11 signaling (transgenic expression of activated Gαq* and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cβ stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in Gq/11-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on Gq/11-mediated phospholipase Cβ activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in Gq/11-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.
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Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy
Journal of Biological Chemistry, 2005Co-Authors: Wei Zhang, Thomas Anger, Jialin Su, Agnieszka Gach, Xiaomei Xu, Ronglih Liao, Ulrike MendeAbstract:Abstract Alterations in cardiac G protein-mediated signaling, most prominently Gq/11 signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced Gq/11 signaling (transgenic expression of activated Gαq* and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cβ stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in Gq/11-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on Gq/11-mediated phospholipase Cβ activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in Gq/11-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.
Stephan L. M. Peters - One of the best experts on this subject based on the ideXlab platform.
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Sphingosine-1-phosphate regulates RGS2 and RGS16 mRNA expression in vascular smooth muscle cells
European Journal of Pharmacology, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Regulator of G protein signalling (RGS) protein expression is altered under growth promoting conditions in vascular smooth muscle cells (VSMCs). Since sphingosine-1-phosphate (S1P) is an important growth stimulatory factor, we investigated whether stimulation of VSMCs with S1P results in alterations in mRNA expression levels of several RGS proteins and which signalling components are involved. VSMCs were stimulated with S1P and mRNA expression levels of RGS2, RGS3, RGS4, RGS5 and RGS16 were measured by real-time polymerase chain reaction. S1P caused a time-dependent up-regulation of RGS2 and RGS16 mRNA expression. FTY720-P, a S1P1/S1P3–5 agonist, did not regulate RGS2 mRNA levels although it did up-regulate RGS16 mRNA expression. Pertussis toxin treatment revealed that the S1P-induced RGS16 expression was Gi/o-dependent whereas up-regulation of RGS2 mRNA was not. Phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase kinase apparently were not involved in the S1P-induced up-regulation of both RGS proteins. The present study demonstrates that S1P induces RGS2 and RGS16 mRNA expression but uses distinct S1P receptor subtypes and signalling pathways to regulate expression of these RGS proteins.
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LPS differentially affects vasoconstrictor responses: a potential role for RGS16?
Journal of physiology and biochemistry, 2009Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Astrid E. Alewijnse, M. Tjon-atsoi, Stephan L. M. PetersAbstract:The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to α1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (α1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.
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S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells.
European Journal of Pharmacology, 2008Co-Authors: Mariëlle C. Hendriks-balk, Najat Hajji, Pieter B. Van Loenen, Martin C. Michel, Stephan L. M. Peters, Astrid E. AlewijnseAbstract:Abstract Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells (VSMCs). Therefore, we investigated the role of altered expression levels of RGS proteins in S1P receptor function in VSMCs and transfected CHO cells. The mRNA expression of the S1P 1 receptor, RGS4 and RGS16 were down-regulated in VSMCs during phenotypic modulation induced by culturing, whereas mRNA levels of RGS2, RGS3, S1P 2 and S1P 3 receptors were unchanged. Interestingly, the expression level of RGS5 was transiently up-regulated. Despite major alterations in RGS levels, S1P-induced calcium elevation in VSMCs was not altered. Co-transfection of RGS2, RGS3, RGS4, RGS5 and RGS16 into CHO-Flp-In cells stably expressing the S1P 1 or S1P 3 receptor did not modify S1P-induced inhibition of cAMP accumulation to a major extent. Similar results were obtained with SEW2871, a selective S1P 1 receptor agonist. However, the inhibition of cAMP accumulation by the agonist FTY720-P via the S1P 1 receptor was significantly decreased by co-transfection with RGS5. These results indicate that mRNA of the S1P 1 receptor, RGS4, RGS5 and RGS16 is differentially regulated during phenotypic modulation. However, major alterations in RGS protein expression have only limited effect on S1P receptor function.