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Aldosterone Stimulates Elastogenesis in Cardiac Fibroblasts via Mineralocorticoid Receptor-independent Action Involving the Consecutive Activation of Gα13, c-Src, the Insulin-like Growth Factor-I Receptor, and Phosphatidylinositol 3-Kinase/Akt*

  • Severa Bunda
    Affiliations
    From the Physiology and Experimental Medicine Program, The Hospital for Sick Children, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario M5G 1X8, Canada
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  • Yanting Wang
    Affiliations
    From the Physiology and Experimental Medicine Program, The Hospital for Sick Children, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario M5G 1X8, Canada
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  • Thomas F. Mitts
    Affiliations
    From the Physiology and Experimental Medicine Program, The Hospital for Sick Children, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario M5G 1X8, Canada
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  • Peter Liu
    Affiliations
    Heart and Stroke/Richard Lewar Centre for Excellence, University of Toronto, Toronto, Ontario M5G 1X8, Canada

    Toronto General Hospital/University Health Network, Toronto, Ontario M5G 1X8, Canada
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  • Sara Arab
    Affiliations
    Toronto General Hospital/University Health Network, Toronto, Ontario M5G 1X8, Canada
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  • Majid Arabkhari
    Affiliations
    From the Physiology and Experimental Medicine Program, The Hospital for Sick Children, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario M5G 1X8, Canada
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  • Aleksander Hinek
    Correspondence
    To whom correspondence should be addressed: The Hospital for Sick Children, 555 University Ave., Toronto ON M5G 1X8, Canada. Tel.: 416-813-6725; Fax: 416-813-7480;
    Affiliations
    From the Physiology and Experimental Medicine Program, The Hospital for Sick Children, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario M5G 1X8, Canada

    Heart and Stroke/Richard Lewar Centre for Excellence, University of Toronto, Toronto, Ontario M5G 1X8, Canada
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  • Author Footnotes
    * This work was supported by the Canadian Institute of Health Research through Grant PG 13920, by the Heart and Stroke Foundation of Ontario through Grants NA 5435 and NA 4381, and by Career Investigator Award CI 4198 (to A. H.).
Open AccessPublished:April 16, 2009DOI:https://doi.org/10.1074/jbc.M109.008748
      We previously demonstrated that aldosterone, which stimulates collagen production through the mineralocorticoid receptor (MR)-dependent pathway, also induces elastogenesis via a parallel MR-independent mechanism involving insulin-like growth factor-I receptor (IGF-IR) signaling. The present study provides a more detailed explanation of this signaling pathway. Our data demonstrate that small interfering RNA-driven elimination of MR in cardiac fibroblasts does not inhibit aldosterone-induced IGF-IR phosphorylation and subsequent increase in elastin production. These results exclude the involvement of the MR in aldosterone-induced increases in elastin production. Results of further experiments aimed at identifying the upstream signaling component(s) that might be activated by aldosterone also eliminate the putative involvement of pertussis toxin-sensitive Gαi proteins, which have previously been shown to be responsible for some MR-independent effects of aldosterone. Instead, we found that small interfering RNA-dependent elimination of another heterotrimeric G protein, Gα13, eliminates aldosterone-induced elastogenesis. We further demonstrate that aldosterone first engages Gα13 and then promotes its transient interaction with c-Src, which constitutes a prerequisite step for aldosterone-dependent activation of the IGF-IR and propagation of consecutive downstream elastogenic signaling involving phosphatidylinositol 3-kinase/Akt. In summary, the data we present reveal new details of an MR-independent cellular signaling pathway through which aldosterone stimulates elastogenesis in human cardiac fibroblasts.
      Aldosterone is a major component of the renin-angiotensin-aldosterone system, which plays an important role in the regulation of electrolyte and fluid balance (
      • Jennings D.L.
      • Kalus J.S.
      • O'Dell K.M.
      ,
      • Connell J.M.
      • Davies E.
      ). The majority of aldosterone-induced effects occur after it binds to the intracellular MR. The activated aldosterone-MR complex translocates to the nucleus, where it modulates the transcription and translation of “aldosterone-induced” proteins involved in blood pressure homeostasis.
      Aldosterone has also been implicated in the stimulation of collagen synthesis and myocardial fibrosis through a process that is independent of its effect on blood pressure (
      • Weber K.T.
      • Brilla C.G.
      ,
      • Brilla C.G.
      • Matsubara L.S.
      • Weber K.T.
      ,
      • Young M.
      • Fullerton M.
      • Dilley R.
      • Funder J.
      ). Two clinical studies, the Randomized Aldactone Evaluation Study (
      • Pitt B.
      • Zannad F.
      • Remme W.J.
      • Cody R.
      • Castaigne A.
      • Perez A.
      • Palensky J.
      • Wittes J.
      ) and the Eplerenone Post-acute Myocardial Infarction Heart Failure Efficacy and Survival Study (
      • Pitt B.
      • Remme W.
      • Zannad F.
      • Neaton J.
      • Martinez F.
      • Roniker B.
      • Bittman R.
      • Hurley S.
      • Kleiman J.
      • Gatlin M.
      ), demonstrated that low doses of MR
      The abbreviations used are: MR
      mineralocorticoid receptor
      ECM
      extracellular matrix
      IGF-I
      insulin-like growth factor-I
      IGF-IR
      IGF-I receptor
      BSA
      bovine serum albumin
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      GPCR
      G protein-coupled receptor
      JNK
      c-Jun N-terminal kinase
      Rb
      retinoblastoma protein
      siRNA
      small interfering RNA
      PI
      phosphatidylinositol 3-kinase
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      RT
      reverse transcription
      MEK
      MAPK/ERK kinase.
      2The abbreviations used are: MR
      mineralocorticoid receptor
      ECM
      extracellular matrix
      IGF-I
      insulin-like growth factor-I
      IGF-IR
      IGF-I receptor
      BSA
      bovine serum albumin
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      GPCR
      G protein-coupled receptor
      JNK
      c-Jun N-terminal kinase
      Rb
      retinoblastoma protein
      siRNA
      small interfering RNA
      PI
      phosphatidylinositol 3-kinase
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      RT
      reverse transcription
      MEK
      MAPK/ERK kinase.
      antagonists lead to a dramatic reduction in the mortality rate of patients who suffered acute myocardial infarctions. Despite the suggestion that these MR antagonists may alleviate maladaptive remodeling of the extracellular matrix (ECM) of post-infarct hearts (
      • Zannad F.
      • Alla F.
      • Dousset B.
      • Perez A.
      • Pitt B.
      ,
      • Zannad F.
      • Dousset B.
      • Alla F.
      ), the molecular mechanisms by which they improve overall heart function in those patients have not been fully resolved.
      It has been also shown that aldosterone can induce numerous effects in a wide range of nonepithelial tissues, including heart, and that it may act through membrane receptors other than the traditional MR (alternative receptors) in epithelial and nonepithelial tissue in a nongenomic manner (
      • Christ M.
      • Sippel K.
      • Eisen C.
      • Wehling M.
      ,
      • Haseroth K.
      • Gerdes D.
      • Berger S.
      • Feuring M.
      • Günther A.
      • Herbst C.
      • Christ M.
      • Wehling M.
      ,
      • Chai W.
      • Garrelds I.M.
      • de Vries R.
      • Batenburg W.W.
      • van Kats J.P.
      • Danser A.H.
      ,
      • Gekle M.
      • Silbernagl S.
      • Wünsch S.
      ).
      Although the classical genomic model of aldosterone action has long been accepted, the rapid, nongenomic mechanism of aldosterone action is not yet fully elucidated (
      • Connell J.M.
      • Davies E.
      ). However, it has been proposed that some of these nongenomic effects of aldosterone also require the presence of MR or a closely related protein (
      • Mihailidou A.S.
      ). In contrast, other studies have shown that the nongenomic aldosterone effects still occur in cell lines lacking the classical MR and in yeast devoid of MR or in normal cells treated with MR antagonists (
      • Connell J.M.
      • Davies E.
      ,
      • Haseroth K.
      • Gerdes D.
      • Berger S.
      • Feuring M.
      • Günther A.
      • Herbst C.
      • Christ M.
      • Wehling M.
      ,
      • Böhmer S.
      • Carapito C.
      • Wilzewski B.
      • Leize E.
      • Van Dorsselaer A.
      • Bernhardt R.
      ). Such results strongly suggest the involvement of other receptor(s), distinct from the classic MR, that may interact with aldosterone and trigger the nongenomic effects of this hormone. Although full structural characterization of this putative receptor (or receptors) has not been completed yet (
      • Eisen C.
      • Meyer C.
      • Christ M.
      • Theisen K.
      • Wehling M.
      ), data suggest that some MR-independent effects of aldosterone occur after activation of the pertussis toxin-sensitive heterotrimeric G proteins (
      • Gekle M.
      • Silbernagl S.
      • Wünsch S.
      ,
      • Urbach V.
      • Harvey B.J.
      ).
      Results of our previous studies have revealed a novel mechanism in which aldosterone and its antagonists modulate the production of elastin, an important ECM component that provides resilience to many tissues, including stroma of the heart. We discovered that aldosterone can stimulate elastogenesis in cultures of human cardiac fibroblasts via an MR-independent mechanism involving IGF-IR activation (
      • Bunda S.
      • Liu P.
      • Wang Y.
      • Liu K.
      • Hinek A.
      ). We have therefore uncovered another level of complexity in which aldosterone in conjunction with MR antagonists may modulate the remodeling of the injured heart.
      In the present study we provide compelling evidence demonstrating that cultured cardiac fibroblasts, in which the production of MR has been inhibited by siRNA, still exhibit the aldosterone-induced increase in elastin production. We also present the first evidence that this MR-independent elastogenic effect of aldosterone can be triggered by a signaling pathway that involves initial activation of the heterotrimeric G protein Gα13 and consecutive activation of c-Src, IGF-IR, and PI 3-kinase/Akt signaling.

      DISCUSSION

      We previously reported that aldosterone stimulates elastogenesis via IGF-IR signaling in both fetal and adult and cultures of human cardiac fibroblasts, even in the presence of the MR-antagonist spironolactone (
      • Bunda S.
      • Liu P.
      • Wang Y.
      • Liu K.
      • Hinek A.
      ). Results of the experiments presented in this report additionally demonstrate that aldosterone still induces elastogenesis in cardiac fibroblast cultures in which the synthesis of MR protein is inhibited by the use of MR-specific siRNA oligonucleotides. Thus, these data further confirm that the elastogenic effect of aldosterone is executed via an MR-independent mechanism. Moreover, we have established that membrane-impermeable, BSA-conjugated aldosterone produces the same magnitude of IGF-IR phosphorylation as equimolar concentrations of free aldosterone (Fig. 1). This suggests that the signaling pathway leading to the MR-independent elastogenic effect of aldosterone may be initiated after the interaction of this steroid hormone with a certain moiety residing on the cell surface of cardiac fibroblasts. This assumption is further supported by other studies that have demonstrated the existence of high affinity membrane-binding sites for aldosterone in human vascular endothelium (
      • Wildling L.
      • Hinterdorfer P.
      • Kusche-Vihrog K.
      • Treffner Y.
      • Oberleithner H.
      ) human mononuclear leukocytes (
      • Wehling M.
      • Eisen C.
      • Christ M.
      ) and in pig kidneys (
      • Christ M.
      • Sippel K.
      • Eisen C.
      • Wehling M.
      ) and livers (
      • Meyer C.
      • Christ M.
      • Wehling M.
      ). It has also been suggested that a 50-kDa protein may meet the criteria for the alternative cell surface receptor for aldosterone (
      • Wehling M.
      • Eisen C.
      • Christ M.
      ). However, this putative aldosterone receptor that mediates MR-independent action has not been characterized yet.
      Because previous reports have suggested that G protein-coupled receptors (GPCRs) are involved in the propagation of certain steroid receptor-independent effects of other steroid hormones in animals (
      • Losel R.M.
      • Wehling M.
      ,
      • Zhu Y.
      • Rice C.D.
      • Pang Y.
      • Pace M.
      • Thomas P.
      ) and humans (
      • Filardo E.J.
      • Quinn J.A.
      • Bland K.I.
      • Frackelton Jr., A.R.
      ,
      • Filardo E.J.
      • Quinn J.A.
      • Frackelton Jr., A.R.
      • Bland K.I.
      ,
      • Filardo E.J.
      • Quinn J.A.
      • Sabo E.
      ,
      • Zhu Y.
      • Bond J.
      • Thomas P.
      ) and that some MR-independent effects of aldosterone can also be mediated through pertussis toxin-sensitive Gαi proteins (
      • Gekle M.
      • Silbernagl S.
      • Wünsch S.
      ,
      • Urbach V.
      • Harvey B.J.
      ), we first investigated whether Gαi would propagate the elastogenic effect of aldosterone. However, the results of our experiments, as depicted in Fig. 2, excluded the possibility that activation of Gαi may be involved in aldosterone-induced elastogenesis. Instead, we demonstrated for the first time that another heterotrimeric Gα protein, a member of the G12 subfamily, Gα13, participates in a cellular response to aldosterone that involves IGF-IR activation and a consequent enhancement of elastogenesis. This conclusion was based on data indicating that the elimination of Gα13 in cultured cardiac fibroblasts by MR-specific siRNA oligonucleotides completely attenuated the aldosterone-induced increase in IGF-IR phosphorylation and subsequent elastin production (FIGURE 3, FIGURE 4). At the same time we also demonstrated that the absence of the Gα13 protein did not eliminate the elastogenic response of IGF-I (Fig. 4). This also reinforced our belief that Gα13 is located upstream of the IGF-IR in the elastogenic signaling pathway triggered by aldosterone.
      Although previous studies have also shown that Gα13 can stimulate the activation of the cytosolic tyrosine kinase c-Src in various cell types, including cardiac fibroblast cultures (
      • Nishida M.
      • Onohara N.
      • Sato Y.
      • Suda R.
      • Ogushi M.
      • Tanabe S.
      • Inoue R.
      • Mori Y.
      • Kurose H.
      ,
      • Kim S.
      • Jin J.
      • Kunapuli S.P.
      ,
      • Adarichev V.A.
      • Vaiskunaite R.
      • Niu J.
      • Balyasnikova I.V.
      • Voyno-Yasenetskaya T.A.
      ,
      • Kim M.
      • Nozu F.
      • Kusama K.
      • Imawari M.
      ,
      • Minuz P.
      • Fumagalli L.
      • Gaino S.
      • Tommasoli R.M.
      • Degan M.
      • Cavallini C.
      • Lecchi A.
      • Cattaneo M.
      • Lechi Santonastaso C.
      • Berton G.
      ,
      • Dib K.
      • Melander F.
      • Andersson T.
      ,
      • Klages B.
      • Brandt U.
      • Simon M.I.
      • Schultz G.
      • Offermanns S.
      ), the results of our co-immunoprecipitation experiments demonstrated that treatment with aldosterone enhances the transient interaction between Gα13 and c-Src (Fig. 6). Because the inactivation of c-Src (by its specific PP2 inhibitor) eliminated the elastogenic effect of aldosterone, we concluded that the action of this kinase constitutes a prerequisite for the propagation of the aldosterone-dependent elastogenic signal (FIGURE 5, FIGURE 6).
      It has been previously shown that Gα13 can directly bind and activate various proteins (
      • Kurose H.
      ), including cytosolic tyrosine kinases such as Pyk2 (
      • Shi C.S.
      • Sinnarajah S.
      • Cho H.
      • Kozasa T.
      • Kehrl J.H.
      ). Currently, we do not know whether the aldosterone-triggered interaction between Gα13 and c-Src is direct or whether it requires other proteins, such as Pyk2, that might bind and facilitate phosphorylation of c-Src (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ,
      • Li J.
      • Avraham H.
      • Rogers R.A.
      • Raja S.
      • Avraham S.
      ). We have established, however, that in aldosterone-treated cardiac fibroblasts, Gα13 stimulates phosphorylation of c-Src, via the Rho-independent pathway and that the consecutive steps of elastogenic signaling involve increased phosphorylation of the IGF-IR and its downstream PI 3-kinase/Akt signaling pathway (Fig. 7).
      It has been previously shown as well that c-Src may not only phosphorylate the IGF-IR on ligand-induced auto-phosphorylation sites but also significantly increase the phosphorylation of this receptor on Tyr1316 (
      • Peterson J.E.
      • Kulik G.
      • Jelinek T.
      • Reuter C.W.
      • Shannon J.A.
      • Weber M.J.
      ), which has been implicated as a potential PI 3-kinase-binding site (
      • Liu D.
      • Zong C.S.
      • Wang L.H.
      ,
      • Jiang Y.
      • Chan J.L.
      • Zong C.S.
      • Wang L.H.
      ). We may therefore speculate that aldosterone-induced Gα13/c-Src activation facilitates IGF-IR signaling by enhancing its Tyr1316 phosphorylation. This in turn selectively promotes the downstream PI 3-kinase/Akt pathway needed for elastogenesis but not the alternative IGF-IR-propagated mitogenic MAPK/ERK signaling pathway. Our speculation seems to be additionally endorsed by data indicating that the aldosterone-induced elastogenic effect was enhanced in the presence of the MEK inhibitor PD 98059. Also, treatment with an inhibitor (SP600125) inactivating JNK, another MAPK family member, did not diminish the elastogenic effect of aldosterone (Fig. 7, A and B).
      Because phosphorylation on Tyr1316 of the insulin receptor, which is closely related to the IGF-IR, has been shown to play an inhibitory role in mitogenic signaling (
      • Ando A.
      • Momomura K.
      • Tobe K.
      • Yamamoto-Honda R.
      • Sakura H.
      • Tamori Y.
      • Kaburagi Y.
      • Koshio O.
      • Akanuma Y.
      • Yazaki Y.
      ), we also speculate that the aldosterone-induced signaling enhancing phosphorylation of Tyr1316 on the IGF-IR may contribute to the mechanism maintaining the balance between signals stimulating differentiation and mitogenesis. Further studies are needed to confirm this concept.
      In this study we did not investigate further the already well disclosed elastogenic mechanism in which IGF-I induces an increase in elastin gene expression. It has been previously documented that in aortic smooth muscle cells (
      • Wolfe B.L.
      • Rich C.B.
      • Goud H.D.
      • Terpstra A.J.
      • Bashir M.
      • Rosenbloom J.
      • Sonenshein G.E.
      • Foster J.A.
      ,
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ,
      • Conn K.J.
      • Rich C.B.
      • Jensen D.E.
      • Fontanilla M.R.
      • Bashir M.M.
      • Rosenbloom J.
      • Foster J.A.
      ) IGF-I induces an increase in elastin gene expression via a derepressive mechanism involving the abrogation of Sp3, a retinoblastoma protein (Rb)-associated element, that allows for activation of the elastin promoter by Rb on its retinoblastoma control element (
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ,
      • Conn K.J.
      • Rich C.B.
      • Jensen D.E.
      • Fontanilla M.R.
      • Bashir M.M.
      • Rosenbloom J.
      • Foster J.A.
      ). Because Rb lies downstream of the PI 3-kinase/Akt/mammalian target of rapamycin signaling pathway (
      • Mita M.M.
      • Mita A.
      • Rowinsky E.K.
      ), we may speculate that the aldosterone-dependent activation of this signaling pathway also modulates the interaction between Rb and pro-elastogenic transcription factors (
      • Wolfe B.L.
      • Rich C.B.
      • Goud H.D.
      • Terpstra A.J.
      • Bashir M.
      • Rosenbloom J.
      • Sonenshein G.E.
      • Foster J.A.
      ,
      • Jensen D.E.
      • Rich C.B.
      • Terpstra A.J.
      • Farmer S.R.
      • Foster J.A.
      ,
      • Conn K.J.
      • Rich C.B.
      • Jensen D.E.
      • Fontanilla M.R.
      • Bashir M.M.
      • Rosenbloom J.
      • Foster J.A.
      ), leading to an increase in elastin gene expression in cardiac fibroblasts. Because we found that inhibition of the promitogenic MAPK/ERK signaling pathway further enhanced the effect of aldosterone on elastin production (Fig. 7, A and B), we may also suggest that the PI 3-kinase/Akt signaling pathway induces elastogenesis by altering the phosphorylation state of Rb, whereas the mitogenic MAPK/ERK pathway antagonizes this effect. Interestingly, a similar pro-elastogenic effect involving the PI 3-kinase/Akt signaling pathway has been reported in lung fibroblasts after exposure to transforming growth factor-β (
      • Kuang P.P.
      • Zhang X.H.
      • Rich C.B.
      • Foster J.A.
      • Subramanian M.
      • Goldstein R.H.
      ).
      In summary, the data presented in this study suggest that the elastogenic effect of aldosterone in cardiac fibroblasts is propagated through the MR-independent action of this hormone. This novel mechanism likely involves a still unidentified GPCR (or GPCRs) that couples to Gα13 to stimulate c-Src, which in turn facilitates the activation of tyrosine kinase-dependent phosphorylation of the IGF-IR and its downstream PI 3-kinase signaling pathway (Fig. 8). This signaling pathway ultimately leads to the up-regulation of the elastin gene and the efficient production of elastic fibers by cardiac fibroblasts. We speculate that the heightened production of elastic fibers that results from the MR-independent action of aldosterone may counterbalance MR-mediated maladaptive fibrosis in the post-infarct heart in patients using MR antagonists, thus providing resilience to the cardiac stroma and facilitating normal ventricular function.
      Figure thumbnail gr8
      FIGURE 8Proposed mechanism by which aldosterone increases elastin production in cardiac fibroblast cultures. Aldosterone interacts with a still unidentified GPCR that causes the activation of Gα13. Activated Gα13, in turn, interacts with cytosolic c-Src. This interaction facilitates the activation of IGF-IR-IRS/PI 3-kinase/Akt signaling, which occurs even in the presence of sub-physiological levels of IGF-I, and subsequently induces increased elastin transcription and production. This effect of aldosterone is not dependent on the presence of the MR.

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