Aldosterone stimulates elastogenesis in cardiac fibroblasts via mineralocorticoid receptor-independent action involving the consecutive activation of Galpha13, c-Src, the insulin-like growth factor-I receptor, and phosphatidylinositol 3-kinase/Akt

Abstract

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 Galphai 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, Galpha13, eliminates aldosterone-induced elastogenesis. We further demonstrate that aldosterone first engages Galpha13 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.

FIGURE 1.

Eliminating the production of MR with siRNA-specific oligonucleotides in cultures of human cardiac fibroblast does not affect aldosterone-induced increases in elastin production. A, representative Western blots of cellular lysates from cultures that were transfected for either 96 or 144 h with scrambled and MR siRNA-specific oligonucleotides. B, immunohistochemistry with anti-MR antibody confirmed that production of MR was completely attenuated in cultures that were transfected for 144 h with MR siRNA. C, results of a quantitative assay of newly deposited insoluble elastin metabolically labeled with [³H]valine in cultures that were initially transfected for 72 h with scrambled or Gα₁₃ siRNA and then transfected again for an additional 72 h and kept in the presence or absence of 50 nm of aldosterone. D, representative photomicrographs immunostained with anti-elastin antibody confirm the results presented in C. E and F, the cultures were either transfected for 96 h with scrambled siRNA control and Gα₁₃ siRNA-specific oligonucleotides and treated for 10 min with or without 50 nm of aldosterone (E) or treated for 10 min with or without 50 nm of aldosterone, 50 nm of aldosterone conjugated to BSA (Aldo-BSA), or 100 ng/ml of IGF-I (F). The cell lysates were immunoprecipitated with an IGF-IR antibody and probed with an anti-phosphotyrosine (anti-phospho-Tyr) antibody or anti-IGF-IR antibody. The graphs depict the means ± S.D. of data from three individual experiments, expressed as percentages of control phosphorylation values obtained by normalizing to the corresponding total level of IGF-IR. ctr, control.

FIGURE 2.

The Gα_i inhibitor pertussis toxin (PTX) does not attenuate aldosterone-induced increases in elastin production in human cardiac fibroblast cultures. A, results of one-step RT-PCR analysis assessing elastin mRNA transcripts (normalized for GAPDH) in cultures treated for 24 h with or without 50 nm of aldosterone prior to 1 h of preincubation with 0.5 mg/ml of PTX. B, results of a quantitative assay of [³H]valine-labeled insoluble elastin. C, immunohistochemistry with anti-elastin antibody of 1-h pretreated cultures with 0.5 mg/ml of PTX, following 72 h of incubation with 50 nm of aldosterone.

FIGURE 3.

Silencing Gα₁₃ expression/production in human cardiac fibroblast cultures. A, one-step RT-PCR analysis assessing Gα₁₃ and GAPDH transcript levels in a negative control culture, a scrambled siRNA control culture, and cultures containing Gα₁₃ and GAPDH siRNA-specific oligonucleotides, 24 h after transfection. GAPDH siRNA served as a positive control. B, representative Western blot of cellular lysates obtained from cultures that were transfected for either 48 h or for 48 h and then transfected again for an additional 72 h (120 h) with scrambled and Gα₁₃ siRNA-specific oligonucleotides and immunoblotted with anti-Gα₁₃ antibody. The blots were then stripped and reprobed with anti-Gα₁₂ and anti-β-actin antibodies. The graphs depict the densitometric evaluation of results obtained from three individual experiments. The means ± S.D. of data were expressed by normalizing Gα₁₃ mRNA levels to the corresponding levels of GAPDH mRNA transcripts (A) and as a percentage of scrambled control Gα₁₃ protein levels (B). ctr, control.

FIGURE 4.

Silencing Gα₁₃ expression/production in cardiac fibroblast cultures attenuates the aldosterone-induced increase in elastin production and IGF-IR phosphorylation but does not affect the collagen production. A, results of a one-step RT-PCR analysis assessing Gα₁₃, elastin, and GAPDH mRNA transcript levels of cultures transfected for 72 h with scrambled siRNA control and Gα₁₃ siRNA-specific oligonucleotides and treated for the last 24 h with or without 50 nm of aldosterone or 100 ng/ml of IGF-I. B, results of a quantitative assay of newly deposited [³H]valine-labeled insoluble elastin in cultures that were initially transfected for 48 h with scrambled or Gα₁₃ siRNA and then transfected again for an additional 72 h and kept in the presence or absence of 50 nm of aldosterone or 100 ng/ml of IGF-I. C, representative photomicrographs of cultures immunostained with anti-elastin antibody confirm the results presented in B. D, cultures transfected for 72 h with scrambled siRNA control and Gα₁₃ siRNA oligonucleotides were treated for 10 min with or without 50 nm of aldosterone or 100 ng/ml of IGF-I. The cell lysates were immunoprecipitated with an IGF-IR antibody and probed with anti-phosphotyrosine antibody or anti-IGF-IR antibody. E, results of a one-step RT-PCR analysis assessing collagen type I and GAPDH mRNA transcript levels of cultures transfected for 72 h with scrambled siRNA control and Gα₁₃ siRNA-specific oligonucleotides and treated for the last 24 h with or without 50 nm of aldosterone. F, representative photomicrographs of cultures immunostained with anti-collagen antibody that were initially transfected for 48 h with scrambled or Gα₁₃ siRNA and then transfected again for an additional 72 h and kept in the presence or absence of 50 nm of aldosterone. ctr, control.

FIGURE 5.

c-Src tyrosine kinase inhibitor PP2 eliminates aldosterone-induced increases in elastin production in human cardiac fibroblast cultures. A, results of a one-step RT-PCR analysis assessing elastin and GAPDH mRNA transcripts in cultures maintained for 24 h in the presence or absence of 50 nm aldosterone, with or without 1 h pretreatment with 10 μm of PP2. B, levels of [³H]valine-labeled insoluble elastin in cultures 1 h pretreated with PP2 following 72 h of incubation with 50 nm of aldosterone. C, representative photomicrographs of confluent cultures immunostained with anti-elastin antibody confirm the results presented in B. ctr, control.

FIGURE 6.

Aldosterone treatment increases the interaction between Gα₁₃ and c-Src, leading to activation of c-Src in human cardiac fibroblast cultures. A, cellular lysates of cardiac fibroblast cultures treated with or without 50 nm of aldosterone for 1, 5, and 10 min were immunoprecipitated with anti c-Src antibody and probed with an anti-Gα₁₃ antibody or anti-c-Src antibody. B, cellular lysates obtained from cultures treated with or without aluminum fluoride for 30 min were immunoprecipitated with anti-Gα₁₃ antibody (left panel) or anti-c-Src antibody (right panel) and probed with an anti-c-Src antibody or anti-Gα₁₃ antibody, respectively. C and D, Western blot analysis of cellular lysates obtained from cultures treated with or without 50 nm of aldosterone for 10 min (C) after they were preincubated for 1 h in the presence or absence of 10 μm of PP2 or (D) following 72 h of transfection with scrambled siRNA control and Gα₁₃ siRNA oligonucleotides and immunoblotted with anti-phospho-c-Src (Tyr⁴¹⁶) and then reprobed with anti-c-Src antibody. ctr, control.

FIGURE 7.

Aldosterone- or IGF-I-induced increases in elastin production in human cardiac fibroblast cultures is propagated via the PI 3-kinase/Akt signaling pathway. A and B, results of a quantitative assay of [³H]valine-labeled insoluble elastin (A) and immunohistochemistry with anti-elastin antibody of cultures pretreated for an hour with or without 10 μm of PD 98059 or SP600125 prior to 72 h treatment with 50 nm of aldosterone (B). C, results of a one-step RT-PCR analysis assessing elastin and GAPDH mRNA transcripts in cultures maintained for 24 h in the presence or absence of 50 nm aldosterone or 100 ng/ml of IGF-I, prior to 1 h pretreatment with 1 μm of wortmannin. D, results of a quantitative assay of [³H]valine-labeled insoluble elastin of cultures treated for 72 h with 50 nm of aldosterone or with 100 ng/ml of IGF-I prior to 1 h pretreatment with 1 μm of wortmannin. E and F, Western blot analysis of cellular lysates obtained from cultures treated for 10 min with or without 50 nm of aldosterone after they were preincubated for 1 h in the presence or absence of 10 μm of PP2 (E) or treated for 10 min with aldosterone or 100 ng/ml of IGF-I following 72 h of transfection with scrambled siRNA control and Gα₁₃ siRNA-specific oligonucleotides (F) and immunoblotted using anti-phospho-Akt (Ser⁴⁷³) antibody and then stripped and reprobed with anti-Akt antibody. G, one-step RT-PCR analysis assessing elastin and GAPDH mRNA transcripts in cultures treated for 30 min or 1, 3, 6, or 16 h with or without 50 nm of aldosterone. ctr, control.

FIGURE 8.

Proposed 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.