The anti-fibrotic actions of relaxin are mediated through AT2R-associated protein phosphatases via RXFP1-AT2R functional crosstalk in human cardiac myofibroblasts

Chao Wang, Anita A. Pinar, Robert E. Widdop, Mohammed A. Hossain, Ross A.D. Bathgate, Kate M. Denton, Barbara K. Kemp-Harper, Chrishan S. Samuel

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT2R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT2R-agonist, Compound 21 (C21; 1 μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 μM) or AT2R antagonist (0.1 μM) to confirm RXFP1-AT2R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT2R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 μM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT2R, confirming RXFP1-AT2R crosstalk in these cells. HCMFs were found to express AT2R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P <.05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT2R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT2R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.

Original languageEnglish
Number of pages17
JournalThe FASEB Journal
DOIs
Publication statusAccepted/In press - 16 Apr 2020

Keywords

  • AT receptor
  • cardiac fibrosis
  • myofibroblasts
  • phosphatases
  • RXFP1

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