FP25-2 Relaxin Signals Through Constitutive Heterodimers of Its Cognate Receptor, RXFP1, and the Angiotensin II Type 2 (AT2) Receptor to Abrogate Renal Fibrosis

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP25-Signaling Originating from Membrane Receptors
Basic/Translational
Sunday, June 16, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 10:50 AM
Room 133 (Moscone Center)

Poster Board SUN-390
Chrishan S Samuel*1, Bryna SM Chow2, Martina Kocan1, Sanja Bosnyak1, Mohsin Sarwar1, Emma S Jones1, Robert E Widdop1, Roger J Summers1, Ross A Bathgate2 and Tim D Hewitson3
1Monash University, Melbourne, Australia, 2Florey Institute for Neuroscience and Mental Health, Melbourne, Australia, 3Royal Melbourne Hospital, Melbourne, Australia
Background: The anti-fibrotic hormone, relaxin, disrupts the pro-fibrotic actions of TGF-β1 by signaling through its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation (pERK)1/2 and a nitric oxide (NO)-dependent pathway to abrogate Smad2 phosphorylation (pSmad2) in renal myofibroblasts. As angiotensin (Ang)-II also antagonizes TGF-β1 activity through its AT2 receptor (AT2R), we investigated for the first time, the extent to which relaxin interferes with the Ang II-TGF-β1 axis at the AT2R level.

Methods: Various markers of relaxin activity: pERK1/2, nNOS phosphorylation (pnNOS), TGF-β1, pSmad2, α-smooth muscle actin (SMA), matrix metalloproteinase (MMP)-13, MMP-2, MMP-9 and collagen concentration in primary renal myofibroblasts isolated from injured rats, and from kidney tissues isolated from relaxin-treated wild-type (AT2R+/+) and AT2R knockout (AT2R‒/‒) mice that were subjected to unilateral ureteric obstruction, in the absence or presence of the AT2R antagonist PD123319, were assessed by Western blotting, gelatin zymography and hydroxyproline assay. Additionally, the ability of relaxin and RXFP1 to interact with the AT2R was assessed by competition binding and bioluminescence resonance energy transfer (BRET) assays, respectively.

Results: Relaxin’s (100ng/ml; 16.8nM) ability to increase pERK, pnNOS, MMP-13, MMP-2 and MMP-9 levels, and inhibit TGF-β1, pSmad2 and myofibroblast differentiation (all p<0.01 vs that in untreated cells) was significantly blocked by PD123319 (0.1µM) in vitro (all p<0.01 vs relaxin-treated cells). Likewise, the collagen-inhibitory and anti-fibrotic actions of relaxin (0.5mg/kg/day) in vivo were completely lost when it was administered to AT2R‒/‒ mice or to AT2R+/+ mice in the presence of PD123319 (3mg/kg/day) (all p<0.05 vs that in relaxin-treated AT2R+/+ mice). While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs or affect the dynamics of pre-formed RXFP1-AT2R complexes, but mediated cross-talk through this receptor dimerization, at the pERK1/2 level, to induce its anti-fibrotic actions.

Conclusions: The AT2R is obligatory for relaxin to signal through a RXFP1/AT2R-pERK1/2-nNOS-NO-cGMP-dependent pathway to disrupt TGF-β1-mediated renal fibrosis. The relaxin-RXFP1-AT2R interaction represents a novel therapeutic target that may be regulated to control fibrosis progression.

Nothing to Disclose: CSS, BSC, MK, SB, MS, ESJ, REW, RJS, RAB, TDH

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: National Health & Medical Research Council (NHMRC) of Australia Project Grants: 436713, 454375, 628634 and APP1045848; and Senior Research Fellowships to C.S.S and R.A.B.