pubs.acs.org/biochemistry Published on Web 01/13/2011 r 2011 American Chemical Society 1368 Biochemistry 2011, 50, 1368–1375 DOI: 10.1021/bi1013968 H3 Relaxin Demonstrates Antifibrotic Properties via the RXFP1 Receptor † Mohammed Akhter Hossain, ‡,§,# Bryna Chow Suet Man, ‡, ) ,# Chongxin Zhao, ‡ Qi Xu, ^ Xiao-Jun Du, ^ John D. Wade, ‡,§ and Chrishan S. Samuel* ,‡, ) ‡ Howard Florey Institute, § School of Chemistry, and ) Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia, and ^ Baker IDI Heart and Diabetes Institute, St. Kilda Road Central, Melbourne, Victoria 8008, Australia. # M.A.H. and B.C.S.M. contributed equally to the manuscript Received August 30, 2010; Revised Manuscript Received January 11, 2011 ABSTRACT: Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor β1 (TGF-β1) administration to ventricular fibroblasts significantly increased Smad2 phos- phorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metallopro- teinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-β1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-β1- stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-β1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-β1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin. Three nonallelic relaxin genes have been identified in humans, which produce H1, H2, 1 and H3 relaxin, respectively (1). Most other mammals including rodents, however, contain only two relaxin genes, which produce relaxin (equivalent to H2 relaxin) and relaxin-3 (equivalent to H3 relaxin) (1). H2 relaxin (and relaxin in other species) represent the major stored and circulat- ing forms of relaxin and have been the most investigated to date (1-7). A plethora of studies have demonstrated that H2 relaxin has several biological actions in the body, which are centered around its antiinflammatory, antifibrotic, antiapopto- tic, antihypertrophic, cardioprotective, vasodilatory, and proan- giogenic actions (1-7), many of which are mediated through its primary G-protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1) (8). On the other hand, comparatively little is known about the more recently discovered H3 relaxin (9, 10) and its highly conserved species equivalent, relaxin-3 (11). While the dis- tribution of H3 relaxin in humans remains unknown, anato- mical studies of relaxin-3 in rodents and primates suggest that it is predominantly expressed in the brain, particularly within neurons of the nucleus insertus (9, 10, 12, 13) in addition to nerve fibers and terminals within the cortex, hippocampus, thalamus, hypothalamus, and midbrain. Thus, H3 relaxin and relaxin-3 are thought to primarily function as neuromodula- tors and have been implicated in regulating arousal, feeding, learning, and memory and central responses to physiological stressors (14-16). Studies using recombinant (17) and chemically synthesized (18) H3 relaxin have identified it as the cognate ligand for relaxin family peptide receptor 3 (RXFP3; formerly known as GPCR- 135), which is also widely distributed in the brain (12, 13, 17). However, it has been demonstrated that H3 relaxin can bind to all four defined receptors of the relaxin family of peptides, including RXFP1 (18), RXFP2 (the primary receptor for insulin-like peptide 3) (18), and RXFP4 (the primary receptor for insulin- like peptide 5) (19, 20), suggesting that it may have additional roles through these receptors, other than its neuromodulatory actions via RXFP3. † This study was supported by a John T. Reid Charitable Trusts Fellowship to M.A.H., National Health and Medical Research Council (NHMRC) of Australia Senior Research Fellowships to X.-J.D. and J.D.W., a National Heart Foundation of Australia/NHMRC RD Wright Fellowship to C.S.S., and a NHMRC Project Grant (508995) to J.D.W. and by the Victorian Government’s Operational Infrastruc- ture Support Program. *To whom correspondence should be addressed at the Howard Florey Institute, The University of Melbourne. Phone: þ 61 3 8344 0416. Fax: þ61 3 9348 1707. E-mail: chrishan.samuel@florey.edu.au. 1 Abbreviations: AR, adrenergic receptors; H2, human gene 2; H3, human gene 3; GPCR, G-protein-coupled receptor; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; MMP, matrix metalloproteinase; RP-HPLC, reverse-phase high-performance liquid chromatography; RXFP, relaxin family peptide receptor; SMA, smooth muscle actin; TGF-β1, transforming growth factor β1; TIMP, tissue inhibitor of metalloproteinase.