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Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2

Ishanee Mookerjee^*,, Tim D. Hewitson^,, Michelle L. Halls^||, Roger J. Summers^||, Michael L. Mathai^*,¶, Ross A. D. Bathgate^*,, Geoffrey W. Tregear^*, and Chrishan S. Samuel^*,,1

^* Howard Florey Institute and

Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia;

Department of Nephrology and

Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Victoria, Australia;

^|| Department of Pharmacology, Monash University, Victoria, Australia; and

^¶ School of Biomedical and Health Sciences, Victoria University, Victoria, Australia

¹Correspondence: Howard Florey Institute, University of Melbourne, Parkville, VIC 3010, Australia. E-mail: chrishan.samuel{at}florey.edu.au

The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-β1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-β1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene-2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing 60–70% myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of -smooth muscle actin (-SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited -SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of G_i/o inhibition, and a sustained increase in extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on -SMA and Smad2 phosphorylation, while the NO inhibitor, L-nitroarginine methyl ester (hydrochloride) (L-NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS-NO-cGMP-dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF-β1-mediated renal myofibroblast differentiation and collagen production.—Mookerjee, I., Hewitson, T. D., Halls, M. L., Summers, R. J., Mathai, M. L., Bathgate, R. A. D., Tregear, G. W., Samuel, C. S. Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2.

Key Words: tubulointerstitial fibrosis • transforming growth factor-β1 • -smooth muscle actin • cell signaling