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

The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-beta1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-beta1/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 approximately 60-70 myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited alpha-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 Gi/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 alpha-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-beta1-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.