The antifibrotic effects of relaxin in human renal fibroblasts are mediated in part by inhibition of the Smad2 pathway.
BACKGROUND
The peptide hormone relaxin has been demonstrated to exert antifibrotic effects in renal and extrarenal tissues. The aims of this study were to identify potential anti-fibrotic effects of relaxin on human renal fibroblasts in vitro and to analyze their mechanisms.


METHODS
All experiments were performed in established renal fibroblast cell lines and in primary cortical fibroblasts. Effects of relaxin were analyzed on cell proliferation, apoptosis, activation of renal fibroblasts, synthesis and secretion of collagen type I and fibronectin, as well as on the secretion of matrix metalloproteinases (MMPs). Effects on transforming growth factor-beta1 (TGF-beta1) receptor binding were analyzed by flow cytometry and on TGF-beta1 signal transduction by immunoblot analyses for Smad4 and 7, translocation from cytosol to nucleus for Smad2 and 3 as well as for phosphorylated and unphosphorylated forms of p38, c-Jun NH2 terminal kinase (JNK) and extracellular-regulated protein kinase (ERK). Finally, specific siRNAs for Smad2 and 3 were applied to assess the signal transduction pathway.


RESULTS
After stimulation with relaxin, tyrosine phosphorylation of a 220 kD protein was demonstrated, indicating interaction with the receptor. Relaxin had only modest inhibitory effects on cell proliferation, and no effects on apoptosis. Conversely, relaxin exerted robust effects on TGF-beta1-induced fibroblast to myofibroblast transformation as well as on matrix synthesis and secretion even at the smallest dose tested. The secretion of MMP-2 and MMP-9 was induced noticeably by all investigated relaxin concentrations. TGF-beta1 receptor binding was not influenced by relaxin; however, it prevented Smad2 phosphorylation, translocation to nucleus, and complex formation between Smad2 and 3 indicating a possible interaction with TGF-beta1 signaling. These findings were corroborated by studies using siRNAs to Smad2 and 3 where siRNA to Smad2 but not to Smad3 inhibited the TGF-beta1 induction of fibronectin synthesis. There was no influence of relaxin on intracellular Smad3, Smad4, and Smad7 translocation or phosphorylation of mitogen-activated protein (MAP) kinases.


CONCLUSIONS
Relaxin is a potent inhibitor of TGF-beta1-induced extracellular matrix (ECM) synthesis and secretion as well as fibroblast activation. Furthermore, it induces ECM degradation by induction of MMP-2 and MMP-9. These effects are mediated, at least in part, by inhibition of TGF-beta1 signaling.
Heeg
Malte H J
MH
Department of Nephrology and Rheumatology, Georg-August-University, Göttingen, Germany.
Koziolek
Michael J
MJ
Vasko
Radovan
R
Schaefer
Liliana
L
Sharma
Kumar
K
Müller
Gerhard A
GA
Strutz
Frank
F
D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005355:Fibrosis; D006801:Humans; D007668:Kidney / Q000473:pathology*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009336:Necrosis; D010766:Phosphorylation / Q000187:drug effects; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D012065:Relaxin / Q000494:pharmacology*; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014443:Tyrosine; D012065:Relaxin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9