THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE
INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cycl...
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doaj-067a8e7a4f664c2bae20e57d81baaff92020-11-24T22:25:12ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122016-03-01710.3389/fphar.2016.00091190302THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATEChao eWang0Barbara Kathryn Kemp-Harper1Martina eKocan2Sheng Yu eAng3Tim David Hewitson4Chrishan S Samuel5Monash UniversityMonash UniversityDrug Discovery Biology, Monash Institute of Pharmacurtical Sciences, Monash UniversityDrug Discovery Biology, Monash Institute of Pharmacurtical Sciences, Monash UniversityRoyal Melbourne HospitalMonash UniversityINTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin’s effects with that of an NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5uM) or the combined effects of RLX (16.8nM) and DEA/NO (5uM) over 72 hours. The effects of RLX (16.8nM) and DEA/NO (5uM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100uM) or sGC inhibitor, ODQ (5uM) over 72 hours. Furthermore, the effects of RLX (30nM), DEA/NO (5uM) and RLX (30nM)+DEA/NO (5uM) on cGMP levels were directly measured, in the presence or absence of ODQ (5uM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p<0.05 vs untreated and vehicle-treated cells). However, 5uM of DEA/NO was required to produce the effects seen with 16.8nM of RLX over 72 hours. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p<0.01 vs RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p<0.05 vs RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p<0.01 vs vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p<0.05 vs the respective treatments alone). CONCLUSIONS: These findings confirmed that RLX mediates its TGF-beta1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO.http://journal.frontiersin.org/Journal/10.3389/fphar.2016.00091/fullFibrosisMyofibroblastsNitric OxideRelaxinCell signalingcGMP |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chao eWang Barbara Kathryn Kemp-Harper Martina eKocan Sheng Yu eAng Tim David Hewitson Chrishan S Samuel |
spellingShingle |
Chao eWang Barbara Kathryn Kemp-Harper Martina eKocan Sheng Yu eAng Tim David Hewitson Chrishan S Samuel THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE Frontiers in Pharmacology Fibrosis Myofibroblasts Nitric Oxide Relaxin Cell signaling cGMP |
author_facet |
Chao eWang Barbara Kathryn Kemp-Harper Martina eKocan Sheng Yu eAng Tim David Hewitson Chrishan S Samuel |
author_sort |
Chao eWang |
title |
THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE |
title_short |
THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE |
title_full |
THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE |
title_fullStr |
THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE |
title_full_unstemmed |
THE ANTI-FIBROTIC ACTIONS OF RELAXIN ARE MEDIATED THROUGH A NO-sGC-cGMP-DEPENDENT PATHWAY IN RENAL MYOFIBROBLASTS IN VITRO AND ENHANCED BY THE NO DONOR, DIETHYLAMINE NONOATE |
title_sort |
anti-fibrotic actions of relaxin are mediated through a no-sgc-cgmp-dependent pathway in renal myofibroblasts in vitro and enhanced by the no donor, diethylamine nonoate |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Pharmacology |
issn |
1663-9812 |
publishDate |
2016-03-01 |
description |
INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin’s effects with that of an NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5uM) or the combined effects of RLX (16.8nM) and DEA/NO (5uM) over 72 hours. The effects of RLX (16.8nM) and DEA/NO (5uM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100uM) or sGC inhibitor, ODQ (5uM) over 72 hours. Furthermore, the effects of RLX (30nM), DEA/NO (5uM) and RLX (30nM)+DEA/NO (5uM) on cGMP levels were directly measured, in the presence or absence of ODQ (5uM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p<0.05 vs untreated and vehicle-treated cells). However, 5uM of DEA/NO was required to produce the effects seen with 16.8nM of RLX over 72 hours. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p<0.01 vs RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p<0.05 vs RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p<0.01 vs vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p<0.05 vs the respective treatments alone). CONCLUSIONS: These findings confirmed that RLX mediates its TGF-beta1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO. |
topic |
Fibrosis Myofibroblasts Nitric Oxide Relaxin Cell signaling cGMP |
url |
http://journal.frontiersin.org/Journal/10.3389/fphar.2016.00091/full |
work_keys_str_mv |
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