PMID- 30927912 OWN - NLM STAT- MEDLINE DCOM- 20190809 LR - 20200225 IS - 1471-2474 (Electronic) IS - 1471-2474 (Linking) VI - 20 IP - 1 DP - 2019 Mar 30 TI - Investigating the effects of Pirfenidone on TGF-beta1 stimulated non-SMAD signaling pathways in Dupuytren's disease -derived fibroblasts. PG - 135 LID - 10.1186/s12891-019-2486-3 [doi] LID - 135 AB - BACKGROUND: Dupuytren's disease (DD) is a progressive, debilitating condition of the hand that can eventually cause contractures of the affected fingers. Transforming growth factor- beta1 (TGF-beta1) has been reported to play a key role in DD pathology. Increased expression of TGF-beta1 has shown to be the main stimulator of myofibroblast activity and in DD contractures. Pirfenidone (PFD), a small active molecule possess the ability to inhibit TGF-beta1-mediated action in various fibrotic disorders. Our recent published findings show that PFD reduced TGF-beta1-mediated cellular functions implicated in DD through SMAD signaling pathways. In the present study, the effect of PFD on TGF-beta1-mediated non-SMAD signaling pathways were investigated in both carpal tunnel (CT) - and DD-derived fibroblasts. METHODS: Fibroblasts harvested from Dupuytren's disease (DD) and carpal tunnel (CT) tissues were cultured in the presence or absence of TGF-beta1 (10 ng/ml) and/or PFD (800 mug/ml). Cell lysates were analyzed using Western blots. Equal amounts of proteins were loaded to determine the phosphorylation levels of phosphatidylinositol-3 kinase (PI3K/AKT), extracellular regulated kinases (ERK1/2), p38 mitogen-activated protein kinase and Rho family related myosin light chain (MLC). RESULTS: We show that the TGF-beta1-induced phosphorylation of AKT was significantly decreased by the addition of PFD (800 mug/mL) in both CT- and DD-derived fibroblasts. Interestingly, there was no significant difference in the phosphorylation levels of both ERK and p38 on TGF-beta1- induced cells in both CT-and DD-derived fibroblasts. But, PFD significantly decreased the TGF- beta1-induced phosphorylation levels of ERK1/2 in both CT- and DD- cells. In contrast, PFD significantly decreased the basal and TGF- beta1-induced phosphorylation levels of p38 in DD-derived fibroblasts. TGF- beta1-induced phosphorylation levels of MLC was decreased by PFD in DD-derived fibroblasts. CONCLUSIONS: These in-vitro results indicate for the first time that PFD has the potential to inhibit TGF-beta1-induced non-SMAD signaling pathways in both CT- and DD-derived fibroblasts but pronounced statistically significant inhibition on all molecules was observed only in DD-derived fibroblasts. Our previous studies show that PFD can inhibit TGF-beta1- induced SMAD signaling pathway proteins, namely p- SMAD2/SMAD3. These broad and complementary actions suggest PFD as a promising candidate to inhibit the TGF-beta1- mediated molecular mechanisms leading to DD fibrosis. FAU - Zhou, Chaoming AU - Zhou C AD - Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, 15261, USA. FAU - Zeldin, Yael AU - Zeldin Y AD - Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, 15261, USA. FAU - Baratz, Mark E AU - Baratz ME AD - Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, 15261, USA. FAU - Kathju, Sandeep AU - Kathju S AD - McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 15219, USA. AD - Lumix Biomedical and Surgical Consulting, Pittsburgh, PA, USA. FAU - Satish, Latha AU - Satish L AD - Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, 15261, USA. lsatish@shrinenet.org. AD - Shriners Hospitals for Children-Cincinnati, Cincinnati, OH, 45229, USA. lsatish@shrinenet.org. AD - Department of Pathology and Laboratory Medicine, University of Cincinnati, 3229 Burnet Avenue, Cincinnati, OH, 45229, USA. lsatish@shrinenet.org. LA - eng PT - Journal Article DEP - 20190330 PL - England TA - BMC Musculoskelet Disord JT - BMC musculoskeletal disorders JID - 100968565 RN - 0 (Pyridones) RN - 0 (TGFB1 protein, human) RN - 0 (Transforming Growth Factor beta1) RN - D7NLD2JX7U (pirfenidone) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.11.24 (Mitogen-Activated Protein Kinases) MH - Carpal Tunnel Syndrome/pathology MH - Cells, Cultured MH - Dupuytren Contracture/*drug therapy/pathology/surgery MH - Fascia/cytology MH - Fibroblasts/*drug effects/metabolism MH - Humans MH - Mitogen-Activated Protein Kinases/metabolism MH - Phosphorylation/drug effects MH - Primary Cell Culture MH - Proto-Oncogene Proteins c-akt/metabolism MH - Pyridones/*pharmacology/therapeutic use MH - Signal Transduction/*drug effects MH - Transforming Growth Factor beta1/antagonists & inhibitors/*metabolism PMC - PMC6441192 OTO - NOTNLM OT - AKT OT - Carpal tunnel OT - Dupuytren's contracture OT - ERK1/2 OT - Palmar fascia fibrosis OT - p38MAPK and myosin light chain (MLC) COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: DD cord and CT fascial tissues were obtained during surgical resection from Division of Upper Extremity Surgery, Department of Orthopaedic Surgery, Allegheny General Hospital, Pittsburgh, PA. Allegheny-Singer Research Institute's Institutional Review Board (IRB protocol no. RC-4040) approved the protocol. Before the surgical procedure, all subjects signed the written informed consent forms. The study protocol strictly conformed to the ethical guidelines of the 1975 Declaration of Helsinki. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EDAT- 2019/04/01 06:00 MHDA- 2019/08/10 06:00 PMCR- 2019/03/30 CRDT- 2019/04/01 06:00 PHST- 2018/09/05 00:00 [received] PHST- 2019/03/03 00:00 [accepted] PHST- 2019/04/01 06:00 [entrez] PHST- 2019/04/01 06:00 [pubmed] PHST- 2019/08/10 06:00 [medline] PHST- 2019/03/30 00:00 [pmc-release] AID - 10.1186/s12891-019-2486-3 [pii] AID - 2486 [pii] AID - 10.1186/s12891-019-2486-3 [doi] PST - epublish SO - BMC Musculoskelet Disord. 2019 Mar 30;20(1):135. doi: 10.1186/s12891-019-2486-3.