Estradiol attenuates the TGF-β1-induced conversion of primary TAFs into myofibroblasts and inhibits collagen production and myofibroblast contraction by modulating the Smad and Rho/ROCK signaling pathways

Jiang,He‑Song; Zhu,Lei‑Lei; Zhang,Zheng; Chen,Hai; Chen,Yun; Dai,Yu‑Tian

doi:10.3892/ijmm.2015.2288

Estradiol attenuates the TGF-β1-induced conversion of primary TAFs into myofibroblasts and inhibits collagen production and myofibroblast contraction by modulating the Smad and Rho/ROCK signaling pathways

Authors:
- He‑Song Jiang
- Lei‑Lei Zhu
- Zheng Zhang
- Hai Chen
- Yun Chen
- Yu‑Tian Dai
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Affiliations: Medical School, Nanjing University, Nanjing, Jiangsu, P.R. China, Department of Andrology, Nanjing Drum Tower Hospital, Nanjing Medical University, Nanjing, Jiangsu, P.R. China
Published online on: July 16, 2015 https://doi.org/10.3892/ijmm.2015.2288
Pages: 801-807

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Abstract

The transformation of tunica albuginea-derived fibroblasts (TAFs) into myofibroblasts plays an important role in the pathological progress of Peyronie's disease (PD). However, no treatment which addresses this transformation is currently available. Estrogen has been shown to inhibit the progression of fibrosis in a number of fibrotic diseases. The aim of this study was to determine whether estrogen [17β‑estradiol (E2)] suppresses the diffentiation of primary rat TAFs into myofibroblasts in vitro. TAFs obtained from male Sprague‑Dawley rats were stimulated with either transforming growth factor‑β1 (TGF‑β1) or E2. Western blot analysis and immunofluorescence staining were used to assess changes in the expression levels of α‑smooth muscle actin (αSMA). The expression levels of additional proteins (GAPDH, p‑Smad2, Smad2, Smad4, RhoA, Rac1, ROCK1 and ROCK2) were also measured by western blot analysis. We used collagen gel assays to assess cell contractility. Additionally, the concentration of hydroxyproline in the TAF cell culture medium was detected using commercially available kits. We found that E2 reduced αSMA expression which was induced by TGF‑β1. E2 also suppressed the TGF‑β1‑induced increase in the concentration of hydroxyproline (a marker of collagen) in addition to suppressing the contraction of TAFs. The key processes affected by TGF‑β1 treatment included the phosphorylation of Smad2, ras homolog gene family, member A (RhoA) and Rho‑associated, coiled-coil containing protein kinase 2 (ROCK2); this increase in phosphorylation was inhibited by treatment with E2. Collectively, these results demonstrate that by modulating the activation of the TGF‑β1‑Smad and RhoA‑ROCK2 signaling pathways, E2 inhibited the transformation of TAFs into myofibroblasts, decreased the expression of collagen and suppressed the contraction of myofibroblasts in response to TGF-β1 stimulation.

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1	Langston JP and Carson CC III: Peyronie's disease: review and recent advances. Maturitas. 78:341–343. 2014. View Article : Google Scholar : PubMed/NCBI
2	Jarow JP and Lowe FC: Penile trauma: an etiologic factor in Peyronie's disease and erectile dysfunction. J Urol. 158:1388–1390. 1997. View Article : Google Scholar : PubMed/NCBI
3	El-Sakka AI, Hassoba HM, Chui RM, Bhatnagar RS, Dahiya R and Lue TF: An animal model of Peyronie's-like condition associated with an increase of transforming growth factor beta mRNA and protein expression. J Urol. 158:2284–2290. 1997. View Article : Google Scholar : PubMed/NCBI
4	Valente EGA, Vernet D, Ferrini MG, Qian A, Rajfer J and Gonzalez-Cadavid NF: L-arginine and phosphodiesterase (PDE) inhibitors counteract fibrosis in the Peyronie's fibrotic plaque and related fibroblast cultures. Nitric Oxide. 9:229–244. 2003. View Article : Google Scholar
5	Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI and West AB: Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol. 277:C1–C9. 1999. View Article : Google Scholar : PubMed/NCBI
6	Gelbard M: Myofibroblasts and mechanotransduction: do forces in the tunica albuginea contribute to Peyronie's disease? J Sex Med. 5:2974–2976. 2008. View Article : Google Scholar : PubMed/NCBI
7	Vernet D, Ferrini MG, Valente EG, Magee TR, Bou-Gharios G, Rajfer J and Gonzalez-Cadavid NF: Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie's fibrotic plaque and in its rat model. Nitric Oxide. 7:262–276. 2002. View Article : Google Scholar : PubMed/NCBI
8	Derynck R and Zhang YE: Smad-dependent and Smad- independent pathways in TGF-beta family signalling. Nature. 425:577–584. 2003. View Article : Google Scholar : PubMed/NCBI
9	Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ and Lan HY: Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation. J Am Soc Nephrol. 13:1464–1472. 2002. View Article : Google Scholar : PubMed/NCBI
10	Schofield AV and Bernard O: Rho-associated coiled-coil kinase (ROCK) signaling and disease. Crit Rev Biochem Mol Biol. 48:301–316. 2013. View Article : Google Scholar : PubMed/NCBI
11	Di Martino V, Lebray P, Myers RP, Pannier E, Paradis V, Charlotte F, Moussalli J, Thabut D, Buffet C and Poynard T: Progression of liver fibrosis in women infected with hepatitis C: long-term benefit of estrogen exposure. Hepatology. 40:1426–1433. 2004. View Article : Google Scholar : PubMed/NCBI
12	Yang JD, Abdelmalek MF, Pang H, Guy CD, Smith AD, Diehl AM and Suzuki A: Gender and menopause impact severity of fibrosis among patients with nonalcoholic steatohepatitis. Hepatology. 59:1406–1414. 2014. View Article : Google Scholar :
13	Smyk DS, Rigopoulou EI, Pares A, Billinis C, Burroughs AK, Muratori L, Invernizzi P and Bogdanos DP: Sex differences associated with primary biliary cirrhosis. Clin Dev Immunol. 2012:6105042012. View Article : Google Scholar : PubMed/NCBI
14	Pedram A, Razandi M, O'Mahony F, Lubahn D and Levin ER: Estrogen receptor-beta prevents cardiac fibrosis. Mol Endocrinol. 24:2152–2165. 2010. View Article : Google Scholar : PubMed/NCBI
15	Morani A, Barros RPA, Imamov O, Hultenby K, Arner A, Warner M and Gustafsson JA: Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta^−/−) mice. Proc Natl Acad Sci USA. 103:7165–7169. 2006. View Article : Google Scholar
16	Dixon A and Maric C: 17beta-Estradiol attenuates diabetic kidney disease by regulating extracellular matrix and transforming growth factor-beta protein expression and signaling. Am J Physiol Renal Physiol. 293:F1678–F1690. 2007. View Article : Google Scholar : PubMed/NCBI
17	Novotný M, Vasilenko T, Varinská L, Smetana K Jr, Szabo P, Sarišský M, Dvořánková B, Mojžiš J, Bobrov N, Toporcerová S, et al: ER-α agonist induces conversion of fibroblasts into myofibroblasts, while ER-β agonist increases ECM production and wound tensile strength of healing skin wounds in ovariectomised rats. Exp Dermatol. 20:703–708. 2011. View Article : Google Scholar
18	Ahuja SK, Sikka SC and Hellstrom WJG: Stimulation of collagen production in an in vitro model for Peyronie's disease. Int J Impot Res. 11:207–212. 1999. View Article : Google Scholar : PubMed/NCBI
19	Illsley MC, Peacock JH, McAnulty RJ and Yarnold JR: Increased collagen production in fibroblasts cultured from irradiated skin and effect of TGF beta(1)- clinical study. Br J Cancer. 83:650–654. 2000. View Article : Google Scholar : PubMed/NCBI
20	Wong M and Mudera V: Feedback inhibition of high TGF-beta1 concentrations on myofibroblast induction and contraction by Dupuytren's fibroblasts. J Hand Surg Br. 31:473–483. 2006. View Article : Google Scholar : PubMed/NCBI
21	Kumar A, Ruan M, Clifton K, Syed F, Khosla S and Oursler MJ: TGF-β mediates suppression of adipogenesis by estradiol through connective tissue growth factor induction. Endocrinology. 153:254–263. 2012. View Article : Google Scholar :
22	Woessner JF Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys. 93:440–447. 1961. View Article : Google Scholar : PubMed/NCBI
23	Sun Z, Wang C, Shi C, Sun F, Xu X, Qian W, Nie S and Han X: Activated Wnt signaling induces myofibroblast differentiation of mesenchymal stem cells, contributing to pulmonary fibrosis. Int J Mol Med. 33:1097–1109. 2014.PubMed/NCBI
24	Lauriol J, Keith K, Jaffré F, Couvillon A, Saci A, Goonasekera SA, McCarthy JR, Kessinger CW, Wang J, Ke Q, et al: RhoA signaling in cardiomyocytes protects against stress-induced heart failure but facilitates cardiac fibrosis. Sci Signal. 7:ra1002014. View Article : Google Scholar : PubMed/NCBI
25	Ji H, Tang H, Lin H, Mao J, Gao L, Liu J and Wu T: Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation. Biomed Rep. 2:787–792. 2014.PubMed/NCBI
26	Manickam N, Patel M, Griendling KK, Gorin Y and Barnes JL: RhoA/Rho kinase mediates TGF-β1-induced kidney myofibroblast activation through Poldip2/Nox4-derived reactive oxygen species. Am J Physiol Renal Physiol. 307:F159–F171. 2014. View Article : Google Scholar : PubMed/NCBI
27	Castiglione F, Hedlund P, Van der Aa F, Bivalacqua TJ, Rigatti P, Van Poppel H, Montorsi F, De Ridder D and Albersen M: Intratunical injection of human adipose tissue-derived stem cells prevents fibrosis and is associated with improved erectile function in a rat model of Peyronie's disease. Eur Urol. 63:551–560. 2013. View Article : Google Scholar
28	Gonzalez-Cadavid NF and Rajfer J: Experimental models of Peyronie's disease. Implications for new therapies. J Sex Med. 6:303–313. 2009. View Article : Google Scholar : PubMed/NCBI
29	McKenna TJ: Oestrogens for women: the risk/benefit ratio. Ir Med J. 80:219–221. 1987.PubMed/NCBI
30	Studd J and Zamblera D: Estrogen therapy in women over 60 years of age. Gynecol Endocrinol. 8:191–196. 1994. View Article : Google Scholar : PubMed/NCBI
31	Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS and Lubahn DB: A role for oestrogens in the male reproductive system. Nature. 390:509–512. 1997. View Article : Google Scholar : PubMed/NCBI
32	Hill RA, Pompolo S, Jones MEE, Simpson ER and Boon WC: Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial preoptic area and arcuate nucleus of male mice. Mol Cell Neurosci. 27:466–476. 2004. View Article : Google Scholar : PubMed/NCBI
33	Gonzalez-Cadavid NF and Rajfer J: Mechanisms of Disease: new insights into the cellular and molecular pathology of Peyronie's disease. Nat Clin Pract Urol. 2:291–297. 2005. View Article : Google Scholar
34	Wu M, Han M, Li J, Xu X, Li T, Que L, Ha T, Li C, Chen Q and Li Y: 17beta-estradiol inhibits angiotensin II-induced cardiac myofibroblast differentiation. Eur J Pharmacol. 616:155–159. 2009. View Article : Google Scholar : PubMed/NCBI
35	Imaoka M, Jindo T and Takasaki W: The process and development mechanism of age-related fibrosis in the pancreatic islets of Sprague-Dawley rats: Immunohistochemical detection of myofibroblasts and suppression effect by estrogen treatment. J Toxicol Pathol. 26:1–10. 2013. View Article : Google Scholar : PubMed/NCBI
36	Matsuo SE, Fiore APZP, Siguematu SM, Ebina KN, Friguglietti CU, Ferro MC, Kulcsar MA and Kimura ET: Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues. Arq Bras Endocrinol Metabol. 54:406–412. 2010. View Article : Google Scholar : PubMed/NCBI
37	Yuan S-M, Wang J, Hu X-N, Li D-M and Jing H: Transforming growth factor-β/Smad signaling function in the aortopathies. Rev Bras Cir Cardiovasc. 26:393–403. 2011. View Article : Google Scholar : PubMed/NCBI
38	Dong C, Li H-J, Chang S, Liao HJ, Zhang ZP, Huang P and Tang HH: A disintegrin and metalloprotease with thrombos-pondin motif 2 may contribute to cirrhosis in humans through the transforming growth factor-β/SMAD pathway. Gut Liver. 7:213–220. 2013. View Article : Google Scholar : PubMed/NCBI
39	Thumkeo D, Watanabe S and Narumiya S: Physiological roles of Rho and Rho effectors in mammals. Eur J Cell Biol. 92:303–315. 2013. View Article : Google Scholar : PubMed/NCBI
40	Fukata Y, Kimura K, Oshiro N, Saya H, Matsuura Y and Kaibuchi K: Association of the myosin-binding subunit of myosin phosphatase and moesin: dual regulation of moesin phos-phorylation by Rho-associated kinase and myosin phosphatase. J Cell Biol. 141:409–418. 1998. View Article : Google Scholar : PubMed/NCBI
41	Sopko NA, Hannan JL and Bivalacqua TJ: Understanding and targeting the Rho kinase pathway in erectile dysfunction. Nat Rev Urol. 11:622–628. 2014. View Article : Google Scholar : PubMed/NCBI