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Attenuation of EMT in RPE cells and subretinal fibrosis by an RAR-γ agonist

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Abstract

Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-β2 (TGF-β2)–induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-β2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-β2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-β2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD.

Key message

  • RAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells.

  • Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667.

  • RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells.

  • RAR-γ agonists may attenuate AMD-associated fibrosis.

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References

  1. van Lookeren Campagne M, LeCouter J, Yaspan BL, Ye W (2014) Mechanisms of age-related macular degeneration and therapeutic opportunities. J Pathol 232:151–164

    Article  PubMed  Google Scholar 

  2. Hong YJ, Miura M, Ju MJ, Makita S, Iwasaki T, Yasuno Y (2014) Simultaneous investigation of vascular and retinal pigment epithelial pathologies of exudative macular diseases by multi-functional optical coherence tomography. Invest Ophthalmol Vis Sci. doi:10.1167/iovs.14-14005

    Google Scholar 

  3. Miller JW (2010) Treatment of age-related macular degeneration: beyond VEGF. Jpn J Ophthalmol 54:523–528

    Article  CAS  PubMed  Google Scholar 

  4. Lim WK, Chee SP, Sng I, Nussenblatt RB, Chan CC (2004) Immunopathology of progressive subretinal fibrosis: a variant of sympathetic ophthalmia. Am J Ophthalmol 138:475–477

    Article  PubMed  Google Scholar 

  5. Saika S, Kono-Saika S, Tanaka T, Yamanaka O, Ohnishi Y, Sato M, Muragaki Y, Ooshima A, Yoo J, Flanders KC et al (2004) Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice. Lab Investig J Tech Methods Pathol 84:1245–1258

    Article  CAS  Google Scholar 

  6. Kalluri R, Neilson EG (2003) Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 112:1776–1784

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Lee H, O’Meara SJ, O’Brien C, Kane R (2007) The role of gremlin, a BMP antagonist, and epithelial-to-mesenchymal transition in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 48:4291–4299

    Article  PubMed  Google Scholar 

  8. Simoni D, Tolomeo M (2001) Retinoids, apoptosis and cancer. Curr Pharm Des 7:1823–1837

    Article  CAS  PubMed  Google Scholar 

  9. Conde I, Paniagua R, Fraile B, Ruiz A, Arenas MI (2004) Expression of vitamin D3 receptor and retinoid receptors in human breast cancer: identification of potential heterodimeric receptors. Int J Oncol 25:1183–1191

    CAS  PubMed  Google Scholar 

  10. Soderpalm AK, Fox DA, Karlsson JO, van Veen T (2000) Retinoic acid produces rod photoreceptor selective apoptosis in developing mammalian retina. Invest Ophthalmol Vis Sci 41:937–947

    CAS  PubMed  Google Scholar 

  11. Clagett-Dame M, McNeill EM, Muley PD (2006) Role of all-trans retinoic acid in neurite outgrowth and axonal elongation. J Neurobiol 66:739–756

    Article  CAS  PubMed  Google Scholar 

  12. Liu Y, Kimura K, Orita T, Teranishi S, Suzuki K, Sonoda KH (2014) Inhibition by all-trans-retinoic acid of transforming growth factor-beta-induced collagen gel contraction mediated by human tenon fibroblasts. Invest Ophthalmol Vis Sci 55:4199–4205

    Article  CAS  PubMed  Google Scholar 

  13. Jo YJ, Sonoda KH, Oshima Y, Takeda A, Kohno R, Yamada J, Hamuro J, Yang Y, Notomi S, Hisatomi T et al (2011) Establishment of a new animal model of focal subretinal fibrosis that resembles disciform lesion in advanced age-related macular degeneration. Invest Ophthalmol Vis Sci 52:6089–6095

    Article  PubMed  Google Scholar 

  14. Kimura K, Orita T, Fujitsu Y, Liu Y, Wakuta M, Morishige N, Suzuki K, Sonoda KH (2014) Inhibition by female sex hormones of collagen gel contraction mediated by retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 55:2621–2630

    Article  CAS  PubMed  Google Scholar 

  15. Ngo P, Ramalingam P, Phillips JA, Furuta GT (2006) Collagen gel contraction assay. Methods Mol Biol 341:103–109

    CAS  PubMed  Google Scholar 

  16. Ramachandran C, Patil RV, Combrink K, Sharif NA, Srinivas SP (2011) Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells. Mol Vis 17:1877–1890

    CAS  PubMed Central  PubMed  Google Scholar 

  17. Lan HY, Chung AC (2011) Transforming growth factor-beta and Smads. Contrib Nephrol 170:75–82

    CAS  PubMed  Google Scholar 

  18. Zhang H, Liu ZL (2012) Transforming growth factor-beta neutralizing antibodies inhibit subretinal fibrosis in a mouse model. Int J Ophthalmol 5:307–311

    CAS  PubMed Central  PubMed  Google Scholar 

  19. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12:2730–2741

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Liu Y, Yanai R, Lu Y, Kimura K, Nishida T (2006) Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts. Exp Eye Res 83:1196–1204

    Article  CAS  PubMed  Google Scholar 

  21. Kischer CW, Shetlar MR, Chvapil M (1982) Hypertrophic scars and keloids: a review and new concept concerning their origin. Scan Electron Microsc :1699–1713

  22. Hammer M, Konigsdorffer E, Liebermann C, Framme C, Schuch G, Schweitzer D, Strobel J (2008) Ocular fundus auto-fluorescence observations at different wavelengths in patients with age-related macular degeneration and diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol 246:105–114

    Article  PubMed  Google Scholar 

  23. Stodtler M, Mietz H, Wiedemann P, Heimann K (1994) Immunohistochemistry of anterior proliferative vitreoretinopathy. Report of 11 cases. Int Ophthalmol 18:323–328

    Article  PubMed  Google Scholar 

  24. Eckes B, Nischt R, Krieg T (2010) Cell-matrix interactions in dermal repair and scarring. Fibrogenesis Tissue Repair 3:4

    Article  PubMed Central  PubMed  Google Scholar 

  25. Suzuki R, Miyazaki Y, Takagi K, Torii K, Taniguchi H (2004) Matrix metalloproteinases in the pathogenesis of asthma and COPD: implications for therapy. Treat Respir Med 3:17–27

    Article  CAS  PubMed  Google Scholar 

  26. Giambernardi TA, Sakaguchi AY, Gluhak J, Pavlin D, Troyer DA, Das G, Rodeck U, Klebe RJ (2001) Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells. Matrix Biol J Int Soc Matrix Biol 20:577–587

    Article  CAS  Google Scholar 

  27. Kijlstra A, La Heij E, Hendrikse F (2005) Immunological factors in the pathogenesis and treatment of age-related macular degeneration. Ocul Immunol Inflamm 13:3–11

    Article  CAS  PubMed  Google Scholar 

  28. Kaiser PK, Gragoudas ES (1996) The subretinal fibrosis and uveitis syndrome. Int Ophthalmol Clin 36:145–152

    Article  CAS  PubMed  Google Scholar 

  29. Baudouin C, Fredj-Reygrobellet D, Brignole F, Negre F, Lapalus P, Gastaud P (1993) Growth factors in vitreous and subretinal fluid cells from patients with proliferative vitreoretinopathy. Ophthalmic Res 25:52–59

    Article  CAS  PubMed  Google Scholar 

  30. Benson MT, Shepherd L, Rees RC, Rennie IG (1992) Production of interleukin-6 by human retinal pigment epithelium in vitro and its regulation by other cytokines. Curr Eye Res 11(Suppl):173–179

    Article  PubMed  Google Scholar 

  31. King RE, Kent KD, Bomser JA (2005) Resveratrol reduces oxidation and proliferation of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition. Chem Biol Interact 151:143–149

    Article  CAS  PubMed  Google Scholar 

  32. Yao HW, Xie QM, Chen JQ, Deng YM, Tang HF (2004) TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro. Life Sci 76:29–37

    Article  CAS  PubMed  Google Scholar 

  33. Liu Y, Ko JA, Yanai R, Kimura K, Chikama T, Sagara T, Nishida T (2008) Induction by latanoprost of collagen gel contraction mediated by human tenon fibroblasts: role of intracellular signaling molecules. Invest Ophthalmol Vis Sci 49:1429–1436

    Article  PubMed  Google Scholar 

  34. Lotan R (1997) Roles of retinoids and their nuclear receptors in the development and prevention of upper aerodigestive tract cancers. Environ Health Perspect 105(Suppl 4):985–988

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Schambach F, Schupp M, Lazar MA, Reiner SL (2007) Activation of retinoic acid receptor-alpha favours regulatory T cell induction at the expense of IL-17-secreting T helper cell differentiation. Eur J Immunol 37:2396–2399

    Article  CAS  PubMed  Google Scholar 

  36. Chen F, Desai TJ, Qian J, Niederreither K, Lu J, Cardoso WV (2007) Inhibition of Tgf beta signaling by endogenous retinoic acid is essential for primary lung bud induction. Development 134:2969–2979

    Article  CAS  PubMed  Google Scholar 

  37. Morales SA, Mareninov S, Prasad P, Wadehra M, Braun J, Gordon LK (2007) Collagen gel contraction by ARPE-19 cells is mediated by a FAK-Src dependent pathway. Exp Eye Res 85:790–798

    Article  CAS  PubMed  Google Scholar 

  38. Huang Z, Yan DP, Ge BX (2008) JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin. Cell Signal 20:2002–2012

    Article  CAS  PubMed  Google Scholar 

  39. Kamato D, Burch ML, Piva TJ, Rezaei HB, Rostam MA, Xu S, Zheng W, Little PJ, Osman N (2013) Transforming growth factor-beta signalling: role and consequences of Smad linker region phosphorylation. Cell Signal 25:2017–2024

    Article  CAS  PubMed  Google Scholar 

  40. Thomas PE, Peters-Golden M, White ES, Thannickal VJ, Moore BB (2007) PGE(2) inhibition of TGF-beta1-induced myofibroblast differentiation is Smad-independent but involves cell shape and adhesion-dependent signaling. Am J Physiol Lung Cell Mol Physiol 293:L417–L428

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This study was supported by Takeda Science Foundation. We thank Yukari Mizuno and Shizuka Murata for technical assistance.

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The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi, 755-8505, Japan

    Kazuhiro Kimura, Tomoko Orita, Yang Liu, Kazuhiro Tokuda, Taishi Kurakazu, Takeshi Noda, Ryoji Yanai, Naoyuki Morishige & Koh-Hei Sonoda

  2. Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Yang Yang, Atsunobu Takeda & Tatsuro Ishibashi

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  1. Kazuhiro Kimura
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  2. Tomoko Orita
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  3. Yang Liu
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  4. Yang Yang
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Correspondence to Kazuhiro Kimura.

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Kimura, K., Orita, T., Liu, Y. et al. Attenuation of EMT in RPE cells and subretinal fibrosis by an RAR-γ agonist. J Mol Med 93, 749–758 (2015). https://doi.org/10.1007/s00109-015-1289-8

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  • DOI: https://doi.org/10.1007/s00109-015-1289-8

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