Rho/myocardin関連転写因子A（MRTF-A）経路は，ヒト網膜色素上皮細胞でTGF-β2による上皮間葉移行において重要な働きをする

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(1)JIMA Vol. 72, No. 5（Dec. 2020）pp. 217-230．. Original. Rho/myocardin-related transcription factor A (MRTF-A) pathway plays an important role in TGF-β-induced epithelial mesenchymal transition in retinal pigment epithelial cells Umi Tanaka 1), Daijiro Kurosaka 1), Kenichi Murai 1), Kouhei Hashizume 1), Kazuhiro Fukuda 1) , Sora Itsukaichi 1) and Atsushi Sanbe 2) 1). Department of Ophthalmology, School of Medicine, Iwate Medical University, Yahaba, Japan 2). Division of Pharmacotherapeutics, Department of Pathophysiology and Pharmacology, School of Pharmacy, Iwate Medical University, Yahaba, Japan. （Received on January 20, 2020 & Accepted on February 18, 2020） Abstract Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is associated with ocular fibrotic conditions such as proliferative vitreoretinopathy and wet/neovascular type agerelated macular degeneration. Upon transforming growth factor-β (TGF-β) stimulation, myocardinrelated transcription factor A (MRTF-A) is translocated from the cytoplasm into the nucleus via Rho activation to promote EMT in various cells. However, it is unknown whether the Rho/MRTF pathway is involved in the EMT of TGF-β-induced RPE cells. In this study, TGF-β2 led to an increase in mRNA expression of α-smooth muscle actin (αSMA), a biomarker of EMT, and type I collagen,. repressed by MRTF-A knockdown, in human retinal pigment epithelial (ARPE- 19 ) cells. CCG- 203971 , an MRTF-A inhibitor, also downregulated the mRNA expression of α-SMA and type I collagen in TGFβ 2 -treated ARPE- 19 cells via a reduction in MRTF-A nuclear translocation. Moreover, Y- 27632 , a Rho-associated protein kinase inhibitor, inhibited the expression of α-SMA and type I collagen and suppressed the translocation of MRTF-A to the nucleus in TGF-β-treated ARPE- 19 cells. These results suggest that the Rho/MRTF pathway plays an important role in the EMT of TGF-β-induced RPE cells.. Key words： retinal pigment epithelial cell, epithelial mesenchymal transition, Rho/MRTF pathway, α-smooth muscle actin, type I collagen . I. Introduction. like cells in ocular fibrosis conditions such. Some retinal pigment epithelial (RPE). as proliferative vitreoretinopathy (PVR) 1-3). cells transdifferentiate into myofibroblast-. and wet/neovascular type age-related macular degeneration (AMD). 4-6). . These. myofibroblast-like RPE cells express α-smooth. Corresponding author: Daijiro Kurosaka,. [email protected]. muscle actin (α-SMA) 217. 4-7). , a myofibroblast.

(2) Umi Tanaka, et al.. 218. biomarker 8), and produce extracellular matrix 2, 3, 9). 2, 3). 32,34,35). . Thus, TGF-β-induced. .. nuclear accumulation of MRTF-A is Rho-. This transdifferentiation is referred to as the. associated protein kinase (ROCK)-dependent.. components. such as type I collagen. translocation. 10). epithelial-mesenchymal transition (EMT) .. This Rho/MRTF pathway plays an important. The deposition of these extracellular matrices. role in the TGF-β-induced EMT of various. 2, 9). leads to fibrotic membrane formation. . In. PVR, the contraction of these membranes often results in the failure of retinal detachment 1,11-14). surgery. . In AMD, these fibrotic membranes 9,15). cells, such as cardiac. 36). 39). , lung. scleral , and dermal fibroblasts. 37). 40). and lens epithelial cells (LECs). , colonic. 38). and kidney 29). ,. 36). . However,. the role of the Rho/MRTF pathway in the. . Thus,. TGF-β-induced EMT of RPE cells is unknown.. the prevention of EMT in RPE cells has. Y-27632, a Rho kinase inhibitor, inhibited the. therapeutic implications.. EMT of various TGF- β -induced cells because. Transforming growth factor-β (TGF-. it inhibits F-actin stress ﬁber formation, which. β ) is a major factor during the EMT of. in turn increases G-actin expression32,34), thereby. RPE cells 9,16) and renal tubular 17-19), lens 20,21),. reducing the nuclear translocation of MRTF-A.. cause severe visual disturbances. 18,19,22-24). epithelial cells. Notably,. Although Y-27632 inhibits the induction of COL1. TGF-β induces α-SMA and collagen production. mRNA via TGF-β in RPE cells 33), the role of. and mammary in these cells. 17,22,25,26). . Recent studies have. MRTF activity on ROCK inhibition is unknown.. indicated that myocardin-related transcription. To determine whether the Rho/MRTF. factor A (MRTF-A) plays an important role in. pathway is involved in the EMT of TGF-β -. 21,22,27,28). ,. induced RPE cells, we examined the effects. epithelial cells.. of Y-27632 on α-SMA and type I collagen. MRTF-A is a transcriptional co-activator. expression levels in TGF-β -induced RPE cells,. TGF-β -induced EMT in renal tubular lens. 20,29). , and mammary. 18,19,23,24). that is sequestered in the cytoplasm. 30). ,. and has been recently reported to play an important role in the EMT of RPE cells. 31). after which MRTF-A localization was linked to said expression levels.. .. Upon TGF-β stimulation, MRTF-A trans-. II. Materials and methods. locates from the cytoplasm into the nucleus. ARPE-19 cell culture: Human retinal. and interacts with the serum response factor. pigmented epithelium cells (ARPE-19). 32). and. were purchased from the American Type. type I collagen . The localization of MRTF-A. Culture Collection (Rockville, MD, USA). To. is controlled by monomeric globular actin. authenticate the cell line, a short tandem. (SRF) to promote the expression of α-SMA 33). 32,34,35). . Decreased cytoplasmic G-actin. repeat (STR) analysis was performed by. expression promotes G-actin dissociation from. the BEX Co., Ltd., (Tokyo, Japan) using the. MRTF-A, which translocates MRTF-A into. GenePrint 10 System (Promega, Madison,. (G-actin). 32,34,35). . TGF-β promotes filamentous. WI, USA). ARPE-19 cells were cultured in. actin (F-actin) stress fiber formation via Rho. Dulbecco’ s modified Eagle’ s medium (DMEM). the nucleus. 32). . This decreases cytoplasmic. (Nacalai Tesque, Inc., Kyoto, Japan) containing. G-actin and promotes MRTF-A nuclear. 10% (v/v) fetal bovine serum (FBS) (GE. activation.

(3) Original: Rho/ MRTF-A pathway in EMT in RPE cells. 219. Table 1. Primer sets Protein. Oligonucleotide. Expected PCR product size. human smooth muscle actin. Sense ATTGCCGACCGAATGCAGAAG Antisense AGAGGCCAGGATGGAGCCAC. 120bp. human collagen type 1A. Sense TGGACCAGCAGACTGGCAAC Antisense TCGTGCAGCCATCGACAGTGGAC. 120bp. GAPDH. Sense AGGTCATCATCCATGACAACTTTG Antisense TTCAGCTCAGGGATGACCTT. 190bp. Healthcare Life Sciences, South Logan, UT,. RNA preparation and RT-PCR analysis:. USA) in a humidified atmosphere of 5% CO2 at. Total RNA from ARPE-19 cells was prepared. 37 ℃. Confluent ARPE-19 cells were cultured. using ISOGEN reagent (NIPPON GENE,. in DMEM containing 0% FBS. Then, the cells. Toyama, Japan). cDNAs were prepared from. were treated with TGF-β2 (12.5 ng/ml; Sigma,. 1 µg of total RNA using PrimeScript reverse. St. Louis, MO, USA) for 24 h. CGG-203971 was. transcriptase (Takara Bio, Kyoto, Japan). dissolved in dimethyl sulfoxide (DMSO) and. according to the manufacturer’s instructions.. diluted with DMEM to 3, 10 or 30 µM (0.1%. PCR amplification was performed using. DMSO). Either 3, 10 or 30 µM CGG-203971. ExTaq polymerase (Takara Bio) for 30 cycles;. or 0.1% DMSO (vehicle) or 3, 10, and 30 µM. each cycle consisted of denaturation at 94 ℃. Y-27632 was added to confluent ARPE-19 cells. for 0.5 min, annealing at 60℃ for 0.5 min, and. 1 h after TGF-β2 treatment.. extension at 72℃ for 1 min. Quantitative real-. siRNA oligonucleotides: The following. time-PCR (qRT-PCR) was performed using the. 21-nucleotide siRNA duplexes were synthesized. EcoTM RT-PCR System (illumina, San Diego,. by Nippon Gene Material Co, Ltd. (Toyama,. CA, USA) according to the manufacturer’s. Japan): 5 ′ -AAGAACATCCTTCCTGTTGAG-. protocol. The primers used for qRT-PCR are. 3 ′for human MRTF a siRNA. The target. listed in Table 1.. sequence for the control Photinus pyralis. Immunocytochemistry: Immunocytochemical. luciferase siRNA was 5-AAGCCATTCTAT. analysis was performed using the anti-. CCTCTAGAG-3 ′ , which has no significant. MRTF-A antibody (ab49311, abcam). All cell-. homology to any mammalian gene sequence. culture media were from Life Technologies,. by BLAST searches.. Inc. An Alexa 488-conjugated anti-rabbit. siRNA transfection: Cells were transfected. antibody and DAPI were obtained from. with siRNA oligonucleotides using the. Molecular Probes (Eugene, OR, USA) and. Lipofectamine 2000 reagent (Invitrogen,. used for nuclear staining. Confluent ARPE-19. Carlsbad, CA, USA) according to the. cells were cultured in DMEM containing 0%. manufacturer’ s protocol and cultured for 48 h. FBS. Then, the cells were treated with TGF-. before subsequent analyses, such as real-time-. β (12.5 ng/ml; Sigma, St. Louis, MO, USA). PCR (RT-PCR) and western blotting.. for 24 h. Leptomycin B (LMB 50 nM) was.

(4) Umi Tanaka, et al.. 220. Relative MRTF-A levels TGF-β2. Protein expression analysis: Sample preparation. **. *. for western blotting, gel preparation, and. **. electrophoretic conditions were performed as described previously 41). Western blot analyses were performed using an anti-MRTF-A antibody (ab49311; Abcam) and anti-GAPDH −. +. siLuc. −. + siMRTF-A. Fig. 1. Effect of MRTF-A siRNA (siMRTF-A) and TGF-β2 on the MRTF-A mRNA expression in ARPE-19 cells (p < 0.0001, ANOVA). Transfection with siMRTF-A decreased MRTF-A mRNA expression in ARPE-19 cells compared with that in control siRNA (siLuc) transfectants. In siLuc transfectants, the expression of MRTF-A mRNA increased slightly by treatment with 12.5 ng/ml TGFβ2. Expression levels were normalized by GAPDH. Data are presented as means ± SEMs of 3 independent experiments. *p < 0.001, ** p < 0.0001 (post-hoc Tukey's multiple comparison tests).. antibody (Chemicon International, Temecula, CA, USA). Band intensity in the immunoblot was semi-quantified using Image J1.38x. Statistics: Data are expressed as means ± standard error of 3 or 6 independent experiments. Statistical analyses were performed using the unpaired Student’ s t-test and one-way analysis of variance (ANOVA) followed by post-hoc Tukey's multiple comparison tests. Statistical tests were performed using Kaleida Graph version 4.1 (Synergy Software, Reading, PA, USA).. III. Results To determine whether MRTF-A plays an added to inhibit nuclear excretion factor for. important role in the EMT of TGF- β -induced. 1 h before cell fixation. After fixation with. ARPE-19 cells, MRTF-A mRNA was knocked. 4% paraformaldehyde solution, cells were. down in the ARPE-19 cells. ARPE-19 cells. incubated with anti-MRTF-A antibody at 4 ℃. transfected with MRTF-A siRNA exhibited. for 6 h. Following incubation with the primary. lower MRTF-A mRNA (Fig. 1) and protein. antibody, the cells were further incubated. expression levels (Fig. 2A, B) than those in. for 1.5 h with Alexa Fluor 488 conjugate goat. control siRNA transfectants (mRNA: 0.55 ±. anti-rabbit secondary antibody (Molecular. 0.04 vs. 1.00 ± 0.03; protein: 0.41 ± 0.05 vs. 1.00. Probes). Immunofluorescent pictures of the. ± 0.00, respectively; p < 0.0001 and p < 0.05).. cells were analyzed using ImageJ software.. TGF-β2 treatment resulted in slightly higher. Integrated intensities were measured from. MRTF-A mRNA and protein expression levels. a specified rectangular area. Measurements. in control siRNA transfectants than those. were obtained from three random cytoplasmic. without TGF-β2 treatment (mRNA: 1.34. sites and the nucleus of each cell, and 300–500. 0.02 vs. 1.00 ± 0.03; protein: 1.58 ± 0.18 vs. 1.00. cells were enumerated per category. The. ± 0.00, respectively; p < 0.001 and p < 0.05).. ±. ratio of the average nuclear and cytoplasmic. To examine whether MRTF-A affects α -. intensities was determined and categorized as. SMA and type I collagen expression, ARPE-. previously reported. 20). .. 19 cells were transfected with control or.

(5) Original: Rho/ MRTF-A pathway in EMT in RPE cells. TGF-β2. MRTF-A. −TGF-β2 +. B. siMRTF-A siLuc. −− +. * *. siMRTF-A. +−. +. MRTF-A. −. TGF-β2. **. **. Relative MRTF-A levels. siLuc. Relative MRTF-A levels. . A. 221. siLuc. +. **. −. +. siMRTF-A. Fig. 2. Effect of MRTF-A siRNA (siMRTF-A) and TGF-β2 TGF-β2on MRTF-A -in ARPE-19 + − protein+expression− cells. siLuc siMRTF-A (A) Transfection with siMRTF decreased MRTF-A protein expression in ARPE-19 cells compared with that in control siRNA (siLuc) transfects by western blotting. In ARPE-19 cells transfected with siLuc, MRTF-A protein expression was increased with treatment of 12.5 ng/ml TGF-β2. (B) Densitometric quantification of MRTF-A/GAPDH (p < 0.05, ANOVA). Densitometric quantification was normalized by GAPDH. Data are presented as means ± SEMs of 3 independent experiments. *p < 0.05, ** p < 0.0001 (post-hoc Tukey's multiple comparison tests). A. * *. B. B Relative Type 1 collagen levels. *. **. Relative Type 1 collagen levels. Relative smooth muscle actin levels. *. Relative smooth muscle actin levels. A. A. *. * *. B *. − − +− − + + + − −+ − + + + TGFTGF TGF-β2 siL siMRT siLuc-β2 siMRTF-A si siMRTF-A siLuc siMR β2 uc F-A Lu TF-A Fig. 3. Effects of MRTF-A on cα -SMA and type I collagen expressions in ARPE-19 cells treated with TGF-β2 −. TGF-β2 (p < 0.0001, p < 0.0001, ANOVA, respectively). ARPE-19 cells transfected with control siRNA (siLuc) or MRTF-A siRNA (siMRTF-A) were cultured with or without 12.5 ng/ml TGF-β2 for 24 h. TGF-β2 treatment increased α -SMA (A) and type I collagen (B) expression in ARPE-19 cells transfected with control siRNA. This increase was partially inhibited by treatment with MRTF-A siRNA. Expression levels were normalized by GAPDH. Data are presented as means ± SEMs of 3 independent experiments. *p < 0.0001, **p < 0.001 (post-hoc Tukey's multiple comparison tests).. MRTF-A siRNA. Then, they were cultured. 0.13 vs. 1.00 ± 0.02; type I collagen expression:. with or without 12.5 ng/ml TGF-β2 for 24 h.. 2.87 ± 0.18 vs. 1.00 ± 0.02, respectively; all. TGF-β2 increased α-SMA and type I collagen. p < 0.0001). These increases were partially. expression levels in ARPE-19 cells transfected. inhibited by treatment with MRTF-A siRNA. with control siRNA ( α -SMA expression: 1.63. (α-SMA expression: 1.10 ± 0.09 vs. 1.63 ± 0.13;.

(6) Umi Tanaka, et al.. 222. A. A. CCG203971(μM). *. * Relative Type 1 collagen levels. Relative smooth muscle actin levels TGF-β2(ng/ml). **. *. B. B. 0. 12.5 12.5 12.5 12.5 0. 3. 10. 30. TGF-β2(ng/ml) CCG203971(μM). 0. 12.5 12.5 12.5 12.5 0. 3. 10. 30. Fig. 4. Effect of CCG-203971 on α-SMA and type I collagen expressions in ARPE-19 cells treated with TGF-β2 (p < 0.001, p < 0.0001, ANOVA, respectively). Treatment with 12.5 ng/ml TGF-β2 increased α-SMA (A) and type I collagen (B) expression in ARPE-19 cells, and CCG203971 abolished these increases in a dose-dependent manner. Expression levels were normalized by GAPDH. Data are presented as means ± SEMs of 3 independent experiments. *p < 0.05, **p < 0.001 (post-hoc Tukey's multiple comparison tests).. type I collagen expression: 1.62 ± 0.18 vs. 2.87. To examine whether TGF-β2 promotes the. ± 0.18, respectively; p < 0.001 and p < 0.0001,. nuclear translocation of MRTF-A and CCG-. Fig. 3A, B).. 203971 inhibits this promotion, ARPE-19 cells. To determine whether CCG-203971, a. were cultured with or without 12.5 ng/ml TGF-. MRTF-A inhibitor, could also reduce the. β2 or a combination of TGF-β2 and 30 µM. expression of α-SMA and type I collagen,. CCG-203971 for 24 h. Thereafter, the cells were. we cultured TGF-β2-treated (12.5 ng/ml). treated with LMB, a nuclear export inhibitor,. ARPE-19 cells in the presence or absence. for 1 h. MRTF-A remained in the cytosol. of CCG-203971 (3, 10, and 30 µM). Although. without TGF-β2 treatment (Fig. 5A) but was. treatment with TGF-β2 increased expressions. translocated to the nucleus after TGF-β 2. of α-SMA and type I collagen in ARPE-19. treatment (Fig. 5B). However, joint treatment. cells relative to the control group (α-SMA. with TGF-β2 and 30 µM CCG-203971 inhibited. expression: 1.55 ± 0.11 vs. 1.00 ± 0.00; type. this nuclear translocation (Fig. 5C).. I collagen expression: 20.38 ± 2.63 vs. 1.00. To quantify the changes in MRTF-A. ± 0.00, respectively; all p < 0.01), CCG-. compartmentalization, the fluorescent intensity. 203971 diminished these increases in a dose-. of MRTF-A in the cytoplasm and nucleus. dependent manner ( α -SMA expression: 1.55. was measured. In controls treated only with. ± 0.11, 1.21 ± 0.11, 1.25 ± 0.13, and 0.84 ± 0.09,. LMB, the number of cells with cytoplasmic. for 0, 3, 10, and 30 µM CCG-20397; type I. MRTF-A was 57.0 ± 3.32%; however, this. collagen expression: 20.38 ± 2.63, 9.76 ± 3.40,. number significantly decreased upon TGF-β2. 10.30 ± 3.73, and 3.65 ± 1.21, for 0, 3, 10, and. treatment (12.5 ng/ml) for 24 h to 11.9 ± 1.91%. 30 µM CCG-20397, respectively, Fig. 4A, B).. (p < 0.05). Correspondingly, the number of cells.

(7) Original: Rho/ MRTF-A pathway in EMT in RPE cells. A. B. C. D. 223. E. Subcellular localization of MRTF-A (%). Fig. 5. Translocation of MRTF-A in ARPE-19 cells stimulated by TGF-β2. ARPE-19 cells were cultured with or without 12.5 ng/ml TGF-β2 for 24 h. Either 30 µM CGG203971 or 10 and 30 µM Y27632 was added 1 h after TGF-β2 treatment. Leptomycin B (LMB 50 nM) was added to inhibit nuclear excretion factor for 10 mins before cell fixation. ARPE-19 cells were immunostained using an MRTF-A-specific antibody (green) and a nuclear stain (DAPI, blue). MRTF-A remained in the cytosol in the absence of TGF-β2 (A), but MRTF-A was translocated to the nucleus after TGF-β2 treatment (B). This nuclear translocation was inhibited by CGG203971 (C). Y27632 also inhibited nuclear translocation of MRTF-A at each concentration (D, E). Scale bar = 100 μm. 100%. *. *. *. *. 80%. *. 60%. カテゴリ 3 Nuclear. * 40%. カテゴリ 2 Pan-cellular カテゴリ 1 Cytoplasmic. *. 20%. * 0%. TGF-β2 (ng/ml). 0. 系列 1. 系列 2 12.5. 系列 3 12.5. 系列 4 12.5. 系列 5 12.5. CCG203971(μM). 0. 30. 0. 0. Y27632 (μM). 0. 0. 10. 30. Fig. 6. Quantification and comparison of intracellular MRTF-A translocation in ARPE-19 cells stimulated by TGF-β2. MRTF-A is primarily cytoplasmic in the control. Nuclear MRTF-A significantly increased after TGF-β2 treatment but significantly decreased upon treatment with 30 µM CCG203971 relative to the TGF-β2-treated cells. Similarly, Y27632 treatment significantly decreased nuclear MRTF-A in a dose-dependent manner. Gene expression levels were normalized to those of GAPDH. Data are presented as the mean ± SEMs of 6 independent experiments. *p < 0.05 (post-hoc Tukey's multiple comparison tests).. with nuclear MRTF-A significantly increased. with 30 µM CCG-203971, this increase was. relative to the control (68.0 ± 3.29% vs. 18.4. significantly attenuated (4.42 ± 1.72% vs. 68.0. ± 1.95%, p < 0.05). However, upon treatment. ± 3.29%, p < 0.05, Fig. 6)..

(8) Umi Tanaka, et al.. 224. TGF-β2(ng/ml) Y27632(μM). B *. *. 0. 12.5 12.5 12.5 12.5 0. 3. 10. 30. TGF-β2(ng/ml) Y27632(μM). *. ***. Relative Type 1 collagen levels. Relative smooth muscle actin levels. A. 0. 12.5 0. **. ***. 12.5 12.5 12.5 3. 10. 30. Fig. 7. Effect of Y27632 on α -SMA and type I collagen expressions in ARPE-19 cells treated with TGFβ2 (p < 0.05, p < 0.001, ANOVA, respectively). Treatment with 12.5 ng/ml TGF-β2 increased α-SMA (A) and type I collagen (B) expression in ARPE-19 cells, and Y27632 abolished these increases in a dose-dependent manner. Expression levels were normalized by GAPDH. Data are presented as means ± SEMs of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (post-hoc Tukey's multiple comparison tests).. To determine whether Y-27632, a ROCK. MRTF-A were examined. ARPE-19 cells were. inhibitor, inhibits the EMT of TGF- β. cultured with or without 12.5 ng/ml TGF- β. -induced RPE cells, TGF-β2-treated (12.5 ng/. 2 or a combination of TGF-β2 and 10 or 30µM. ml) ARPE-19 cells were cultured with or. Y-27632 for 24 h. ARPE-19 cells were then. without Y-27632 (3, 10, and 30 µM). Although. treated with LMB for 1 h. MRTF-A remained. treatment with TGF-β2 increased the. in the cytosol without TGF-β2 treatment. expression levels of α-SMA and type I collagen. (Fig. 5A) but was translocated to the nucleus. in ARPE-19 cells relative to the control (α-. after TGF-β2 treatment (Fig. 5B). However,. SMA expression: 1.43 ± 0.03 vs. 1.00 ± 0.02;. treatment with a combination of TGF-β2 and. type I collagen expression: 16.99 ± 1.91 vs. 1.00. 10 or 30µM Y-27632 inhibited this nuclear. ± 0.00, respectively; p < 0.05 and p < 0.0001),. translocation (Fig. 5D, E).. Y-27632 diminished these increases in a dose-. Furthermore, we investigated the effect. dependent manner (α-SMA expression: 1.43 ±. of Y-27632 on the changes in MRTF-A. 0.03, 1.29 ± 0.11, 1.10 ± 0.08 and 1.01 ± 0.09, for. c o m p a r t m e n t a l i z a t i o n . A g a i n , T G F - β2. 0, 3, 10, and 30 µM Y-27632; type I collagen. treatment significantly increased the number. expression: 16.99 ± 1.91, 11.34 ± 0.40, 9.97 ±. of cells with nuclear MRTF-A relative to the. 0.80, and 7.82 ± 0.28, for 0, 3, 10, and 30 µM. control (68.0 ± 3.29% vs. 18.4 ± 1.95%, p < 0.05);. Y-27632, respectively, Fig. 7A, B).. however, Y-27632 treatment (10 and 30 µM). To determine whether the Rho/MRTF. significantly attenuated this increase in a dose-. pathway is involved in the EMT of TGF-. dependent manner (30.7 ± 3.04% and 20.7 ±. β-induced RPE cells, the localizations of. 3.92%, respectively; p < 0.05, Fig. 6)..

(9) Original: Rho/ MRTF-A pathway in EMT in RPE cells. 225. IV. Discussion. bleb injections were required for the first 9. In this study, TGF-β2 led to increases in α-. days post-surgery followed by twice-weekly. SMA and type I collagen mRNA expression. injections for 3 weeks. Kobayashi et al.. in ARPE-19 cells, which were repressed. reported that intravitreal CCG-1423 injection. by MRTF-A knockdown. CCG-203971, an. markedly suppressed the development of. MRTF-A inhibitor, also diminished the. photocoagulation-induced subretinal fibrosis.. promotion of α-SMA and type I collagen. Although the residence time of the drug in. mRNA expressions in TGF-β2-treated ARPE-. the vitreous cavity was longer than usual 44),. 19 cells with a reduction of MRTF-A nuclear. CCG-1423 had to be injected into the vitreous. translocation. Y-27632, a ROCK inhibitor,. cavity twice a week. In this study, Y-27632,. also inhibited α-SMA and type I collagen. a ROCK inhibitor, also inhibited α-SMA and. expression and suppressed the translocation. type I collagen expression and suppressed. of MRTF-A to the nucleus in TGF-β2-treated. the translocation of MRTF-A to the nucleus. ARPE-19 cells. These results suggest that the. in TGF-β2-induced ARPE-19 cells. Moreover,. Rho/MRTF pathway plays an important role. Y-27632 also inhibited α-SMA and type I. in the EMT of TGF-β-induced RPE cells.. collagen expression in TGF-β2-induced LECs. 31). also. Our results also indicated that CCG-203971,. and topical Y-27632 administration prevented. a MRTF-A inhibitor, represses α-SMA and. anterior subcapsular cataract induced by. type I collagen mRNA expression and TGF-. UV-B irradiation, which had been developed. β2-induced MRTF-A nuclear translocation in. by the EMT of LECs 45). A topically instilled. ARPE-19 cells. These results are consistent. radiolabeled ROCK inhibitor can be detected. 31). with recent work by Kobayashi et al. , who. in the retina and choroid up to 4 h later 46).. reported that CCC-1423, a first-generation. Ripasudil, another ROCK inhibitor applied as. MRTF-A inhibitor, repressed the expression. eye drops, was used clinically in Japan for the. of α-SMA and type I collagen mRNA and. treatment of glaucoma 47,48). Although topical. TGF- β2-mediated nuclear translocation of. administration of ripasudil is required twice a. MRTF-A in RPE-1 cells, another cell line of. day via eye drops, this administration method. RPE cells. CCG-203971 is a second-generation. is easier and safer than a subconjunctival or. inhibitor with far less cytotoxicity than CCG-. intravitreal injection, which in rare cases can. 1423. 30,42). . Notably, CCG-222740, a novel MRTF. cause endophthalmitis, a complication that. inhibitor, was recently developed and has. can have devastating visual outcomes. These. exhibited a greater inhibitory effect on α-SMA. findings highlight the promising potential. 43). expression than CCG-203971 .. of topical ROCK inhibitor administration. Local administration of these MRTF-A. as a novel PVR and AMD treatment.. inhibitors prevented ocular fibrosis in vivo.. In this study, MRTF-A knockdown repressed. Particularly, local administration of CCG-. α-SMA mRNA expression induced by TGF-. 222740 prevented subconjunctival scarring. β2 in ARPE-19 cells. MRTF-A in conjunction. in rabbits after in vivo glaucoma filtration. with SRF. surgery. 43). . However, daily subconjunctival. 49,50). can induce α -SMA mRNA. expression after MRTF-A translocation from.

(10) Umi Tanaka, et al.. 226. . In ARPE-. hepatocyte growth factor 22), epidermal growth. 19 cells, TGF-β2 promoted MRTF-A nuclear. factor 1,9,12,15), and other members of the TGF-. transport. Moreover, TGF-β2 increased the. β superfamily such as activin 57) can induce. expression of MRTF-A mRNA and protein in. EMT in RPE cells. Vascular endothelial. ARPE-cells. In MS-1 endothelial cells, TGF-. growth factor (VEGF) is a key factor in. β promoted α -SMA expression not only by. choroidal neovascularization, which is a main. stimulating the nuclear transport of MRTF-A. pathological AMD lesion 5,58,59). VEGF activates. but also by enhancing MRTF-A transcription,. the nuclear translocation of MRTF-A in mouse. which promoted MRTF-A accumulation in. retinal endothelial cells 60), while PDGF post-. the nucleus 51). Further research is required. transcriptionally activates MRTF-A expression. to determine the effect of enhanced MRTF-A. in cultured hepatic stellate cells 61). Therefore,. transcription on TGF-β2-induced α -SMA. cells and growth factors other than RPE cells. expression in ARPE-19 cells.. and TGF-β may potentially play critical roles. the cytoplasm to the nucleus. 32). In this study, the Rho/MRTF pathway was. in PVR or AMD. Further investigations using. found to play an important role in the EMT. in vivo models are required to elucidate the. of TGF-β-induced RPE cells. However, Smad. role of MRTF-A in these diseases.. pathways are canonical TGF-β signaling. In conclusion, our results demonstrate that. pathways and play important roles in. TGF-β2 promoted the expression of α-SMA. 52,53). EMT. . Its cross-talk with the Rho/MRTF. and type I collagen in ARPE-19 cells via the. pathway has also been shown to be critical. Rho/MRTF pathway. These results suggest. during TGF-β induced EMT. TGF-β-induced. that MRTF-A may be a critical mediator of. translocation of MRTF-A in LECs was. EMT in human RPE cells.. recently found to be inhibited in the presence. . of SIS3, a Smad3-specific inhibitor. 54). . Such. cross-talk with the Rho/MRTF pathway may also play an important role in the EMT of RPE cells. In this study, we examined the activity of MRTF-A in TGF-β2-treated ARPE-19 cells. Morphological and histochemical studies of proliferative membranes in PVR 2,3,12,13,55) and AMD. 5,9,15,56). showed that a variety of cells,. such as RPE cells, fibroblasts, glial cells, and inflammatory cells, participate in the pathogenesis of these diseases. Although TGFβ plays a critical role in the EMT of RPE cells. 1,22). , various growth factors, such as. connective tissue growth factor 9,13), plateletderived growth factor (PDGF) 1,9,12,13,15,22) ,. Acknowledgements We thank Ms. Rieko Higashio for technical assistance with the real-time PCR analysis. This work was supported by a Japan Society for the Promotion of Science KAKENHI grant (no. 17K11465). Conflict of interest: The authors have no conflict of interest to declare.. .

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(14) 岩手医誌 72 巻，5 号（令和 2 年 12 月）217-230 頁．. Rho/myocardin 関連転写因子 A（MRTF-A）経路は，ヒト網膜色素上皮細胞で TGF-β2 による上皮間葉移行において重要な働きをする田中うみ 1），黒坂大次郎 1），村井憲一 1），橋爪公平 1），福田一央 1），五日市そら 1），三部篤 2） 1). 2）. 岩手医科大学医学部，眼科学講座岩手医科大学薬学部，病態薬理学講座薬剤治療学分野. （Received on January 20, 2020 & Accepted on February 18, 2020）. 要旨網膜色素上皮細胞（ RPE ）における上皮間葉移行（ EMT ）は，増殖硝子体網膜症や滲出型加齢黄斑変性に関与する．トランスフォーミング増殖因子β（ TGFβ）刺激により，Rho 活性化を介し myocardin 関連転写因子 A（ MRTF-A ）が細胞質から核内に移行し EMT が生じるが，RPE における Rho/MRTF 経路の働きは知られていない．本研究で，TGF-β2 はヒト RPE（ ARPE- 19）において EMT の指標であるα平滑. 筋アクチン（α-SMA ）と I 型コラーゲン発現を促進し，これは MRTF のノックダウンにより阻害された． MRTF 阻害薬である CCG- 203971 は MRTF-A の核内移行を阻害し，両者の発現を阻害した．Rho 関連タンパクキナーゼ阻害薬である Y- 27632 も，同様に核内移行と両者の発現を阻害した．Rho/MRTF 経路が TGF-β2 による RPE での EMT で重要な役割を果たすことが示唆された．. 230.

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