Effect of transforming growth factor β1 on Smad ubiquitination regulatory factor 2 and collagen type Ⅰ α1 in human fetal scleral fibroblasts

doi:10.3877/cma.j.issn.2095-2007.2019.01.003

Abstract

Abstract:

Objective

The aim of this study was to investigate the expression of Smad ubiquitination regulatory factor 2 (Smurf 2) in human fetal scleral fibroblasts (HFSFs) cells, and the changes of Smurf 2 and collagen type Ⅰ α₁ (COLIA1) in HFSFs induced by transforming growth factor-beta 1 (TGF-β₁).

Methods

After recovery and stable passage of HFSFs, expression of Smurf2 in cells was detected by immunocytochemistry. HFSFs were treated with different concentrations of TGF-β₁(0 , 1 ng/ml, 5 ng/ml, 10 ng/ml) for 24 hours, respectively, and co-cultured with TGF-β₁ (10 ng/ml) for 1 hour, 6 hours, 12 hours, and 24 hours. Then, the expression of COLIA1 messenger RNA (mRNA) and Smurf2 mRNA were detected by reverse transcription-polymerase chain reaction. Data was described by (±s ). One-way ANOVA was used for comparison between groups, then SNK test was used if difference was statistically significant.

Results

The results showed that COLIA1 mRNA and Smurf2 protein expressed in HFSFs. COLIA1 mRNA expressed an upward trend in 10 ng/ml TGF-β₁ group compared with control group (F=3.17, P<0.05), as well as Smurf2 mRNA in 5 ng/ml TGF-β₁ group and 10 ng/ml TGF-β₁ group (F=2.35, 2.00; P<0.05). After 1 hour, 6 hours, 12 hours, 24 hours for HFSFs co-cultured with 10 ng/ml TGF-β₁, the expression of COLIA1 mRNA increased significantly in 24 hours group (F=29.20, P<0.05). While, the expression of Smurf2 mRNA increased first and then decreased, reaching the highest level at 6 hours (F=10.65, P<0.05).

Conclusion

TGF-β₁ may induce the synthesis of HFSFs COLIA1 by regulating the expression of HFSFs Smurf2.

Key words: Smad ubiquitination regulatory factor 2 (Smurf2), Human fetal scleral fibroblasts, Transforming growth factor-β₁ (TGF-β₁) Smad pathway, Myopia

Lijuan Chen, Ting Chen, Xuelan Chen, Wenwen Ye, Lijuan Huang, Jianmin Hu. Effect of transforming growth factor β₁ on Smad ubiquitination regulatory factor 2 and collagen type Ⅰ α₁ in human fetal scleral fibroblasts[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(01): 14-20.

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Figures/Tables 5

References 39

[1]	李翯，周翔天，瞿佳. 碱性成纤维细胞生长因子和转化生长因子-β在实验性近视中的研究进展[J]. 眼视光学杂志，2004，6(3)：193-195，198.
[2]	Praveen K, Gal LC, Michael B. Smurfs in protein homeostasis, signaling, and cancer[J]. Front Oncol, 2018, 8: 295.
[3]	杨俊侠，曹述任，张敏. Smad泛素化调节因子2在转化生长因子β1诱导人肺成纤维细胞活化中的作用及其分子机制[J]. 吉林大学学报(医学版)，2015，41(5)：891-897.
[4]	Fukasawa H, Yamamoto T, Togawa A, et al. Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury[J]. Kidney Int, 2006, 69(10): 1733-1740.
[5]	Cai Y, Shen XZ, Zhou CH, et al. Abnormal expression of Smurf2 during the process of rat liver fibrosis[J]. Chin J Dig Dis, 2006, 7(4): 237-245.
[6]	McBrien NA, Gentle A. Role of the sclera in the development and pathological complications of myopia[J]. Prog Retin Eye Res, 2003, 22(3): 307-338.
[7]	Norton TT, Rada JA. Reduced extracellular matrix in mammalian sclera with induced myopia[J]. Vision Res, 1995, 35(9): 1271-1281.
[8]	McBrien NA, Jobling AI, Gentle A. Biomechanics of the sclera in myopia: extracellular and cellular factors[J]. Optom Vis Sci, 2009, 86(1): E23-E30.
[9]	Lin HJ, Wan L, Tsai Y, et al. Sclera-related gene polymorphisms in high myopia[J]. Mol Vis, 2009, 15(176-178): 1655-1663.
[10]	Kusakari T, Sato T, Tokoro T. Visual deprivation stimulates the exchange of the fibrous sclera into the cartilaginous sclera in chicks[J]. Exp Eye Res, 2001, 73(4): 533-546.
[11]	Jobling AI, Gentle A, Metlapally R, et al. Regulation of scleral cell contraction by transforming growth factor-beta and stress: competing roles in myopic eye growth[J]. J Biol Chem, 2009, 284(4): 2072-2079.
[12]	Mcbrien NA, Jobling AI, Gentle A. Biomechanics of the sclera in myopia: extracellular and cellular factors[J]. Optom Vis Sci, 2009, 86(1): E23-E30.
[13]	Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-beta 2 in experimental myopia in chicks[J]. Invest Ophthalmol Vis Sci, 1995, 36(6): 1183-1187.
[14]	Honda S, Fujii S, Sekiya Y, et al. Retinal control on the axial length mediated by transforming growth factor-beta in chick eye[J]. Invest Ophthalmol Vis Sci, 1996, 37(12): 2519-2526.
[15]	曾爱萍，曾水清，程扬. 转化生长因子β1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响[J]. 眼科新进展，2006，26(2)：81-84.
[16]	Xiaofen Z, Renyuan C. Effects of TGF-β on human embryonic retinal pigment epithelium cells[J]. Chinese Ophthalmic Research, 2007, 25(1): 14-17.
[17]	Siegwart J, Norton TT. Selective regulation of MMP and TIMP mRNA levels in tree shrew sclera during minus lens compensation and recovery[J]. Invest Ophthalmol Vis Sci, 2005, 46(10): 3484-3492.
[18]	Liu R, Ahmed KM, Nantajit D, et al. Therapeutic effects of α-lipoic acid on bleomycin-induced pulmonary fibrosis in rats[J]. Int J Mol Med, 2007, 19(6): 865-873.
[19]	刘海霞，杨坤禹，项楠，等. 转化生长因子-β1转基因小鼠巩膜厚度研究[J]. 华中科技大学学报(医学版)，2007，36(5)：655-657，670.
[20]	Edwards DR, Murphy G, Reynolds JJ, et al. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor[J]. EMBO J, 1987, 6(7): 1899-1904.
[21]	Hu J, Cui D, Yang X, et al. Bone morphogenetic protein-2: a potential regulator in scleral remodeling[J]. Mol Vis, 2008, 14(12): 2373-2380.
[22]	邢凯，吴宁玲，亢泽峰，等. 高度近视眼巩膜细胞外基质中相关胶原分子机制的研究进展[J]. 中华眼科医学杂志(电子版)，2018，8(1)：44-48.
[23]	Shelton L, Rada JS. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Exp Eye Res, 2007, 84(2): 314-322.
[24]	蔡瑜，徐奕，沈锡中. Smad泛素化调节因子2在肝纤维化过程中对结缔组织生长因子的作用研究[J]. 内科理论与实践，2012，7(4)：305-309.
[25]	Inoue Y, Imamura T. Regulation of TGF-beta family signaling by E3 ubiquitin ligases[J]. Cancer Sci, 2008, 99(11): 2107-2112.
[26]	Izzi L, Attisano L. Regulation of the TGFbeta signalling pathway by ubiquitin-mediated degradation[J]. Oncogene, 2004, 23(11): 2071-2078.
[27]	Itoh S, ten Dijke P. Negative regulation of TGF-beta receptor/Smad signal transduction[J]. Curr Opin Cell Biol, 2007, 19(2): 176-184.
[28]	Ito I, Hanyu A, Wayama M, et al. Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation[J]. J Biol Chem, 2010, 285(19): 14747-14755.
[29]	Bizet AA, Tran-Khanh N, Saksena A, et al. CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function[J]. J Cell Biochem, 2012, 113(1): 238-246.
[30]	Yang Q, Chen SP, Zhang XP, et al. Smurf2 participates in human trophoblast cell invasion by inhibiting TGF-beta type I receptor[J]. J Histochem Cytochem, 2009, 57(6): 605-612.
[31]	Chong PA, Lin H, Wrana JL, et al. An expanded WW domain recognition motif revealed by the interaction between Smad7 and the E3 ubiquitin ligase Smurf2[J]. J Biol Chem, 2006, 281(25): 17069-17075.
[32]	Morén A, Imamura T, Miyazono K, et al. Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases[J]. J Biol Chem, 2005, 280(23): 22115-22123.
[33]	Nomura N, Sasamoto S, Ishii S, et al. Isolation of human cDNA clones of ski and the ski-related gene, sno[J]. Nucleic Acids Res, 1989, 17(14): 5489-5500.
[34]	Debigaré R, Price SR. Proteolysis, the ubiquitin-proteasome system, and renal diseases[J]. Am J Physiol Renal Physiol, 2003, 285(1): F1-F8.
[35]	Asano Y, Ihn H, Yamane K, et al. Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts[J]. J Clin Invest, 2004, 113(2): 253-264.
[36]	刘晓政，田春阳，夏永欣. Smurf在肝纤维化中的表达及其对TGF-β/Smad信号传导的影响[J]. 广东医学，2017，38(6)：834-838.
[37]	Ohashi N, Yamamoto T, Uchida C, et al. Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta[J]. FEBS Lett, 2005, 579(12): 2557-2563.
[38]	潘红卫，高岩，曾骏文. 转化生长因子β1对小鼠巩膜成纤维细胞DNA和胶原合成的影响及其在近视形成中的作用[J]. 中国临床康复，2005，9(18)：160-162.
[39]	Li H, Cui D, Zhao F, et al. BMP-2 is involved in scleral remodeling in myopia development[J]. PLoS One, 2015, 10(5): e0125219.

引物	引物序列(5′-3′)
Smurf2	上游：5′-TTGCTAAGGTGGTGGTTG-3′
Smurf2	下游：5′-AAGCGTATTCTTCACAGTATCT-3′’
COLIA1	上游：5′-CGGCTCCTGCTCCTCTTA-3′
COLIA1	下游：5′-GGTGGGATGTCTTCGTCTT-3′
β肌动蛋白	上游：5′-TGACGTGGACATCCGCAAAG-3′
β肌动蛋白	下游：5′-CTGGAAGGTGGACAGCGAGG-3′

引物	引物序列(5′-3′)
Smurf2	上游：5′-TTGCTAAGGTGGTGGTTG-3′
Smurf2	下游：5′-AAGCGTATTCTTCACAGTATCT-3′’
COLIA1	上游：5′-CGGCTCCTGCTCCTCTTA-3′
COLIA1	下游：5′-GGTGGGATGTCTTCGTCTT-3′
β肌动蛋白	上游：5′-TGACGTGGACATCCGCAAAG-3′
β肌动蛋白	下游：5′-CTGGAAGGTGGACAGCGAGG-3′

组别	COLIA1 mRNA相对表达量	Smurf2 mRNA相对表达量
空白对照组	1.00±0.02	1.00±0.02
1 ng/ml组	0.80±0.01	0.87±0.10
5 ng/ml组	1.20±0.06	1.35±0.02^*
10 ng/ml组	2.40±0.06^*	1.19±0.02^*
F值	2.45	2.34
P值	<0.05	<0.05

组别	COLIA1 mRNA相对表达量	Smurf2 mRNA相对表达量
空白对照组	1.00±0.02	1.00±0.02
1 ng/ml组	0.80±0.01	0.87±0.10
5 ng/ml组	1.20±0.06	1.35±0.02^*
10 ng/ml组	2.40±0.06^*	1.19±0.02^*
F值	2.45	2.34
P值	<0.05	<0.05

组别	COLIA1 mRNA相对表达量	Smurf2 mRNA相对表达量
空白对照组	1.00±0.31	1.00±0.48
1 h组	1.51±0.18	0.92±0.16
6 h组	1.48±0.13	1.60±0.18^*
12 h组	1.90±0.21	1.36±0.11
24 h组	2.42±0.18^*	1.12±0.17
F值	4.45	3.32
P值	<0.05	<0.05

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