TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响

doi:10.11843/j.issn.0366-6964.2023.08.019

Abstract

Abstract: The study aimed to explore the mechanism of the TGF-β/Smad signaling pathway mediated by TGFβR1 regulating the function of sheep granulosa cells. In this study, sheep ovarian granulosa cells were collected, and pcDNA3.1(+)-TGFβR1 and pcDNA3.1(+)-SMAD4 overexpressed plasmid vectors and shRNA-TGFβR1 and shRNA-SMAD4 interfering plasmid vectors were constructed to overexpress and interfere with TGFβR1 and SMAD4 genes in sheep granulosa cells, respectively. The proliferation, cell cycle and apoptosis of sheep granulosa cells were detected by CCK-8 cell proliferation assay, flow cytometry and Annexin-V FITC/PI double staining. The mRNA levels of TGFβR1 and SMAD4, the protein expression levels of apoptosis-related proteins BAX, BCL2, and Caspase3 and the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway were detected by Real-Time PCR and Western blot. The results showed that overexpression of TGFβR1 and SMAD4 extremely significantly promoted the ability of granulosa cells to enter the S phase from the G0/G1 phase (P<0.01), the proliferation of granulosa cells and the expression of anti-apoptotic protein BCL2 (P<0.01), and inhibited the expression of apoptotic protein BAX and Caspase3 (P<0.01). Interference with TGFβR1 and SMAD4 both promoted granulosa cell apoptosis and the expressions of apoptotic protein BAX and Caspase3 (P<0.01) and inhibited the expression of anti-apoptotic protein BCL2 (P<0.01). Further studies showed that overexpression of TGFβR1 could promote the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway (P<0.01), and extremely significantly promote the protein expression level of SMAD4 (P<0.01). Interference with TGFβR1 significantly inhibited the phosphorylation levels of TGFβR1, TGFβR2 (P<0.01) and SMAD2/3 (P<0.05), and inhibited the protein expression of SMAD4 (P<0.01). Overexpression of SMAD4 could significantly promote the expression of TGFβR1 protein (P<0.01). The results showed that TGFβR1 promoted the proliferation and cycle of sheep granulosa cells, inhibited granulosa cell apoptosis, positively regulated the proliferation and cycle of sheep granulosa cells, and negatively regulated the apoptosis of sheep granulosa cells by mediating the expression of SMAD2/3 and SMAD4 in TGF-β/Smad signaling pathway.

Key words: TGFβR1, sheep, granulosa cell, TGF-β/Smad signaling pathway

CLC Number:

S826.3

LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.

References 42

[1]	付强, 岳巧娴, 锡建中, 等.绵羊miR-127/FOXO4反馈环路及其对卵泡颗粒细胞凋亡相关基因表达的影响[J].畜牧兽医学报, 2022, 53(1):66-75.FU Q, YUE Q X, XI J Z, et al.Effect of Oar-miR-127/FOXO4 feedback loop on genes associated with apoptosis of sheep granulosa cells[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(1):66-75.(in Chinese)
[2]	FANG X H, XIA W, LI S, et al.SIRT2 is critical for sheep oocyte maturation through regulating function of surrounding granulosa cells[J].Int J Mol Sci, 2022, 23(9):5013.
[3]	LI M N, LIANG W W, ZHU C Y, et al.Smad4 mediates Bmf involvement in sheep granulosa cell apoptosis[J].Gene, 2022, 817:146231.
[4]	LI Y X, MA X F, GAO T, et al.Differential expression and functional prediction of mRNA in the ovaries of Hanper sheep of high and low fecundity[J].Reprod Domest Anim, 2022, 57(12):1623-1635.
[5]	ROSAIRO D, KUYZNIEREWICZ I, FINDLAY J, et al.Transforming growth factor-β:its role in ovarian follicle development[J]. Reproduction, 2008, 136(6):799-809.
[6]	LIU J Y, DU X, ZHOU J L, et al.MicroRNA-26b functions as a proapoptotic factor in porcine follicular granulosa cells by targeting Sma-and Mad-related protein 4[J].Biol Reprod, 2014, 91(6):146.
[7]	DU X, ZHANG L F, LI X Y, et al.TGF-β signaling controls FSHR signaling-reduced ovarian granulosa cell apoptosis through the SMAD4/miR-143 axis[J].Cell Death Dis, 2016, 7(11):e2476.
[8]	ZHOU J L, LIU J Y, PAN Z X, et al.The let-7g microRNA promotes follicular granulosa cell apoptosis by targeting transforming growth factor-β type 1 receptor[J].Mol Cell Endocrinol, 2015, 409:103-112.
[9]	YAO G D, YIN M M, LIAN J, et al.MicroRNA-224 is involved in transforming growth factor-β-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4[J].Mol Endocrinol, 2010, 24(3):540-551.
[10]	YAO W, PAN Z X, DU X, et al.miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-β signaling by interacting with the TGFBR1 promoter[J].J Cell Physiol, 2018, 233(9):6807-6821.
[11]	ZHANG J Q, GAO B W, GUO H X, et al.miR-181a promotes porcine granulosa cell apoptosis by targeting TGFBR1 via the activin signaling pathway[J].Mol Cell Endocrinol, 2020, 499:110603.
[12]	LI Y X, JIN Y J, LIU Y X, et al.SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway[J].Reproduction, 2013, 146(2):169-179.
[13]	YU M M, LIU J X.MicroRNA-30d-5p promotes ovarian granulosa cell apoptosis by targeting Smad2[J].Exp Ther Med, 2020, 19(1):53-60.
[14]	DENG M, HOU S Y, TONG B D, et al.The Smad2/3/4 complex binds miR-139 promoter to modulate TGFβ-induced proliferation and activation of human Tenon's capsule fibroblasts through the Wnt pathway[J].J Cell Physiol, 2019, 234(8):13342-13352.
[15]	CHEN L, TOKE N H, LUO S, et al.A reinforcing HNF4-SMAD4 feed-forward module stabilizes enterocyte identity[J].Nat Genet, 2019, 51(5):777-785.
[16]	NEWMAN A C, KEMP A J, DRABSCH Y, et al.Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins[J].Nat Commun, 2017, 8(1):1537.
[17]	DING Z H, WU C J, CHU G C, et al.SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression[J]. Nature, 2011, 470(7333):269-273.
[18]	DU X, LI Q Q, YANG L, et al.SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis[J].Cell Death Dis, 2020, 11(5):373.
[19]	LEE K P, SHIN Y J, PANDA A C, et al.MiR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4[J].Genes Dev, 2015, 29(15):1605-1617.
[20]	WANDZIOCH E, ZARET K S.Dynamic signaling network for the specification of embryonic pancreas and liver progenitors[J]. Science, 2009, 324(5935):1707-1710.
[21]	LIU K, JI F, YANG G, et al.SMAD4 defect causes auditory neuropathy via specialized disruption of cochlear ribbon synapses in mice[J].Mol Neurobiol, 2016, 53(8):5679-5691.
[22]	YAO W, DU X, ZHANG J B, et al.SMAD4-induced knockdown of the antisense long noncoding RNA BRE-AS contributes to granulosa cell apoptosis[J].Mol Ther Nucleic Acids, 2021, 25:251-263.
[23]	韩红叶.CTSB基因对绵羊卵巢颗粒细胞功能的影响[D].保定:河北农业大学, 2021.HAN H Y.Effect of CTSB gene on the function of ovarian granulosa cells in sheep[D].Baoding:Hebei Agricultural University, 2021.(in Chinese)
[24]	MATSUDA F, INOUE N, MANABE N, et al.Follicular growth and atresia in mammalian ovaries:regulation by survival and death of granulosa cells[J].J Reprod Dev, 2012, 58(1):44-50.
[25]	DAVID C J, MASSAGUÉ J.Contextual determinants of TGFβ action in development, immunity and cancer[J].Nat Rev Mol Cell Biol, 2018, 19(7):419-435.
[26]	赵金.BMP15通过BMPR1B调控陕北白绒山羊卵泡颗粒细胞功能的分子机制[D].杨凌:西北农林科技大学, 2021.ZHAO J.The molecular mechanism of BMP15 regulates the function of follicular granulosa cells through BMPR1B in Shanbei white cashmere goat[D].Yangling:Northwest A&F University, 2021.(in Chinese)
[27]	CHANG H, BROWN C W, MATZUK M M.Genetic analysis of the mammalian transforming growth factor-β superfamily[J]. Endocr Rev, 2002, 23(6):787-823.
[28]	LI Q L, AGNO J E, EDSON M A, et al.Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function[J].PLoS Genet, 2011, 7(10):e1002320.
[29]	ZHAO Y, FENG H W, ZHANG Y H, et al.Current understandings of core pathways for the activation of mammalian primordial follicles[J].Cells, 2021, 10(6):1491.
[30]	王伟.BMP/Smad信号通路对猪卵泡颗粒细胞的影响[D].南京:南京农业大学, 2010.WANG W.Effects of BMP/Smad signaling on porcine follicular granulosa cells[D].Nanjing:Nanjing Agricultural University, 2010.(in Chinese)
[31]	LARSSON J, GOUMANS M J, SJÖSTRAND L J, et al.Abnormal angiogenesis but intact hematopoietic potential in TGF-β type I receptor-deficient mice[J].EMBO J, 2001, 20(7):1663-1673.
[32]	徐梦思.TGF β-SMAD信号通路对猪颗粒细胞和繁殖性状的作用研究[D].石河子:石河子大学, 2015.XU M S.The influence of TGF beta SMAD signaling pathway on the pig granulosa cells and reproductive traits[D]. Shihezi:Shihezi University, 2015.(in Chinese)
[33]	UMENO K, SASAKI A, KIMURA N.The impact of oocyte death on mouse primordial follicle formation and ovarian reserve[J]. Reprod Med Biol, 2022, 21(1):e12489.
[34]	PEREZ G I, ROBLES R, KNUDSON C M, et al.Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency[J]. Nat Genet, 1999, 21(2):200-203.
[35]	LU X R, DUAN A Q, MA X Y, et al.Knockdown of CYP19A1 in buffalo follicular granulosa cells results in increased progesterone secretion and promotes cell proliferation[J].Front Vet Sci, 2020, 7:539496.
[36]	SAI T, GOTO Y, YOSHIOKA R, et al.Bid and Bax are involved in granulosa cell apoptosis during follicular atresia in porcine ovaries[J].J Reprod Dev, 2011, 57(3):421-427.
[37]	VAN OPDENBOSCH N, LAMKANFI M.Caspases in cell death, inflammation, and disease[J].Immunity, 2019, 50(6):1352-1364.
[38]	庞雪利, 李矿发, 魏兰, 等.IL-8通过上调Bcl-2的表达和下调caspase-3的表达抑制MCF-7乳腺癌细胞凋亡[J].细胞与分子免疫学杂志, 2015, 31(3):307-311.PANG X L, LI K F, WEI L, et al.IL-8 inhibits the apoptosis of MCF-7 human breast cancer cells by up-regulating Bcl-2 and down-regulating caspase-3[J].Chinese Journal of Cellular and Molecular Immunology, 2015, 31(3):307-311.(in Chinese)
[39]	ZHANG Y M, JIANG Q, WANG N, et al.Effects of taspine on proliferation and apoptosis by regulating caspase-3 expression and the ratio of Bax/Bcl-2 in A431 cells[J].Phytother Res, 2011, 25(3):357-364.
[40]	ZHOU J, JIN X J, SHENG Z M, et al.miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis[J].Exp Ther Med, 2021, 21(3):179.
[41]	YANG L, ROH Y S, SONG J Y, et al.Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice[J].Hepatology, 2014, 59(2):483-495.
[42]	PANGAS S A, LI X H, ROBERTSON E J, et al. Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice[J].Mol Endocrinol, 2006, 20(6):1406-1422.

Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 42

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LÜ Shiqi, ZHOU Rongyan, TIAN Shujun, CHEN Xiaoyong. Study on the Physiological Mechanism of Mitochondrial tRNA-Lys(T7719G) Gene Variation Affecting Apoptosis of Ovine Granulosa Cell [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2011-2021.
[2]	DONG Shucan, MAO Shuaixiang, WU Cuiying, LI Yaokun, SUN Baoli, GUO Yongqing, DENG Ming, LIU Dewu, LIU Guangbin. The Effect of the Androgen Receptor Inhibitor Enzalutamide on Proliferation and Apoptosis of Goat Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2022-2031.
[3]	PENG Peiya, CHEN Yuhan, YANG Long, WANG Ming, ZHAO Ruiting, HE Jun, YIN Yulong, LIU Mei. Research Progress of Copy Number Variation in Livestock [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1356-1369.
[4]	ZHANG Shaohua, WANG Shuai, ZOU Yang, LIU Zhongli, CAI Xuepeng. Advances in Detection Approaches for Ovine Haemonchosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1499-1510.
[5]	YANG Yang, YU Qian, LIU Yucheng, YANG Hua, ZHAO Zhuo, WANG Limin, ZHOU Ping, YANG Qingyong, DAI Rong. Identification and Tissue Expression Analysis of the Sheep MYL Gene Family [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1551-1564.
[6]	CHANG Xindan, HU Fan, WU Zhiwu, YE Bingsen, LIU Tiehai, LIN Jie, HE Zhixiong, TAN Zhiliang. Effect of High Proportion Rumen Bypass Fat Diet on Feeding Behavior of Growing Mutton Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1077-1084.
[7]	PIAN Huifang, DU Xubin, LI Yan, ZHANG Yuchen, HE Hui, YU Debing. Effects of Betaine on Performance, Egg Quality and Antioxidant Capacity of Late-phase Laying Hens [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1085-1094.
[8]	LIU Yangguang, ZHANG Huibin, WEN Haoyu, XIE Fan, ZHAO Shiming, DING Yueyun, ZHENG Xianrui, YIN Zongjun, ZHANG Xiaodong. SNP/Indel Screening Analysis of Porcine Ovarian Granulosa Cells Treated with Follicular Fluid Exosomes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 576-586.
[9]	GAO Hui, FANG Min, JIANG Lingling, MA Yaoyu, LIU Qiang, ZHANG Gang, NIU Xiaoxia, WANG Pu, LI Yong, ZHANG Sinong. Meta-analysis of Bluetongue Virus Prevalence in Sheep Flocks in China from 2012—2022 [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 706-717.
[10]	WANG Haibo, ZHAN Jinshun, GU Zhiyong, CHEN Xinfeng, PAN Yue, JIA Haobin, ZHONG Xiaojun, LI Kairong, ZHAO Shengguo, HUO Junhong. Comparative Study on Meat Quality Characteristics of Three-Way Hybrid Sheep Charolais×Duper×Hu and Charolais×Australian White×Hu and Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 110-119.
[11]	SHI Shengjie, WANG Liguang, GAO Lei, CAI Chuanjiang, HE Weixian, CHU Guiyan. Effect of miR-24-3p on Estradiol Synthesis in Porcine Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 169-178.
[12]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[13]	ZHANG Hanyue, ZHAO Dan, LIANG Yu, ZHAO Bishi, FAN Mengdan, QIAO Liying, LIU Jianhua, YANG Kaijie, PAN Yangyang, LIU Wenzhong. miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3262-3274.
[14]	LIU Linli, PENG Xuelan, LI Bo, CHENG Lefan, CIREN Lamu, ZHANG Enping. Effect of Overexpression of UCP3 Gene on the Differentiation of Sahu Sheep Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3275-3285.
[15]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.