PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway

in Journal of Molecular Endocrinology
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Xiaodan LiCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Xiaolei YaoCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Yongjin BaoCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Kaiping DengCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Mingtian DengCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Fan YangCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Xuan SunCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Peihua YouCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Qingxian CaiCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Feng WangCollege of Animal Science and Technology, Nanjing Agricultural University, No. 1 Weigang, Nanjing, 210095, China

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Correspondence should be addressed to F Wang: caeet@njau.edu.cn
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The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

 

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  • Akerfelt M, Henriksson E, Laiho A, Vihervaara A, Rautoma K, Kotaja N & & Sistonen L 2008 Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2. PNAS 105 1122411229. (https://doi.org/10.1073/pnas.0800620105)

    • Search Google Scholar
    • Export Citation
  • An SY, Zhang GM, Liu ZF, Zhou C, Yang PC & & Wang F 2019 MiR-1197-3p regulates testosterone secretion in goat Leydig cells via targeting PPARGC1A. Gene 710 131139. (https://doi.org/10.1016/j.gene.2019.05.057)

    • Search Google Scholar
    • Export Citation
  • Andreu-Fernández V, Sancho M, Genovés A, Lucendo E, Todt F, Lauterwasser J, Funk K, Jahreis G, Pérez-Payá E, Mingarro I, et al.2017 Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes. PNAS 114 310315. (https://doi.org/10.1073/pnas.1612322114)

    • Search Google Scholar
    • Export Citation
  • Chen F, Wang Y, Liu Q, Hu J, Jin J, Ma Z & & Zhang J 2020 ERO1α promotes testosterone secretion in hCG-stimulated mouse Leydig cells via activation of the PI3K/AKT/mTOR signaling pathway. Journal of Cellular Physiology 235 56665678. (https://doi.org/10.1002/jcp.29498)

    • Search Google Scholar
    • Export Citation
  • Chen Y, Wang J, Xu D, Xiang Z, Ding J, Yang X, Li D & & Han X 2021 m6A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells. Autophagy 17 457475. (https://doi.org/10.1080/15548627.2020.1720431)

    • Search Google Scholar
    • Export Citation
  • Cui R, Jiang N, Zhang M, Du S, Ou H, Ge R, Ma D & & Zhang J 2021 AMOTL2 inhibits JUN Thr239 dephosphorylation by binding PPP2R2A to suppress the proliferation in non-small cell lung cancer cells. Biochimica et Biophysica Acta. Molecular Cell Research 1868 118858. (https://doi.org/10.1016/j.bbamcr.2020.118858)

    • Search Google Scholar
    • Export Citation
  • Dong T, Zhang X, Liu Y, Xu S, Chang H, Chen F, Pan L, Hu S, Wang M & & Lu M 2021 Opa1 prevents apoptosis and cisplatin-induced ototoxicity in murine cochleae. Frontiers in Cell and Developmental Biology 9 744838. (https://doi.org/10.3389/fcell.2021.744838)

    • Search Google Scholar
    • Export Citation
  • Du M, Yuan L, Zhang Z, Zhang C, Zhu M, Zhang Z, Li R, Zhao X, Liang H, Li Y, et al.2021 PPP2R1B is modulated by ubiquitination and is essential for spermatogenesis. FASEB Journal 35 e21564. (https://doi.org/10.1096/fj.202002810R)

    • Search Google Scholar
    • Export Citation
  • Dudiki T, Kadunganattil S, Ferrara JK, Kline DW & & Vijayaraghavan S 2015 Changes in carboxy methylation and tyrosine phosphorylation of protein phosphatase PP2A are associated with epididymal sperm maturation and motility. PLoS One 10 e0141961. (https://doi.org/10.1371/journal.pone.0141961)

    • Search Google Scholar
    • Export Citation
  • Edlich F 2018 BCL-2 proteins and apoptosis: recent insights and unknowns. Biochemical and Biophysical Research Communications 500 2634. (https://doi.org/10.1016/j.bbrc.2017.06.190)

    • Search Google Scholar
    • Export Citation
  • Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, et al.2006 OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126 177189. (https://doi.org/10.1016/j.cell.2006.06.025)

    • Search Google Scholar
    • Export Citation
  • Gao LL, Liu XQ, Xu BQ, Jiang SW, Cui YG & & Liu JY 2013 SET/PP2A system regulates androgen production in ovarian follicles in vitro. Molecular and Cellular Endocrinology 374 108116. (https://doi.org/10.1016/j.mce.2013.04.012)

    • Search Google Scholar
    • Export Citation
  • Gogvadze V, Orrenius S & & Zhivotovsky B 2006 Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochimica et Biophysica Acta 1757 639647. (https://doi.org/10.1016/j.bbabio.2006.03.016)

    • Search Google Scholar
    • Export Citation
  • Gorowska-Wojtowicz E, Dutka P, Kudrycka M, Pawlicki P, Milon A, Plachno BJ, Tworzydlo W, Pardyak L, Kaminska A, Hejmej A, et al.2018 Regulation of steroidogenic function of mouse Leydig cells: G-coupled membrane estrogen receptor and peroxisome proliferator-activated receptor partnership. Journal of Physiology and Pharmacology 69 373390. (https://doi.org/10.26402/jpp.2018.3.04)

    • Search Google Scholar
    • Export Citation
  • Gu G, Hu C, Hui K, Zhang H, Chen T, Zhang X & & Jiang X 2021 Exosomal miR-136-5p derived from anlotinib-resistant NSCLC cells confers anlotinib resistance in non-small cell lung cancer through targeting PPP2R2A. International Journal of Nanomedicine 16 63296343. (https://doi.org/10.2147/IJN.S321720)

    • Search Google Scholar
    • Export Citation
  • Guo H, Luo X, Sun L, Li J & & Cui S 2021 Cyclin-dependent kinase inhibitor 1B acts as a novel molecule to mediate testosterone synthesis and secretion in mouse Leydig cells by luteinizing hormone (LH) signaling pathway. In Vitro Cellular and Developmental Biology. Animal 57 742752. (https://doi.org/10.1007/s11626-021-00545-x)

    • Search Google Scholar
    • Export Citation
  • Guran T, Yesil G, Turan S, Atay Z, Bozkurtlar E, Aghayev A, Gul S, Tinay I, Aru B, Arslan S, et al.2019 PPP2R3C gene variants cause syndromic 46,XY gonadal dysgenesis and impaired spermatogenesis in humans. European Journal of Endocrinology 180 291309. (https://doi.org/10.1530/EJE-19-0067)

    • Search Google Scholar
    • Export Citation
  • He X, Li M, Yu H, Liu G, Wang N, Yin C, Tu Q, Narla G, Tao Y, Cheng S, et al.2020 Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex. PLOS Biology 18 e3000803. (https://doi.org/10.1371/journal.pbio.3000803)

    • Search Google Scholar
    • Export Citation
  • Hong Y & & Sarge KD 1999 Regulation of protein phosphatase 2A activity by heat shock transcription factor 2. Journal of Biological Chemistry 274 1296712970. (https://doi.org/10.1074/jbc.274.19.12967)

    • Search Google Scholar
    • Export Citation
  • Jones PM, Sayed SB, Persaud SJ, Burns CJ, Gyles S & & Whitehouse BJ 2000 Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities. Journal of Molecular Endocrinology 24 233239. (https://doi.org/10.1677/jme.0.0240233)

    • Search Google Scholar
    • Export Citation
  • Kauko O & & Westermarck J 2018 Non-genomic mechanisms of protein phosphatase 2A (PP2A) regulation in cancer. International Journal of Biochemistry and Cell Biology 96 157164. (https://doi.org/10.1016/j.biocel.2018.01.005)

    • Search Google Scholar
    • Export Citation
  • Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE & & Oltvai ZN 1993 Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death. Seminars in Cancer Biology 4 327332.

    • Search Google Scholar
    • Export Citation
  • Li X, Yao X, Xie H, Deng M, Gao X, Deng K, Bao Y, Wang Q & & Wang F 2021a Effects of SPATA6 on proliferation, apoptosis and steroidogenesis of Hu sheep Leydig cells in vitro. Theriogenology 166 920. (https://doi.org/10.1016/j.theriogenology.2021.02.011)

    • Search Google Scholar
    • Export Citation
  • Li X, Yao X, Xie H, Zhang G, Deng M, Deng K, Gao X, Bao Y, Li K & & Wang F 2021b PPP2R2A affects embryonic implantation by regulating the proliferation and apoptosis of Hu sheep endometrial stromal cells. Theriogenology 176 149162. (https://doi.org/10.1016/j.theriogenology.2021.09.026)

    • Search Google Scholar
    • Export Citation
  • Luan Z, Fan X, Song H, Li R, Zhang W & & Zhang J 2019 Testosterone promotes GPX5 expression of goat epididymal epithelial cells cultured in vitro. In Vitro Cellular and Developmental Biology. Animal 55 677685. (https://doi.org/10.1007/s11626-019-00391-y)

    • Search Google Scholar
    • Export Citation
  • Martínez-Alonso D & & Malumbres M 2020 Mammalian cell cycle cyclins. Seminars in Cell and Developmental Biology 107 2835. (https://doi.org/10.1016/j.semcdb.2020.03.009)

    • Search Google Scholar
    • Export Citation
  • Matsuo M, Onuma TA, Omotezako T & & Nishida H 2020 Protein phosphatase 2A is essential to maintain meiotic arrest, and to prevent Ca(2+) burst at spawning and eventual parthenogenesis in the larvacean Oikopleura dioica. Developmental Biology 460 155163. (https://doi.org/10.1016/j.ydbio.2019.12.005)

    • Search Google Scholar
    • Export Citation
  • Meng K, Wang X, He Y, Yang J, Wang H, Zhang Y & & Quan F 2019 The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cellsdagger. Biology of Reproduction 100 13441355. (https://doi.org/10.1093/biolre/ioz003)

    • Search Google Scholar
    • Export Citation
  • Panicker N, Coutman M, Lawlor-O'Neill C, Kahl RGS, Roselli S & & Verrills NM 2020 Ppp2r2a knockout mice reveal that protein phosphatase 2A regulatory subunit, PP2A-B55α, is an essential regulator of neuronal and epidermal embryonic development. Frontiers in Cell and Developmental Biology 8 358. (https://doi.org/10.3389/fcell.2020.00358)

    • Search Google Scholar
    • Export Citation
  • Reynhout S & & Janssens V 2019 Physiologic functions of PP2A: lessons from genetically modified mice. Biochimica et Biophysica Acta. Molecular Cell Research 1866 3150. (https://doi.org/10.1016/j.bbamcr.2018.07.010)

    • Search Google Scholar
    • Export Citation
  • Rossmann MP, Dubois SM, Agarwal S & & Zon LI 2021 Mitochondrial function in development and disease. Disease Models and Mechanisms 14 dmm048912. (https://doi.org/10.1242/dmm.048912)

    • Search Google Scholar
    • Export Citation
  • Roy S, Tomaszowski KH, Luzwick JW, Park S, Li J, Murphy M & & Schlacher K 2018 p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLθ pathways. eLife 7 e31723. (https://doi.org/10.7554/eLife.31723)

    • Search Google Scholar
    • Export Citation
  • Shouse G, de Necochea-Campion R, Mirshahidi S, Liu X & & Chen CS 2016 Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest. Oncotarget 7 6108161092. (https://doi.org/10.18632/oncotarget.11209)

    • Search Google Scholar
    • Export Citation
  • Sun J, Zhong L, Zhu Y & & Liu G 2011 Research on the isolation of mouse Leydig cells using differential digestion with a low concentration of collagenase. Journal of Reproduction and Development 57 433436. (https://doi.org/10.1262/jrd.10-123n)

    • Search Google Scholar
    • Export Citation
  • Wang H, Wang X, Li T, An X, Yin D, Chen N & & Ma Y 2022 Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity. Gene Expression Patterns 43 119218. (https://doi.org/10.1016/j.gep.2021.119218)

    • Search Google Scholar
    • Export Citation
  • Wang J, Wu J, Zhang Y, Zhang J, Xu W, Wu C & & Zhou P 2021a Growth hormone protects against ovarian granulosa cell apoptosis: alleviation oxidative stress and enhancement mitochondrial function. Reproductive Biology 21 100504. (https://doi.org/10.1016/j.repbio.2021.100504)

    • Search Google Scholar
    • Export Citation
  • Wang TE, Minabe S, Matsuda F, Li SH, Tsukamura H, Maeda KI, Smith L, O'Hara L, Gadella BM & & Tsai PS 2021b Testosterone regulation on quiescin sulfhydryl oxidase 2 synthesis in the epididymis. Reproduction 161 593602. (https://doi.org/10.1530/REP-20-0629)

    • Search Google Scholar
    • Export Citation
  • White JP, Gao S, Puppa MJ, Sato S, Welle SL & & Carson JA 2013 Testosterone regulation of Akt/mTORC1/FoxO3a signaling in skeletal muscle. Molecular and Cellular Endocrinology 365 174186. (https://doi.org/10.1016/j.mce.2012.10.019)

    • Search Google Scholar
    • Export Citation
  • Yang H, Wan Z, Jin Y, Wang F & & Zhang Y 2021a SMAD2 regulates testicular development and testosterone synthesis in Hu sheep. Theriogenology 174 139148. (https://doi.org/10.1016/j.theriogenology.2021.08.027)

    • Search Google Scholar
    • Export Citation
  • Yang M, Guan S, Tao J, Zhu K, Lv D, Wang J, Li G, Gao Y, Wu H, Liu J, et al.2021b Melatonin promotes male reproductive performance and increases testosterone synthesis in mammalian Leydig cells†. Biology of Reproduction 104 13221336. (https://doi.org/10.1093/biolre/ioab046)

    • Search Google Scholar
    • Export Citation
  • Zarazaga LA, Guzmán JL, Domínguez C, Pérez MC & & Prieto R 2009 Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats. Theriogenology 71 13161325. (https://doi.org/10.1016/j.theriogenology.2009.01.007)

    • Search Google Scholar
    • Export Citation
  • Zeng LP, Hu ZM, Li K & & Xia K 2016 miR-222 attenuates cisplatin-induced cell death by targeting the PPP2R2A/Akt/mTOR Axis in bladder cancer cells. Journal of Cellular and Molecular Medicine 20 559567. (https://doi.org/10.1111/jcmm.12760)

    • Search Google Scholar
    • Export Citation
  • Zhang M, Wang S, Yi A & & Qiao Y 2020 microRNA-665 is down-regulated in gastric cancer and inhibits proliferation, invasion, and EMT by targeting PPP2R2A. Cell Biochemistry and Function 38 409418. (https://doi.org/10.1002/cbf.3485)

    • Search Google Scholar
    • Export Citation
  • Zhao X, Xu W, Wu J, Zhang D, Abou-Shakra A, Di L, Wang Z, Wang L, Yang F & & Qiao Z 2018 Nicotine induced autophagy of Leydig cells rather than apoptosis is the major reason of the decrease of serum testosterone. International Journal of Biochemistry and Cell Biology 100 3041. (https://doi.org/10.1016/j.biocel.2018.05.001)

    • Search Google Scholar
    • Export Citation
  • Zhao Z, Kurimchak A, Nikonova AS, Feiser F, Wasserman JS, Fowle H, Varughese T, Connors M, Johnson K, Makhov P, et al.2019 PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55alpha/PP2A reconstitution that triggers centrosome destabilization. Oncogenesis 8 72. (https://doi.org/10.1038/s41389-019-0180-9)

    • Search Google Scholar
    • Export Citation
  • Zheng W, Zhang Y, Sun C, Ge S, Tan Y, Shen H & & Yang P 2021 A multi-omics study of human testis and epididymis. Molecules 26 3345. (https://doi.org/10.3390/molecules26113345)

    • Search Google Scholar
    • Export Citation
  • Zhou R, Wu J, Liu B, Jiang Y, Chen W, Li J, He Q & & He Z 2019 The roles and mechanisms of Leydig cells and myoid cells in regulating spermatogenesis. Cellular and Molecular Life Sciences 76 26812695. (https://doi.org/10.1007/s00018-019-03101-9)

    • Search Google Scholar
    • Export Citation