NEK2 plays an essential role in porcine embryonic development by maintaining mitotic division and DNA damage response via the Wnt/β-catenin signalling pathway

Abstract

NIMA-related kinase 2 (NEK2) is a serine/threonine protein kinase that regulates mitosis and plays pivotal roles in cell cycle regulation and DNA damage repair. However, its function in porcine embryonic development is unknown. In this study, we used an NEK2-specific inhibitor, JH295 (JH), to investigate the role of NEK2 in embryonic development and the underlying regulatory mechanisms. Inhibition of NEK2 after parthenogenesis activation or in vitro fertilization significantly reduced the rates of cleavage and blastocyst formation, the numbers of trophectoderm and total cells and the cellular survival rate compared with the control condition. NEK2 inhibition delayed cell cycle progression at all stages from interphase to cytokinesis during the first mitotic division; it caused abnormal nuclear morphology in two- and four-cell stage embryos. Additionally, NEK2 inhibition significantly increased DNA damage and apoptosis, and it altered the expression levels of DNA damage repair- and apoptosis-related genes. Intriguingly, NEK2 inhibition downregulated the expression of β-catenin and its downstream target genes. To validate the relationship between Wnt/β-catenin signalling and NEK2 during porcine embryonic development, we cultured porcine embryos in JH-treated medium with or without CHIR99021, a Wnt activator. CHIR99021 co-treatment strongly restored the developmental parameters reduced by NEK2 inhibition to control levels. Our findings suggest that NEK2 plays an essential role in porcine embryonic development by regulating DNA damage repair and normal mitotic division via the Wnt/β-catenin signalling pathway.

References

[1] SfakianoudisK, Maziotis E, KarantzaliE, et al. Molecular drivers of developmental arrest in the human preimplantation embryo: a systematic review and critical analysis leading to mapping future research. Int J Mol Sci. 2021;22(15):8353.

[2] PaonessaM, BoriniA, CoticchioG. Genetic causes of preimplantation embryo developmental failure. Mol Reprod Dev. 2021;88(5):338-348.

[3] O’ConnellMJ, Walworth NC, CarrAM. The G2-phase DNA-damage checkpoint. Trends Cell Biol. 2000;10(7):296-303.

[4] BohgakiT, Bohgaki M, HakemR. DNA double-strand break signaling and human disorders. Genome Integr. 2010;1(1):15.

[5] MyungK, SmithS, KolodnerRD. Mitotic checkpoint function in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2004;101(45):15980-15985.

[6] HanZ, Chatterjee D, HeDM, et al. Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation. Mol Cell Biol. 1995;15(11):5849-5857.

[7] CuddihyAR, O’Connell MJ. Cell-cycle responses to DNA damage in G2. Int Rev Cytol. 2003;222:99-140.

[8] WangY, JiP, LiuJ, Broaddus RR, XueF, ZhangW. Centrosome-associated regulators of the G(2)/M checkpoint as targets for cancer therapy. Mol Cancer. 2009;8:8.

[9] MonizL, DuttP, HaiderN, Stambolic V. Nek family of kinases in cell cycle, checkpoint control and cancer. Cell Div. 2011;6:18.

[10] LuKP, KempBE, MeansAR. Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J Biol Chem. 1994;269(9):6603-6607.

[11] PavanICB, Peres de Oliveira A, DiasPRF, et al. On broken Ne(c)ks and broken DNA: the role of human NEKs in the DNA damage response. Cells. 2021;10(3):507.

[12] O’ReganL, BlotJ, FryAM. Mitotic regulation by NIMA-related kinases. Cell Div. 2007;2:25.

[13] DanaD, DasT, ChoiA, et al. Nek2 kinase signaling in malaria, bone, immune and kidney disorders to metastatic cancers and drug resistance: Progress on Nek2 inhibitor development. Molecules. 2022;27(2):347.

[14] FangY, ZhangX. Targeting NEK2 as a promising therapeutic approach for cancer treatment. Cell Cycle. 2016;15(7):895-907.

[15] FryAM, Meraldi P, NiggEA. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J. 1998;17(2):470-481.

[16] BachusS, GravesD, FulhamL, et al. In mitosis you are not: the NIMA family of kinases in aspergillus, yeast, and mammals. Int J Mol Sci. 2022;23(7):4041.

[17] FujiokaT, Takebayashi Y, ItoM, UchidaT. Nek2 expression and localization in porcine oocyte during maturation. Biochem Biophys Res Commun. 2000;279(3):799-802.

[18] SonnS, KhangI, KimK, RheeK. Suppression of Nek2A in mouse early embryos confirms its requirement for chromosome segregation. J Cell Sci. 2004;117(Pt 23):5557-5566.

[19] RheeK, Wolgemuth DJ. The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes. Development. 1997;124(11):2167-2177.

[20] RimEY, Clevers H, NusseR. The Wnt pathway: from signaling mechanisms to synthetic modulators. Annu Rev Biochem. 2022;91:571-598.

[21] AlbrechtLV, Tejeda-Munoz N, De RobertisEM. Cell biology of canonical Wnt signaling. Annu Rev Cell Dev Biol. 2021;37:369-389.

[22] WallingfordJB, HabasR. The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development. 2005;132(20):4421-4436.

[23] MacDonaldBT, TamaiK, HeX. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009;17(1):9-26.

[24] SidratT, RehmanZU, JooMD, Lee KL, KongIK. Wnt/beta-catenin pathway-mediated PPARdelta expression during embryonic development differentiation and disease. Int J Mol Sci. 2021;22(4):1854.

[25] ZhangY, WangX. Targeting the Wnt/beta-catenin signaling pathway in cancer. J Hematol Oncol. 2020;13(1):165.

[26] MbomBC, Siemers KA, OstrowskiMA, NelsonWJ, BarthAI. Nek2 phosphorylates and stabilizes beta-catenin at mitotic centrosomes downstream of Plk1. Mol Biol Cell. 2014;25(7):977-991.

[27] BahmanyarS, KaplanDD, DelucaJG, et al. Beta-catenin is a Nek2 substrate involved in centrosome separation. Genes Dev. 2008;22(1):91-105.

[28] NealCP, FryAM, MoremanC, et al. Overexpression of the Nek2 kinase in colorectal cancer correlates with beta-catenin relocalization and shortened cancer-specific survival. J Surg Oncol. 2014;110(7):828-838.

[29] XuT, ZengY, ShiL, et al. Targeting NEK2 impairs oncogenesis and radioresistance via inhibiting the Wnt1/beta-catenin signaling pathway in cervical cancer. J Exp Clin Cancer Res. 2020;39(1):183.

[30] ShenH, YanW, YuanJ, Wang Z, WangC. Nek2B activates the wnt pathway and promotes triple-negative breast cancer chemothezrapy-resistance by stabilizing beta-catenin. J Exp Clin Cancer Res. 2019;38(1):243.

[31] HeniseJC, Taunton J. Irreversible Nek2 kinase inhibitors with cellular activity. J Med Chem. 2011;54(12):4133-4146.

[32] BurruelV, Klooster K, BarkerCM, PeraRR, MeyersS. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage. Sci Rep. 2014;4:6598.

[33] MorrisNR. Mitotic mutants of Aspergillus nidulans. Genet Res. 1975;26(3):237-254.

[34] OsmaniAH, McGuire SL, OsmaniSA. Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans. Cell. 1991;67(2):283-291.

[35] BelhamC, RoigJ, CaldwellJA, et al. A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases. J Biol Chem. 2003;278(37):34897-34909.

[36] O’ConnellMJ, Krien MJ, HunterT. Never say never. The NIMA-related protein kinases in mitotic control. Trends Cell Biol. 2003;13(5):221-228.

[37] SchultzSJ, FryAM, SutterlinC, Ried T, NiggEA. Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans. Cell Growth Differ. 1994;5(6):625-635.

[38] FryAM, Schultz SJ, BartekJ, NiggEA. Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans. J Biol Chem. 1995;270(21):12899-12905.

[39] BarbagalloF, Paronetto MP, FrancoR, et al. Increased expression and nuclear localization of the centrosomal kinase Nek2 in human testicular seminomas. J Pathol. 2009;217(3):431-441.

[40] SonnS, JeongY, RheeK. Nip2/centrobin may be a substrate of Nek2 that is required for proper spindle assembly during mitosis in early mouse embryos. Mol Reprod Dev. 2009;76(6):587-592.

[41] KnouseKA, LopezKE, BachofnerM, Amon A. Chromosome segregation fidelity in epithelia requires tissue architecture. Cell. 2018;175(1):200-211 e213.

[42] BartekJ, LukasJ. DNA damage checkpoints: from initiation to recovery or adaptation. Curr Opin Cell Biol. 2007;19(2):238-245.

[43] WangH, LuoY, ZhaoMH, et al. DNA double-strand breaks disrupted the spindle assembly in porcine oocytes. Mol Reprod Dev. 2016;83(2):132-143.

[44] LuoD, YuC, YuJ, SuC, LiS, LiangP. p53-mediated G1 arrest requires the induction of both p21 and Killin in human colon cancer cells. Cell Cycle. 2022;21(2):140-151.

[45] FletcherL, Cerniglia GJ, NiggEA, YendTJ, Muschel RJ. Inhibition of centrosome separation after DNA damage: a role for Nek2. Radiat Res. 2004;162(2):128-135.

[46] BrandsmaI, GentDC. Pathway choice in DNA double strand break repair: observations of a balancing act. Genome Integr. 2012;3(1):9.

[47] ScullyR, XieA. Double strand break repair functions of histone H2AX. Mutat Res. 2013;750(1–2):5-14.

[48] HeadDR, Jacobberger JW, MosseC, et al. Innovative analyses support a role for DNA damage and an aberrant cell cycle in myelodysplastic syndrome pathogenesis. Bone Marrow Res. 2011;2011:1-9.

[49] ZhouW, YangY, XiaJ, et al. NEK2 induces drug resistance mainly through activation of efflux drug pumps and is associated with poor prognosis in myeloma and other cancers. Cancer Cell. 2013;23(1):48-62.

[50] de Jaime-SogueroA, Abreu de OliveiraWA, LluisF. The pleiotropic effects of the canonical Wnt pathway in early development and pluripotency. Genes. 2018;9(2):93.

[51] ZhaoY, TaoL, YiJ, SongH, ChenL. The role of canonical Wnt signaling in regulating Radioresistance. Cell Physiol Biochem. 2018;48(2):419-432.

[52] BaiR, YuanC, SunW, et al. NEK2 plays an active role in tumorigenesis and tumor microenvironment in non-small cell lung cancer. Int J Biol Sci. 2021;17(8):1995-2008.

[53] HuangY, OuyangH, XieW, et al. Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development. Cell Signal. 2013;25(4):778-785.

[54] WangB, KhanS, WangP, et al. A highly selective GSK-3beta inhibitor CHIR99021 promotes osteogenesis by activating canonical and autophagy-mediated Wnt signaling. Front Endocrinol. 2022;13:926622.

[55] ChoiKH, LeeDK, KimSW, Woo SH, KimDY, LeeCK. Chemically defined media can maintain pig pluripotency network in vitro. Stem Cell Reports. 2019;13(1):221-234.

[56] KwonJ, LiYH, JoYJ, OhY, NamgoongS, Kim NH. Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes. PeerJ. 2019;6:e5840.

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2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.