Department of Production Animal Health, Faculty of Veterinary Medicine, Heritage Medical Research Building RM 400, 3330 Hospital Drive NW, University of Calgary, Calgary, AB, Canada.
Biol Reprod. 2017 Feb 1;96(2):456-468. doi: 10.1095/biolreprod.116.141267.
Interaction of Na/K-ATPase with its ligand ouabain has been implicated in the regulation of various biological processes. The objective was to investigate roles of Na/K-ATPase isoforms in formation and function of junctional complexes in Sertoli cells. Primary cultures of Sertoli cells were obtained by enzymatic digestion of 20-day-old rat testes and grown on Matrigel-coated dishes for 7 days. Sertoli cells predominantly expressed the ubiquitous isoform of Na/K-ATPase (ATP1A1), confirmed by immunoblotting, PCR, immunofluorescence, and mass spectrometry. Treatment of Sertoli cells with 50 nM ouabain increased transepithelial electrical resistance (TER) and expression of claudin 11 (tight junctions) and connexin 43 (gap junctions), whereas 1 mM ouabain had opposite effects. Involvement of Src-EGFR-ERK1/2-CREB pathway in ouabain-mediated expression of claudin 11 and connexin 43 was evaluated. Incubation of Sertoli cells with 50 nM ouabain increased content of p-Src, p-EGFR, p-ERK1/2, and p-CREB; in contrast, 1 mM ouabain decreased phosphorylation of these signaling molecules. Preincubation of Sertoli cells with inhibitors of Src and MAPK pathways inhibited ouabain-induced effects on these signaling molecules, TER, and expression of claudin 11 and connexin 43. In conclusion, we inferred that ATP1A1 regulated Sertoli cell tight junctions and gap junctions through the Src-EGFR-ERK1/2-CREB pathway. Ouabain is an endogenous steroid; therefore, its interaction with ATP1A1 may be a critical signaling mechanism for the regulation of Sertoli cell function and male fertility.
钠钾-ATP 酶与其配体哇巴因的相互作用被认为参与了各种生物过程的调节。本研究旨在探讨钠钾-ATP 酶同工型在支持细胞连接复合体形成和功能中的作用。通过酶消化 20 日龄大鼠睾丸获得支持细胞原代培养物，并在 Matrigel 包被的培养皿中培养 7 天。免疫印迹、PCR、免疫荧光和质谱分析证实，支持细胞主要表达普遍存在的钠钾-ATP 酶同工型（ATP1A1）。用 50 nM 哇巴因处理支持细胞可增加跨上皮电阻（TER）和紧密连接蛋白 11（claudin 11）以及缝隙连接蛋白 43（connexin 43）的表达，而 1 mM 哇巴因则产生相反的效果。评估了Src-EGFR-ERK1/2-CREB 通路在哇巴因介导的 claudin 11 和 connexin 43 表达中的作用。孵育支持细胞 50 nM 哇巴因可增加 p-Src、p-EGFR、p-ERK1/2 和 p-CREB 的含量；相反，1 mM 哇巴因可降低这些信号分子的磷酸化。支持细胞预先孵育 Src 和 MAPK 通路抑制剂可抑制哇巴因对这些信号分子、TER 和 claudin 11 和 connexin 43 表达的诱导作用。综上所述，我们推断 ATP1A1 通过 Src-EGFR-ERK1/2-CREB 通路调节支持细胞紧密连接和缝隙连接。哇巴因是一种内源性甾体；因此，它与 ATP1A1 的相互作用可能是调节支持细胞功能和男性生育力的关键信号机制。
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