Reproduction, Mother and Child Health, Centre de recherche du centre hospitalier universitaire de Québec, Université Laval, Québec City, QC, G1V 4G2, Canada.
Centre de recherche en Reproduction, Développement et Santé Intergénérationnelle, Department of Obstetrics, Gynecology, and Reproduction, Faculty of Medicine, Université Laval, Québec City, QC, G1V 0A6, Canada.
Endocrinology. 2023 Aug 1;164(9). doi: 10.1210/endocr/bqad120.
Leydig cells produce hormones required for the development and maintenance of sex characteristics and fertility in males. MEF2 transcription factors are important regulators of Leydig cell gene expression and steroidogenesis. ERK5 is an atypical member of the MAP kinase family that modulates transcription factor activity, either by direct phosphorylation or by acting as a transcriptional coactivator. While MEF2 and ERK5 are known to cooperate transcriptionally, the presence and role of ERK5 in Leydig cells remained unknown. Our goal was to determine whether ERK5 is present in Leydig cells and whether it cooperates with MEF2 to regulate gene expression. We found that ERK5 is present in Leydig cells in testicular tissue and immortalized cell lines. ERK5 knockdown in human chorionic gonadotrophin-treated MA-10 Leydig cells reduced steroidogenesis and decreased Star and Nr4a1 expression. Luciferase assays using a synthetic reporter plasmid containing 3 MEF2 elements revealed that ERK5 enhances MEF2-dependent promoter activation. Although ERK5 did not cooperate with MEF2 on the Star promoter in Leydig cell lines, we found that ERK5 and MEF2C do cooperate on the Nr4a1 promoter, which contains 2 adjacent MEF2 elements. Mutation of each MEF2 element in a short version of the Nr4a1 promoter significantly decreased the ERK5/MEF2C cooperation, indicating that both MEF2 elements need to be intact. The ERK5/MEF2C cooperation did not require phosphorylation of MEF2C on Ser387. Taken together, our data identify ERK5 as a new regulator of MEF2 activity in Leydig cells and provide potential new insights into mechanisms that regulate Leydig cell gene expression and function.
Leydig 细胞产生雄性生殖特征和生育所必需的激素。MEF2 转录因子是 Leydig 细胞基因表达和类固醇生成的重要调节因子。ERK5 是 MAP 激酶家族的非典型成员，可通过直接磷酸化或作为转录共激活因子来调节转录因子活性。虽然已知 MEF2 和 ERK5 在转录上合作，但 ERK5 在 Leydig 细胞中的存在和作用尚不清楚。我们的目标是确定 ERK5 是否存在于 Leydig 细胞中，以及它是否与 MEF2 合作调节基因表达。我们发现 ERK5 存在于睾丸组织和永生化细胞系中的 Leydig 细胞中。在人绒毛膜促性腺激素处理的 MA-10 Leydig 细胞中敲低 ERK5 会减少类固醇生成并降低 Star 和 Nr4a1 的表达。使用包含 3 个 MEF2 元件的合成报告质粒进行的荧光素酶测定表明，ERK5 增强了 MEF2 依赖性启动子激活。尽管 ERK5 并未在 Leydig 细胞系中的 Star 启动子上与 MEF2 合作，但我们发现 ERK5 和 MEF2C 在 Nr4a1 启动子上合作，该启动子包含 2 个相邻的 MEF2 元件。Nr4a1 启动子短版本中每个 MEF2 元件的突变显着降低了 ERK5/MEF2C 合作，表明两个 MEF2 元件都需要完整。ERK5/MEF2C 合作不要求 MEF2C 的丝氨酸 387 磷酸化。总之，我们的数据将 ERK5 鉴定为 Leydig 细胞中 MEF2 活性的新调节剂，并为调节 Leydig 细胞基因表达和功能的机制提供了新的见解。
