β-catenin regulates FSHβ induction by GnRH: next generation RNA-Sequencing identifies Brms1L as a mediator of beta-catenin regulation of FSHβ gene expression. Mus musculus

The regulation of gonadotropin synthesis by GnRH (Gonadotropin-releasing hormone) plays an essential role in the neuroendocrine control of reproduction. The known signaling mechanisms involved in gonadotropin synthesis have been expanding. For example, involvement of β-catenin in LHβ induction by GnRH has been discovered. We examined the role of β-catenin in FSHβ gene expression in LβT2 gonadotrope cells. GnRH caused a sustained increase in nuclear β-catenin levels, which was significantly reduced by JNK inhibition. siRNA-mediated knockdown of β-catenin mRNA demonstrated that induction of FSHβ mRNA by GnRH depended on β-catenin and that regulation of FSHβ by β-catenin occurred independently of the JNK-c-jun pathway. β-catenin depletion had no impact on FSHβ mRNA stability. In LβT2 cells transfected with FSHβ promoter luciferase fusion constructs, GnRH responsiveness was conferred by the proximal promoter (-944/-1), and was markedly decreased by β-catenin knockdown. However, none of the TCF/LEF binding sites in that region were required for promoter activation by GnRH. Chromatin immunoprecipitation further corroborated the absence of direct interaction between β-catenin and the 1.8 kb FSHβ promoter. To elucidate the mechanism for the β-catenin effect, we analyzed ~1 billion reads of next generation RNA sequencing β-catenin knockdown assays and selected the nuclear cofactor Brms1L as one candidate for further study. Subsequent experiments confirmed that Brms1L mRNA expression was decreased by β-catenin knockdown as well as by JNK inhibition. Furthermore, knockdown of Brms1L significantly attenuated GnRH-induced FSHβ expression. Thus, our findings indicate that the expression of Brms1L depends on β-catenin activity and contributes to FSHβ induction by GnRH. Overall design: A total of 24 samples were analyzed, namely 6 different experimental conditions, each comprised of 4 replicates. Control samples are included in the analysis. For the RNA-Seq assay, samples were multiplexed in the form of 3 samples per lanes, resulting in a total of 8 lanes.