Biphasic Roles of Clock Genes and Bone Morphogenetic Proteins in Gonadotropin Expression by Mouse Gonadotrope Cells

Roles of Clock genes and the bone morphogenetic protein (BMP) system in the regulation of gonadotropin secretion by gonadotropin-releasing hormone (GnRH) were investigated using mouse gonadotropin L beta T2 cells. It was found that luteinizing hormone (LH)beta mRNA expression level in L beta T2 cells changed gradually over time, with LH beta expression being suppressed in the early phase up to 12 h and then elevated in the late phase 24 h after GnRH stimulation. In addition, the mRNA expression levels of Clock genes, including Bmal1, Clock, Per2, and Cry1, also showed temporal changes mimicking the pattern of LH beta expression in the presence and absence of GnRH. Notably, the expression levels of Bmal1 and Clock showed strong positive correlations with LH beta mRNA expression levels. Moreover, a functional link of the ERK signaling of mitogen-activated protein kinases (MAPKs) in the suppression of LH beta mRNA expression, as well as Bmal1 and Clock mRNA expression by GnRH at the early phase, was revealed. Inhibition of Bmal1 and Clock expression using siRNA was involved in the reduction in LH beta mRNA levels in the late phase 24 h after GnRH stimulation. Furthermore, in the presence of BMP-6 and -7, late-phase Bmal1 and LH beta mRNA expression after GnRH stimulation was significantly attenuated. Collectively, the results indicated that LH expression in gonadotrope cells exhibits Bmal1/Clock-dependent fluctuations under the influence of GnRH and that the fluctuations are regulated by ERK and BMPs in the early and late stages, respectively, in a phase-dependent manner after GnRH stimulation.