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

Abstract

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 LβT2 cells. It was found that luteinizing hormone (LH)β mRNA expression level in LβT2 cells changed gradually over time, with LHβ 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 LHβ expression in the presence and absence of GnRH. Notably, the expression levels of Bmal1 and Clock showed strong positive correlations with LHβ mRNA expression levels. Moreover, a functional link of the ERK signaling of mitogen-activated protein kinases (MAPKs) in the suppression of LHβ 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 LHβ mRNA levels in the late phase 24 h after GnRH stimulation. Furthermore, in the presence of BMP-6 and -7, late-phase Bmal1 and LHβ 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.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells

“…The mutual effects of orexin and GnRH responsiveness in relation to clock gene expression were further evaluated. Among the core clock genes expressed in gonadotrope cells [ 20 ], Bmal1 and Clock are considered to be functional regulators for LHβ mRNA expression by LβT2 cells as we earlier reported [ 11 ]. As shown in Figure 1 B, it was revealed that orexin A (100 nM) treatment significantly suppressed GnRH receptor (GnRHR) mRNA expression, whereas GnRH (10 nM) increased OX1R and OX2R mRNA expression by LβT2 cells for 24 h in serum-free conditions.…”

“…The involvement of BMP signaling in orexin receptor signaling and clock gene expression was then examined. The BMP system has also been postulated to be a functional modulator of LH expression by LβT2 cells [ 11 , 18 ]. As shown in Figure 2 A, treatments with BMP-6 and -15 (30 ng/mL) resulted in upregulation of both OX1R and OX2R mRNA expression in LβT2 cells.…”

“…Among the transcriptional activators, Clock and Bmal1 proteins form a heterodimer and positively regulate the expression of the Period (Per1, Per2) and Cryptochrome (Cry1, Cry2) genes, and then Per and Cry gene products dimerize and translocate into the nucleus to interact with Clock and Bmal1 proteins, leading to repression of their own transcription [ 7 ]. We previously reported that among the canonical clock genes, Bmal1 and Clock functionally regulate LHβ mRNA expression under the influence of GnRH by interacting with bone morphogenetic proteins (BMPs) in mouse gonadotrope cells [ 11 ].…”

“…It was also revealed that BMP-6 modulates gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) production by altering the responsiveness to somatostatin analogs [ 18 ]. It has also been shown that intracellular signaling of BMPs interacted with intracellular signaling of mitogen-activated protein kinase (MAPK) in the regulation of LH expression via clock gene expression in a phase- and GnRH-dependent manner [ 11 ]. However, the regulatory mechanism involving BMPs, orexins and circadian rhythm in gonadotropin synthesis is still unclear.…”

Molecular Mechanisms Underlying Stress Response and Resilience

“…Pituitary BMPs are involved in the secretory control of follicle-stimulating hormone produced by gonadotroph cells. Soejima et al (2021) elucidated the effects of Clock gene suppression and BMPs on the expression of luteinizing hormone ( Lh ) and Clock genes induced by gonadotropin-releasing hormone in gonadotroph cells [ 5 ]. Control of Clock gene expression by regulating the extracellular signal-regulated kinase pathway in the early phase and the BMP-Smad signal in the chronic phase could be a future strategy for modulating the LH surge [ 5 ].…”