OR01-07 Lhb expression is indirectly regulated by GnRH-induced RNA-binding protein ZFP36 binding with AU-rich elements of mRNAs essential to LH synthesis and secretion.

Abstract Disclosure: T. Terasaka: None. K. Inagaki: None. K. Yamaoka: None. T. Miyazaki: None. S. Fujisawa: None. M.A. Lawson: None. J. Wada: None. RNA-binding protein (RBP) function in the production of secreted hormones has not been fully studied. Gonadotropin secretion, stimulated by gonadotropin-releasing hormone (GnRH), is a critical feature of reproductive control and maintenance of fertility. Recently, using RIP-Chip analysis, we have shown that GnRH receptor (Gnrhr) expression depends on the interaction of its mRNA with the RBP ELAVL1. Also, Gnrhr mRNA is among the most highly represented ELAVL1-associated mRNAs (1). Odle et al. showed RBP Musashi1 regulates GnRH receptor mRNA translation by directly binding with the Gnrhr 3’UTR (2). Further, immediate-early transcription factor mRNAs encoding proteins essential to gonadotropin gene expression show increased binding to ELAVL1 after GnRH treatment. Knockdown of ELAVL1 results in the decrease of Gnrhr expression and of Egr1 expression, a transcription factor essential to Lhb transcription. Interestingly, Lhb mRNA does not contain an ARE in 3’UTR. We are investigating the role of the RBP ZFP36, which competes with ELAVL1 for binding to AU-rich elements (ARE) but has the opposing effect of directing mRNA to the degradation pathway. The ZFP36 protein was upregulated by pulsatile GnRH induction in LβT2 cells, a cell model of mouse gonadtropes. We have examined the role of ZFP36 protein in GnRH-induced gene expression. Interestingly, Gnrhr, SF1, and Lhb mRNA levels decreased by ZFP36 overexpression in LbetaT2 cells. Also, GnRH-stimulated Egr1 mRNA and EGR1 production were decreased by ZFP36 overexpression. Basal and GnRH-induced LH secretion was concomitantly decreased by ZFP36 overexpression. These observations are consistent with previous results with ELAVL1 knockdown showing decreased mRNA stability. Moreover, GnRH-stimulated EGR1 production increased in ZFP36 KO cells. Also, SF1 expression levels were increased by ZFP36 KO. Consistently, Lhb mRNA expression levels and basal and GnRH-induced LH secretion were increased by ZFP36 KO. To evaluate the direct effect of ZFP36, luciferase reporter activity with Egr1, Gnrhr, and Zfp36 3’UTRs was executed. These Egr1, Gnrhr, and Zfp36 3’UTR contained plasmids were transfected in both ZFP36 overexpression and control cells, and luciferase activity was monitored. The results showed that luciferase expression from reporters bearing the target 3’UTR was significantly reduced in ZFP36 overexpressed cells. And luciferase activity with the specific mutant ARE-contained 3’UTR was increased.Collectively, our studies show that ZFP36 opposes ELAVL1 action in a gonadotrope cell model and their target mRNAs are regulated by GnRH. Thus, competing activities of ELAVL1 and ZFP36 indirectly regulates LH secretion in gonadotropes thorough modulation of mRNA levels of the factors essential for expression of the LHβ subunit. 1) Terasaka T et al. Endocrinology 160(8): 1999-2014, 2019 2) Odle KA et al. Endocrinology 159(2): 883-894, 2018 Presentation: Saturday, July 12, 2025