New Mechanisms of Steroidogenesis
Session: Poster Session C
Alison F. Ermisch, PhD
Graduate Research Assistant
University of Nebraska-Lincoln
Lincoln, Nebraska, United States
Methylation of RNA at the N6 position of adenosine (N6-methyladeonsine; m6A) is a critical post-transcriptional regulator of mRNA metabolic fate. A recent focus of our lab is to understand how oxidative stress and inflammation impact this reversible modification and thereby affect transcript stability and translation. Oxidative stress and inflammation also alter ovarian steroid production, which is critical for oocyte development and ovulation. The preponderance of studies examining regulation of steroidogenesis emphasize transcriptional mechanisms of mRNA synthesis and/or post-translational modifications that affect protein activity. Much less is known about how post-transcriptional m6A modification regulates mRNA, protein, and subsequent steroid production in the somatic cells of the ovary. Our current hypothesis is that inhibiting m6A methylation or demethylation will alter steroid production in granulosa cells. To test this hypothesis, we performed cell culture experiments with the KGN cell line, which are ovarian granulosa-like tumor cells capable of producing progesterone (P4) and estradiol (E2). KGN cells were cultured in serum-free DMEM/F12 base media containing cAMP to boost steroid production and androstenedione (A4) as a substrate for E2 synthesis. One of two inhibitors was added to this base (Control) media: 1) STM2457, which inhibits METTL3, the catalytic subunit of the methyltransferase complex, and thus decreases m6A by inhibiting methylation of RNA, or 2) FB23-2, which inhibits the demethylase FTO and thereby increases m6A modification on RNA by inhibiting its removal. Inhibition of FTO-mediated RNA demethylation reduced (p< 0.05) E2 production in spent media, as measured by ELISA, compared to Control (220.2±63.55 pg/mL, 703.1±6.9 pg/mL, respectively). Similarly, inhibition of both METTL3 methylation (3.9±0.2 ng/mL) and FTO demethylation (4.1±0.2 ng/mL) significantly reduced P4 concentrations compared to Control (5.8±0.6 ng/mL). Given that altering m6A impacts steroid hormone production, we next wanted to assess changes in mRNA and steroidogenic enzyme abundance in cells subjected to the same inhibitor treatments. Changes in methylation may alter the stability, degradation, or translation of a particular mRNA due to changes in the interaction of RNA binding proteins (i.e., reader proteins) with that transcript. Several enzymes that regulate steroidogenesis are predicted to have multiple sites for potential m6A methylation on mRNA, including STAR, which is responsible for shuttling cholesterol into the mitochondria, CYP11A1, which converts cholesterol to pregnenolone, and CYP19A1, which converts androgens to estrogens. Therefore, transcript abundance was assessed via RT-qPCR and protein abundance was measured via Western blot. When m6A mRNA methylation was inhibited with STM2457, transcript abundance of STAR was significantly (P< 0.05) decreased. Additionally, protein levels of CYP11A1 also tended (P=0.06) to be decreased. When m6A mRNA demethylation was inhibited with FB23-2, there was a significant 2-fold increase in transcript abundance of both CYP11A1 and STAR. Interestingly, altered m6A had no effect on transcript abundance of CYP19A1 or aromatase expression, indicating that m6A may play a role in the first stages of steroid biosynthesis by regulating cholesterol conversion into early steroid hormone precursors. Taken together, these data suggest that m6A is an important post-transcriptional regulator of genes involved in ovarian steroid production, and changes in methylation status either directly or indirectly alter steroidogenesis within granulosa cells. Future studies will focus on the causative effect of methylation status on steroidogenic mRNA storage, degradation, and translation. The results of these experiments will provide new information about how m6A dysregulation may contribute to pathologies of altered steroidogenesis.