(298) Protein Kinase A (PKA) Mediated Chromatin Accessibility and Transcriptomic Alterations in Granulosa Cells

Emilia Przygrodzka, Sullivan Haine, Lindsey Do, Maggie Weisman, Crisla Glasper, Emily Murphy

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland

Abstract Text:

Protein kinase A (PKA) signaling is one of the main pathways triggered by gonadotropins and regulates the growth and differentiation of highly steroidogenic granulosa cells. To better understand the PKA-induced transcriptomic landscape and chromatin accessibility changes, we performed ATAC- (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq (RNA sequencing) in the primary granulosa cells. Both analyses were performed on the bovine granulosa cells isolated from healthy ovarian follicles (6-9 mm) and cultured in the absence (Control group; n=4) or presence of PKA activator- Forskolin (Forskolin Group; n=4; 10 μM) for 8- and 24h. Afterward, cells were collected to further ATAC- and RNA-seq analysis performed by NovoGene Corporation Inc. Additionally, we performed further in vitro experiments to determine the effects of PKA signaling and downstream metabolic pathways inhibition on the proliferation and metabolic activity of bovine granulosa cells. Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc test. RNA-seq found 2573 and 2393 up- and down-regulated genes (adjusted p-value < 0.05) in granulosa cells treated with Forskolin for 8h. Gene Set Enrichment Analysis (GSEA) identified galactose metabolism; autophagy; various types of N-glycans biosynthesis; amino sugar and nucleotide sugar metabolism; and HIF-1 alpha signaling pathway among the top five (FDR q-value < 0.05) activated pathways in Forskolin-treated cells. After 24h, 2714 and 2676 genes were indicated as up- and down-regulated in Forskolin Group. According to GSEA, in addition to the HIF1 alpha signaling, there was prominent activation of starch and sucrose metabolism; glycolysis/gluconeogenesis; glycosaminoglycan biosynthesis; and fructose and mannose metabolism in granulosa cells at 24h post-Forskolin treatment. ATAC-seq showed that enhanced activity of PKA signaling elevated the level of open chromatin regions in the bovine granulosa cells. Peaks representing increased or decreased chromatin accessibility were primarily identified in the promoter (< =1kb; ~20%), distal intergenic (~27%), and intronic (~27%) regions in Control and Forskolin-treated granulosa cells. KEGG enrichment analysis for differential peak genes showed significant changes in metabolic pathways, MAPK-, or PI3K/Akt signaling. Motif enrichment analysis determined enrichment for transcription factors such as AP-1, ATF3, BATF, JUNB, FOSL2, FRA1, or FRA2. Due to enrichment in metabolic pathways identified by both omics analyses, we further inhibited PKA-signaling and enzymes that catalyze glucose metabolism using small molecule inhibitors. Cell proliferation assay measuring DNA content using fluorescence method (CyQUANT Direct Assay) and western blotting analysis showed a significant drop (p< 0.05; 30-60% reduction) in FSK-induced cell proliferation and content of Proliferating Cell Nuclear Antigen (PCNA), a known marker of cell proliferation, in granulosa cells treated with an inhibitor of PKA signaling (H89; 10 and 20 μM), or crucial enzymes in Pentose Phosphate Pathway i.e., glucose-6-phosphate-dehydrogenase (G6PD; 6-aminonicotinamide; 0.5 and 2.5 μM), and pyruvate synthesis i..e, pyruvate kinase M2 (PKM2; Shikonin; 0.5 and 1 μM). In addition, treatment with G6PD or PKM2 inhibitor dropped (p< 0.05; 40-50% reduction) FSK-mediated metabolic activity of granulosa cells measured by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. G6PD supplies cells with nicotinamide adenine dinucleotide phosphate (NADPH), a known cofactor required for the proper activity of steroidogenic machinery proteins and intermediates necessary for nucleotide synthesis. PKM2 catalyzes the synthesis of pyruvate, an essential intermediate for energy production. In conclusion, PKA signaling induces alterations of chromatin accessibility and the transcriptomic landscape, increasing signaling and metabolic pathways supplying cells with building blocks and cofactors required for the proliferation and proper steroidogenic activity of granulosa cells.