Genome-wide shifts in gene expression induced in theca-interstitial cells by inflammatory stimuli

Abstract

Excessive androgen production and hyperplasia of theca cells are central features of polycystic ovary syndrome (PCOS). Since PCOS is associated with systemic inflammation, this study evaluated the effects of inflammatory stimuli on gene expression in theca-interstitial cells (TICs). In vitro study evaluating effects of lipopolysacchride (LPS) and interleukin 1ß (IL1ß) on gene expression in TICs. Isolated rat TICs were cultured without or with LPS (1μg/ml) or IL1ß (1 ng/ml). RNAseq analysis was conducted by standard methods using Illumina HiSeq. Reads were mapped to Rat genome and differential expression was analyzed using DESeq2 package in R. We report on differentially expressed genes shifted in expression by at least 1.5-fold (log2) with an adjusted p-value (q-value) of ≤0.05. Enriched functional categories were identified using gene ontology (GO) analysis and Ingenuity® Pathway Analysis (IPA®). Using the stringent criterion described above, we identified 104 (Log2 fold change > 2.5) differentially expressed genes (DEGs) between the LPS-treated and control cells, 125 DEGs (Log2 fold change > 2.5) between the IL1β-treated and control cells, and 48 DEGs (Log2 fold change > 1.5) between the LPS-treated and IL1β-treated cells. Enriched functional categories of genes shifted in expression by LPS and by IL1ß treatment include those involved in the inflammatory response, the IL-17 signaling pathway, the humoral immune response, cell proliferation, cell cycle progression, inhibition of apoptosis, and hormone biosynthesis. LPS treatment as well as IL1ß treatment upregulated the inflammasome pathway (including Tlr4, NF-kB, Nlrp1, Nlrp3 and Caspase 1), key pathways associated with proliferation (including PI3K/Akt and Erk1/2), as well genes relevant to inhibition of apoptosis (Bcl-2 and Bcl-x). LPS and IL1ß, each, also stimulated expression of key genes involved in androgen biosynthesis (Cyp11, Hsd3b and Cyp17a1). Also upregulated by LPS and by IL1ß were several genes in the mevalonate pathway, including those encoding enzymes involved in synthesis of isoprenylation substrates, which would be predicted to activate small GTPases such as RAS and RHO. RNAseq analysis revealed that inflammatory stimuli profoundly affect the pattern of gene expression in TICs. Among the categories of genes significantly affected were those involved in the regulation of growth and androgen production. Given that excessive growth and androgen production by theca cells is a hallmark of PCOS, our results support the hypothesis that chronic inflammation in PCOS contributes to the altered function of theca cells and may play a role in the pathophysiology of PCOS.