Abstract
We investigated the effects of tumor necrosis factor alpha (TNFalpha) on the transcriptional activity of the porcine P-45011A (P450scc) insulin-like growth factor response element (IGFRE). TNFalpha inhibited insulin-like growth factor-I (IGF-I)-stimulated P450scc mRNA concentrations in cultures of porcine granulosa cells. Transient transfection experiments in granulosa cells with deletion P450scc/luciferase constructs showed that TNFalpha inhibited the transcriptional activity of the IGFRE. IGF-I binding and IGF-I receptor mRNA concentrations in porcine granulosa cells were not inhibited by TNFalpha. Electrophoretic mobility shift assay with nuclear extract protein from porcine granulosa cells treated with IGF-I and TNFalpha showed that Sp1 and a second transcription factor, P2, bound to the IGFRE. While IGF-I treatment increased the binding activity of both factors, TNFalpha specifically inhibited the IGF-I-stimulated binding activity of P2. Transient transfection studies done in mouse fibroblasts overexpressing the IGF-I receptor (NWTb3) with the porcine IGFRE (three repeats) in an SV40/luciferase construct also showed TNFalpha inhibited IGF-I-stimulated reporter gene expression. We conclude that TNFalpha inhibits the transcriptional activity of the porcine P450scc IGFRE by preventing IGF-I-stimulated binding of P2.
Highlights
Tumor necrosis factor ␣ (TNF␣)1 is a cytokine that regulates steroidogenesis in the ovary
TNF␣ Effects on the Porcine P450scc IGF-responsive region (IGFRE) in Porcine Granulosa Cells—Transient transfections of deletion constructs of the upstream region of the porcine P450scc gene were done in porcine granulosa cells during treatment with TNF␣ to determine whether TNF␣ inhibited the activity of the porcine IGFRE
This study found that TNF␣ inhibited Insulin-like growth factor I (IGF-I)-stimulated P450scc mRNA concentrations and progesterone production in porcine granulosa cells by suppressing the transcriptional activity of the porcine P450scc IGFRE
Summary
TNF␣, tumor necrosis factor ␣; IGF-I, insulin-like growth factor I; IGFRE, insulin-like growth factor response element; P450scc, P-450 cholesterol side-chain cleavage enzyme; P450scc/luc, endogenous P450scc promoter/luciferase plasmid; CL, corpus luteum. Other transcription factor, P2 [6]. This study determined that TNF␣ inhibits the function of the porcine P450scc IGFRE in porcine granulosa by preventing IGF-I-stimulated binding of P2. This finding presents a mechanism whereby TNF␣ can induce luteolysis by inhibiting IGFI-supported steroidogenesis in the CL. It establishes P2 as the transcription factor mediating the IGF-I response
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