Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells

Abstract

Hormone-induced Leydig cell steroidogenesis requires rapid changes in gene expression in response to various hormones, cytokines, and growth factors. These proteins act by binding to their receptors on the surface of Leydig cells leading to activation of multiple intracellular signaling cascades, downstream of which are several kinases, including protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase I (CAMKI), and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). These kinases participate in hormone-induced steroidogenesis by phosphorylating numerous proteins including transcription factors leading to increased steroidogenic gene expression. How these various kinases and transcription factors come together to appropriately induce steroidogenic gene expression in response to specific stimuli remains poorly understood. In the present work, we compared the effect of PKA, CAMKI and ERK1/2 on the transactivation potential of 15 transcription factors belonging to 5 distinct families on the activity of the Star gene promoter. We not only validated known cooperation between kinases and transcription factors, but we also identified novel cooperations that have not yet been before reported. Some transcription factors were found to respond to all three kinases, whereas others were only activated by one specific kinase. Differential responses were also observed within a family of transcription factors. The diverse response to kinases provides flexibility to ensure proper genomic response of steroidogenic cells to different stimuli.

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells

“…MA-10 Leydig cells were validated by morphology and by quantifying steroidogenic output, as previously described [ 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. MA-10 cells were transiently transfected using polyethylenimine hydrochloride (PEI) (Sigma-Aldrich Canada, Oakville, ON, Canada), as previously described [ 61 , 63 , 64 ], or the calcium phosphate co-precipitation method, as described in [ 22 , 35 , 48 , 65 ]. Briefly, MA-10 cells were transfected 24 h after plating at a density of 100,000 cells/well, by using 0.5 μg of Insl3 promoter construct fused to the Firefly luciferase reporter gene, 20 ng of phRL-TK Renilla luciferase expression vector used as an internal control for transfection efficiency, and pSP64 as carrier DNA up to 1.5 μg/well.…”