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.

Hormones and Signaling Pathways Involved in the Stimulation of Leydig Cell Steroidogenesis

“…In MA-10 Leydig cells, ERK1/2 has been found to functionally cooperate with STAT5B and GATA4 to activate the Star promoter [29]. The ERK1/2-GATA4 cooperation on Star is consistent with a study conducted in rat primary cardiomyocyte cells, which revealed that ERK1/2 phosphorylates GATA4 at Ser105 [66].…”

“…In the mouse MA-10 Leydig cell line, active PKA translocates to the nucleus, where it phosphorylates multiple transcription factors, such as GATA4 and bZIP family members. Phosphorylation of GATA4 at Ser261 leads to increased GATA4-dependent activation of several gene promoters such as Star, Cyp17a1, aromatase, and Inha (inhibin α) [28,29]. More recently, we have shown that PKA also cooperates with COUP-TFII (NR2F2) and STAT5B to activate the Star promoter in MA-10 Leydig cells [29].…”

“…Phosphorylation of GATA4 at Ser261 leads to increased GATA4-dependent activation of several gene promoters such as Star, Cyp17a1, aromatase, and Inha (inhibin α) [28,29]. More recently, we have shown that PKA also cooperates with COUP-TFII (NR2F2) and STAT5B to activate the Star promoter in MA-10 Leydig cells [29].…”

“…Although our understanding of CAMKI action in Leydig cell function is still in its early stages, emerging evidence suggests it is involved in crucial cellular processes. Previous studies have revealed that CAMKI acts as an activator, collaborating with multiple proteins, including transcription factors, to enhance the expression of genes associated with steroid hormone production [29] (reviewed in [42,43]). Some of the transcription factors found to cooperate with CAMKI include members of the nuclear receptor family NR4A (NUR77/NR4A1, NURR1/NR4A1, NOR1/NR4A3), NRF2 members (COUP-TFI/NR2F1 and COUP-TFII/ NR2F2), GATA4, STAT5B, CREB, and cJUN.…”

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.…”