GATA4 Is a Key Regulator of Steroidogenesis and Glycolysis in Mouse Leydig Cells

Presentation Number: THR-116
Date of Presentation: March 5th, 2015

Anja Schrade*1, Antti Kyrönlahti1, Marjut Pihlajoki1, Jorma Toppari2, David B Wilson3 and Markku Heikinheimo1
1University of Helsinki, Helsinki, Finland, 2University of Turku, Turku, Finland, 3Washington University in St. Louis, Saint Louis, MO

Abstract

Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells (1). The role of GATA4 in Leydig cell development, however, remains controversial because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses, as reviewed in (2). We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are downregulated or upregulated following siRNA-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1 (Gata4 mRNA levels were reduced by 80±8%; P<0.01; n=3). Among the downregulated genes were enzymes of the androgen biosynthetic pathway [Cyp11a1, fold change (FC)=0.30±0.03; Hsd3b1, FC=0.14±0.01; Cyp17a1, FC=0.34±0.07; Srd5a, FC=0.49±0.13; for all P<0.01 and n=3-4]. These same genes were dysregulated when primary cultures of Gata4flox/flox adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Notably, the level of androstendione, the immediate progenitor of testosterone, was marked reduced in the knockdown cells (Gata4 siRNA: 24.2±4.8 nM vs. non-targeting siRNA: 172.7±48.1 nM; P<0.01; n=3). Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed disruption of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, FC=0.59±0.10; Gpi1, FC=0.85±0.10; Pfkp, FC=0.81±0.16; Pgam1, FC=0.68±0.27; for all P<0.05 and n=8), lower intracellular levels of ATP (30±12%; P<0.01; n=4) through impaired glycolysis, and increased extracellular levels of glucose (Gata4 siRNA: 3.57±0.043 g/L vs. non-targeting siRNA: 3.28±0.025 g/L; P<0.001; n=8). Altogether, our results suggest that GATA4 is linked to cell metabolism and regulation of steroid production in adult Leydig cells.

 

Nothing to Disclose: AS, AK, MP, JT, DBW, MH