SP600125 decreases cAMP/PKA-dependent steroid production through ATF4/DDIT3 activation in MA-10 Leydig cells

Leydig cells, located between seminiferous tubules within the testis, are the primary source of testosterone in males. In these cells, the luteinizing hormone (LH)/cAMP/protein kinase A (PKA) signaling pathway mainly regulates androgen biosynthesis. We have previously reported that the connexin43 (Gja) promoter can be activated in mouse MA-10 Leydig cells as a result of a cooperation between the AP-1 transcription factors JUN and FOS. The mitogen-activated protein kinases (MAPK) signaling pathway, through the JUN N-terminal kinase (JNK), can phosphorylate members of the AP-1 family of transcription factors, modulating their activities. Hence, MA-10 Leydig cells were treated for 4 h with the JNK inhibitor SP600125 (pyrazolanthrone) at 25 μM, in the absence or presence of the activator of adenylate cyclase forskolin (FSK) at 10 μM, followed by RNA extractions and 3'Tag RNA-Seq analysis of the transcriptome. Interestingly, SP600125 decreases cAMP/PKA dependent expression of genes related to cholesterol and steroid biosynthetic/metabolic processes, resulting in decreased cAMP/PKA dependent progesterone production in MA-10 cells. Moreover, SP600125 increases the expression of genes involved in the endoplasmic reticulum stress response related to ATF4, resulting in activation of DDIT3 and apoptosis as indicated with cleaved caspase 3. Overall, our results suggest that SP600125 increases the ATF4/DDIT3-dependent endoplasmic reticulum stress response independently of MAPK9 (JNK2) inhibition, and inhibits LH/cAMP/PKA-dependent androgen synthesis in Leydig cells.