Activation of mitochondrial ERK1/2 is required for PKA-dependent steroidogenesis in MA-10 celIs

CECILIA PODEROSO', DANIELA CONVERSO., VICTORIA RODRIGUEZ'. CRISTINA PAZ'.ERNESTÓ J PODESTÁ '. JUAN J PODEROSO'

Niza, Francia

Congreso; 4th Annual Meeting of ELSO; 2004

ELSO

MA-10 is a murine cell line transformed from Leydig cells that produces progesterone as final steroid in response to luteinizing hormone being cAMP one of the accepted second messenger. Early steps of progesterone synthesis occur in mitochondria and involve activation of Steroidogenic Ácute Regulatory protein (StAR) leading to cholesterol import to the inner mitochondrial membrane. The rate-limiting step in steroid biosynthesis, Recently, our group and orthers reporrted that members of MAP kinases family, the extracellular signal-related kinases 1/2 (ERK1/2) are activated by phosphorylation in mitochondria, To investigate relationship between a putative non-genomic ERK mitochondrial effect and steroidogenesis, MA-10 cells were arrested in G1 phase (by transferring them to a serum-free medium) and stimulated with the permeable cAMP analogue 8Br-cAMP (0.5 mM) in the presence or .absence of two inhibitors of MEK1/2 (MAP kinase kínase, upstream ERK kinase), PD98059 and U0126 and progesterone production was evaluated by radioimmunoanalysis, Both compounds inhibited 70-100% 8Br-cAMP-stimulaled-steroid production neither affecting hormonal basal level nor PKA activity, To analyze the effect of steroidogenic stimuli on ERK .activation, kinase phosphorylation was followed by Western blot with specific antibodies in cells exposed to 8Bc-cAMP at different times, MEK1/2 an ERK1/2 resulted phosphorylated with maximal activity after 5 min of stimulation that decreased up to 1 h, without modifying total kinase expression. A similar time-coursed activation of constitutive ERK1/2 was found in purified non-contaminated mitochondria isolated from stimulated cells as assessed by Westem blot or by co-Iocalization in immunocitochemistry with mitotracker red, a mitochondrial dye, The pattem of 8Br-cAMP activation of MEK1/2 and ERK1/2 was abrogated when MA-10 cells were pre-incubated with H-89, a permeable PKA inhibitor, indicating that these events are PKA-dependent in this cell type. For comparison, celIs were stimulated with EGF (10ng/ml) that promotes ERK activation via Ras/Raf, but not steroidogenesis. In this case, a substantial cytosolic ERK phosphorylation was only associated to a prompt but extremely brief kinase activation in mitochondria (~3 min). It is surmised that sustained mitochondrial ERK activity via PKA is required to StAR-dependent cholesterol transport and steroidogenesis in cAMP-stimulated MA-10 cells,