INVESTIGADORES
PODEROSO Juan Jose
congresos y reuniones científicas
Título:
Activation of mitochondrial ERK1/2 is required for PKA-dependent steroidogenesis in MA-10 cells.
Autor/es:
PODEROSO C; CONVERSO D; RODRÍGUEZ V; PAZ C; PODESTÁ E; PODEROSO JJ
Lugar:
Niza, Francia
Reunión:
Congreso; ELSO; 2004
Resumen:
M-10 is a murine cell line transformed from Leydig cells that produces progesterone as final steroid in response to luteinnizing hormone being camp one of the accepted second messenger. Early steps of progesterone synthesis occur in mitochondria and involve activation of Steroidogenic Acute Regulatory protein (STAR) leading to cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis. Recently, our group and others reported that members of MAP Kinases family, the extracellular signal-regulated kinases ½ (ERK ½), are activated by phosphorilation in mitochondria. To investigate a 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 (0.5mM) in the presence or absence of two inhibitors of MEK1/2 (MAP Kinase Kinase, upstream ERK Kinase), PD980059 and U0126, and progesterone production was evaluated by radioimmunoanalysis. Both compounds inhibited 70-100% 8Br-cAMP stimulated-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 8Br-cAMP, at different times. MEK ½ and ERK ½ 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 Western blot or by co-localization in immunocytochemistry with mitotracker red, a mitochondrial dye. The pattern of 8Br-cAMP activation of MEK ½ and ERK ½ was abrogated when MA-10 cells were pre-incubated with H-8.9, a permeable PKA inhibitor, indicating that these events are PKA-dependent in this cell type. For comparison, cells 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 PAK is required to STAR-dependent cholesterol transport and steroidogenesis in cAMP-stimulated MA-10 cells.