Interdependence of steroidogenesis and shape changes in Y1 adrenocortical cells: studies with inhibitors of phosphoprotein phosphatases.

Y1 adrenocortical cells respond to activators of the cyclic AMP-dependent protein kinase (PKA) signalling pathway not only with increases in steroid secretion but also with a characteristic change in cell morphology from flat and adherent to round and loosely attached. This change of shape, which may facilitate cholesterol transport to the mitochondrion, requires tyrosine dephosphorylation of the focal adhesion protein, paxillin, and can be blocked by inhibitors of phosphotyrosine phosphatase (PTP) activity. In a previous study we demonstrated that inhibition of phosphoserine/threonine phosphatase 1 and 2A (PP1/2A) activities caused a similar morphological response to PKA activation whilst opposing the effects on steroid production. We have now investigated the responses to PKA activation and inhibition of PP1/2A and used PTP inhibitors to examine the relationship between the morphological changes and enhanced steroid production. Both forskolin (FSK) and the PP1/2A inhibitor, calyculin A (CA), caused rapid and extensive rounding of Y1 cells. FSK-induced cell rounding was reversible and accompanied by a reduction in the tyrosine phosphorylation of paxillin. Rounding was prevented by the PTP inhibitors pervanadate (PV) and calpeptin (CP) and was associated with the maintained tyrosine phosphorylation of paxillin. In contrast, CA-induced cell rounding was not reversible over a 2-h period and was not affected by the presence of PTP inhibitors, and CA had no effect on the tyrosine phosphorylation of paxillin. Although neither CA nor FSK produced any gross changes in cell viability as judged by Trypan Blue exclusion or mitochondrial activity, CA-treated cells showed a marked reduction in total protein synthesis assessed by (35)S-incorporation. The effects of FSK and the PTP inhibitors on cell rounding were reflected in their effects on steroid production since PV and CP also inhibited FSK-stimulated steroid production. These results suggest that the mechanism through which inhibition of PP1/2A activities induces morphological changes in Y1 cells is fundamentally different from that seen in response to activation of PKA. They are consistent with PKA-induced shape changes in adrenocortical cells being mediated through increased PTP activity and the dephosphorylation of paxillin, and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are intimately linked.

ERKs regulate cyclic AMP-induced steroid synthesis through transcription of the steroidogenic acute regulatory (StAR) gene.

Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSK-stimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

Phosphoprotein phosphatases regulate steroidogenesis by influencing StAR gene transcription.

The rate-limiting step in steroidogenesis is the transport of cholesterol into the mitochondria, and this is controlled by the steroidogenic acute regulatory (StAR) protein. We have previously shown that inhibition of phosphoprotein phosphatase 1 and 2A (PP1/2A) activities with the PP1/2A inhibitor calyculin A selectively reduces StAR protein expression and thus inhibits the synthesis of steroid hormones. The aim of this study was to determine whether this inhibition of StAR protein expression occurs at the level of transcription of StAR mRNA. We have used a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique to determine whether inhibition of PP1/2A activities has any effect on the levels of StAR mRNA. Exposure of Y1 cells to forskolin significantly increased the expression of StAR mRNA and this forskolin-induced increase was reduced after exposure to Cal A at levels similar to those seen in the controls. These results suggest that cyclic AMP-induced increases in StAR mRNA levels are dependent upon phosphoprotein phosphatase activities.

Inhibition of protein tyrosine phosphatase activity blocks shape change & steroidogenesis in Y1 cells.

Y1 adrenocortical cells respond to forskolin stimulation with increases in steroid secretion and change of shape. The rapid rounding of flat, adherent cells which occurs is known to involve dephosphorylation of the focal adhesion protein, paxillin. We have investigated the effects of a tyrosine phosphatase inhibitor, calpeptin (CP) on steroidogenesis and shape change in Y1 cells. Forskolin treatment (FSK, 2 microM) caused marked rounding of Y1 cells (FSK = 76.3 +/- 1.5% cells rounded after 30 minutes, untreated = 2.9 +/- 0.7 % rounded); calpeptin pretreatment (CP; 100 ug/ml) had little effect on shape (9.6 +/- 2.4% rounded) but blocked the rounding response to FSK (32.1 +/- 2.1% rounded. Calpetin also eliminated the steroidogenic response to FSK ( FSK = 242 +/- 14% control ; FSK + CP = 113 +/- 18% control ) without affecting production of steroid from membrane permeant 22R-OH-cholesterol. The results support the view that dephosphorylation of paxillin is important in the rounding response and provide evidence for the involvement of tyrosine-phosphatase activity in cyclic AMP-stimulated steroidogenesis in Y1 cells.