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

Abstract

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.

Regulation of adrenocortical steroid hormone production by RhoA–diaphanous 1 signaling and the cytoskeleton

“…For example, using coherent anti-Stokes Raman Scattering Microscopy to visualize live cells, lipid droplets were shown to move along microtubule tracts in Y1 murine adrenocortical cells (Nan et al, 2006). The effect of chemical agents that modulate the stability of microtubules and microfilaments on both adrenal and gonadal steroid hormone biosynthesis has also been an area of active investigation for several decades (Cortese and Wolff, 1978, Hall and Almahbobi, 1997, Lee et al, 2001, Rainey et al, 1985, Rainey et al, 1984, Rajan and Menon, 1985, Sackett and Wolff J, 1986, Whitehouse et al, 2002). In fact, dating back to the late 1970s Cortese and Wolf demonstrated that cytochalasins B, D and E, inhibitors of actin polymerization that disrupt the formation of microfilaments, stimulate steroid hormone production in Y1 adrenocortical cells (Cortese and Wolff, 1978).…”

Regulation of Steroid Hormone Biosynthesis by the Cytoskeleton

“…In fact, four decades ago observations made on Y1 mouse adrenal cells that underwent morphological changes in response to adrenocorticotropin (ACTH) provided clues into the role of the cytoskeleton architecture in controlling the response of steroidogenic cells to trophic hormone stimulation [3,4]. These marked changes in steroidogenic cell morphology have been confirmed by several researchers [5][6][7] and have been subsequently attributed to cAMP-dependent rapid dephosphorylation of the focal adhesion protein paxillin [8,9].…”

Blocking BRE expression in Leydig cells inhibits steroidogenesis by down-regulating 3β-hydroxysteroid dehydrogenase

“…The cytoskeleton in Leydig (and adrenocortical) cells changes in response to stimulation with hCG or cAMP and is linked to mobilization of cellular cholesterol, including its transport to a mitochondrion (Bilinska et al 1999, Whitehouse et al 2002. Therefore it seems BRE may also have an impact on the mobilization of intracellular cholesterol.…”