Irina Artemenko

Regulation of cholesterol movement to mitochondrial cytochrome P450scc in steroid hormone synthesis

Transfer of cholesterol to cytochrome P450scc is generally the rate-limiting step in steroid synthesis. Depending on the steroidogenic cell, cholesterol is supplied from low or high density lipoproteins (LDL or HDL) or de novo synthesis. ACTH and gonadotropins stimulate this cholesterol transfer prior to activation of gene transcription, both through increasing the availability of cytosolic free cholesterol and through enhanced cholesterol transfer between the outer and inner mitochondrial membranes. Cytosolic free cholesterol from LDL or HDL is primarily increased through enhanced cholesterol ester hydrolysis and suppressed esterification, but increased de novo synthesis can be significant. Elements of the cytoskeleton, probably in conjunction with sterol carrier protein(2) (SCP(2)), mediate cholesterol transfer to the mitochondrial outer membranes. Several factors contribute to the transfer of cholesterol between mitochondrial membranes; steroidogenesis activator peptide acts synergistically with GTP and is supplemented by SCP(2). 5-Hydroperoxyeicosatrienoic acid, endozepine (at peripheral benzodiazepine receptors), and rapid changes in outer membrane phospholipid content may also contribute stimulatory effects at this step. It is suggested that hormonal activation, through these factors, alters membrane structure around mitochondrial intermembrane contact sites, which also function to transfer ADP, phospholipids, and proteins to the inner mitochondria. Cholesterol transfer may occur following a labile fusion of inner and outer membranes, stimulated through involvement of cardiolipin and phosphatidylethanolamine in hexagonal phase membrane domains. Ligand binding to benzodiazepine receptors and the mitochondrial uptake of 37 kDa phosphoproteins that uniquely characterize steroidogenic mitochondria could possibly facilitate these changes. ACTH activation of rat adrenals increases the susceptibility of mitochondrial outer membranes to digitonin solubilization, suggesting increased cholesterol availability. Proteins associated with contact sites were not solubilized, indicating that this part of the outer membrane is resistant to this treatment. Two pools of reactive cholesterol within adrenal mitochondria have been distinguished by different isocitrate- and succinate-supported metabolism. These pools appear to be differentially affected in vitro by the above stimulatory factors.

Relationship of Star Expression to Mitochondrial Cholesterol Transfer and Metabolism

Experiments in Y-1 and primary adrenal cells have established that basal StAR mRNA is sufficient for maximum cAMP-stimulated cholesterol metabolism providing that newly synthesized p37 StAR precursor is phosphorylated, transferred to the matrix and proteolytically cleaved to pp30. This form is active at the inner membrane. The majority of mitochondrial StAR redistributes, perhaps with cholesterol, to matrix vesicles but no longer facilitates intermembrane transfer even when appropriately phosphorylated. MA10 cells utilize a similar to Y01 cells mechanism, but sustain a higher rate of cholesterol metabolism at comparable StAR levels and exhibit much higher maximum rates. In Y-1 adrenal cells cholesterol metabolism is fully activated prior to increased StAR expression which then does not affect the rate. Thus factors other than StAR are at least as important in determining overall rates of cholesterol delivery. Following cAMP stimulation StAR is predominantly expressed as the 3.5kb form which arises from alternative polyadenylation following transcription of an extended exon 7. This form contains an AU-rich regulatory element at the 3′-end that potentially mediates the relatively rapid turnover of this form. The 1.6kb form is more stable and reaches a steady state at later time points. Turnover of both forms is coupled tightly to ongoing transcription and translation. In addition to enhanced transcription cAMP appears to direct enhanced turnover of the 3.5kb form. StAR participation in cholesterol metabolism functions at very low levels of mRNA and high efficiency at each step.

Novel signaling stimulated by arsenite increases cholesterol metabolism through increases in unphosphorylated steroidogenic acute regulatory (StAR) protein.

Cholesterol metabolism to pregnenolone is dependent on the steroidogenic acute regulatory protein (StAR), which activates mitochondrial transfer of cholesterol to cytochrome CYP450scc. In mouse Y-1 adrenal cells and testis MA10 cells stimulation by 8-Bromo-cAMP (Br-cAMP) is augmented by a novel signaling initiated by low concentrations of arsenite (3-20 microM) and anisomycin (0.2 microM), a more selective stress agent. Each elevated StAR mRNA (three-fold after 6 h treatment) even with simultaneous stimulation by Br-cAMP. Arsenite produced parallel increases in StAR protein expression and cholesterol metabolism, but not for P450scc-mediated metabolism of 20alpha-hydroxycholesterol. Although arsenite and anisomycin each stimulated the phosphorylation of p38, the p38 inhibitor SB203580 (SB) produced additive increases in StAR expression. Cholesterol metabolism increased in parallel but without the increased StAR protein phosphorylation produced by Br-cAMP. Arsenite and anisomycin each elevated StAR mRNA but preferentially increased the 3.5 kb form relative to the 1.6 kb form. Arsenite and anisomycin each enhanced the stability of the more labile 3.5 kb mRNA which contains AU-rich elements that control mRNA stability. Although there were increases in both forms of StAR mRNA, arsenite did not stimulate a StAR promoter-reporter that exhibited a typical three-fold response to Br-cAMP. Arsenite and anisomycin may therefore activate a novel SB-independent MAP kinase which in part increases StAR expression through stabilizing the 3.5 kb mRNA but which may also activate a mechanism that by-passes transcription factors detected by the reporter. SB stimulation, which was completely blocked by a MEK inhibitor, was also selective towards the 3.5 kb StAR mRNA suggesting a second pathway for mRNA stabilization. These activations contrast with inhibition of StAR expression by arsenite at higher concentrations or longer incubation times.

Phosphoinositide signalling in human neuroblastoma cells: Biphasic effect of Li+ on the level of the inositolphosphate second messengers

Lithium has a biphasic effect of the agonist-dependent accumulation of Ins(1,4,5)P3 in human neuroblastoma SH-SY5Y cells. These effects consist of a transient reduction, followed by a long-lasting increase in Ins(1,4,5)P3 as compared to controls. The Li+ effects are dose dependent, and were observed at concentrations used in the treatment of bipolar disorders, and thus may have therapeutic implications. The mechanism of the Li+ effect on Ins(1,4,5)P3 accumulation requires further investigation. The transient reduction of Ins(1,4,5)P3 was observed under conditions where Li+ causes only a moderate increase in the inositol mono- and bi-phosphates. Supplementation with exogenous inositol had no effect on the level of Ins(1,4,5)P3, indicating that the mechanism of the Li(+)-dependent reduction of Ins(1,4,5)P3 is not due to inositol depletion. Li+ did not interfere with degradation of Ins(1,4,5)P3 after receptor-blockage with atropine, suggesting that Li+ has no direct effect on the Ins(1,4,5)P3 metabolizing enzymes. A direct effect of Li+ on the phospholipase C is also unlikely. Entry of Ca2+ into the cells is an important factor, which affects agonist-stimulated accumulation of Ins(1,4,5)P3, as well as absolute values of Li(+)-dependent increase in Ins(1,4,5)P3; however, it is not essential for the manifestation of Li+ effects. Our results also show that manifestation of Li+ effects in human neuroblastoma cells requires the stimulation of muscarinic receptors and activation of PLCs, PKCs, and/or that other staurosporine/H-7/GF 109203X-sensitive protein kinases are involved in the regulation of Ins(1,4,5)P3 during the plateau phase of ACh-stimulation. We also suggest an important role for these enzymes in the Li(+)-dependent elevation of Ins(1,4,5)P3.

Multiple Contributions from Long‐Chain Fatty Acid Metabolism in Y‐1 and MA‐10 Cells

Acute steroidogenesis in either Y-1 or MA-10 cells is sensitive to different effects of fatty acids compare to a chronic stimulation. A 3-h stimulation of StAR expression in both cell types was completely blocked by NDGA and AA861, each functioning as lipoxygenase inhibitors. However, the acute 15-min stimulation in Y-1 cells was inhibited by these agents by distinct mechanisms. The inhibition by NDGA was reversed by arachidonic, linoleic, and oleic acids. The inhibition by AA861 was insensitive to reversal by these acids. StAR expression was not affected by these short-term inhibitor treatments. These more rapid fatty acid reversible effects of NDGA are consistent with previously reported inhibition of mitochondrial Acyl CoA ligase. This may function in cooperation with StAR and PBR in providing fatty acid regulation of mitochondrial cholesterol transport. The acute effect of AA861 supports the involvement of an NDGA-insensitive lipoxygenase in the acute stimulation of mitochondrial cholesterol metabolism. The activity of MA-10 cells during prolonged treatments with cAMP appears to utilize each of these processes, which depend on different metabolism of fatty acids.