Metabolism of acid-soluble nucleotides in starved-refed-synchronized Tetrahymena pyriformis GL.
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Adenine↗
Biomedical subjects
D M Stocco
Publications and source records attributed to D M Stocco.
Effect of anoxia on nucleotide metabolism in encysted embryos of the brine shrimp.
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Adenine Nucleotides↗
Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis.
The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T(3)) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T(3) response requires both on-going mRNA and protein synthesis. In addition, the effects of T(3) were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T(3). Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T(3)-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5'-flanking region of the mouse StAR gene, which identified a region between -254 and -110 bp that was essential for T(3) function. Importantly, it was found that the SF-1 binding site at position -135 bp of the 5'-flanking region was greatly involved in T(3)-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T(3) function. The relevance of T(3)-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.
Animals↗
Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein.
The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.
Animals↗
Expression of the steroidogenic acute regulatory (StAR) protein: a novel LH-induced mitochondrial protein required for the acute regulation of steroidogenesis in mouse Leydig tumor cells.
The acute response of steroidogenic cells to hormone stimulation is the mobilization of cholesterol from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane and the cholesterol side-chain cleavage complex (CSCC) where the first enzymatic reaction occurs. It has been well established that the translocation of cholesterol from the outer to the inner mitochondrial membrane requires de novo protein synthesis and that this process is the rate-limiting, regulated step in steroidogenesis. We have purified a novel mitochondrial protein (named StAR) from the MA-10 mouse Leydig tumor cells which we have previously proposed represents a strong candidate for the newly synthesized regulatory protein in steroidogenesis. The cDNA for StAR was cloned and we have demonstrated that expression of the StAR protein in MA-10 cells in the absence of hormone stimulation results in a 3 fold increase in progesterone production compared to mock transfected cells. These studies indicate a direct indicate a direct relationship between StAR expression and steroidogenesis, therefore, we conclude that StAR is required in the acute regulation of steroidogenesis.
Amino Acid Sequence↗
Ethane dimethane sulfonate and NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 Leydig cells and rat Sertoli cells.
Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (delta psi). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of delta psi; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of delta psi.
Animals↗
Acute in vivo effects of ACTH on the expression of steroidogenic acute regulatory protein in rat adrenal.
We have studied the effects of adrenocorticotropin (ACTH) on the expression of steroidogenic acute regulatory protein (StAR) in rat adrenals in vivo. Following ACTH stimulation, the level of StAR mRNA was increased within 1 h in zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with a maximum increase at 3 h. The increase in StAR protein was delayed when compared to its mRNA. The increase in the mitochondrial StAR protein at 3 h was concomitant with that of the homogenate indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. In conclusion, we showed that ACTH increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that post-translational modifications of StAR precursor occur during the early stimulatory phase and this occurs before the apparent translation of the newly formed mRNA.
Adrenocorticotropic Hormone↗
Comparison of cellular and secreted proteins of macrophages from the testis and peritoneum on two-dimensional polyacrylamide gels: evidence of tissue specific function.
It has been shown that the testis contains a population of cells with many characteristics typical of macrophages of other tissues. However, these macrophages are unique in that they secrete a product(s) that is responsible for stimulating testosterone secretion by Leydig cells while peritoneal macrophages have no similar effect. The purpose of the present study was to compare the pattern of cellular and secreted proteins of rat testicular macrophages to those of peritoneal macrophages using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Cellular and secreted proteins of cultured macrophages from the testis and peritoneum were labeled with 35S-methionine and 35S-cystine and then analyzed by 2D-PAGE using a computer-assisted image analysis system. It was found that the patterns of both cellular and secreted proteins of testicular macrophages were vastly different than those of peritoneal macrophages. One striking difference was the presence of a family of proteins in the 47 kd range, with a range of pI from 5.7-6.3 that represented approximately 57% of the total protein secreted by testicular macrophages. Proteins of similar molecular weight and pI represented only approximately 15% of the total peritoneal macrophage secreted proteins. Many other differences were observed in less abundant cellular and secreted proteins. These studies strongly support the concept that macrophages are functionally heterogeneous and secrete a group of proteins characteristic to the histological location of these cells.
Animals↗
The cellular mechanisms of corticotropin-releasing hormone (CRH)-stimulated steroidogenesis in mouse Leydig cells are similar to those for LH.
Previous reports have demonstrated that corticotropin-releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of steroidogenesis, probably by acting through the cAMP/protein kinase A second messenger pathway. Based on this observation, the mechanism of CRH-stimulated steroidogenesis is now further investigated and compared to trophic hormone stimulation. Both cell types were treated with human chorionic gonadotropin (hCG) or CRH in the absence and presence of the following agents: the translation inhibitor cycloheximide, the transcription inhibitor actinomycin D, the protonophore carbonyl cyanide m-chlorophenylhydrozone (mCCCP), which disrupts the mitochondrial electrochemical gradient or the phorbol ester, phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C. Cortico-releasing hormone-stimulated steroidogenesis was completely blocked by cycloheximide in both cell types, indicating that CRH-stimulated steroidogenesis in mouse Leydig cells requires ongoing protein synthesis. Actinomycin D had profound inhibitory effects on CRH-stimulated steroidogenesis in MA-10 cells, and this inhibition was greater than that seen in mouse primary Leydig cells. mCCCP severely inhibited CRH-stimulated steroid production in both cell types, indicating that an electrochemical gradient across the inner mitochondrial membrane is required for CRH-stimulated steroidogenesis. In addition, PMA inhibited hCG- and CRH-stimulated steroidogenesis in MA-10 cells and CRH-stimulated steroidogenesis in primary Leydig cells, suggesting that activation of the protein kinase C pathway can influence protein kinase A stimulated steroidogenesis. Results of these studies suggest that the mouse Leydig cell steroidogenic response to CRH shares many similarities to that of the LH response.
Animals↗