Human Cholesterol Side-chain Cleavage Research Articles

CYP11A1 expression in bone is associated with aromatase inhibitor-related bone loss.

Aromatase inhibitors (AIs) used as adjuvant therapy in postmenopausal women with hormone receptor-positive breast cancer cause diverse musculoskeletal side effects that include bone loss and its associated fracture. About half of the 391 patients treated with AIs in the Barcelona-Aromatase induced bone loss in early breast cancer cohort suffered a significant bone loss at lumbar spine (LS) and/or femoral neck (FN) after 2 years on AI-treatment. In contrast, up to one-third (19.6% LS, 38.6% FN) showed no decline or even increased bone density. The present study aimed to determine the genetic basis for this variability. SNPs in candidate genes involved in vitamin D and estrogen hormone-response pathways (CYP11A1, CYP17A1, HSD3B2, HSD17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, DHCR7, GC, CYP2R1, CYP27B1, VDR and CYP24A1) were genotyped for association analysis with AI-related bone loss (AIBL). After multiple testing correction, 3 tag-SNPs (rs4077581, s11632698 and rs900798) located in the CYP11A1 gene were significantly associated (P<0.005) with FN AIBL at 2 years of treatment. Next, CYP11A1 expression in human fresh bone tissue and primary osteoblasts was demonstrated by RT-PCR. Both common isoforms of human cholesterol side-chain cleavage enzyme (encoded by CYP11A1 gene) were detected in osteoblasts by western blot. In conclusion, the genetic association of CYP11A1 gene with AIBL and its expression in bone tissue reveals a potential local function of this enzyme in bone metabolism regulation, offering a new vision of the steroidogenic ability of this tissue and new understanding of AI-induced bone loss.

Hormonal regulation of the steroidogenic acute regulatory protein (StAR) in gonadal tissues of the Atlantic croaker ( Micropogonias undulatus)

The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62–85% identity) than to the related START protein MLN-64 (28–31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24 h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24 h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6 h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20β,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels.

Salt-inducible Kinase Represses cAMP-dependent Protein Kinase-mediated Activation of Human Cholesterol Side Chain Cleavage Cytochrome P450 Promoter through the CREB Basic Leucine Zipper Domain

Salt-inducible kinase (SIK), one of the serine/threonine protein kinases, was transiently expressed in Y1 cells during the early phase of the ACTH/cAMP-dependent protein kinase (PKA)-mediated signal transduction. The overexpression of SIK(N), the SIK's N-terminal kinase domain, repressed the expression of the side chain cleavage cytochrome P450 (CYP11A) gene. To elucidate the mechanism of the repression by SIK, several CYP11A promoter constructs were tested for the promoter activities in the presence of PKA and/or SIK(N). A cAMP-response element (CRE)-like sequence present in the promoter was shown to be responsible not only for the PKA-mediated promoter activation but also for the SIK(N)-mediated repression. When the Gal4 DNA binding domain-linked full-length CRE-binding protein (CREB) construct was cotransfected with Gal4 reporter gene, SIK(N) repressed the PKA-induced reporter gene expression. However, SIK(N) could not repress the PKA-induced reporter activity conferred by Gal4 DNA binding domain-linked basic leucine zipper (bZIP)-less CREB or bZIP-disrupted CREB. On the other hand, SIK(N) could repress the kinase-inducible domain-disrupted CREB-dependent reporter gene expression in the presence of PKA. The in vitro kinase reaction studies showed that SIK(N) could not phosphorylate CREB, and PKA failed to phosphorylate SIK(N). Taken together, these results suggest that SIK(N), cooperating with PKA, may act on the CREB's bZIP domain and repress the CREB-mediated transcriptional activation of the CYP11A gene.

Differential regulation of aldosterone synthase and 11beta-hydroxylase transcription by steroidogenic factor-1

11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.

Salt-Inducible Kinase Is Involved in the ACTH/cAMP-Dependent Protein Kinase Signaling in Y1 Mouse Adrenocortical Tumor Cells

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system.

A fusion construct for the human cholesterol side-chain cleavage enzyme system termed F2 (H(2)N-P450scc-adrenodoxin reductase-adrenodoxin-COOH), was stably expressed in nonsteroidogenic COS-1 cells. Multiple clones were obtained and analyzed, identifying the clone COS-F2-130 as the most active in converting 22R-hydroxycholesterol (22R-OH-C) to pregnenolone. The F2 fusion construct was properly transcribed and translated in COS-F2-130 cells, indicating that these cells did not proteolytically cleave the F2 protein. Steroid analyses show that the COS-F2-130 cells do not convert appreciable quantities of pregnenolone to other steroids. Isolated COS-F2-130 mitochondria showed enhanced steroidogenesis when incubated with biosynthetic N-62 StAR protein in vitro. The cells were easily transfectable with StAR expression vectors, showing that COS-F2-130 cells exhibited both StAR-independent and StAR-dependent activity. Transient expression of either full-length or N-62 StAR stimulated steroidogenesis to approximately 45% of the maximal steroidogenic capacity, as indicated by incubation with 22R-OH-C. Single, double, and triple transfections of individual vectors expressing P450scc, adrenodoxin reductase, and adrenodoxin demonstrated that the P450 moiety of the F2 fusion protein could only receive electrons from the covalently linked adrenodoxin moiety, but that free adrenodoxin reductase could foster activity of the fusion enzyme. COS-F2-130 cells provide a useful system for studying steroidogenesis, as these are the only cells described to date that convert cholesterol to pregnenolone but lack downstream enzymes that catalyze other steroidogenic reactions.

Creation and Activity of COS-1 Cells Stably Expressing the F2 Fusion of the Human Cholesterol Side-Chain Cleavage Enzyme System

Creation and Activity of COS-1 Cells Stably Expressing the F2 Fusion of the Human Cholesterol Side-Chain Cleavage Enzyme System

Phosphorylation of Steroidogenic Acute Regulatory Protein (StAR) Modulates Its Steroidogenic Activity

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis.

MLN64 is a protein that is highly expressed in certain breast carcinomas. The C terminus of MLN64 shares significant homology with the steroidogenic acute regulatory protein (StAR), which plays a key role in steroid hormone biosynthesis by enhancing the intramitochondrial translocation of cholesterol to the cholesterol side-chain cleavage enzyme. We tested the ability of MLN64 to stimulate steroidogenesis by using COS-1 cells cotransfected with plasmids expressing the human cholesterol side-chain cleavage enzyme system and wild-type and mutant MLN64 proteins. Wild-type MLN64 increased pregnenolone secretion in this system 2-fold. The steroidogenic activity of MLN64 was found to reside in the C terminus of the protein, because constructs from which the C-terminal StAR homology domain was deleted had no steroidogenic activity. In contrast, removal of N-terminal sequences increased MLN64's steroidogenesis-enhancing activity. MLN64 mRNA was found in many human tissues, including the placenta and brain, which synthesize steroid hormones but do not express StAR. Western blot analysis revealed the presence of lower molecular weight immunoreactive MLN64 species that contain the C-terminal sequences in human tissues. Homologs of both MLN64 and StAR were identified in Caenorhabditis elegans, indicating that the two proteins are ancient. Mutations that inactivate StAR were correlated with amino acid residues that are identical or similar among StAR and MLN64, indicating that conserved motifs are important for steroidogenic activity. We conclude that MLN64 stimulates steroidogenesis by virtue of its homology to StAR.

Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.

Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.

Characterization of human cholesterol side chain cleavage enzyme (EC 1.14.15x) of human term placental mitochondria

The cholesterol side chain cleavage enzyme (EC 1.14.15x) in mitochondria of a human term placenta was partially characterized. Enzyme activity was determined by separation of [26- 14C]-cholesterol and [5- 14C]-isocaproic acid formed by side chain cleavage. Since the amounts of unlabeled cholesterol were too large, a K M of cholesterol could not be determined. The apparent K M value of NADPH is 6.25 × 10 −4 M. A pH optimum was found at pH 9.5 (Tris-buffer) and a temperature optimum at 40°C. The metal ions Sr 2+ and Ba 2+ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg 2+ and Ca 2+ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn 2+, Cd 2+, Cu 2+ and by 10 and 100 mM Fe 2+, Mn 2+, Co 2+ and Ni 2+. During preincubation of the enzyme without radioactive substrate, a rapid loss in enzyme activity in relation to enzyme concentration was observed (initial activity = 100%) (preincubation time in hours): 0.5 h (97%), 1 h (55%) and 1.5 h (34%). A dose-dependent inhibition of the enzyme by the following proteins was achieved: bovine serum protein, human serum protein, human immunoglobulin G and ovalbumin. Furthermore, a dose-dependent inhibition was found with the membrane lipids lecithin and sphingosine.