p53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin-inhibitor with anti-HIV properties.

Previous reports have indicated that benzothiophenes exhibit broad anti-inflammatory properties and inhibit human immunodeficiency virus-type 1 (HIV-1) replication. We show that the immunosuppressant cyclosporin A (CsA) and benzothiophene-2-carboxamide, 5-methoxy-3-(1-methyl ethoxy)-1-oxide (PD 144795) block the induction of p53 and NF-kappaB binding to the HIV-1 long terminal repeat (LTR) by the T cell receptor activator phytohemagglutinin. CsA and PD 144795 also inhibit the induction by phytohemagglutinin of the transcription mediated by an HIV-1 LTR fragment containing the p53 and NF-kappaB sites. These effects of PD 144795 on HIV-1 transcription correlate with its ability to inhibit the phosphatase activity of calcineurin and are similar to those previously described for CsA. Moreover, a constitutive active form of calcineurin is able to induce expression from the HIV-1 LTR in a p53- and NF-kappaB-dependent manner and PD 144795 is able to block this induction. These results demonstrate that the DNA binding of p53 to the HIV-1 LTR can be modulated by calcineurin and provide a framework to understand the anti-HIV properties of benzothiophene derivatives.

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) by androgens.

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.

Tumor necrosis factor-alpha inhibition of 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) expression.

Testosterone biosynthesis in Leydig cells is dependent on the action of 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450c17), which is encoded by the Cyp17 gene. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, inhibits cAMP-stimulated testosterone production in mouse Leydig cells. The inhibition of testosterone production is parallel to the inhibition of P450c17 messenger RNA and protein levels. To examine the mechanism of TNF alpha-mediated inhibition of steroidogenesis, the effect of TNF alpha on cAMP-stimulated induction of Cyp17 expression was investigated. To determine whether the protein kinase C (PKC) signaling pathway is involved in TNF alpha inhibition of steroidogenesis, the effects of the PKC activator, phorbol 12-myristate 13-acetate (PMA), and the PKC inhibitor, calphostin C, were examined. Treatment of normal mouse Leydig cells in primary culture with 50 microM 8-bromo-cAMP (cAMP) plus 1 ng/ml TNF alpha or 10 nM PMA caused a similar (approximately 90%) decrease in testosterone accumulation and cAMP-stimulated P450c17 messenger RNA levels compared to those after treatment with cAMP alone. To determine whether TNF alpha inhibits the cAMP-induced expression of the Cyp17 gene, plasmids containing two different size fragments of the 5'-flanking region of the Cyp17 gene upstream of the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into MA-10 tumor Leydig cells, and the effect of TNF alpha on cAMP-induced CAT activity was determined. Treatment of cells, transfected with either plasmid, with 500 microM cAMP plus increasing concentrations (0.1, 1.0, and 10 ng/ml) of TNF alpha resulted in a dose-dependent repression of cAMP-stimulated CAT activity. Higher concentrations of TNF alpha (up to 100 ng/ml) did not result in greater inhibition. Treatment of transfected cells with 10 nM PMA resulted in a 51 +/- 6.6% inhibition of cAMP-stimulated CAT activity. Calphostin C (1 microM) completely reversed the inhibitory effect of TNF alpha or PMA. Calphostin C alone had no effect on promoter activity. TNF alpha-stimulated PKC alpha translocation was quantitated by Western blot. After treatment for 3 h, the distribution of immunoreactive PKC alpha in cytosol vs. nucleus was 55%/45%, 60%/40%, and 29%/71% in control, cAMP-treated, and TNF alpha-treated cells, respectively. TNF alpha-stimulated PKC alpha translocation was further demonstrated by indirect immunofluorescence assay. PMA, a known activator of PKC, and TNF alpha had a similar inhibitory effect on P450c17 expression, testosterone production, and Cyp17-CAT activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of expression of steroidogenic enzymes in Leydig cells.

The Leydig cell of the testis is the only cell in the male that has the capacity to synthesize testosterone from cholesterol. Testosterone is critical during fetal development for male sexual differentiation, and postnatally for initiation and maintenance of spermatogenesis and the expression of the male secondary sex characteristics. The biosynthesis of testosterone requires the activities of four enzymes, cholesterol side-chain cleavage enzyme (P450scc), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)), and 17-ketosteroid reductase. The expression of these enzymes appears to be regulated by different mechanisms. The recent isolation of the mouse cDNAs and structural genes that encode these enzymes has enabled us to begin to investigate the regulation of their expression at the molecular level. This review discusses the regulation by cAMP and steroids of three enzymes in Leydig cells, P450scc, P450(17 alpha), and 3 beta HSD, as well as characterization of the promoters of the mouse genes that encode P450scc and P450(17 alpha).

Isolation and characterization of the mouse P450 17 alpha-hydroxylase/C17-20-lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells.

The biosynthesis in Leydig cells of the C19 steroid testosterone from the C21 precursor progesterone requires the activities of the enzyme cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)). Previous studies from this laboratory demonstrated that the de novo synthesis of the P450(17 alpha) protein and the accumulation of P450(17 alpha) mRNA in mouse Leydig cell cultures is absolutely dependent on cAMP stimulation. To investigate further the cAMP regulation of P450(17 alpha) expression in Leydig cells, the structural gene encoding P450(17 alpha) (Cyp17) was isolated from a mouse genomic library using a full-length mouse P450(17 alpha) cDNA. Two overlapping genomic clones were isolated and characterized by restriction mapping and partial sequencing. The two clones together contain the entire coding region and approximately 10 kilobases of 5'-flanking sequences of Cyp17. To identify regions necessary for cAMP-induced transcription, 5'-flanking regions of Cyp17 were fused with the chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into MA-10 tumor Leydig cells. Studies localized the cAMP-responsive region of the gene to a region between -346 and -245 basepairs relative to the transcription initiation site. Transient transfections of MA-10 cells with a construct consisting of the -346/-245 sequences fused to a heterologous promoter, thymidine kinase, and the CAT reporter gene demonstrated a marked increase in cAMP stimulation of CAT expression, providing additional evidence that the -346/-245 sequences of the Cyp17 5'-flanking region confer cAMP-induced expression of Cyp17. This cAMP-responsive region of mouse Cyp17 bears no apparent homology to the cAMP-responsive regions identified in the human and bovine Cyp17 genes.

Hormonal regulation of steroidogenic enzyme gene expression in Leydig cells.

In normal mouse Leydig cells, steady state levels of mRNA of CYP11A, 3ß-hydroxysteroid dehydrogenase Δ5- >Δ4-isomerase (3ßHSD), and CYP17 are differentially regulated. There is high basal expression of 3ßHSD and CYP11A mRNA, while expression of CYP17 mRNA is absolutely dependent on cAMP stimulation. cAMP is required for maximal expression of all three enzymes. The expression of CYP11A in normal mouse Leydig cells is repressed by glucocorticoids. Glucocorticoids also repress both basal and cAMP-induced expression of 3ßHSD mRNA, but do not repress the synthesis or mRNA levels of CYP17. cAMP induction of 3ßHSD mRNA can be observed only when aminoglutethimide (AG), an inhibitor of cholesterol metabolism, is added to the Leydig cell cultures. The addition of AG also markedly increases cAMP induction of CYP17 mRNA levels. Addition of testosterone or the androgen agonist, mibolerone, to cAMP plus AG treated cultures reduced 3ßHSD and CYP17 mRNA levels to levels comparable to those observed when cells were treated with cAMP only. These data indicate that testosterone acting via the androgen receptor represses expression of both CYP17 and 3ßHSD. The role of protein synthesis in mediating the cAMP induction of 3ßHSD, CYP17 and CYP11A was examined. The addition of cycloheximide, an inhibitor of protein synthesis, to cAMP treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3ßHSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of CYP17 to 12% of levels observed in the absence of cycloheximide. In sharp contrast, treatment for 24 h with cycloheximide did not suppress cAMP induction of CYP11A mRNA, but reduced basal levels by approx. 50%. These data indicate that newly synthesized protein(s) are required for cAMP induction of CYP17 and 3ßHSD mRNA levels, but not for CYP11A mRNA. A mouse Cyp17 genomic clone containing the entire coding region plus 10 kb of 5' flanking region has been isolated. Fragments of 5' flanking sequences were subcloned into vectors containing the CAT reporter gene and transfected into MA-10 Leydig cells. Transfected cells were treated with cAMP and expression was determined by measuring CAT activity. A cAMP responsive element was identified in a region between -245 and -346 bp relative to the transcription initiation site of Cyp17. Cotransfection into MA-10 Leydig cells of constructs containing 4.5 kb of Cyp17 5' flanking sequences together with a mouse androgen receptor expression vector demonstrate a dose dependent repression of cAMP-induced Cyp17 transcription by the androgen receptor. Studies with the mouse Cyp11a gene demonstrate that the 5' flanking region of the gene contains sequences between 2.5 and 5 kb that are necessary for expression of mouse Cyp11a in Leydig cells but not in adrenal cells.

Isolation, characterization, and chromosomal mapping of mouse P450 17 alpha-hydroxylase/C17-20 lyase.

Cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P45017 alpha) catalyzes the conversion of C-21 steroids to C-19 steroids in gonads. A full-length mouse cDNA encoding P450 17 alpha was isolated from a mouse Leydig cell library and characterized by restriction mapping and sequencing. The predicted amino acid sequence has 83% homology to rat, 66% homology to human, and 62% homology to bovine P45017 alpha amino acid sequences. The protein is 507 amino acids in length, which is 1 amino acid shorter than the human protein and 2 amino acids shorter than the bovine protein. The structural gene encoding P450 17 alpha (Cyp17) was localized utilizing an interspecific testcross to mouse chromosome 19, distal to Got-1. Another cytochrome P450, P4502c (Cyp2c), also is located at the distal end of chromosome 19. CYP17, CYP2c, and GOT1 have been mapped to human chromosome 10, with CYP2C and GOT1 mapped to the distal region, q24.3 and q25.3, respectively. The data in the present study indicate conserved syntenic loci on mouse chromosome 19 and human chromosome 10 and predict that the structural gene encoding P45017 alpha will be found distal to GOT1 on human chromosome 10.

The structural genes encoding P450scc and P450arom are closely linked on mouse chromosome 9.

The chromosomal location of the two genes that encode the cytochrome P450 enzymes, P450SCC (cholesterol side-chain cleavage) and P450arom (aromatase), was identified in the mouse. Genomic DNA from several progenitor strains of recombinant inbred (RI) strains of mice was tested with various restriction endonucleases for restriction fragment length variations. Variation in Bam HI fragment length was detected between A/J and C57BL/6J. Genomic DNA from 43 RI strains derived from A/J and C57BL/6J was analyzed in a similar manner. Complete concordance of the strain distribution pattern for P450SCC and that of P450arom was observed for 43 RI strains. The lack of recombination indicates that the structural genes encoding P450SCC and P450arom are closely linked. The strain distribution patterns of the P450SCC and P450arom genes were compared with other markers previously mapped in these RI lines. The results demonstrate that both P450SCC and P450arom are found on mouse chromosome 9. Of the other loci on mouse chromosome 9, P450SCC and P450arom are most closely linked to the gene encoding P1450. Among 31 RI strains for which the three loci were analyzed, only one example of discordance was found. Human P450SCC, P450arom and P1450 have been mapped to human chromosome 15. However, the distance between the human P450SCC gene and other loci has not been determined. The information presented in this report, along with other studies, indicate conservation between homologous human and mouse chromosomal regions and suggest that human P450SCC will be found to be closely linked with human P450arom.