Matthew C. Lorence

3β-Hydroxysteroid dehydrogenase/Δ5→4-isomerase expression in rat and characterization of the testis isoform

Abstract The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3β-hydroxysteroid dehydrogenase/Δ5→4-isomerase (3β-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3β-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3β-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3β-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 μM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3β-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3β-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3β-HSD content, a 3β-HSD is also expressed in liver in a sex-specific manner.

Nucleotide sequence of a cDNA encoding porcine testis 17α-hydroxylase cytochrome P-450

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17α-hydroxylase/C-17,20-lyase cytochrome P -450. The deduced amino acid sequence is 509 amino acids in length.

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.

Expression of cytochrome P-45017α, 3β-hydroxysteroid dehydrogenase/Δ5 → 4-isomerase, and steroid 5α-reductase in rat H540 Leydig tumor cells

Abstract The rat H540 Leydig tumor cell is established as a model for acute lutropin action on the initial step of steroidogenesis, namely the conversion of cholesterol to pregnenolone. Herein, we demonstrate that H540 cells express high levels of three steroid-metabolizing enzymes which are involved in the further processing of pregnenolone in the endoplasmic reticulum of the steroidogenic cell. In particular, in addition to expressing 17α-hydroxylase cytochrome P -450 ( P -450 17 α ) and β-hydroxysteroid dehydrogenase/ Δ 5 → 4 -isomerase (3β-HSD), H540 cells also showed high levels of steroid 5α-reductase mRNA and activity. The H540 cells therefore exhibit similarity to Leydig cells from sexually immature animals which also demonstrate high 5α-reductase activity. Thus, after 3β-HSD-catalyzed formation from pregnenolone, progesterone was efficiently converted to 5α-pregnan-3,20-dione (5α-dihydroprogesterone) and subsequent metabolism to the corresponding 17α-hydroxylated derivative and 5α-androstan-3,17-dione in a reaction catalyzed by P -450 17 α . H540 cells have apparently very low 17-ketosteroid reductase activity and, therefore, a principal end-product of the steroidogenic pathway in these cells was 5α-androstan-3,17-dione. H540 cells maintained in primary culture under serum-free conditions accumulated demonstrable levels of mRNA species for P -540 17 α (1.7 kb), 3β-HSD (1.6 kb) and 5α-reductase (2.7 kb). This finding suggests that the H540 tumor cell model will not only be of utility in the study of acute lutropin action but also in the elucidation of mechanisms involved in the regulation of expression of various families of microsomal steroid-metabolizing enzymes.

Expression of P-450 enzyme activities in heterologous cells by transfection.

The product of the A gene of simian virus 40 (SV40), T antigen, is synthesized and accumulates in the nucleus of infected cells. This regulartory protein acts to initiate viral DNA replication as well as regulating SV40 gene transcription (Tjian 1981). Following transformation of CV-1 cells with an origin-defective mutant of SV40, the resultant monkey kidney cell line (called COS 1) produces T antigen (Gluzman 1981). Transfection of COS 1 cells with plasmid vectors which contain an SV40 origin of replication leads to replication of the plasmid DNA under the influence of T antigen. Accordingly the number of plasmid vectors is amplified in transfected COS 1 cells and the amplified plasmid DNA can then be transcribed leading to production of relatively large quantities of RNA derived from the plasmid. This RNA can, of course, be translated by the endogenous protein synthetic machinery in the COS 1 cells. When the plasmid vector contains a cDNA insert encoding a specific protein, readily detectable quantities of the protein encoded by the insert can be produced in COS 1 cells. A convenient vector for such expression studies is the pcD vector constructed by Okayama and Berg (1982) which contains an SV40 origin of replication and SV40 promotor sequence. A similar vector (pSVL) can be purchased from Pharmacia. pSVL contains a limited multiple cloning site which facilitates insertion of different cDNAs into this expression vector.