Human cytochromes P450

In situ hybridization studies reveal novel sites of expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) during murine embryonic development. In addition to fetal adrenals and testes, P450scc transcripts localize in situ to the primitive gut and to a subset of unidentified cells in the dermal mesenchyme of embryonic skin. In the gut, transcripts are most abundant in luminal epithelia of the hindgut, which will form the colon. P450scc transcript abundance at these novel sites is a fraction of that in fetal adrenals or testes, suggesting a local rather than an endocrine function. Immunocytochemical analyses localize P450scc protein to the fetal hindgut, indicating that the transcripts are translated in vivo. RNA isolated from microdissected embryonic hindgut and skin was reverse transcribed and amplified by polymerase chain reaction. DNA sequence analyses of polymerase chain reaction products confirmed that specific hybridization in situ represents authentic P450scc gene (Cyp11A) transcripts and that 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4-isomerase transcripts are also present, demonstrating the potential of these fetal tissues to produce pregnenolone and progesterone. P450scc transcripts are also detectable by in situ hybridization in primitive gut and skin of Fushi tarazu factor 1 null mice, which lack the nuclear receptor steroidogenic factor 1, proving that steroidogenic factor 1 is not required for steroid hydroxylase gene expression at these sites. The capacity for C21 steroid biosynthesis in primitive gut and skin during organogenesis raises the question whether local production of steroid hormones may be required for normal cellular growth and differentiation of these tissues during embryogenesis.

We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.