Microsomal P450c17 Research Articles

Androgen biosynthesis from cholesterol to DHEA

Androgens and estrogens are made from dehydroepiandrosterone (DHEA), which is made from cholesterol via four steps. First, cholesterol enters the mitochondria with the assistance of the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia. Second, within the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc. Third, pregnenolone undergoes 17α-hydroxylation by microsomal P450c17. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. The ratio of the 17,20 lyase to 17α-hydroxylase activity of P450c17 determines the ratio of C21 to C19 steroids produced. This ratio is regulated post-translationally by at least three factors: the abundance of the electron-donating protein P450 oxidoreductase, the presence of cytochrome b 5, and the serine phosphorylation of P450c17. Study of these and related factors may yield important information about the pathophysiology of adrenarche and the polycystic ovary syndrome (PCOS).

Regulation of microsomal P450, redox partner proteins, and steroidogenesis in the developing testes of the neonatal pig.

Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17alpha-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b(5) in testicular microsomes. Testes were collected from 1-24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1-14. Both 17/20-lyase and aromatase activities increased from d 1-7 but not thereafter, and 17-20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b(5) expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b(5) expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b(5) expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells

Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3β-HSD type II (3β-HSD II), P450c17, P450c21 and P45011β, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 μM of the flavonoids 6-hydroxyflavone, 4′-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 μM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 μg/ml (24 μM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3β-HSD II activity at concentrations from 1 to 25 μM, and I 50 values were estimated to be 1.3, 2, 1, 0.5 and 2.7 μM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 μM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011β at 12.5 and/or 25 μM. Results of Lineweaver–Burk’s plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3β-HSD II and P450c21. K m and V max values of 3β-HSD II for DHEA were estimated to be 6.6 μM and 328 pmol/min mg protein, respectively. K m and V max values of P450c21 for progesterone were estimated to be 2.8 μM and 16 pmol/min mg protein, respectively. K i values of 3β-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 μM, respectively.

Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3β-Hydroxysteroid Dehydrogenase

Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.

Early steps in androgen biosynthesis: From cholesterol to DHEA

Sex steroids, both androgens and oestrogens, are made from dehydroepiandrosterone (DHEA). The biosynthesis of DHEA from cholesterol entails four steps. First, cholesterol enters the mitochondria with the assistance of a recently described factor called the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia. Next, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc. Mutations in the gene for P450scc and for its electron transfer partners, ferredoxin reductase and ferredoxin, have not been described and are probably incompatible with term gestation. Third, pregnenolone undergoes 17 alpha-hydroxylation by microsomal P450c17. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. Isolated 17,20 lyase deficiency is rare, but the identification of its genetic basis and the study of P450c17 enzymology have recently clarified the mechanisms by which DHEA synthesis may be regulated in adrenarche, and have suggested that the lesion underlying polycystic ovary syndrome might involve a serine kinase.

NADPH-flavodoxin reductase and flavodoxin from Escherichia coli: characteristics as a soluble microsomal P450 reductase.

In addition to their endogenous roles as an activation system for various Escherichia coli metabolic pathways, the soluble flavoproteins flavodoxin (Fld) and NADPH-flavodoxin (ferredoxin) reductase (Fpr) can serve as an electron-transfer system for microsomal cytochrome P450s. Furthermore, since Fld and Fpr are structurally similar to the functional domains (FMN binding and NADPH/FAD binding domains, respectively) of NADPH-cytochrome P450 reductases (P450 reductases), these bacterial proteins represent a potentially useful model system for eukaryotic P450 reductases. Here we delineate similarities and differences between the E. coli Fpr-Fld system and rat P450 reductase as electron donors to bovine 17alpha-hydroxylase/17,20-lyase P450 (P450c17). Importantly, recombinant Fpr, in combination with recombinant Fld, supports both the hydroxylase and lyase activities of P450c17 to the same proportional extent (hydroxylase-to-lyase ratio) as does P450 reductase. Maximum P450c17 turnover [5-6 mol of 17alpha-OH-progesterone (mol of P450c17)-1 min-1] was achieved using a large molar excess (50-100-fold over P450c17) of a 1:1 ratio of Fpr-Fld, although this rate was an order of magnitude less than the maximal P450 reductase-supported activity. Using these conditions, we have examined the effects of increasing ionic strength and the presence of cytochrome b5 (b5) on these two systems. Critical Fld-P450c17 electrostatic interactions are disrupted at moderate ionic strength (>100 mM NaCl) as evidenced by significant inhibition (>50%) of Fpr-Fld-supported P450c17 activity while much higher ionic strength (300 mM NaCl) is required to disrupt P450 reductase-P450c17 interactions to the same extent. Interestingly, cytochrome b5 was found to dramatically inhibit both P450 reductase- and Fpr-Fld-supported P450c17 progesterone 17alpha-hydroxylase activity while in contrast 17alpha-OH-pregnenolone lyase activity was stimulated by b5. Investigation of the fate of reducing equivalents from NADPH added to Fpr under aerobic conditions revealed that the majority of the protein-bound FAD of Fpr is converted to the hydroquinone form. In constrast, the FMN of Fld is reduced by Fpr to a stable blue, neutral semiquinone which serves as the predominant electron donor to P450c17 in reconstitution assays. Thus, while the Fpr-Fld system and P450 reductase are fundamentally different with respect to their electrostatic interactions with P450c17, their ability to support maximal P450c17 turnover, and the FMN redox states (one-electron-reduced for Fld and two-electron-reduced for P450 reductase) capable of transferring electrons to microsomal cytochrome P450s, these differences do not appear to influence the relative catalytic efficiency of the P450c17 hydroxylase and lyase reactions.

Expression in Escherichia coli of Functional Cytochrome P450c17 Lacking Its Hydrophobic Amino-Terminal Signal Anchor

A truncated form of bovine microsomal 17α-hydroxylase cytochrome P450 (P450c17) lacking its amino-terminal hydrophobic signal anchor sequence (Δ2-17) was expressed in Escherichia coli to give more than 600 nmol P450 per liter using XL1-blue as the host. The expression level of the truncated P450c17 showed variation among different host strains. When intact E. coli cells were used for analysis, the truncated P450c17 showed the typical cytochrome P450 CO-difference spectrum and type I substrate binding spectra for pregnenolone, 17α-hydroxypregnenolone, progesterone, and 17α-hydroxyprogesterone. The apparent K8 values for these steroids are comparable to those obtained under the same conditions with wild type P450c17. The truncated P450c17 is primarily associated with the membrane fraction from E. coli and remained with this fraction after treatment with 0.1 M Na2CO3. The 17α-hydroxylase activity of the truncated form was observed using intact E. coli. Using isolated membrane fractions, the truncated P450c17 also showed 17,20-lyase activity upon reconstitution with rat liver microsomal NADPH-cytochrome P450 reductase. These results indicate that P450c17 can be associated with E. coli membranes not only via the amino-terminal hydrophobic region as expected for the wild type, nontruncated form, but also through other sequences within the polypeptide chain and that integration of the amino- terminal region into the membrane is not essential for the folding pathway leading to functional P450c17 in E. coli. This is in contrast to mammalian cells where the same truncated P450c17 is found to be inactive and the folding pathway leading to functional P450c17 appears to require a signal anchor sequence.