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Originally published In Press as doi:10.1074/jbc.M414673200 on February 1, 2005

J. Biol. Chem., Vol. 280, Issue 14, 13265-13271, April 8, 2005
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Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17*

Amit V. Pandey and Walter L. Miller{ddagger}

From the Department of Pediatrics and The Metabolic Research Unit, University of California San Francisco, San Francisco, California 94143-0978

Received for publication, December 30, 2004 , and in revised form, January 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450c17 catalyzes the 17{alpha}-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17{alpha}-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450c17 is an essential steroid biosynthetic enzyme required for reproduction in all vertebrates. In mammals (1–4), fish (5), birds (6), and amphibians (7), P450c17 catalyzes the steroid 17{alpha}-hydroxylase and 17,20 lyase activities needed to produce the 19-carbon precursors of sex steroids. The 17{alpha}-hydroxylase activity typically converts pregnenolone to 17{alpha}-hydroxypregnenolone (17OH-Preg)1 and progesterone to 17{alpha}-hydroxyprogesterone (17OH-Prog), and the 17,20 lyase activity converts 17OH-Preg to dehydroepi- and rosterone (DHEA) and 17OH-Prog to androstenedione (Fig. 1). In human adrenal steroidogenesis, P450c17 is the qualitative regulator that determines the class of steroids synthesized in different cell types: in its absence, mineralocorticoids are produced; if only its 17{alpha}-hydroxylase activity is present, glucocorticoids are produced; if both its 17{alpha}-hydroxylase and 17,20 lyase activities are present, precursors of sex steroids are produced (8). The single human CYP17 gene (9) on chromosome 10q24 (10) produces a single species of mRNA (11) encoding P450c17, which catalyzes both reactions on a single active site, apparently using the same chemical reaction mechanism (12). However, there are species-specific differences in these activities, especially concerning the 17,20 lyase reaction: the rodent and guinea pig enzymes use both 17OH-Preg and 17OH-Prog as substrates, the bovine enzyme mainly uses 17OH-Prog, and the human enzyme uses 17OH-Preg almost exclusively (1, 2, 4, 13, 14).



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  		FIG. 1.
Early steps of sex steroid biosynthesis. P450scc converts cholesterol to pregnenolone, a C21 {Delta}5-steroid. Human P450c17 performs the 17{alpha}-hydroxylase reaction equally well using pregnenolone and progesterone as substrates, but the 17,20-lyase reaction occurs 50–100 times more efficiently using 17OH-Preg as substrate rather than 17OH-Prog. Thus conversion of 17OH-Prog to androstenedione is insignificant, and DHEA is the major precursor of human sex steroid synthesis.

 
Similar to all other microsomal forms of cytochrome P450, the catalytic activities of P450c17 require electron donation from NADPH through the intermediacy of a membrane-bound flavoprotein termed P450 oxidoreductase (POR) (15–17). All cytochrome P450 enzymes catalyze multiple reactions, but the ratios of these multiple reactions are determined primarily by substrate concentrations. By contrast, P450c17 appears to be unique in that the ratio of its two principal activities is developmentally regulated. Human serum concentrations of cortisol, an index of 17{alpha}-hydroxylase activity, remain constant as a function of age, but concentrations of DHEA, an index of 17,20 lyase activity, rise over 100-fold during adrenarche (18), an event that is contemporaneous with, but independent of puberty (19–22). The ratio of 17,20 lyase activity to 17{alpha}-hydroxylase activity can be regulated by three distinct post-translational mechanisms. First, the presence of high molar ratios of POR to P450c17 favor the 17,20 lyase reaction (14, 23). Second, 17,20 lyase activity can be enhanced by the presence of cytochrome b5 (24–26), which acts allosterically to foster interactions with POR (13, 27). Third, serine/threonine phosphorylation of P450c17 increases 17,20 lyase activity but does not affect 17{alpha}-hydroxylase activity (28, 29). In some P450-mediated drug metabolism reactions, cytochrome b5 appears to act as an alternative electron donor that can substitute for POR in the donation of second electron in the P450 cycle (30–32) but it does not function in this fashion to foster 17,20 lyase activity, as apo b5, which is devoid of heme, is as effective as holo b5 (13, 32). Mutations in the POR binding site of P450c17 selectively reduce 17,20 lyase activity (33); when cytochrome b5 is added, 17,20 lyase activity is partially restored, increasing the Vmax but not influencing the Km of the mutants (27), thus confirming the allosteric action of cytochrome b5. The mechanism by which P450c17 phosphorylation augments 17,20 lyase activity is not known, nor is it known whether the state of P450c17 phosphorylation influences the allosteric action of cytochrome b5. Using purified, catalytically active, bacterially-expressed human P450c17, POR, and b5 in vitro and RNA interference knockdown of cytochrome b5 in human adrenal NCI-H295A cells, we now show that these two mechanisms of augmenting 17,20 lyase activity function independently.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Microsome Preparation—NCI-H295A cells (34), an adherent population of human adrenocortical carcinoma NCI-H295 cells (35, 36), were grown in 150-mm Petri dishes as described (34). HepG2, JEG3 (37), and HEK293 (38) cells were grown as described. NCI-H295A cells from several plates were collected by scraping, washed with chilled phosphate-buffered saline (PBS), suspended in 50 mM sodium phosphate containing 150 mM KCl, and lysed by sonication. Unbroken cells and mitochondria were removed by centrifugation at 10,000 x g for 15 min, and microsomes were collected by ultracentrifugation at 100,000 x g for 90 min in a Beckman T-100 rotor. Microsomes were resuspended in 50 mM potassium phosphate buffer containing 20% glycerol.

Cytochrome b5 Expression Analysis—Expression of cytochrome b5 in various cell lines was analyzed by RT-PCR using GAPDH as control. The primer sequences used were for cytochrome b5: sense, 5'-ATGGCAGAGCAGTCGGACGA-3' and antisense, 5'-TCAGTCCTCTGCCATGTATAG-3'; for OMb5: sense, 5'-ATGGCGACTGCGGAAGCTA-3' and antisense, 5'-TCAGGAGGATTTGCTTTCCGA-3'; for GAPDH: sense, 5'-GTATCGTGGAAGGACTCAT-3' and antisense, 5'-TACTCCTTGGTGGCCATGT-3'. All PCR reactions were performed for 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C 45s followed by 1 cycle of 72 °C for 10 min, and products were separated on 10% Tris borate/EDTA acrylamide gel, stained with ethidium bromide and visualized on a Kodak EDS 290 gel documentation system.

Expression and Purification of Human P450c17—Human P450c17 was expressed in Escherichia coli and purified as described (39). The plasmid pCWH17-mod(His)4 containing the cDNA for N-terminally modified human P450c17 (40) was transformed into E. coli JM109, colonies were selected on LB agar plates containing 100 µg/ml carbenicillin, and a single colony was grown to A600 of 0.4–0.6 in LB broth containing 100 µg/ml carbenicillin. From this culture, 1 ml of bacteria were seeded into terrific broth containing 40 µM FeCl3,4 µM ZnCl2,2 µM CoCl2, 2 µM Na2MoO4, 2 µM CaCl2, 2 µM CuCl2, 2 µM H3BO3, and 10% v/v potassium phosphate solution (0.17 M KH2PO4, 0.72 M K2HPO4, pH 7.4) and 100 µg/ml carbenicillin and grown at 30 °C with shaking at 250 rpm. At A600 of 0.5–0.7, 0.4 mM isopropyl-1-thio-{beta}-D-galactopyranoside (IPTG) and 0.4 mM {delta}-amino levulinic acid were added, and the culture was shaken at 125 rpm for 36 h at 30 °C. The bacteria were then chilled on ice, pelleted by centrifugation at 5000 x g for 10 min at 4 °C, washed once with PBS, and resuspended in 100 mM Tris-HCl, pH 7.8 containing 500 mM sucrose and 0.5 mM EDTA (10 ml/g of pellet). Lysozyme (0.1 mg/ml) was added to the bacterial suspension, and the cells were kept on ice for 30 min with occasional stirring. Spheroplasts were harvested by centrifugation at 12,000 x g for 15 min at 4 °C and resuspended in 100 mM potassium phosphate pH 7.6 containing 6 mM magnesium acetate, 20% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride. The resulting spheroplasts were sonicated on ice with 15–20 cycles of 20-s pulses followed by 30 s cooling at 50% power using a Fisher scientific 550 sonicator with a microprobe. The lysate was cleared of unbroken cells and debris by centrifugation at 10,000 x g for 10 min at 4 °C, and membranes were pelleted by ultracentrifugation in a Beckman T-100 rotor at 100,000 x g for 90 min. The resultant pellet containing human P450c17 was used for enzyme assays and purification of P450c17; cytochrome P450 content was measured by reduced carbon monoxide binding spectra (41). P450c17 was purified from the E. coli membranes by solubilization in 0.7% Triton X-114 followed by chromatography on Ni-NTA and hydrophobic interaction chromatography as described (29, 39). Purified P450c17 was resuspended in 100 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.1% Triton X-100.

Phosphatase Treatment of Bacterially Expressed Human P450c17— Purified preparations of P450c17 (5 µg) were treated with 5 units of alkaline phosphatase or protein phosphatase 2A (PP2A) for 15 min at 25 °C, and the reaction was stopped by adding 1 mM sodium orthovanadate, 50 mM NaF, and/or 50 µM okadaic acid (Calbiochem) in the case of protein PP2A. P450c17 treated with phosphatases was repurified on a Ni-NTA spin column (Amersham Biosciences) and used for enzyme assays. The amount of phosphate released into the supernatant was monitored by malachite green reaction (BioMol Laboratories).

Expression and Purification of Human POR—Human POR was expressed in bacteria by transforming E. coli BL21(DE3)pLysS cells with a pET22b vector (Novagen) containing human POR cDNA (17). Freshly transformed E. coli were selected on a LB agar plate with 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol, and a single colony was seeded into LB media containing 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol and grown at 28 °C to A600 of about 0.5. From this culture, 1 ml of bacteria were seeded into 150 ml of terrific broth supplemented with 40 µM FeCl3, 4 µM ZnCl2, 2 µM CoCl2, 2 µM Na2MoO4, 2 µM CaCl2, 2 µM CuCl2, 2 µM H3BO3, and 10% v/v potassium phosphate solution (0.17 M KH2PO4, 0.72 M K2HPO4, pH 7.4), 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol and grown at 30 °C to A600 of 0.4–0.7, 0.4 mM IPTG and 0.1 mg/ml riboflavin were added, and the bacteria were shaken at 125 rpm for another 16 h before harvesting by centrifugation at 5000 x g for 10 min at 4 °C. The bacteria were then washed with PBS, treated with lysozyme (0.1 mg/ml) and EDTA (0.1 mM, pH 8.0), and membranes were prepared as described for P450c17. Membranes were dissolved in 100 mM phosphate buffer containing 100 mM NaCl, 1.5% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride, and POR was purified by affinity chromatography on 2'-5' ADP-agarose as described (42). The catalytic activity of this POR preparation was assessed by assays using either cytochrome c or NADPH as substrates as described (17).

Expression and Purification of Cytochrome b5—Human cytochrome b5 was prepared in E. coli strain BL21(DE3) transformed with plasmid pLW01b5 containing the cDNA for human cytochrome b5 and selected over several cycles, as described (43). A single colony of transformed E. coli was inoculated into LB media containing 100 µg/ml carbenicillin, grown to an A260 of 0.5–0.7, and IPTG was added to a final concentration of 0.4 mM. Cell density was measured every 30 min; when the A600 nm started to rise again (90–180-min postinduction), 100 µl of cells diluted 1:50 were plated on LB agar plates containing 100 µg/ml carbenicillin and 0.4 mM IPTG and allowed to grow overnight at 37 °C. A single colony was grown the next day and the process of induction and selection with carbenicillin and IPTG was repeated three more times. After four cycles of selection, the E. coli were transformed with plasmid pLysS Rare, which encodes lysozyme and six rare tRNAs that assist the expression of mammalian proteins by complementing the codon bias of E. coli. The resultant E. coli were used for expression of cytochrome b5. Expression and purification was performed as described previously (44).

Suppression of Cytochrome b5 by Short Hairpin RNA (shRNA) in NCI-H295A Cells—NCI-H295A cells were transfected with the retroviral vector pSUPERretro (45, 46) expressing shRNA targeted against both types 1 and 2 human cytochrome b5. The pSUPERretro vector was digested with BglI and HindIII and ligated to the 64-mer oligonucleotide duplexes (sense 5'-GATCCCCCAAGCTGGAGGTGACGCTATTCAAGAGATAGCGTCACCTCCAGCTTGTTTTTGGAAA-3'and antisense 3'-GGGGTTCGACCTCCACTGCGATAAGTTCTCTATCGCAGTGGAGGTCGAACAAAAACCTTTTCGA-5') encoding small interfering RNA (siRNA) against cytochrome b5 and a 9-nucleotide loop region. The recombinant plasmid was purified from bacterial cultures and digested with EcoRI and HindIII to check the inserts. Purified plasmid was transformed into NCI-H295A, and cells containing the virus were selected by puromycin and used for cytochrome b5 measurement and P450c17 enzyme assays.

Measurement of Cytochrome b5 Protein and mRNA—The cytochrome b5 content of NCI-H295A cells was measured by differential spectroscopy (47). Cells were homogenized in sodium phosphate buffer (50 mM, pH 7.4) containing 150 mM KCl; the homogenate was cleared by centrifugation at 5000 x g for 10 min, placed into reference and sample cuvettes and baseline spectra were recorded between 400 and 500 nm at a speed of 120 nm/min. Cytochrome b5 in the sample cuvette was reduced with 1 mg sodium dithionite, and the spectra were recorded again. Cytochrome b5 content was estimated by the difference in absorbance at 423 and 490 nm using a millimolar extinction coefficient of 181 mmol cm–1 (47). Cytochrome b5 mRNA was estimated by RT-PCR.

In Vitro Phosphorylation of P450c17—To prepare a cytoplasmic fraction enriched for the kinases that phosphorylate P450c17 and devoid of phosphatase activity, NCI-H295A cells were lysed, and cytosol was passed over a {gamma}-ATP-Sepharose column (Upstate Biotechnologies); the column was washed with NAD, NADP, ADP, and AMP, and ATP-binding proteins were eluted with 10 mM ATP (29). Kinase activity was checked by phosphorylation of microsomes or purified bacterially expressed recombinant human P450c17 by incorporation of [{gamma}-32P]ATP. Purified P450c17 was phosphorylated in vitro using 6 µg of purified P450c17 incubated with 10 µg of kinase fraction in the presence of 50 mM HEPES buffer, 10 mM ATP, 60 mM MgCl2, and 10 µM okadaic acid at 25 °C for 30 min and phosphorylated P450c17 was purified on mini Ni-NTA columns (29).

P450c17 Enzyme Assays—The 17{alpha}-hydroxylase and 17,20 lyase activities of P450c17 were assayed as described (13, 39). Purified P450c17 (10 pmol) and POR (20 pmol) were incubated with 100 mM potassium phosphate, 6 mM potassium acetate, 10 mM MgCl2, 1 mM reduced glutathione, 20% glycerol, 20 µg phosphatidylcholine, 3 units of glucose-6-phosphate dehydrogenase, 0.1 mM glucose-6-phosphate and radiolabeled substrate ([14C]progesterone for hydroxylase assay and [3H]17OH-Preg for lyase assay) with or without 10 pmol of cytochrome b5 for 5 min at 25 °C in a total volume of 200 µl. The reaction was started by addition of 20 µl of 10 mM NADPH and incubations at 37 °C were carried out for various times and stopped by adding ethyl acetate/iso-octane (3:1) to extract the steroids. Steroids from different reactions were spotted on silica gel 60 F-254 thin layer chromatography plates (Merck) and developed with ethyl acetate/chloroform (3:1) (4). Plates were dried, and steroids were quantitated by autoradiography on a Storm 860 phosphorimager using Image Quant software. Kinetic behavior was approximated as a Michaelis-Menten system. Curve fitting and calculations of maximum velocity (Vmax) and apparent Michaelis constant (Km) values were performed using LEONORA (48).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isoforms of Cytochrome b5 in NCI-H295A Cells—Three forms of cytochrome b5 have been described: the 98-amino acid soluble and 134-amino acid microsomal form are encoded by one gene (49, 50) and a 146-amino acid form associated with the outer mitochondrial membrane (OMb5) is encoded by a second gene (51–53). To identify the forms of cytochrome b5 found in NCI-H295A cells, we performed RT-PCR with two pairs of oligonucleotide primers that will amplify the three products of the two genes. In human liver HepG2 cells, the mRNAs for microsomal cytochrome b5 and for OMb5 were found in approximately equal amounts, but in human kidney HEK293, human placenta JEG-3 cells, and especially in human adrenal NCI-H295A cells, the mRNA for microsomal cytochrome b5 was much more abundant than the mRNA for OMb5 (Fig. 2A). The mRNA for the soluble form, generally associated with erythropoietic tissues, was not detected in the cell lines tested. As human P450c17 is a microsomal enzyme and as the mRNA for the microsomal form of cytochrome b5 was most abundant in adrenal cells, we focused attention on microsomal cytochrome b5.



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  		FIG. 2.
Expression and suppression of cytochrome b5 in NCI-H295A cells. A, RT-PCR of cytochrome b5 mRNA from the human cell lines shown. Left, RT-PCR using oligonucleotides that amplify mitochondrial OMb5 but not microsomal b5. Right, RT-PCR using oligonucleotides that amplify both microsomal and soluble b5. The soluble form would yield a 300-bp band not seen in any sample. GAPDH mRNA is amplified as a control in each lane. B, agarose gel of PCR products stained with ethidium bromide showing reduction in cytochrome b5 mRNA but no change in GAPDH control at the times shown (in hours) after transfecting cells with the vector producing shRNA. C, reduction in cytochrome b5 protein content at various times after transfection as in B; maximal inhibition (80%) was achieved in 72 h. D, thin layer chromatograms from an experiment assaying lyase and hydroxylase activities in NCI-H295A cells transformed with shRNA against cytochrome b5. E, activities of P450c17 in NCI-H295A cells expressing shRNA against cytochrome b5. Open bars, 17{alpha}-hydroxylase activity (conversion of Prog to 17OH-Prog), solid bars, 17,20 lyase activity (conversion of 17OH-Preg to DHEA). For C and E, data are mean ± S.E. of three experiments, each performed in triplicate.

 
Knockdown of Cytochrome b5 in NCI-H295A Cells—To determine the effect of cytochrome b5 on the catalytic activities of P450c17 in steroidogenic cells, we used RNA interference to knock down cytochrome b5 mRNA in human adrenal NCI-H295A cells (Fig. 2B). An shRNA targeted against cytochrome b5 was transfected into human adrenal NCI-H295A cells, and cells were harvested 24, 48, 72, and 96 h later. Expression of this shRNA caused a gradual decrease in spectrally assayable cytochrome b5 over time: cytochrome b5 was reduced by 15% after 24 h, 65% after 48 h, and 85% after 72 h (Fig. 2C). Knockdown of cytochrome b5 had no effect on 17{alpha}-hydroxylase activity in NCI-H295A cells for up to 96 h (Fig. 2, D and E). There was no measurable change in the 17,20 lyase activity in the first 24 h, but 17,20 lyase activity was reduced 15% after 48 h and 30% after 72–96 h (Fig. 2, D and E). This decrease in 17,20 lyase activity was much less than the decrease in cytochrome b5 protein, suggesting that another factor was promoting 17,20 lyase activity.

Increased Phosphorylation of P450c17 Counters the Effect of Reduced Cytochrome b5 on 17,20 Lyase Activity—Because reducing cytochrome b5 in intact cells had only a modest effect on 17,20 lyase activity, we sought to examine the potential interactions between the presence of cytochrome b5 and the phosphorylation of P450c17 on 17,20 lyase activity. Okadaic acid, an inhibitor of serine/threonine phosphatases, specifically increases 17,20 lyase activity in NCI-H295A cells and increases P450c17 phosphorylation (29). Okadaic acid treatment of NCI-H295A cells expressing the shRNA against cytochrome b5 increased the 17,20 lyase activity in cells expressing decreased amounts of cytochrome b5 (Fig. 3). The increase in 17,20 lyase activity in control cells was similar to that in cells with a reduced content of cytochrome b5. Overexpression of cytochrome b5 in NCI-H295A cells moderately increases 17,20 lyase activity. Okadaic acid treatment of NCI-H295A cells overexpressing cytochrome b5 increased 17,20 lyase activity, but the increase was similar to control cells treated with okadaic acid. These studies suggest that cytochrome b5 and P450c17 phosphorylation augment 17,20 lyase activity independently.



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  		FIG. 3.
Protein phosphorylation increases 17,20 lyase activity irrespective of cytochrome b5 content. NCI-H295A cells expressing shRNA against cytochrome b5 (sib5) show a modest reduction in 17,20 lyase activity (solid bars) but no change in 17{alpha}-hydroxylase activity (open bars). Overexpression of cytochrome b5 (b5) in NCI-H295A cells increases17,20 lyase activity about 2-fold, but does not influence hydroxylase activity. Addition of 10 nM okadaic acid (OA) for 6 h to control NCI-H295A cells or to those expressing siRNA directed against cytochrome b5 or to those overexpressing cytochrome b5 increases 17,20 lyase activity 3-fold but does not increase 17{alpha}-hydroxylase activity. Data are mean ± S.E. of three experiments, each performed in triplicate.

 
Expression and Purification of Human P450c17, Cytochrome b5, and POR—To examine the role of the individual components in P450c17-POR-b5 reaction, we used purified preparations of recombinant human P450c17, cytochrome b5, and POR expressed in E. coli. All three proteins were purified to apparent homogeneity, evidenced by Coomassie Blue staining on an SDS-PAGE gel (Fig. 4). The bacterially expressed P450c17 was not phosphorylated as shown in two fashions. First, assay with malachite green indicated that no phosphate was released when the bacterially expressed human P450c17 was treated with either alkaline phosphatase or PP2A (data not shown); by contrast, native human P450c17 isolated from human adrenal cells or NCI-H295A cells releases readily detected phosphate under these conditions. Second, mass spectrometric analysis of tryptic peptides of bacterially expressed human P450c17 showed no phosphopeptides.



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  		FIG. 4.
Characterization of purified proteins. SDS-polyacrylamide gel electrophoresis showing purified P450c17, POR, and cytochrome b5. These purified enzymes were used to reconstitute P450c17-POR-cytochrome b5 system in vitro.

 
Effect of Cytochrome b5 and Phosphorylation on Bacterially Expressed Human P450c17—Using the purified, bacterially expressed human P450c17 and POR, we examined the effects of cytochrome b5 and serine phosphorylation as independent variables on 17{alpha}-hydroxylase and 17,20 lyase activities in vitro. The P450c17-POR-b5 system was reconstituted using phosphatidylcholine (to provide a membrane environment) and an NADPH regeneration system consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase. When recombinant human P450c17 was incubated with POR at a molar ratio of 1:2 and cytochrome b5 was added in molar ratios of cytochrome b5 to P450c17 ranging from 0.1 to 100, an effect of cytochrome b5 on 17,20 lyase activity was first seen at a ratio of 0.5. A 1:1 ratio doubled the 17,20 lyase activity and a 3:1 ratio tripled it, but the maximal effect was seen until a molar ratio of about 30:1 (Fig. 5A). This high ratio for a maximal effect is consistent with previous results (13). Although the molar ratios of cytochrome b5 to P450c17 in human androgen-producing cells are not known, it is clear that a substantial effect is achieved with low ratios.



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  		FIG. 5.
Dose response of cytochrome b5 on 17,20 lyase activity. A, effect of increasing the ratio of cytochrome b5 to P450c17 using purified, bacterially expressed human b5 and P450c17 combined in vitro. Upper panel, maximal 17,20 lyase activity is achieved at a b5:P450 ratio of 30. Data are mean ± S.D. of three experiments. Lower panel, representative thin layer chromatogram of one of the experiments. B, effect of cytochrome b5 on purified, bacterially expressed human P450c17. In the presence of purified, bacterially expressed human POR (P450c17: POR:: 1:2), P450c17 has a low level of 17,20 lyase activity. The 17,20 lyase activity (solid bars) and 17{alpha}-hydroxylase activity (open bars) under these control conditions are set at 100%. Addition of purified cytochrome b5 (b5) in a 1:1:2 ratio of b5:P450c17: POR increased 17,20 lyase activity 3-fold; phosphorylation of human P450c17 with the kinase fraction from NCI-H295H cell cytoplasm increased 17,20 lyase activity 4-fold (Kinase); and addition of cytochrome b5 in a 1:1:2 ratio of b5: P450c17: POR using the phosphorylated P450c17 increased 17,20 lyase activity 6-fold. The 17{alpha}-hydroxylase activity of P450c17 remained unaffected in all cases. Data are mean ± S.D. of three experiments.

 
To examine the potential interplay of cytochrome b5 and phosphorylation of P450c17 on 17,20 lyase activity, bacterially expressed human P450c17 was phosphorylated in vitro using a kinase-enriched fraction from NCI-H295A cytosol that was devoid of 17{alpha}-hydroxylase or 17,20 lyase activity (29). P450c17 activities were assayed in reconstituted systems containing 10 pmol of P450c17, 20 pmol of POR, and 20 µg of phosphatidylcholine. In the absence of cytochrome b5 or serine phosphorylation, bacterially expressed human P450c17 had 17{alpha}-hydroxylase activity but very little 17,20 lyase activity. When cytochrome b5 was added to bacterially expressed human P450c17 in vitro at a cytochrome b5 to P450c17 ratio of 3:1, 17,20 lyase activity increased 3–4-fold. When bacterially expressed human P450c17 was phosphorylated with the kinase fraction from NCI-H295A cytosol, 17,20 lyase activity increased 4–5-fold in the absence of cytochrome b5 (Fig. 5B). Addition of cytochrome b5 to phosphorylated P450c17 at a cytochrome b5 to P450c17 ratio of 3:1, increased activity further, but the effects of cytochrome b5 and phosphorylation were neither additive nor cooperative (Fig. 5B). To explore these findings further, we performed kinetic analysis of P450c17 activities from non-phosphorylated and phosphorylated P450c17 with and without cytochrome b5 (Table I). Neither phosphorylation nor addition of cytochrome b5 affected the kinetic parameters of the 17{alpha}-hydroxylase reaction (p > 0.5). Addition of cytochrome b5 increased the catalytic efficiency (Vmax/Km) of the 17,20 lyase reaction 4-fold (p = 0.013), and P450c17 phosphorylation increased catalytic efficiency 6-fold (p = 0.003 compared with control). However, addition of cytochrome b5 to phosphorylated P450c17 increased the 17,20 lyase activity only 7-fold. This effect was not significantly different from the action of phosphorylation alone (p = 0.2). Thus the effects of cytochrome b5 and P450c17 phosphorylation are not additive. Most of the effect seems to come from increased reaction velocities, as changes in Km were less than 2-fold.


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  		TABLE I
Kinetics of P450c17 activities with cytochrome b5 and kinase treatments

All date are ±S.D.

 
Effect of POR and Cytochrome b5 on 17,20 Lyase Activity— POR is the obligate electron donor for both activities of P450c17, and high molar ratios of POR to P450c17 increase 17,20 lyase activity (14, 23). To examine the role of POR in modulating 17,20 lyase activity, we fixed the molar ratio of cytochrome b5:P450c17 at 3:1 and varied the amount of POR. As expected, excess POR significantly increased 17,20 lyase activity (Fig. 6A). A dose response was seen for POR either in the absence or presence of cytochrome b5, but 17,20 lyase activity was always greater when cytochrome b5 was present. Addition of POR increased the hydroxylase activity slightly but addition of cytochrome b5 had no effect on the hydroxylase activity. These data support our previous findings that cytochrome b5 augments 17,20 lyase activity by facilitating electron transfer from POR (13, 29).



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  		FIG. 6.
Effects of cytochrome b5 on the 17,20 lyase activity promoted by POR and by P450c17 phosphorylation. A, increased POR favors 17,20 lyase activity. Bacterially expressed P450c17, in the absence (open bars) and presence (solid bars) of a 3-fold molar excess of cytochrome b5 was assayed for 17,20 lyase activity in the presence of 2-, 10-, 20-, or 50-fold molar excess of POR over P450c17. Data are mean ± S.D. of three experiments. B, effect of cytochrome b5 on human P450c17 in microsomes from NCI-H295A cells. The microsomes, which contain POR and P450c17 (with an unknown level of phosphorylation), have a low level of 17,20 lyase activity (solid bars) and substantially higher levels of 17{alpha}-hydroxylase activity (open bars); the level of these activities in the absence of cytochrome b5 is set at 100%. Exogenous addition of purified cytochrome b5 increased 17,20 lyase activity 2.5-fold but had no effect on 17{alpha}-hydroxylase activity. Phosphorylation of microsomal proteins with the kinase fraction from NCI-H295A cell cytoplasm increased 17,20 lyase activity 3.5-fold, and addition of purified cytochrome b5 to the phosphorylated microsomes increased 17,20 lyase activity 5-fold without affecting 17{alpha}-hydroxylase activity. Data are mean ± S.D. of three experiments.

 
Effect of Cytochrome b5 and Phosphorylation on NCI-H295A Microsomes—To determine whether the effects seen on recombinant human P450c17 in vitro also occurred in a native protein environment, we isolated microsomes from NCI-H295A cells and examined the effect of cytochrome b5 and of in vitro protein phosphorylation on 17{alpha}-hydroxylase and 17,20 lyase activities. Using the kinase preparation from NCI-H295A cytosol (29) we phosphorylated microsomes from NCI-H295A cells in vitro and assayed them for hydroxylase and lyase activities in the presence and absence of purified cytochrome b5 (Fig. 6B). NCI-H295A cells normally have a low level of 17,20 lyase activity. Adding cytochrome b5 increased 17,20 lyase activity 2–3-fold but had no effect on 17{alpha}-hydroxylase activity. In vitro phosphorylation of microsomal P450c17 using the kinase fraction from NCI-H295A cells increased 17,20 lyase activity 3.5-fold but had no effect on 17{alpha}-hydroxylase activity. Adding cytochrome b5 to microsomes that had been phosphorylated in vitro increased 17,20 lyase activity 5-fold, but had no effect on 17{alpha}-hydroxylase activity. Thus both protein phosphorylation and cytochrome b5 can enhance 17,20 lyase activity independently of each other, but their effects are neither additive nor cooperative.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Steroidogenesis in the primate adrenal is divided into three morphologically and functionally distinct zones (54). The zona glomerulosa, located just below the adrenal capsule, does not express P450c17 and produces the 17-deoxy 21-carbon steroid aldosterone, the principal mineralocorticoid, under the regulation of angiotensin II. The zona fasciculata, which lies just below the glomerulosa, express P450c17 and has abundant 17{alpha}-hydroxylase but very little 17,20 lyase activity, and produces the 17-hydroxy 21-carbon steroid cortisol, the principal glucocorticoid, under the regulation of corticotropin. The inner zona reticularis, which does not become morphologically identifiable until the onset of adrenarche, expresses P450c17 and has both 17{alpha}-hydroxylase and 17,20 lyase activities and produces 17-hydroxy 19-carbon precursors of sex steroids under ill-defined regulation. The event(s) triggering adrenarche remain unknown (21). Understanding the reticularis-specific activation of the 17,20 lyase activity of P450c17 has been a major challenge as there are no non-primate systems that recapitulate this biology (22). Several mechanisms contribute to the developmental and tissue-specific differential regulation of P450c17 activities (54). Serine phosphorylation of P450c17 increases 17,20 lyase activity without significantly affecting 17{alpha}-hydroxylase activity (28) and treatment with alkaline phosphatase or PP2A eliminates almost all 17,20 lyase activity without changing 17{alpha}-hydroxylase activity (28, 29). Thus the activities of P450c17 can be differentially regulated by protein phosphorylation based on differential expression of protein kinases and/or phosphatases in different cell types or times in development, thus determining the pattern of steroid hormones produced.

Cytochrome b5 also enhances the 17,20 lyase activity of human P450c17 (24, 55–57) by allosteric action that does not involve electron donation (13). Whereas P450c17 is expressed in both the human zona fasciculata and zona reticularis and shows little change as a function of age, the expression of cytochrome b5 increases in the adrenal zona reticularis at the onset of adrenarche (58, 59) when adrenal 17,20 lyase activity and secretion of 19-carbon steroids (DHEA, androstenedione) increases. The adult human (60) and rhesus monkey (61) zona reticularis contains abundant cytochrome b5 as well as P450c17, and adrenal adenomas that produce high levels of DHEA also contain large amounts of cytochrome b5 (62, 63). Similarly, cytochrome b5 is found in gonadal cells that produce sex steroids, including testicular Leydig cells, follicular theca cells, theca lutein cells, and ovarian stroma (60). Thus there is a strong association between the presence of 17,20 lyase activity and expression of cytochrome b5.

There are three forms of cytochrome b5 expressed from two genes. A gene on chromosome 18q23 consists of 6 exons encoding two alternatively spliced mRNAs: exons 1–4 encode the 98-amino acid soluble form while exons 1–3, 5, and 6 encode the 134-amino acid form bound to the endoplasmic reticulum (49, 50). A gene on chromosome 16q22.1 consists of 5 exons encoding a distinct 146-amino acid form of cytochrome b5 bound to the outer mitochondrial membrane (OMb5). The heme-binding N termini of the products of these two genes are about 70% identical. The C-terminal 10 residues of rat OMb5 determine targeting to the endoplasmic reticulum or mitochondria (52). Rat OMb5 can support 17,20 lyase activity (64, 65), but unlike the soluble or endoplasmic reticulum form of cytochrome b5, OMb5 preferentially supports 17{alpha}-hydroxylase activity in studies with rat testicular Leydig cells (65). As the principal form of cytochrome b5 found in the adrenal is the 136-amino acid membrane-bound form, and as OMb5 fosters 17{alpha}-hydroxylase as well as 17,20 lyase activities, it is likely that the major effects of cytochrome b5 on human adrenal synthesis of 19-carbon precursors of sex steroids is mediated by the microsomal form.

Overexpression of cytochrome b5 in non-steroidogenic HEK-293 cells co-transfected with P450c17 and POR dramatically enhances 17,20 lyase activity (64). However, reducing cytochrome b5 expression in NCI-H295A cells by 60% using RNA interference did not affect 17,20 lyase activity, and an 85% reduction in cytochrome b5 only decreased 17,20 lyase activity by 30%. Similarly, overexpression of cytochrome b5 in NCI-H295A cells increased 17,20 lyase activity only modestly. Consistent with this we have found that NCI-H295A cells contain about 5-fold more cytochrome b5 protein than do HEK-293 cells or other non-steroidogenic cell lines. Our data indicate that cytochrome b5 and protein phosphorylation enhance 17,20 lyase activity independently and that each mechanism is sufficient to achieve nearly maximal induction on its own. Their effects do not seem to be cooperative since we did not observe enhanced DHEA production in samples that were both phosphorylated and contained cytochrome b5 when compared with the effect of each factor individually. These results also suggest that endogenous cytochrome b5 in NCI-H295A cells is sufficient to maintain the 17,20 lyase activity and perhaps outer mitochondrial cytochrome b5 supports some of the 17,20 lyase activity that could not be reduced even after knockdown of more than 85% cytochrome b5.

Irrespective of P450c17 phosphorylation or the presence of cytochrome b5, increasing the ratio of POR to P450c17 increases 17,20 lyase activity (14, 23). As both the 17{alpha}-hydroxylase and 17,20 lyase activities require the donation of electrons from P450 oxidoreductase, this observation thus suggests that both the presence of cytochrome b5 and phosphorylation of P450c17 increase 17,20 lyase activity by facilitating the association of POR with P450c17, speeding the lyase reaction by increasing the efficiency of electron transfer. Thus the combination of these factors regulates the activities of P450c17 (Fig. 7). It is likely that the direction of steroid biosynthesis in a particular tissue or at a particular age is regulated by one or more of the factors involved in P450c17 activities. A change in 17,20 lyase activity may come from changes in cytochrome b5 levels, extent and pattern of P450c17 phosphorylation or presence of other enzymes like 3{beta}-hydroxysteroid dehydrogenase. In a disease like polycystic ovary syndrome one or more of these factors might contribute to the overall change in DHEA production.



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  		FIG. 7.
A schematic diagram showing activities of cytochrome P450c17 supported by POR, cytochrome b5, and/or phosphorylation by a kinase. 17{alpha}-Hydroxylation of pregnenolone requires only P450c17 and POR. The conversion of 17OH-Pregnenolone to DHEA requires P450c17, POR, and either the allosteric action of cytochrome b5 (right) or the phosphorylation of P450c17 (left), which is catalyzed by an unidentified kinase and opposed by PP2A.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HD41958 (to W. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pediatrics, Bldg. MR4 Rm. 209, University of California San Francisco, San Francisco, CA 94143-0978. Tel.: 415-476-2598; Fax: 415-476-6286; E-mail: wlmlab{at}itsa.ucsf.edu.

1 The abbreviations used are: 17OH-Preg, 17{alpha}-hydroxypregnenolone; 17OH-Prog, 17{alpha}-hydroxyprogesterone; DHEA, dehydroepiandrosterone; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilo triacetic acid; POR, P450 oxidoreductase; PP2A, protein phosphatase 2A; shRNA, small hairpin RNA; siRNA, small interfering RNA; IPTG, isopropyl-1-thio-{beta}-D-galactopyranoside; HEK, human embryonic kidney; RT-PCR, reverse transcriptase-PCR; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Richard J. Auchus for providing [3H]17OH-Preg, Dr Ningwu Huang for RNA samples and PCR primers, and Dr. Dustin C. Yaworsky for the mass spectrometric analysis of the bacterially expressed human P450c17.



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L. G. Gomes, N. Huang, V. Agrawal, B. B. Mendonca, T. A. S. S. Bachega, and W. L. Miller
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