B5 Expression Research Articles

Structural and biochemical characteristics of the cyclotide kalata B5 from Oldenlandia affinis

Cyclotides are a large family of plant-derived proteins typified by their head-to-tail cyclic backbone and knotted arrangement of three disulfide bonds. Although they display a diverse range of biological activities, their native function is thought to be plant defense. Here we characterized the expression, three-dimensional structure, and hemolytic activity of the cyclotide kalata B5 from the African plant Oldenlandia affinis. Kalata B5 shows an interesting seasonal variation in its expression and can only be isolated during certain times of the year, when the plant is flowering. It displays a typical tightly folded cyclic Scystine knot structure. A range of pH and temperature titrations reveal that a conserved glutamic acid in loop 1 Sof the structure forms a key hydrogen bond network, similar to that reported previously for other cyclotides. However, specific line broadening in the NMR spectra of kalata B5 suggests that the hydrogen bonding network in this peptide is less rigid than in other cyclotides. Notably, the pK9a) of Glu6 of 4.5 is higher than the values for other cyclotides studied so far, which range from 3.0 to 4.0, providing a further indication of a weaker hydrogen bond network. Kalata B5 has only moderate hemolytic activity compared with other highly expressed cyclotides, and this reduced activity probably reflects its more flexible structure. As is the case with other cyclotides, kalata B5 has an exposed hydrophobic region on its surface, supporting suggestions that this hydrophobic patch is a key feature for membrane binding and biological activity of cyclotides.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype–phenotype correlations for hydroxylamine reduction

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

In-Vitro and in-Vivo Evaluation of the Orally Active Proteasome Inhibitor MLN9708 in Hematological Models of Human Cancer.

Abstract 2709Poster Board II-685 Introduction:The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. The successful development of bortezomib for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. MLN9708 was identified in screens for a proteasome inhibitor with greater antitumor activity than bortezomib in preclinical xenograft models. MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Results:MLN2238 inhibited the 20S proteasome b5 site (IC50 of 3.4 nM), an engineered proteasome substrate (4x ubiquitin-luciferase reporter) and blocked TNFa-induced activation of the NFkB pathway. Cell viability studies confirmed that MLN2238 has potent activity against both myeloma and lymphoma cell lines. The proteasome dissociation half life of MLN2238 was determined to be approximately 6-fold faster than bortezomib, consistent with data generated from Proteasome-Glo wash out experiments where proteasome activity recovered more quickly in MLN2238-treated cells compared to bortezomib. In immunocompromised mice MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition and had increased plasma and tumor exposure compared to bortezomib (when dosed at MTD). In Sprague-Dawley rats, MLN2238 had improved plasma exposure, lower plasma clearance, higher blood Vdss, better oral F% and higher plasma protein binding than bortezomib. In WSU-DLCL2 xenografts, a model of diffuse large B cell lymphoma, greater antitumor activity was seen in mice treated with IV or SC doses of MLN2238 compared to bortezomib. For example, MLN2238 dosed SC QD at its MTD resulted in a T/C of 0.29 compared to bortezomib dosed SC QD at its MTD which resulted in a T/C of 0.79. Pharmacodynamic (PD) responses (in mice) were assessed by evaluating tumor 20S b5 site-specific activity and expression levels of GADD34, an unfolded protein response (UPR) pathway gene shown to be upregulated in response to proteasome inhibition. Consistent with the efficacy difference between MLN2238 and bortezomib in WSU-DLCL2 xenografts, MLN2238 demonstrated an improved PD response compared to bortezomib, showing higher levels of tumor proteasome inhibition and pathway marker elevation. MLN2238 and bortezomib were then evaluated for their ability to reduce tumor burden and improve overall survival in a disseminated model of lymphoma. Tumor burden was tracked over time via bioluminescent scans in NOD-SCID mice inoculated with OCI-Ly7-luciferase cells. The strongest antitumor response was seen in mice treated with MLN2238 SC QD at its MTD, and this dosing regimen also significantly prolonged overall survival compared to vehicle treated controls (median survival was 54 vs. 33 days). Weaker antitumor responses were seen following treatment with bortezomib (either at its SC QD MTD or weekly IV MTD) and these dose regimens did not significantly prolong survival in this study. Conclusions:MLN2238 is a potent, reversible and orally bioavailable proteasome inhibitor with improved pharmacokinetics, pharmacodynamics and antitumor activity in preclinical xenograft models compared to bortezomib. MLN9708 is currently in clinical development for a variety of oncology indications. Disclosures:Kupperman:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership. Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Fitzgerald:Millennium Pharmaceuticals: Employment. Berger:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Zhang:Millennium Pharmaceuticals: Employment. Hales:Millennium Pharmaceuticals: Employment. Bruzzese:Millennium Pharmaceuticals: Employment. Liu:Millennium Pharmaceuticals: Employment. Blank:Millennium Pharmaceuticals: Employment. Garcia:Millennium Pharmaceuticals: Employment. Tsu:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Fleming:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Rolfe:Millennium Pharmaceuticals: Employment. Bolen:Millennium Pharmaceuticals: Employment.

Plasticity of the zona reticularis in the adult marmoset adrenal cortex: voyages of discovery in the New World

Adrenarche in humans occurs at the age of 5-7 years, yet the process by which dehydroepiandrosterone (DHEA) biosynthesis in the adrenal zona reticularis (ZR) increases so dramatically remains as a matter of debate. One suggestion is that increased DHEA production by P450c17 (CYP17A1 as listed in HUGO Database) in the ZR results from a coincident fall in the expression of HSD3B, which would otherwise compete for pregnenolone substrate. Nonetheless, studies of human and rhesus adrenal show that cytochrome b5 (CYTB5) expression increases in the ZR with DHEA biosynthesis, and cloned human and rhesus P450c17 show selective increases in 17,20-lyase activity in the presence of CYTB5. The marmoset, a New World primate, expresses a fetal zone during development which regresses after birth. Adult males, however, do not develop an obvious functional ZR, while females develop a ZR in a manner that depends on their social/gonadal status. In all social and physiologic states, changes in marmoset ZR function relate directly to changes in the expression of CYTB5. Recent cloning and expression of marmoset P450c17 also show that while amino acid sequence homology is in the order of approximately 85% of that found in human and rhesus sequences, and basal lyase activity is low compared with rhesus, all previously described amino acids critical to human 17,20-lyase activity are completely conserved. Furthermore, the 17,20-lyase activity of the marmoset P450c17 clone is dramatically increased by addition of CYTB5. We propose that these combined data from the marmoset model provide further compelling evidence that the control of ZR CYTB5 expression is a key determinant of ZR function.

Preparation of a biologically active apo-cytochrome b5via heterologous expression in Escherichia coli

Cytochrome b5 (b5) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b5 on CYP-catalyzed reactions, but also that of the apo-cytochrome b5 (apo-b5). Therefore, the apo-b5 protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b5 was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b5 was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b5 was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b5 protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b5 protein was obtained. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native b5. The prepared recombinant apo-b5 reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b5 is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b5.

Genetic variability of cytochrome b5 and NADH cytochrome b5 reductase: SNP discovery and characterization

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX‐HA) to the parent drug sulfamethoxazole. SMX‐HA and a spontaneous nitroso metabolite are thought to contribute to sulfonamide hypersensitivity. Variability in hydroxylamine reduction could therefore contribute to drug toxicity. The aim of this study was to characterize variability in SMX‐HA reduction in 111 human livers, and investigate the functional significance of non‐synonymous polymorphisms (SNPs) in b5R and b5 cDNA. Hepatic SMX‐HA reduction displayed a 19‐fold variability (0.09‐1.69 nmol/min/mg protein), with the Vmax for the reaction varying 5‐fold between low‐activity and high‐activity outliers. The expression of immunoreactive b5 and b5R were significantly correlated to each other (p&lt;0.0001). SMX‐HA reduction activity increased with increasing b5 and b5R protein content (p=0.003, p=0.0239). A novel S5A SNP in b5 was associated with very low activity and protein expression. Two novel b5R SNPs (R59H, R297H) displayed biphasic SMX‐HA reduction kinetics, with the mutant enzyme contributing to the lower affinity component, and, in the case of the R59H SNP, contributing to a reduction in activity. The b5 S5A and b5R R297H SNPs are associated with reduced SMX‐HA reduction in human liver and work is ongoing to clone, express, and characterize these mutant b5 and b5R proteins.Supported by National Institutes of Health grant GM61753

The Developmental Increase in Adrenocortical 17,20-Lyase Activity (Biochemical Adrenarche) Is Driven Primarily by Increasing Cytochrome b5 in Neonatal Rhesus Macaques

Adrenarche is thought to be experienced only by humans and some Old World primates despite observed regression of an adrenal fetal zone and establishment of a functional zona reticularis (ZR) in other species like rhesus macaques. Adrenal differentiation remains poorly defined biochemically in nonhuman primates. The present studies defined ZR development in the neonatal rhesus by examining androgen synthetic capacity and factors affecting it in rhesus and marmoset adrenals. Western immunoblots examined expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5 (b5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD), among other key enzymes. 17,20-lyase activity was quantified in adrenal microsomes, as was the contribution of b5 to 17,20-lyase activity in microsomes and cell transfection experiments with rhesus and marmoset P450c17. Expression of b5 increased from birth to 3 months, and was positively correlated with age and 17,20-lyase activity in the rhesus. Recombinant b5 addition stimulated 17,20-lyase activity to an extent inversely proportional to endogenous levels in adrenal microsomes. Although 3betaHSD expression also increased with age, P450c17, 21-hydroxylase cytochrome P450, and the redox partner, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase, did not; nor did recombinant cytochrome P450 oxidoreductase augment 17,20-lyase activity. Cotransfection with b5 induced a dose-dependent increase in dehydroepiandrosterone synthesis by both nonhuman primate P450c17 enzymes. We conclude that the increase in 17,20-lyase activity characteristic of an adrenarche in rhesus macaques is driven primarily by increased b5 expression, without the need for a decrease in 3betaHSD, as suggested from human studies. The rhesus macaque is a relevant and accessible model for human ZR development and adrenal function.

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

Morphological adrenarche in rhesus macaques: development of the zona reticularis is concurrent with fetal zone regression in the early neonatal period

Human adrenarche is associated with the establishment of a functional zona reticularis (ZR) and increasing secretion of dehydroepiandrosterone (DHEA) in sulfated form (DS). Like most non-human primates, rhesus macaques are not believed to undergo adrenarche, though they clearly establish a functional ZR after birth. However, the origins of the rhesus ZR are not well defined. Therefore, we investigated the zonal development, steroidogenic enzyme expression and morphology of rhesus adrenals from 1 day to 14 months of age. Immunohistochemistry was conducted to determine expression profiles of the steroidogenic enzymes 17alpha-hydroxylase/17,20-lyase cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), cytochrome P450, family 21, subfamily A, polypeptide 2 (CYP21A2), hydroxy-Delta-5-steroid dehydrogenase, 3beta- and steroid Delta-isomerase 2 (HSD3B2), the redox partner NADPH-cytochrome P450 oxidoreductase (CPR), as well as the accessory protein cytochrome b5 (b5), a marker of the primate ZR. The rhesus ZR is mature by 3 months of age based on differentiation of the innermost zone that lacks HSD3B2, but exhibits increased b5 expression during this period. Further, the ZR develops in neonates from a previously described dense band of cells which we show expresses b5, CYP17A1, CPR, and CYP21A2 throughout maturation. The fetal zone (FZ) is distinguished from the ZR by its lack of CYP21A2, and ZR development proceeded as the FZ regressed with two important implications: neither FZ regression nor ZR maturation can be monitored independently by circulating adrenal androgens, and these events must be induced by different factors in rhesus, and likely humans. Collectively these data demonstrate that ZR development begins before birth in the rhesus, proceeding concomitantly with FZ regression post-natally, suggesting that rhesus experiences morphological adrenarche during the first three months of life.

Doxycycline treatment prevents alveolar destruction in VEGF‐deficient mouse lung

In vivo lung-targeted VEGF gene inactivation results in pulmonary cell apoptosis, airspace enlargement, and increased lung compliance consistent with an emphysema-like phenotype. The predominant hypothesis for the cause of lung destruction in emphysema is an imbalance between active lung protease and anti-protease molecules. Therefore, we investigated the role of protease (e.g., matrix metalloproteinases--MMPs) and anti-protease (e.g., tissue inhibitors of metalloproteinases--TIMPs) expression in contributing to the lung structural remodeling observed in pulmonary-VEGF-deficient mice. VEGFLoxP mice instilled through the trachea with an adeno-associated virus expressing Cre recombinase (AAV/Cre) manifest airspace enlargement and a greater (P < 0.05) mean linear intercept (MLI: 44.2 +/- 4.2 microm) compared to mice instilled with a control virus expressing LacZ (31.3 +/- 2.5 microm). Airspace enlargement was prevented by the continuous administration of the general MMP inhibitor, doxycycline (Dox) (Cre + Dox: 32.6 +/- 2.5 microm), and MLI values were not different from either control (LacZ + Dox: 30.5 +/- 1.2 microm). In situ magnetic resonance imaging of VEGF gene inactivated mouse lungs revealed uneven inflation, residual trapped gas volumes upon oxygen absorption deflation/re-inflation, and loss of parenchymal structure; effects that were largely prevented by Dox. Five weeks after AAV/Cre infection Western blot revealed a 9.9-fold increase in pulmonary MMP-3, and 2-fold increases in MMP-9 and TIMP-2. However, the increase in MMP-3 was prevented by Dox administration and was associated with a 2-fold increase in serpin b5 (Maspin) expression. These results suggest that doxycycline treatment largely prevents the aberrant lung remodeling response observed in VEGF-deficient mouse lungs and is associated with changes in protease and anti-protease expression.

Discovery and characterization of a cytochrome b 5 variant in humans with impaired hydroxylamine reduction capacity

We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals. Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals. One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation. These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.

Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

A New Class of Receptor for Herpes Simplex Virus Has Heptad Repeat Motifs That Are Common to Membrane Fusion Proteins

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an alpha-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat alpha-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.

Effects of Aging on Cytochrome B5 Expression in the Human Adrenal Gland

Aging in humans is characterized by a selective decline in circulating levels of adrenal androgens. The results of in vivo studies are suggestive of reduced adrenal 17,20-lyase activity in aging men and women. We sought to determine whether there are changes in the distribution and/or expression of cytochrome B5 (CytB5), an accessory protein important in the regulation of 17,20-lyase activity, in the adrenals of aging humans. Comparison between younger and older adrenal glands. The study was conducted in a University Center. Adrenal glands obtained at autopsy after sudden death as a result of trauma from 46 young (age 20-40 yr) and 26 older (age 50-91 yr) humans were obtained and fixed within 24 h postmortem. Paraffin sections were stained with hematoxylin and eosin and also were subjected to immunohistochemical staining for CytB5. All sections were quantitatively evaluated using an image capture and analysis program and qualitatively evaluated with respect to staining intensity. To determine whether there are any changes in CytB5 distribution in the adult human adrenal cortex during the aging process using qualitative and quantitative analysis with respect to age, gender, race, and postmortem interval. CytB5 immunoreactivity was found in cells that corresponded to those of the zona reticularis. The percentage of the adrenal cortex immunoreactive for CytB5 decreased with aging (38.6 +/- 7.6% for young and 30.1 +/- 5.9% for older, mean +/- sd; P < 0.0001) as did the percentage of adrenocortical tissue comprising the zona reticularis (36.8 +/- 10.8% for young and 27.2 +/- 5.9% for older; P < 0.001). However, there was no apparent change in the staining intensity of CytB5 among those cells that were immunopositive for this factor with aging. There appears to be a reduction in the proportion of the adrenal cortex that expresses CytB5 with aging, and this likely corresponds to a shrinkage of the zona reticularis. The mechanism and cause for this cortical regression are unknown.

Regulation of Cytochrome b5 Gene Transcription by Sp3, GATA-6, and Steroidogenic Factor 1 in Human Adrenal NCI-H295A Cells

Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.

Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-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 17alpha-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.

Using Human Microarrays to Identify Differentially Expressed Genes Associated with Increased Steroidogenesis in Boars

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.

Biochemical assessment of limits to estrogen synthesis in porcine follicles.

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

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.

Adrenocortical Cytochrome b5 Expression during Fetal Development of the Rhesus Macaque

The developmental expressions of cytochrome b5 (b5), 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), and 3β-hydroxysteroid dehydrogenase were examined in primate fetal adrenals by immunocytochemistry from 50–160 d gestation. The expression of b5 was evident at 50 d in the developing fetal zone (FZ), but decreased markedly through midgestation, then increased again from 150 d to term. Similar changes in the temporal expression was observed for P450c17. Whereas P450c17 was induced largely in the transitional zone (TZ; outer-most FZ), b5 expression was strongest in FZ cells further from the capsule, although overlap between these regions involved a narrow band of cells beneath the TZ that may represent the developing zona reticularis. Thus, the induction of b5 in the FZ and of P450c17 in the TZ of the fetal adrenal late in gestation coincided temporally with the prepartum rise in dehydroepiandrosterone previously reported. These data are consistent with the proposed role of b5 in supporting 17,20-lyase activity of P450c17. However, the lack of cytochrome b5 and P450c17 expression in the FZ of the developing macaque adrenal cortex for much of the second and third trimesters distinguishes it from the mature zona reticularis seen in adult animals.