TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis

Although tumor necrosis factor (alpha) (TNF) exerts proinflammatory activities in a variety of diseases, including inflammatory bowel disease, there is increasing evidence for antiinflammatory actions of TNF. In contrast, glucocorticoids (GCs) are steroid hormones that suppress inflammation, at least in part by regulating the expression and action of TNF. We report that TNF induces extraadrenal production of immunoregulatory GCs in the intestinal mucosa during acute intestinal inflammation. The absence of TNF results in a lack of colonic GC synthesis and exacerbation of dextran sodium sulfate-induced colitis. TNF seems to promote local steroidogenesis by directly inducing steroidogenic enzymes in intestinal epithelial cells. Therapeutic administration of TNF induces GC synthesis in oxazolone-induced colitis and ameliorates intestinal inflammation, whereas inhibition of intestinal GC synthesis abrogates the therapeutic effect of TNF. These data show that TNF suppresses the pathogenesis of acute intestinal inflammation by promoting local steroidogenesis.

Colon cancer cells produce immunoregulatory glucocorticoids

Glucocorticoids (GC) have important anti-inflammatory and pro-apoptotic activities. Initially thought to be exclusively produced by the adrenal glands, there is now increasing evidence for extra-adrenal sources of GCs. We have previously shown that the intestinal epithelium produces immunoregulatory GCs and that intestinal steroidogenesis is regulated by the nuclear receptor liver receptor homolog-1 (LRH-1). As LRH-1 has been implicated in the development of colon cancer, we here investigated whether LRH-1 regulates GC synthesis in colorectal tumors and whether tumor-produced GCs suppress T-cell activation. Colorectal cancer cell lines and primary tumors were found to express steroidogenic enzymes and regulatory factors required for the de novo synthesis of cortisol. Both cell lines and primary tumors constitutively produced readily detectable levels of cortisol, as measured by radioimmunoassay, thin-layer chromatography and bioassay. Whereas overexpression of LRH-1 significantly increased the expression of steroidogenic enzymes and the synthesis of cortisol, downregulation or inhibition of LRH-1 effectively suppressed these processes, indicating an important role of LRH-1 in colorectal tumor GC synthesis. An immunoregulatory role of tumor-derived GCs could be further confirmed by demonstrating a suppression of T-cell activation. This study describes for the first time cortisol synthesis in a non-endocrine tumor in humans, and suggests that the synthesis of bioactive GCs in colon cancer cells may account as a novel mechanism of tumor immune escape.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

Impact of differential P450c17 phosphorylation by cAMP stimulation and by starvation conditions on enzyme activities and androgen production in NCI-H295R cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

Clinical and biochemical consequences of p450 oxidoreductase deficiency

Patients with P450 oxidoreductase (POR) deficiency typically present with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley-Bixler craniosynostosis syndrome. Since our first report in 2004, more than 40 POR mutations have been identified in over 65 patients. POR is the obligate electron donor to all microsomal P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46, XY newborns as well as overvirilization in those who are 46, XX. This may be explained by impaired aromatization of fetal androgens that may cause maternal virilization and low urinary estriol levels during pregnancy. In addition, the alternate 'backdoor' pathway of androgen biosynthesis, which leads to dihydrotestosterone production bypassing androstenedione and testosterone, may also play a role. Functional assays studying the effects of POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. POR variants may also affect skeletal development and drug metabolism. As most drugs are metabolized by hepatic microsomal P450 enzymes, studies of the impact of POR mutations on drug-metabolizing P450s are particularly important.

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.

Exogenous gonadotropins have little impact on follicular but considerable effect on serum cytokine concentrations – a comparison between Natural Cycle and stimulated IVF using a multiplexed assay platform

Introduction: Throughout follicular growth and subsequent corpus luteum formation the leukocyte number increases and follicular vascularisation changes. These processes are enhanced under exogenous stimulation with gonadotropins. Cytokines released by leukocytes contribute to further recruitment and vascularisation of the follicle, and they play an important role in regulating ovarian steroidogenesis by influencing theca and granulosa–lutein cell function. Changes in cytokine and vascular endothelial growth factor (VEGF) concentrations in the ovary as a consequence of gonadotropin stimulation may negatively influence oocyte quality. In this project we have compared the intrafollicular production of inflammatory cytokines and growth factors between natural IVF cycles (NC) and classical, gonadotropin-stimulated IVF cycles (gsIVF). Material and Methods: Serum on the day of oocyte retrieval and follicular fluid (FF) were collected in 37 NC and 39 gsIVF cycles. Thirteen women within this population underwent one NC and one gsIVF cycle each. A total of 14 cytokines from Bio-Plex panels I and II were determined in matched serum and FF samples using Luminex xMAP technology on the Bio-Plex(R) platform, using the serum protocol. Results: Tumour necrosis factor-alpha, RANTES, eotaxin and interferon-gamma-induced protein-10 levels were lower in FF than in serum, and thus not further investigated. Interleukin (IL)-6, -8, -10, -15, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median concentrations in FF than in serum, indicating possible ovarian production. Moreover, most of these showed higher evels in the gsIVF than in the NC groups in the serum, but not in the follicular fluid. IL-8 was reduced in gsIVF cycles. Conclusion: The fact that serum but not FF levels of the studied cytokines were higher in the stimulated than in the natural cycles can be attributed to the increased number of active follicles present after controlled ovarian stimulation.

Biochemical analysis of mutations in P450 oxidoreductase

All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.

Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

Pioglitazone inhibits androgen production in NCI-H295R cells by regulating gene expression of CYP17 and HSD3B2

Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are widely used as insulin sensitizers in the treatment of type 2 diabetes. In diabetic women with polycystic ovary syndrome, treatment with pioglitazone or rosiglitazone improves insulin resistance and hyperandrogenism, but the mechanism by which TZDs down-regulate androgen production is unknown. Androgens are synthesized in the human gonads as well as the adrenals. We studied the regulation of androgen production by analyzing the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells, an established in vitro model of steroidogenesis of the human adrenal cortex. Both TZDs changed the steroid profile of the NCI-H295R cells and inhibited the activities of P450c17 and 3betaHSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor gamma (PPARgamma) is the nuclear receptor for TZDs, suppression of PPARgamma by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPARgamma. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.

P450 oxidoreductase deficiency: a new form of congenital adrenal hyperplasia

PURPOSE OF REVIEW: P450 oxidoreductase deficiency--a newly described form of congenital adrenal hyperplasia--typically presents a steroid profile suggesting combined deficiencies of steroid 21-hydroxylase and 17alpha-hydroxylase/17,20-lyase activities. These and other enzymes require electron donation from P450 oxidoreductase. The clinical spectrum of P450 oxidoreductase deficiency ranges from severely affected children with ambiguous genitalia, adrenal insufficiency and the Antley-Bixler skeletal malformation syndrome to mildly affected individuals with polycystic ovary syndrome. We review current knowledge of P450 oxidoreductase deficiency and its broader implications. RECENT FINDINGS: Since the first report in 2004, at least 21 P450 oxidoreductase mutations have been reported in over 40 patients. The often subtle manifestations of P450 oxidoreductase deficiency suggest it may be relatively common. P450 oxidoreductase deficiency, with or without Antley-Bixler syndrome, is autosomal recessive, whereas Antley-Bixler syndrome without disordered steroidogenesis is caused by autosomal dominant fibroblast growth factor receptor 2 mutations. In-vitro assays of P450 oxidoreductase missense mutations based on P450 oxidoreductase-supported P450c17 activities provide excellent genotype/phenotype correlations. The causal connection between P450 oxidoreductase deficiency and disordered bone formation remains unclear. SUMMARY: P450 oxidoreductase mutations cause combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase. Individuals with an Antley-Bixler syndrome-like phenotype presenting with sexual ambiguity or other abnormalities in steroidogenesis should be analyzed for P450 oxidoreductase deficiency.

Placental ABCA1 expression is reduced in primary antiphospholipid syndrome compared to pre-eclampsia and controls

The ATP binding cassette transporter A1 (ABCA1) mediates cellular cholesterol and phospholipid efflux, and is implicated in phosphatidylserine translocation and apoptosis. Loss of functional ABCA1 in null mice results in severe placental malformation. This study aimed to establish the placental localisation of ABCA1 and to investigate whether ABCA1 expression is altered in placentas from pregnancies complicated by pre-eclampsia and antiphospholipid syndrome. ABCA1 mRNA and protein localisation studies were carried out using in situ hybridization and immunohistochemistry. Comparisons of gene expression were performed using real-time PCR and immunoblotting. ABCA1 mRNA and protein was localised to the apical syncytium of placental villi and endothelia of fetal blood vessels within the villi. ABCA1 mRNA expression was reduced in placentas from women with APS when compared to controls (p<0.001), and this was paralleled by reductions in ABCA1 protein expression. There were no differences in ABCA1 expression between placentas from pre-eclamptic pregnancies and controls. The localisation of ABCA1 in human placenta is consistent with a role in cholesterol and phospholipid transport. The decrease in ABCA1 protein in APS may reflect reduced cholesterol transport to the fetus affecting the formation of cell membranes and decreasing the level of substrate available for steroidogenesis.

Clinical, structural and functional implications of mutations and polymorphisms in human NADPH P450 oxidoreductase

Cytochrome P450 proteins are involved in metabolism of drugs and xenobiotics. In the endoplasmic reticulum a single nicotinamide adenine dinucleotide phosphate (NADPH) P450 oxidoreductase (POR) supplies electrons to all microsomal P450s for catalytic activity. POR is a flavoprotein that contains both flavin mononucleotide and flavin adenine dinucleotide as cofactors and uses NADPH as the source of electrons. We have recently reported a number of POR mutations in the patients with disordered steroidogenesis. In the first report we had described missense mutations (A287P, R457H, V492E, C569Y, and V608F) identified in four patients with defects in steroid production. Each POR variant was produced as recombinant N-27 form of the enzyme in bacteria and as full-length form in yeast. Membranes from bacteria or yeast expressing normal or variant POR were purified and their activities were characterized in cytochrome c and CYP17A1 assays. Later we have published a larger study that described a whole range of POR mutations and characterized the mutants/polymorphisms A115V, T142A, M263V, Y459H, A503V, G539R, L565P, R616X, V631I, and F646del from the sequencing of patient DNA. We also studied POR variants Y181D, P228L, R316W, G413S, and G504R that were available in public databases or published literature. Three-dimensional structure of rat POR is known and we have used this structure to deduce the structure-function correlation of POR mutations in human. The missense mutations found in patients with disordered steroidogenesis are generally in the co-factor binding and functionally important domains of POR and the apparent polymorphisms are found in regions with lesser structural importance. A variation in POR can alter the activity of all microsomal P450s, and therefore, can affect the metabolism of drugs and xenobiotics even when the P450s involved are otherwise normal. It is important to study the genetic and biochemical basis of POR variants in human population to gain information about possible differences in P450 mediated reactions among the individuals carrying a variant or polymorphic form of POR that could impact their metabolism.

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.