Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.

Effects of 1α,25-dihydroxyvitamin D3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells.

The active hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1α,25-dihydroxyvitamin D3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1α,25-dihydroxyvitamin D3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indicated suppression of p70-S6 kinase levels by 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G cells, whereas the levels of PLCγ, a target for phospholipid signaling, was slightly increased. Activation of STAT3, an important regulator of malignancy, was suppressed by 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1α,24-dihydroxyvitamin D3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1α,25-dihydroxyvitamin D3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumorigenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.

Effects of vitamin D in the nervous system: Special focus on interaction with steroid hormone signalling and a possible role in the treatment of brain cancer.

The active vitamin D hormone, 1,25-dihydroxyvitamin D3 , exerts many physiological actions in the body, including effects on the nervous system. Studies of steroidogenesis in cells of the nervous system and elsewhere not only indicate that 1,25-dihydroxyvitamin D3 affects steroidogenic pathways but also suggest varying responses in different cell types. For example, 1,25-dihydroxyvitamin D3 stimulates the expression of aromatase in human glioma but not in human neuroblastoma cells or rat astrocytes. However, in astrocytes, 1,25-dihydroxyvitamin D3 suppresses 3ß-hydroxysteroid dehydrogenase and steroid 17-hydroxylase/lyase. Other studies indicate cross-talk between vitamin D signalling and signalling of oestrogens, progesterone or glucocorticoids. Reported data indicate synergistic effects of combinations of 1,25-dihydroxyvitamin D3 and other steroid hormones on neuroinflammation, neurite outgrowth and neuroprotection. Also, dysregulation of steroid pathways affecting brain cells is found in vitamin D deficiency. Thus, several studies suggest that active vitamin D may affect steroid hormone synthesis and/or signalling in the nervous system, although the potential mechanisms for these responses remain unclear. 1,25-Dihydroxyvitamin D3 suppresses proliferation in several cell types and is therefore of interest in cancer treatment. Also, epidemiological studies associate vitamin D levels with cancer risk or outcomes. Reported data on tumours of the nervous system are mainly on glioma, a common type of brain cancer. Expression of the vitamin D receptor in glioma tumours is associated with improved survival. Several studies show that 1,25-dihydroxyvitamin D3 and vitamin D analogues (synthetic vitamin D-like compounds) suppress proliferation and migration in human vitamin D receptor-expressing glioma cell lines. Studies on mechanisms for actions of 1,25-dihydroxyvitamin D3 or its analogues indicate regulation of cell cycle proteins and senescence markers. These compounds also show synergism in combination with other cancer therapies treating glioma. From the data available, vitamin D analogues emerge as interesting candidates for the future improved treatment of human glioma and possibly also other cancers of the nervous system.

Effects of glucocorticoids on vitamin D3-metabolizing 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts.

Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to bone disorders. Long-term treatment with glucocorticoids results in osteoporosis and increased risk of fractures. Much remains unclear regarding the effects of these compounds in bone cells. In the current study, human osteosarcoma Saos-2 cells and primary human osteoblasts were found to express mRNA for the vitamin D receptor as well as activating and deactivating enzymes in vitamin D3 metabolism. These bone cells exhibited CYP24A1-mediated 24-hydroxylation which is essential for deactivation of the active vitamin form. However, bioactivating vitamin D3 hydroxylase activities could not be detected in either of these cells. Several glucocorticoids, including prednisolone, down regulated CYP24A1 mRNA and CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblasts. Also, prednisolone significantly suppressed a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells co-transfected with the glucocorticoid receptor. Thus, the results of the present study show suppression by glucocorticoids on CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of this study we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid with glucocorticoid activity, which can bind the glucocorticoid receptor. Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblasts suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may interfere with regulation of active vitamin D levels.

Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells.

The active form of vitamin D (1α,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 µM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.

Expression and regulation of CYP17A1 and 3ß-hydroxysteroid dehydrogenase in cells of the nervous system: Potential effects of vitamin D on brain steroidogenesis.

Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). 3ß-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3ß-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3ß-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3ß-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3ß-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3ß-HSD, particularly on the transcriptional level, may play a role in the nervous system.

Drug-Mediated Gene Regulation of Vitamin D3 Metabolism in Primary Human Dermal Fibroblasts.

Vitamin D metabolism was studied in primary human dermal fibroblasts with focus on drug-mediated gene regulation related to adverse side effects of antiretroviral drugs used in HIV therapy. The fibroblasts expressed mRNA for cytochrome P450 (CYP) enzymes catalysing bioactivating (CYP2R1, CYP27A1 and CYP27B1) and catabolic reactions (CYP24A1). The cells produced both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 . The results demonstrate that primary dermal fibroblasts have an active vitamin D3 -metabolizing system. High incidence of low bone mineral density is a concern for HIV-infected patients treated with antiretroviral drugs. Osteomalacia and severe vitamin D deficiency have been reported. We investigated whether drug-mediated gene regulation could be a possible mechanism behind these adverse drug effects. Fibroblasts were treated with different drugs used in HIV therapy, and the 1α,25-dihydroxyvitamin D3 levels and relative mRNA levels for crucial enzymes were determined. Efavirenz, stavudine and ritonavir significantly down-regulated the bioactivating CYP2R1 and up-regulated the catabolic CYP24A1. The drugs reduced bioactivating enzyme activities and cellular levels of 1α,25-dihydroxyvitamin D3 . The current results indicate that effects on gene expression may lead to disturbed vitamin D metabolism and decreased cellular levels of active vitamin D3 . The data are consistent with the impaired bone health in patients treated with certain antiretroviral drugs.

Effects of Osteoporosis-Inducing Drugs on Vitamin D-Related Gene Transcription and Mineralization in MG-63 and Saos-2 Cells.

Vitamin D3 is important for calcium and phosphate homeostasis. To exert its effects, vitamin D3 has to be enzymatically activated into 1,25D3 (1,25-dihydroxyvitamin D3 ). Regulation by endogenous vitamin D metabolites of the activation and inactivation of 1,25D3 is important to maintain adequate amounts of active vitamin D3 . Vitamin D deficiency and low bone mineral density have been linked to treatments with antiretroviral drugs and glucocorticoids. However, the causes of drug-induced osteoporosis remain unclear. The antiretroviral drugs efavirenz and ritonavir as well as the glucocorticoid dexamethasone were included in this study. Their effects on transcription of vitamin D-regulating enzymes in MG-63 cells were investigated. Ritonavir and dexamethasone both induced transcription of CYP27B1, the enzyme responsible for the formation of 1,25D3 . Efavirenz, however, suppressed CYP27B1 expression. When administered together with endogenous vitamin D metabolites, dexamethasone and efavirenz counteracted the 1,25D3 -mediated up-regulation of CYP24A1, which inactivates 1,25D3 . This suggests that the drugs may interfere with local regulation of the vitamin D metabolizing system in osteoblasts. Studies on mineralization were performed in MG-63 cells and Saos-2 cells by measuring calcium concentrations accumulated over time. The effects of efavirenz, ritonavir and dexamethasone and/or vitamin D metabolites were examined. 1,25D3 induced mineralization in both cell lines. Efavirenz administered alone did not affect mineralization but suppressed the inducing effects of 1,25D3 on mineralization in both MG-63 cells and Saos-2 cells. In summary, the results suggest that antiretroviral drugs and glucocorticoids may adversely affect bone by interference with the vitamin D system in osteoblasts.

Motor neuron-like NSC-34 cells as a new model for the study of vitamin D metabolism in the brain.

Vitamin D3 is a pro-hormone, which is sequentially activated by 25- and 1α-hydroxylation to form 25-hydroxyvitamin D3 [25(OH)D3] and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], respectively. Subsequent inactivation is performed by 24-hydroxylation. These reactions are carried out by a series of CYP450 enzymes. The 25-hydroxylation involves mainly CYP2R1 and CYP27A1, whereas 1α-hydroxylation and 24-hydroxylation are catalyzed by CYP27B1 and CYP24A1, respectively, and are tightly regulated to maintain adequate levels of the active vitamin D hormone, 1α,25(OH)2D3. Altered circulating vitamin D levels, in particular 25(OH)D3, have been linked to several disorders of the nervous system, e.g., schizophrenia and Parkinson disease. However, little is known about the mechanisms of vitamin D actions in the neurons. In this study, we examined vitamin D metabolism and its regulation in a murine motor neuron-like hybrid cell line, NSC-34. We found that these cells express mRNAs for the four major CYP450 enzymes involved in vitamin D activation and inactivation, and vitamin D receptor (VDR) that mediates vitamin D actions. We also found high levels of CYP24A1-dependent 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] production, that was inhibited by the well-known CYP enzyme inhibitor ketoconazole and by several inhibitors that are more specific for CYP24A1. Furthermore, CYP24A1 mRNA levels in NSC-34 cells were up-regulated by 1α,25(OH)2D3 and its synthetic analogs, EB1089 and tacalcitol. Our results suggest that NSC-34 cells could be a novel model for the studies of neuronal vitamin D metabolism and its mechanism of actions.

Estrogen-mediated regulation of steroid metabolism in rat glial cells; effects on neurosteroid levels via regulation of CYP7B1-mediated catalysis.

Many neuroactive steroids, including dehydroepiandrosterone (DHEA), pregnenolone, 27-hydroxycholesterol and 17ß-estradiol, are known to affect development and function of the brain and nervous system. These and other steroids can undergo tissue and/or cell-specific enzymatic conversions into steroid metabolites. Carefully regulated production of steroids with various physiological effects is important for cells of the nervous system. Astrocytes express many steroidogenic enzymes and are considered important producers of brain steroids. The quantitative roles of different pathways for steroid metabolism in rat astrocytes are not clear. In the current study we examined effects of estrogens on steroid metabolism catalyzed by CYP7B1 and other enzymes in primary cultures of rat astrocytes. The CYP7B1 enzyme, which has been linked to neurodegenerative disease, is involved in the metabolism of several important neurosteroids. In the present study, we found that 7α-hydroxylation, performed by CYP7B1, is the quantitatively most important pathway for DHEA metabolism in rat astrocytes. In addition, our present experiments on catalytic steroid conversions revealed that estrogens significantly suppress the CYP7B1-catalyzed metabolism of not only DHEA but also of pregnenolone and 27-hydroxycholesterol in rat astrocytes. These novel findings point to a regulatory mechanism for control of the cellular levels of these neurosteroids via CYP7B1. Our hypothesis that estrogens can regulate neurosteroid levels via this enzymatic reaction was supported by experiments using ELISA to assay levels of DHEA and pregnenolone in the presence or absence of estrogen. Furthermore, the present results show that estrogen suppresses CYP7B1-catalyzed 7α-hydroxylation also in primary cultures of rat Schwann cells, indicating that regulation by estrogen via this enzyme may be of relevance in both the CNS and the PNS.

Protective effects of 27- and 24-hydroxycholesterol against staurosporine-induced cell death in undifferentiated neuroblastoma SH-SY5Y cells.

Alterations in cholesterol metabolism have been linked to several neurodegenerative disorders, including Alzheimer's disease, multiple sclerosis and Parkinson's disease. Brain cholesterol is metabolized to the oxysterols 24-hydroxycholesterol and 27-hydroxycholesterol. Disturbed levels of these oxysterols are found in neurodegenerative conditions. In the current study we examined the effects of 27- and 24-hydroxycholesterol on viability of human neuroblastoma SH-SY5Y cells treated with staurosporine, a toxic substance that induces apoptosis. Analyses using MTT assay and measurement of lactate dehydrogenase release showed that presence of 27-hydroxycholesterol counteracted the toxic effects of staurosporine on these cells. Also, 27-hydroxycholesterol significantly decreased the staurosporine-mediated induction of caspase-3 and -7, known to be important in apoptotic events. 24-Hydroxycholesterol had similar effects on viability as 27-hydroxycholesterol in low concentrations, although in higher concentrations this oxysterol exacerbated the toxic effects of staurosporine. From these findings it may be concluded that effects of oxysterols on cellular viability are strongly dependent on the concentration and on the type of oxysterol. Previous studies on oxysterols have reported that these compounds are pro-apoptotic or trigger pathological changes that result in neurodegeneration. The present data indicate that, during some conditions, oxysterols may have neuroprotective effects.

Effects of CYP7B1-related steroids on androgen receptor activation in different cell lines.

The widely expressed steroid hydroxylase CYP7B1 is involved in metabolism of a number of steroids reported to influence estrogen and androgen signaling. Several studies by us and other investigators have linked this enzyme to effects on estrogen receptor activation. In a previous report we examined the effect of CYP7B1-mediated hormone metabolism for estrogen-mediated response in kidney-derived HEK293 cells. In the current study we used an androgen response element (ARE) reporter system to examine androgen-dependent response of some CYP7B1 substrates and CYP7B1-formed metabolites in several cell lines derived from different tissues. The results indicate significantly lower androgen receptor activation by CYP7B1-formed steroid metabolites than by the corresponding steroid substrates, suggesting that CYP7B1-mediated catalysis may decrease some androgenic responses. Thus, CYP7B1-dependent metabolism may be of importance not only for estrogenic signaling but also for androgenic. This finding, that CYP7B1 activity may be a regulator of androgenic signaling by converting AR ligands into less active metabolites, is also supported by real-time RT-PCR experiment where a CYP7B1 substrate, but not the corresponding product, was able to stimulate known androgen-sensitive genes. Furthermore, our data indicate that the effects of some steroids on hormone response element reporter systems are cell line-specific. For instance, despite transfection of the same reporter systems, 5-androstene-3ß,17ß-diol strongly activates an androgen-dependent response element in prostate cancer cells whereas it elicits only ER-dependent responses in kidney HEK293 cells. Potential roles of cell-specific metabolism or comodulator expression for the observed differences are discussed.

Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism.

The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1-mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor ß, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism.

1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism.

It is well-known that 1α,25-dihydroxyvitamin D(3) and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D(3) has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D(3) on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D(3) treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D(3) exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D(3) stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D(3) exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D(3) as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.

Androgen receptor-mediated regulation of the anti-atherogenic enzyme CYP27A1 involves the JNK/c-jun pathway.

CYP27A1, an enzyme with several important roles in cholesterol homeostasis and vitamin D3 metabolism, has been ascribed anti-atherogenic properties. This study addresses an important problem regarding how this enzyme, involved in cholesterol metabolism in the liver and peripheral tissues, is regulated. Our results identify the human CYP27A1 gene as a new target for the JNK/c-jun pathway. Initial experiments showed that an inhibitor of c-Jun N-terminal kinase (JNK) downregulated basal CYP27A1 promoter activity whereas overexpression of JNK slightly enhanced promoter activity. Androgen receptor (AR)-mediated upregulation of mRNA levels and endogenous enzyme activity was recently reported. In the present study, the AR antagonist nilutamide blocked the androgen induction of CYP27A1. The present data revealed that inhibition of the JNK/c-jun pathway abolishes the AR-mediated effect on CYP27A1 transcription and enzyme activity, whereas overexpression of JNK markedly increased androgenic upregulation of CYP27A1. In conclusion, the current results indicate involvement of the JNK/c-jun pathway in AR-mediated upregulation of human CYP27A1. The link to JNK signaling is interesting since inflammatory processes may upregulate CYP27A1 to clear cholesterol from peripheral tissues.

Effects of CYP7B1-mediated catalysis on estrogen receptor activation.

Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.

1alpha,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells.

The current study presents data indicating that 1alpha,25-dihydroxyvitamin D(3) affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1alpha,25-dihydroxyvitamin D(3) suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3beta-hydroxysteroid dehydrogenase (3betaHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1alpha,25-dihydroxyvitamin D(3). Interestingly, the two CYP17A1-mediated activities were influenced reciprocally - the 17alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1alpha,25-dihydroxyvitamin D(3)-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1alpha,25-dihydroxyvitamin D(3)-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

CYP7B1-mediated metabolism of 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol): a novel pathway for potential regulation of the cellular levels of androgens and neurosteroids.

The current study presents data indicating that 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3alpha-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABA(A)) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3alpha-hydroxylated steroids. 3alpha-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. In addition, 3alpha-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. Recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3alpha-Adiol hydroxylation. Furthermore, the observed hydroxylase activity towards 3alpha-Adiol was very low or undetectable in livers of Cyp7b1(-/-) knockout mice. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3alpha-Adiol which may impact intracellular levels of dihydrotestosterone and GABA(A)-modulating steroids.

Regulation of human vitamin D(3) 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells.

In this study, we examined whether 1alpha,25-dihydroxyvitamin D(3) (calcitriol), phenobarbital, and the antiretroviral drug efavirenz, drugs used by patient groups with high incidence of low bone mineral density, could affect the 25-hydroxylase activity or expression of human 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells. Fibroblasts express the 25-hydroxylating enzymes CYP2R1 and CYP27A1. LNCaP cells were found to express two potential vitamin D 25-hydroxylases-CYP2R1 and CYP2J2. The presence in different cells of nuclear receptors vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) was also determined. Phenobarbital suppressed the expression of CYP2R1 in fibroblasts and CYP2J2 in LNCaP cells. Efavirenz suppressed the expression of CYP2R1 in fibroblasts but not in LNCaP cells. CYP2J2 was slightly suppressed by efavirenz, whereas CYP27A1 was not affected by any of the two drugs. Calcitriol suppressed the expression of CYP2R1 in both fibroblasts and LNCaP cells but had no clear effect on the expression of either CYP2J2 or CYP27A1. The vitamin D(3) 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1alpha-hydroxyvitamin D(3)) was used as indicator of metabolism because no 1alpha,25-dihydroxyvitamin D(3) product could be determined. The amount of 1alpha-hydroxyvitamin D(3) remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D(3) was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D(3) may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.

Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling.

The steroid hydroxylase CYP7B1 metabolizes neurosteroids, cholesterol derivatives, and estrogen receptor (ER) ligands. Previous studies identified CYP7B1 as a target for regulation by estrogen. The present study examines the mechanism for estrogen-mediated regulation of the human CYP7B1 gene promoter. Treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), abolished ER-mediated up-regulation of a CYP7B1 promoter-luciferase reporter in HepG2 cells, whereas overexpression of PI3K or Akt significantly increased estrogenic up-regulation of CYP7B1. Overexpression of dominant-negative mutant Akt abolished ER-mediated stimulation of CYP7B1 in HepG2 cells. Data indicated no binding of ER to CYP7B1 promoter sequences, suggesting that ER interacts with the PI3K/Akt pathway without binding to the gene. At low ER levels, overexpression of Akt suppressed CYP7B1 promoter activity, suggesting that its effect on CYP7B1 is different when estrogens are absent. In HEK293 cells, CYP7B1 transcription was much less affected by Akt, indicating that the mechanism for up-regulation of CYP7B1 is different in different cell types. Other experiments indicated that MAPK signalling may affect basal CYP7B1 levels. The current results, indicating that regulation of CYP7B1 by ER can be mediated via the PI3K/Akt signal pathway, a regulatory pathway important for cellular survival and growth, suggest an important role for CYP7B1 in cellular growth, particularly in connection with estrogenic signalling.