RESUMO
Vitamin D signaling is important in regulating calcium homeostasis essential for bone health but also displays other functions in cells of several tissues. Disturbed vitamin D signaling is linked to a large number of diseases. The multiple cytochrome P450 (CYP) enzymes catalyzing the different hydroxylations in bioactivation of vitamin D3 are crucial for vitamin D signaling and function. This review is focused on the progress achieved in identification of the bioactivating enzymes and their genes in production of 1α,25-dihydroxyvitamin D3 and other active metabolites. Results obtained on species- and tissue-specific expression, catalytic reactions, substrate specificity, enzyme kinetics, and consequences of gene mutations are evaluated. Matters of incomplete understanding regarding the physiological roles of some vitamin D hydroxylases are critically discussed and the authors will give their view of the importance of each enzyme for vitamin D signaling. Roles of different vitamin D receptors and an alternative bioactivation pathway, leading to 20-hydroxylated vitamin D3 metabolites, are also discussed. Considerable progress has been achieved in knowledge of the vitamin D3 bioactivating enzymes. Nevertheless, several intriguing areas deserve further attention to understand the pleiotropic and diverse activities elicited by vitamin D signaling and the mechanisms of enzymatic activation necessary for vitamin D-induced responses.
Assuntos
Vitamina D , Vitaminas , Sistema Enzimático do Citocromo P-450/metabolismo , Especificidade por Substrato , HidroxilaçãoRESUMO
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.
Assuntos
Movimento Celular , Proliferação de Células , Isomerases de Dissulfetos de Proteínas , Receptores de Calcitriol , Vitamina D3 24-Hidroxilase , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Humanos , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Linhagem Celular Tumoral , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Calcitriol/farmacologia , Calcitriol/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Vitamina D/metabolismo , Vitamina D/farmacologia , Vitamina D/análogos & derivados , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
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.
Assuntos
Androgênios/farmacologia , Androstenodiol/análogos & derivados , Androstenodiol/farmacologia , Receptores Androgênicos/metabolismo , Esteroide Hidroxilases/metabolismo , Linhagem Celular Tumoral , Família 7 do Citocromo P450 , Expressão Gênica , Células HEK293 , Humanos , Masculino , Especificidade de Órgãos , Neoplasias da Próstata , Receptores Androgênicos/genética , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Esteroide Hidroxilases/genética , Testosterona/biossíntese , TransfecçãoRESUMO
BACKGROUND: 1α,25-Dihydroxyvitamin D(3) has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D(3)-mediated effect on CYP21A2 transcriptional rate. METHODS: We have studied the effects of 1α,25-dihydroxyvitamin D(3) using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA. RESULTS: 1α,25-Dihydroxyvitamin D(3) was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D(3) was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect. GENERAL SIGNIFICANCE: This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D(3).
Assuntos
Esteroide 21-Hidroxilase/genética , Vitamina D/análogos & derivados , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Calcitriol/genética , Receptores de Calcitriol/fisiologia , Receptores X de Retinoides/genética , Receptores X de Retinoides/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica/efeitos dos fármacos , Vitamina D/farmacologia , Vitamina D/fisiologia , Elemento de Resposta à Vitamina D/efeitos dos fármacos , Elemento de Resposta à Vitamina D/fisiologiaRESUMO
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.
Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Androgênios/metabolismo , Neoplasias da Mama/tratamento farmacológico , Estrogênios/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Vitamina D/análogos & derivados , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Aromatase/genética , Aromatase/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/metabolismo , Células Tumorais Cultivadas , Vitamina D/farmacologiaRESUMO
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.
RESUMO
Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.
Assuntos
Mapeamento de Interação de Proteínas , Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Hormônios/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 21-Hidroxilase/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Aldosterona/metabolismo , Androstenodiona/metabolismo , Western Blotting , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Corticosterona/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Desidroepiandrosterona/metabolismo , Sulfato de Desidroepiandrosterona/metabolismo , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 11-beta-Hidroxilase/genética , Esteroide 11-beta-Hidroxilase/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 21-Hidroxilase/genética , Esteroides/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Rim/metabolismo , Microssomos Hepáticos/metabolismo , Esteroide Hidroxilases/metabolismo , Adjuvantes Imunológicos/farmacologia , Anabolizantes/farmacologia , Androstano-3,17-diol/farmacologia , Animais , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Catálise , Células Cultivadas , Família 7 do Citocromo P450 , Desidroepiandrosterona/farmacologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Luciferases/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , RNA Mensageiro/genética , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide Hidroxilases/genética , SuínosRESUMO
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.
Assuntos
Aterosclerose/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Androgênicos/metabolismo , Antracenos/farmacologia , Aterosclerose/genética , Aterosclerose/prevenção & controle , Colestanotriol 26-Mono-Oxigenase/genética , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Androgênios/metabolismo , Androstano-3,17-diol/metabolismo , Redes e Vias Metabólicas , Neurotransmissores/metabolismo , Esteroide Hidroxilases/metabolismo , Androstano-3,17-diol/química , Animais , Biocatálise , Linhagem Celular , Família 7 do Citocromo P450 , Receptor beta de Estrogênio/metabolismo , Humanos , Hidroxilação , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Modelos Biológicos , NADP/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilases/deficiência , Sus scrofaRESUMO
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.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/fisiologia , Vitamina D/metabolismo , Astrócitos/metabolismo , Humanos , Neurônios/metabolismoRESUMO
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.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Anticonvulsivantes/efeitos adversos , Benzoxazinas/efeitos adversos , Colestanotriol 26-Mono-Oxigenase/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Fibroblastos/efeitos dos fármacos , Fenobarbital/efeitos adversos , Alcinos , Calcitriol/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Ciclopropanos , Citocromo P-450 CYP2J2 , Citocromo P-450 CYP3A/biossíntese , Família 2 do Citocromo P450 , Fibroblastos/metabolismo , Humanos , Masculino , Receptor de Pregnano X , Neoplasias da Próstata , Receptores de Calcitriol/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores de Esteroides/biossíntese , Pele/citologia , Fatores de Transcrição/biossínteseRESUMO
Sterol 27-hydroxylase (CYP27A1) is required for the hepatic conversion of cholesterol into bile acids and for production of 27-hydroxycholesterol which affects cholesterol homeostasis in several ways. Dexamethasone increases hepatic bile acid biosynthesis and CYP27A1-mediated enzyme activity in HepG2 cells. This study examines the mechanism of the dexamethasone-induced effect on the human CYP27A1 promoter. Dexamethasone treatment of HepG2 cells overexpressed with glucocorticoid receptor alpha (GRalpha) increased the CYP27A1 promoter activity more than four-fold as compared with untreated cells. The GR-antagonist mifepristone almost completely abolished the dexamethasone-induced effect on the promoter activity. Progressive deletion analysis of the CYP27A1 promoter indicated that sequences involved in GR-mediated induction by dexamethasone are present in a region between -1094 and -792. Several putative GRE sites could be found in this region and EMSA experiments revealed that two of these could bind GR. Site-directed mutagenesis of GR-binding sequences in the CYP27A1 promoter identified a GRE at -824/-819 important for GR-mediated regulation of the transcriptional activity. Endogenous and pharmacological glucocorticoids may have a strong impact on several aspects of cholesterol homeostasis and other processes related to CYP27A1-mediated metabolism. The glucocorticoid-mediated induction of human CYP27A1 transcription is of particular interest due to the anti-atherogenic properties ascribed to this enzyme.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Receptores de Glucocorticoides/metabolismo , Sequência de Bases , Colestanotriol 26-Mono-Oxigenase/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Regulação para CimaRESUMO
The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.
Assuntos
Anticolesterolemiantes/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colestenonas/metabolismo , Linhagem Celular , Colestenonas/química , Cromatografia Líquida de Alta Pressão , Éteres/metabolismo , Humanos , Hidroxilação , Cinética , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
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-2â¯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-2â¯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-2â¯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.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Osteoblastos/enzimologia , Regiões Promotoras Genéticas , Vitamina D3 24-Hidroxilase/biossíntese , Linhagem Celular Tumoral , Colecalciferol/metabolismo , Humanos , Osteoblastos/citologia , Vitamina D3 24-Hidroxilase/genéticaRESUMO
CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3beta-Adiol may play an important role for estrogen receptor beta-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor beta activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3beta-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3beta-Adiol (a CYP7B1 substrate) and 7alpha-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor beta activation. The data obtained indicated that 3beta-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor beta activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3beta-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the K(cat)/K(m) values were in the same order of magnitude. A high dehydroepiandrosterone/3beta-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3beta-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3beta-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3beta-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor beta signaling.
Assuntos
Androstano-3,17-diol/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Desidroepiandrosterona/metabolismo , Estrogênios/metabolismo , Transdução de Sinais , Envelhecimento/fisiologia , Animais , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Hidroxilação , Cinética , Masculino , Especificidade de ÓrgãosRESUMO
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.
Assuntos
Elafina/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esteroide Hidroxilases/metabolismo , Antracenos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Cromonas/farmacologia , Família 7 do Citocromo P450 , Elafina/antagonistas & inibidores , Ativação Enzimática , Flavonoides/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Dados de Sequência Molecular , Morfolinas/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Piridinas/farmacologiaRESUMO
This article aims to give an overview on the characterization, properties and regulation of enzymes, particularly the cytochrome (CYP) P450 enzymes, in the formation of bile acids from cholesterol. Bile acids are biologically active molecules that promote absorption of dietary lipids in the intestine and stimulate biliary excretion of cholesterol. Bile acids and oxysterols, formed from cholesterol, act as ligands to nuclear receptors regulating the expression of important genes in cholesterol homeostasis. Thus, the bioactivation of cholesterol into bile acids is crucial for regulation of cholesterol homeostasis. The primary human bile acids, cholic acid and chenodeoxycholic acid, are formed from cholesterol via several pathways involving many different enzymes. Many of these enzymes are cytochrome P450 (CYP) enzymes, introducing a hydroxyl group in the molecule. The "classic" pathway of bile acid formation starts with a 7alpha-hydroxylation of cholesterol by CYP7A1 in the liver. The "acidic" pathway starts with a hepatic or extrahepatic 27-hydroxylation by CYP27A1. There also exist some quantitatively minor pathways which may be of importance under certain conditions. Formation of cholic acid requires insertion of a 12alpha-hydroxyl group performed by CYP8B1. Oxysterols are precursors to bile acids, participate in cholesterol transport and are known to affect the expression of several genes in cholesterol homeostasis. Enzymes with capacity to form and metabolize oxysterols are present in liver and extrahepatic tissues. The enzymes, nuclear receptors and transcription factors involved in bile acid biosynthesis are potential pharmaceutical targets for the development of new drugs to control hypercholesterolemia and to prevent atherosclerosis and other diseases related to disturbed cholesterol homeostasis. The review will also discuss some inborn errors of bile acid biosynthesis and the recently acquired knowledge on the genetic defects underlying these diseases.
Assuntos
Ácidos e Sais Biliares/biossíntese , Colesterol/metabolismo , Animais , Ácidos e Sais Biliares/química , Colesterol/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Homeostase , Humanos , Modelos Biológicos , Estrutura Molecular , Mutação , Erros Inatos do Metabolismo de Esteroides/genética , Erros Inatos do Metabolismo de Esteroides/metabolismo , Esteróis/metabolismoRESUMO
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.