RESUMEN
The consumption of dairy products, particularly of low fat milk, has been shown to be associated with the occurrence of Parkinson's disease. This association does not necessarily reflect a pathophysiological role of milk intake in the development of Parkinson's disease. Nevertheless, the present review discusses a potential mechanism possibly mediating an effect of milk consumption on Parkinson's disease. The case is made that milk is tailored in part to support bone mineralization of the suckling offspring and is thus rich in calcium and phosphate. Milk intake is thus expected to enhance intestinal calcium phosphate uptake. As binding to fatty acids impedes Ca2+ absorption, low fat milk is particularly effective. Calcium and phosphate uptake inhibit the formation of 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), the active form of vitamin D. Calcium inhibits 1,25(OH)2D3 production in part by suppressing the release of parathyroid hormone, a powerful stimulator of 1,25(OH)2D3 formation. Phosphate excess stimulates the release of fibroblast growth factor FGF23, which suppresses 1,25(OH)2D3 formation, an effect requiring Klotho. 1,25(OH)2D3 is a main regulator of mineral metabolism, but has powerful effects apparently unrelated to mineral metabolism, including suppression of inflammation and influence of multiple brain functions. In mice, lack of 1,25(OH)2D3 and excessive 1,25(OH)2D3 formation have profound effects on several types of behavior, such as explorative behavior, anxiety, grooming and social behavior. 1,25(OH)2D3 is produced in human brain and influences the function of various structures including substantia nigra. In neurons 1,25(OH)2D3 suppresses oxidative stress, inhibits inflammation and stimulates neurotrophin formation thus providing neuroprotection. As a result, 1,25(OH)2D3 is considered to favorably influence the clinical course of Parkinson's disease. In conclusion, consumption of milk could in theory accelerate the downhill course of neuronal function in Parkinson's disease. However, substantial additional experimentation is required to define the putative causal role of 1,25(OH)2D3 in the pathophysiology of Parkinson's disease and its sensitivity to milk consumption.
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Encéfalo/metabolismo , Calcitriol/metabolismo , Calcio de la Dieta/metabolismo , Calcio/metabolismo , Leche/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Calcitriol/antagonistas & inhibidores , Calcio de la Dieta/efectos adversos , Factor-23 de Crecimiento de Fibroblastos , Humanos , Leche/efectos adversos , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/prevención & control , Factores de Riesgo , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Vitamin D-1,25(OH)2 D3 or 1,25D3-maintains healthy osseous tissue, stimulates the production of the antimicrobial peptide cathelicidin and has anti-inflammatory effects, but it can cause hypercalcemia. Evidence links diminished serum levels of 1,25D3 with increased gingival inflammation. Periodontitis progression is associated with increased local production of inflammatory mediators by immune cells and gingival fibroblasts. These include interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8, both regulated by signaling pathways, including NF-κB and MAPK/AP-1. The objectives were to determine the effects of 1,25D3 or a non-calcemic analog, 20-hydroxyvitamin D3 -20(OH)D3 or 20D3-on IL-1ß-stimulated IL-6 and IL-8 production, and NF-κB and MAPK/AP-1 activation, by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were incubated ± IL-1ß, with or without exposure to 1,25D3 or 20D3. IL-6 and IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay. NF-κB (p65) and AP-1 (phospho-cJun) and were measured in nuclear extracts via binding to specific oligonucleotides. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß-stimulated IL-6 and IL-8 levels were both significantly inhibited (40%-60%) (P<.045) by 1,25D3, but not 20D3 (0%-15% inhibition, not statistically significant). Both 1,25D3 and 20D3 significantly and similarly inhibited IL-1ß-stimulated nuclear levels of p65 and phospho-cJun (P<.02). CONCLUSION: Reduction of the activation of NF-κB and AP-1 alone is not able to inhibit strongly the IL-1ß stimulated IL-6 and IL-8 gene expression. 1,25D3 but not 20D3 may affect some of the many other factors/processes/pathways that in turn regulate the expression of these genes. However, the results suggest that topical application of ligands of the vitamin D receptor may be useful in the local treatment of periodontitis while reducing adverse systemic effects.
Asunto(s)
Calcifediol/análogos & derivados , Calcitriol/antagonistas & inhibidores , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Calcifediol/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Encía/metabolismo , Humanos , Interleucina-6/genética , Interleucina-8/genética , FN-kappa B/efectos de los fármacos , Periodontitis/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/efectos de los fármacosRESUMEN
Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxia-induced cell damage in the eye by removing free radicals. However, its role in other tissues is controversial, and the effects of lutein on bone tissues are unknown. To identify a possible role of lutein in bone tissues, we examined the effects of lutein on bone formation and bone resorption and on femoral bone mass in mice. Lutein enhanced the formation of mineralized bone nodules in cultures of osteoblasts. On the other hand, lutein clearly suppressed 1α, 25-dihydroxyvitamin D3-induced bone resorption as measured by pit formation in organ culture of mouse calvaria. In co-cultures of bone marrow cells and osteoblasts, lutein suppressed 1α, 25-dihydroxyvitamin D3-induced osteoclast formation. In cultures of bone marrow macrophages, lutein suppressed soluble RANKL, the receptor activator of nuclear factor-kappaB (NF-κB) ligand, induced osteoclast formation. When five-week-old male mice were orally administered lutein for 4 weeks, the femoral bone mass was clearly enhanced in cortical bone, as measured by bone mineral density in dual X-ray absorptiometry and micro computed tomography (µCT) analyses. The present study indicates that lutein enhances bone mass in growing mice by suppressing bone resorption and stimulating bone formation. Lutein may be a natural agent that promotes bone turnover and may be beneficial for bone health in humans.
Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Resorción Ósea/prevención & control , Huesos/anatomía & histología , Luteína/farmacología , Absorciometría de Fotón , Animales , Densidad Ósea/efectos de los fármacos , Huesos/diagnóstico por imagen , Calcificación Fisiológica/efectos de los fármacos , Calcitriol/antagonistas & inhibidores , Calcitriol/farmacología , Células Cultivadas , Fémur/anatomía & histología , Fémur/efectos de los fármacos , Luteína/uso terapéutico , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , Osteoblastos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Tomografía Computarizada por Rayos XRESUMEN
It is known that Ca therapy may have anti-obesity effects. Since early weaning leads to obesity, hyperleptinaemia and insulin resistance, we studied the effect of dietary Ca supplementation in a rat model. Lactating rats were separated into two groups: early weaning (EW) - dams were wrapped with a bandage to interrupt lactation in the last 3 d of lactation and control (C) - dams whose pups had free access to milk during the entire lactation period (21 d). At 120 d, EW and C offspring were subdivided into four groups: (1) C, received standard diet; (2) CCa, received Ca supplementation (10 g of calcium carbonate/kg of rat chow); (3) EW, received standard diet; (4) EWCa, received Ca supplementation similar to CCa. The rats were killed at 180 d. The significance level was at P < 0·05. Adult EW offspring displayed hyperphagia (28 %), higher body weight (9 %) and adiposity (77 %), hyperleptinaemia (twofold increase), hypertriacylglycerolaemia (64 %), hyperglycaemia (16 %), higher insulin resistance index (38 %) and higher serum 25-hydroxyvitamin D3 (fourfold increase), but lower adiponectinaemia:adipose tissue ratio (44 %). In addition, they showed Janus tyrosine kinase 2 and phosphorylated signal transducer and activator of transcription 3 underexpression in hypothalamus (36 and 34 %, respectively), suggesting leptin resistance. Supplementation of Ca for 2 months normalised these disorders. The EW group had no change in serum insulin, thyroxine or triiodothyronine, and Ca treatment did not alter these hormones. In conclusion, we reinforced that early weaning leads to late development of some components of the metabolic syndrome and leptin resistance. Dietary Ca supplementation seems to protect against the development of endocrine and metabolic disorders in EW offspring, maybe through vitamin D inhibition.
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Calcio de la Dieta/administración & dosificación , Hiperglucemia/prevención & control , Leptina/sangre , Obesidad/prevención & control , Adiposidad , Animales , Glucemia/metabolismo , Calcitriol/antagonistas & inhibidores , Carbonato de Calcio/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Hiperglucemia/etiología , Hiperfagia/etiología , Hiperfagia/prevención & control , Resistencia a la Insulina , Síndrome Metabólico/etiología , Síndrome Metabólico/prevención & control , Obesidad/etiología , Embarazo , Ratas , DesteteRESUMEN
Several non-hypercalcemic analogs of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)(2)D(3) analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/antagonistas & inhibidores , Neoplasias del Colon/metabolismo , Proteínas de Unión al ADN/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Calcitriol/metabolismo , Factores de Transcripción/farmacología , Animales , Antineoplásicos/antagonistas & inhibidores , Cadherinas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunoprecipitación , Ratones , Regiones Promotoras Genéticas/genética , Receptores de Calcitriol/genética , Factores de Transcripción de la Familia SnailRESUMEN
(23S)-25-Dehydro-1alpha(OH)-vitamin D(3)-26,23-lactone (MK) is an antagonist of the 1alpha,25(OH)(2)-vitamin D(3) (1,25D)/human nuclear vitamin D receptor (hVDR) transcription initiation complex, where the activation helix (i.e. helix-12) is closed. To study the mode of antagonism of MK an hVDR mutant library was designed to alter the free molecular volume in the region of the hVDR ligand binding pocket occupied by the ligand side-chain atoms (i.e. proximal to helix-12). The 1,25D-hVDR structure-function studies demonstrate that 1) van der Waals contacts between helix-12 residues Leu-414 and Val-418 and 1,25D enhance the stability of the closed helix-12 conformer and 2) removal of the side-chain H-bonds to His-305(F) and/or His-397(F) have no effect on 1,25D transactivation, even though they reduce the binding affinity of 1,25D. The MK structure-function results demonstrate that the His-305, Leu-404, Leu-414, and Val-418 mutations, which increase the free volume of the hVDR ligand binding pocket, significantly enhance MK antagonist potency. Surprisingly, the H305F and H305F/H397F mutations turn MK into a VDR superagonist (EC(50) approximately 0.05 nm) but do not concomitantly alter MK binding affinity. Molecular modeling studies demonstrate that MK antagonism stems from its side chain energetically preferring a pose in the VDR ligand binding pocket where its terminal C26-methylene atom is far removed from helix-12. MK superagonism results from an energetically favored increase in interaction between Leu-404/Val-418 and C26, resulting in an increase in the stability and population of the closed, helix-12 conformer. Finally, the results/model generated, coupled with application of a VDR ensemble allosterics model, provide an understanding for the species specificity of MK.
Asunto(s)
Calcitriol/antagonistas & inhibidores , Calcitriol/química , Modelos Moleculares , Receptores de Calcitriol/antagonistas & inhibidores , Receptores de Calcitriol/química , Sitios de Unión , Calcitriol/metabolismo , Humanos , Mutación , Estructura Secundaria de Proteína/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Relación Estructura-ActividadRESUMEN
Human vitamin D3 hydroxylase (CYP24) catalyzes the inactivation of 1alpha,25-dihydroxyvitamin D3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3'-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P < 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.
Asunto(s)
Calcitriol/antagonistas & inhibidores , MicroARNs/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Esteroide Hidroxilasas/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Biocatálisis , Western Blotting , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Calcitriol/farmacología , Línea Celular Tumoral , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunohistoquímica , ARN Mensajero/genética , Esteroide Hidroxilasas/genética , Vitamina D3 24-HidroxilasaRESUMEN
To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)2D3 in primary cultured murine renal tubules. Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD(3) (10(-6) M), 1,25-(OH)2D3 was determined. PTH(1-34) stimulated the conversion of 25-OHD3 to 1,25-(OH)2D3, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1alpha-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1alpha or 24-hydroxylase mRNA. These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)2D in patients with severe primary hyperparathyroidism with renal failure.
Asunto(s)
Calcitriol/antagonistas & inhibidores , Túbulos Renales/efectos de los fármacos , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Animales , Calcitriol/biosíntesis , Técnicas In Vitro , Túbulos Renales/metabolismo , RatonesRESUMEN
Matrix gamma-carboxyglutamic acid protein (MGP), a vitamin K-dependent protein, is involved in regulation of tissue calcification. We previously reported that 9-cis retinoic acid (RA) mitigates 1alpha,25-dihydroxycholecalciferol [1,25(OH)(2)D3]-induced renal calcification in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer A/J male mouse model. This raised the question if the mechanism(s) underlying this calcification involves vitamin K. We assessed expression and vitamin K dependent gamma-carboxylation of MGP and vitamin K concentrations [phylloquinone (PK), as well as its conversion product, menaquinone-4 (MK-4)] in tissues obtained from NNK-injected A/J male mice fed 1,25(OH)(2)D3 (2.5 microg/kg diet; D group) +/- RA (15 mg/kg diet) for 20 wk. Renal calcification was only observed in the D group (2/10; 20% of the group). Renal MGP mRNA and uncarboxylated MGP (ucMGP) increased in response to D (P < 0.05) but not in response to RA or RA + D. In contrast, gamma-carboxylated MGP increased to 2.2-fold of the control in response to D+RA (P < 0.05) but not in response to RA or D alone. Although all diets contained equal amounts of PK, the kidney MK-4 concentration was higher in the D group (P < 0.05) and lower in the RA group (P < 0.05) compared with the RA+D or control groups. Renal PK concentrations were lower in the RA and RA+D groups than in the control and D groups (P < 0.05). These data suggest that 9-cis RA mitigated 1,25(OH)(2)D3-induced renal calcification by modifying the 1,25(OH)(2)D3-induced increase in ucMGP. The mechanisms by which 9-cis RA and 1,25(OH)(2)D3 alter vitamin K concentrations warrant further investigation.
Asunto(s)
Calcinosis/prevención & control , Calcitriol/toxicidad , Proteínas de Unión al Calcio/metabolismo , Ligasas de Carbono-Carbono/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Enfermedades Renales/prevención & control , Tretinoina/farmacología , Alitretinoína , Animales , Secuencia de Bases , Calcinosis/etiología , Calcinosis/metabolismo , Calcitriol/administración & dosificación , Calcitriol/antagonistas & inhibidores , Calcitriol/metabolismo , Proteínas de Unión al Calcio/genética , Carcinógenos/administración & dosificación , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Cartilla de ADN/genética , Suplementos Dietéticos , Proteínas de la Matriz Extracelular/genética , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos A , Nitrosaminas/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tretinoina/administración & dosificación , Vitamina K/metabolismo , Proteína Gla de la MatrizRESUMEN
The pathogenesis of preeclampsia (PE) involves a number of biological processes that may be directly or indirectly affected by glucocorticoid (GC) and vitamin D. GC exposure increases the risk of PE, and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) deficiency may result in PE. The purpose of the present study was to confirm the involvement of GC/1,25-(OH)2D3 axis in the pathogenesis of PE. In the study, cortisol levels of PE patients were found to be higher than that of non-complicated pregnancies, while 1,25-(OH)2D3 were decreased in both PE women and GC-induced PE rats. Mechanically, GC reduced 1,25-(OH)2D3 levels via disturbing its biosynthetic and catabolic enzymes, including Cyp3a1,Cyp24a1 and Cyp27b1, especially enhancing the expressions of Cyp3a1, the dominant enzyme for vitamin D degeneration. Moreover, replenishing 1,25-(OH)2D3 ameliorated the symptoms and placental oxidative stress of GC-induced rat PE. The protective actions of 1,25-(OH)2D3 might be explained by its roles in antagonizing the effects of GC on trophoblast proliferation and apoptosis. Together, these findings suggest that GC exposure could lead to PE via dampening 1,25-(OH)2D3 biosynthesis, and GC/1,25-(OH)2D3 axis might represent a common pathway through which PE occurs.
Asunto(s)
Calcitriol/antagonistas & inhibidores , Glucocorticoides/toxicidad , Preeclampsia/inducido químicamente , Preeclampsia/metabolismo , Albuminuria/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Calcitriol/sangre , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP3A/biosíntesis , Citocromo P-450 CYP3A/genética , Femenino , Glucocorticoides/sangre , Humanos , Hidrocortisona/metabolismo , Estrés Oxidativo/efectos de los fármacos , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Trofoblastos/efectos de los fármacosRESUMEN
The possible suppressive effects of 24,25-dihydroxycholecalciferol on secondary hyperparathyroidism and increased bone resorption were investigated in adult rats raised on a diet normal in calcium, phosphorus, and vitamin D, and subjected to acute bilateral nephrectomy. The animals had received subcutaneous radiocalcium 4 wk before the experiment. 5 h after nephrectomy an increase in serum total calcium, (45)Ca-specific activity, citrate, phosphorus, and magnesium concentrations were observed. Serum immunoreactive parathyroid hormone increased, while serum calcitonin decreased. The osteoclast count in the tibial metaphyses was augmented. The biochemical and histological changes observed were partly parathyroid hormone and calcitonin independent, as they also occurred in parathyroidectomized hypocalcemic rats. Pretreatment with 650 pmol of 24,25-dihydroxycholecalciferol 16 h before nephrectomy prevented bone calcium mobilization and diminished the rise in serum total calcium and citrate both in parathyroid-intact and in parathyroidectomized animals. In parathyroid-intact rats, serum immunoreactive parathyroid hormone and calcitonin remained normal in spite of the fall in serum-ionized calcium, and the number of osteoclasts did not increase. In parathyroidectomized rats, 24,25-dihydroxycholecalciferol did not prevent the postnephrectomy rise in the osteoclast count. This latter observation suggests that this metabolite exerts its effect on bone either by acting on cells other than osteoclasts, i.e., the osteocytes, or by inhibiting cell activity. At equimolar dosage 1,25-dihydroxycholecalciferol had a potent stimulatory effect on bone resorption. This effect of 1,25-dihydroxycholecalciferol was partly blocked by the simultaneous administration of 24,25-dihydroxycholecalciferol. The potential clinical significance of these observations remains to be determined.
Asunto(s)
Resorción Ósea/efectos de los fármacos , Dihidroxicolecalciferoles/farmacología , Hidroxicolecalciferoles/farmacología , Nefrectomía , 24,25-Dihidroxivitamina D 3 , Animales , Calcitriol/antagonistas & inhibidores , Calcio/sangre , Glándulas Paratiroides/fisiología , Ratas , Factores de TiempoRESUMEN
Human retinoid X receptor alpha (hRXR alpha) is a member of the nuclear receptor family of transcriptional regulators. It regulates transcription through its association with several heterodimeric partners, including the vitamin D3 receptor (VDR). Signaling through the VDR is essential for normal calcium homeostasis and has been shown to inhibit the proliferation of cancer cells derived from a number of tissues. Here we show that phosphorylation of hRXR alpha in ras-transformed human keratinocytes through the activated Ras-Raf-mitogen-activated protein kinase (Ras-Raf-MAP kinase) pathway results in attenuated transactivation by the VDR and resistance to the growth inhibitory action of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and RXR-specific agonist LG1069 (4-[1-(5,6,7, 8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) ethenyl]-benzoic acid). Phosphorylation of hRXR alpha occurs at serine 260, a consensus MAP kinase site. Inhibition of MAP kinase activity or point mutagenesis of serine 260 of hRXR alpha reverses the observed resistance to 1,25(OH)2D3 and LG1069. Thus, hRXR alpha is a downstream target of MAP kinase, and its phosphorylation may play an important role in malignant transformation.
Asunto(s)
Calcitriol/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Anticarcinógenos/farmacología , Bexaroteno , Células COS , Calcitriol/metabolismo , Calcitriol/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular Transformada , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Resistencia a Antineoplásicos , Genes ras/fisiología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Queratinocitos/metabolismo , Mutagénesis Sitio-Dirigida , Fenotipo , Fosforilación/efectos de los fármacos , Receptores de Calcitriol/metabolismo , Receptores X Retinoide , Serina/genética , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Tetrahidronaftalenos/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.
Asunto(s)
Calcitriol/farmacología , Regulación de la Expresión Génica , Osteocalcina/genética , Receptores de Superficie Celular/fisiología , Receptores de Ácido Retinoico , Receptores de Esteroides/fisiología , Factores de Transcripción , Tretinoina/farmacología , Animales , Calcitriol/antagonistas & inhibidores , Expresión Génica , Células HeLa , Humanos , Ligandos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Ratas , Receptores de Calcitriol , Proteínas Recombinantes/metabolismo , Receptores X RetinoideRESUMEN
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.
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Fármacos Anti-VIH/farmacología , Colecalciferol/metabolismo , Colestanotriol 26-Monooxigenasa/metabolismo , Familia 2 del Citocromo P450/metabolismo , Dermis/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Vitamina D3 24-Hidroxilasa/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Adolescente , Adulto , Alquinos , Benzoxazinas/farmacología , Calcifediol/metabolismo , Calcitriol/antagonistas & inhibidores , Calcitriol/metabolismo , Células Cultivadas , Colestanotriol 26-Monooxigenasa/antagonistas & inhibidores , Colestanotriol 26-Monooxigenasa/genética , Ciclopropanos , Familia 2 del Citocromo P450/antagonistas & inhibidores , Familia 2 del Citocromo P450/genética , Dermis/citología , Dermis/metabolismo , Femenino , Humanos , Masculino , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Ritonavir/farmacología , Estavudina/farmacología , Vitamina D3 24-Hidroxilasa/química , Vitamina D3 24-Hidroxilasa/genética , Adulto JovenRESUMEN
Members of the steroid receptor superfamily are known to alter the transcription of apolipoprotein AI (apo AI), the major apoprotein of high-density lipoprotein (HDL). To assess the role of vitamin D receptor (VDR) in apo AI gene expression, we investigated the effect of 1alpha, 25-dihydroxycholecalciferol (1, 25-(OH)2 D3) as well as the vitamin D antagonist ZK-191784 (ZK), on apo AI gene expression and promoter activity in the human hepatoma cell line HepG2. Apo AI secretion and mRNA levels were both suppressed in a dose-dependent manner in HepG2 cells treated 1, 25-(OH)2 D3. This was accompanied by a similar decrease in apo AI promoter activity. Mapping of the vitamin D response element showed that suppression required a region of the apo AI gene promoter identified previously to contain site A. However, vitamin D treatment had no effect on nuclear factor binding to site A of the apo AI promoter. Treatment with vitamin D receptor antagonist ZK inhibited the ability of 1, 25-(OH)2 D3 to repress apo AI promoter activity, while higher doses of ZK increased apo AI promoter activity. ZK did not alter estradiol stimulated apo AI promoter activity. The VDR antisense ODN had no effect on apo AI promoter activity in control cells, however, it reversed the repression normally seen in cells treated with 1, 25-(OH)2D3. It is concluded that 1, 25-(OH)2 D3 suppresses apo A1 gene expression at the transcriptional level, possibly by altering coactivators or corepressors. This effect requires the VDR as well as a vitamin D response element in the apo AI promoter.
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Apolipoproteína A-I/biosíntesis , Calcitriol/farmacología , Acetiltransferasas/biosíntesis , Apolipoproteína A-I/genética , Calcitriol/análogos & derivados , Calcitriol/antagonistas & inhibidores , Línea Celular Tumoral , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Histona Acetiltransferasas , Humanos , Neoplasias Hepáticas , Coactivador 3 de Receptor Nuclear , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteínas Oncogénicas/biosíntesis , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Calcitriol/efectos de los fármacos , Receptores de Calcitriol/genética , Receptores X Retinoide/biosíntesis , Transactivadores/biosíntesis , Elemento de Respuesta a la Vitamina D/genéticaRESUMEN
A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission- and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-beta protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagy-inducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Calcitriol/análogos & derivados , Proteínas/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Proteínas Reguladoras de la Apoptosis , Autofagia/efectos de los fármacos , Beclina-1 , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadaverina/análogos & derivados , Cadaverina/farmacocinética , Calcitriol/antagonistas & inhibidores , Calcitriol/farmacología , Catepsinas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Microscopía por Crioelectrón , Interacciones Farmacológicas , Genes Supresores de Tumor , Células HeLa , Humanos , Immunoblotting , Proteínas de la Membrana , Proteínas/genética , Proteínas/metabolismo , ARN Interferente Pequeño/genética , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A practical synthetic route to novel vitamin D antagonists of DLAM (1alpha,25-dihydroxyvitamin D(3)-26,23-lactam) was developed from vitamin D(2) via the 1,3-dipolar cycloaddition reaction as a key step. Six DLAM derivatives (24 compounds) with a variety of nitrogen substituents and stereochemistries at C23 and C25 were synthesized. Among these new derivatives, (23S,25S)-DLAM isomers bound effectively to VDRs and showed antagonistic activity in the HL-60 cell differentiation inhibition assay. The importance of the substituent on the nitrogen of DLAMs for antagonistic activity was also suggested by computational docking studies.
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Calcitriol/análogos & derivados , Calcitriol/antagonistas & inhibidores , Lactamas/síntesis química , Lactamas/farmacología , Vitamina D/análogos & derivados , Vitamina D/antagonistas & inhibidores , Animales , Sitios de Unión , Células COS , Calcitriol/síntesis química , Calcitriol/química , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Chlorocebus aethiops , Biología Computacional , Cristalografía por Rayos X , Diseño de Fármacos , Células HL-60 , Humanos , Lactamas/química , Modelos Moleculares , Conformación Molecular , Estructura Secundaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Vitamina D/químicaRESUMEN
Transforming growth factor beta s (TGF-beta s) are a family of polypeptide growth factors that regulate cellular growth, phenotype, and differentiation. TGF-beta s are synthesized as latent high molecular weight complexes that include the NH2-terminal remnant of the TGF-beta precursor (latency-associated protein) and, frequently, latent TGF-beta binding protein. After activation, TGF-beta s act as local mediators of hormonal responses in target tissues. TGF-beta functions as a negative growth regulator for both breast cancer cells and normal mammary epithelial cells. Vitamin D3 is growth inhibitory for the estrogen receptor-negative breast cancer cell line BT-20 and regulates TGF-beta expression in cultured keratinocytes. We studied here the effects of vitamin D3 and its analogues on TGF-beta expression and activity in BT-20 cells. It was found that vitamin D3 enhanced both TGF-beta 1 mRNA and secretion of the protein in a time- and dose-dependent manner. Analyses of the vitamin D3 responses in the presence of cycloheximide or actinomycin D indicated that the TGF-beta 1 mRNA induction was dependent on both protein and RNA synthesis. The amounts of latent TGF-beta binding protein were also increased in the conditioned medium but not in the pericellular matrix of vitamin D3-treated cultures. The amounts of active TGF-beta were enhanced in vitamin D3-treated cultures as well, suggesting autocrine or paracrine functions for the secreted growth factor. Some analogues of vitamin D3 (EB 1089, MC 903, and KH 1060) that are known to be potent inhibitors of breast cancer cell growth both in vitro and in vivo had similar or more pronounced inducing effects on TGF-beta 1 mRNA levels. The present results indicate that vitamin D3 and its analogues are potent inducers of both active and latent forms of TGF-beta 1 in BT-20 breast carcinoma cells and provide evidence for coordinated regulation of latent TGF-beta binding protein and TGF-beta 1.
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Neoplasias de la Mama/metabolismo , Calcitriol/farmacología , Proteínas Portadoras/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Calcitriol/antagonistas & inhibidores , Cicloheximida/farmacología , Dactinomicina/farmacología , Humanos , Proteínas de Unión a TGF-beta Latente , ARN Mensajero/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células Tumorales CultivadasRESUMEN
1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and retinoic acid (RA) induce a high-phagocytic phenotype in the macrophage-like tumor cell line P388D1. A concurrent cultivation of P388D1 cells in the presence of suboptimal concentrations of both agents led to an extent of induction of phagocytic activity that surpassed the additive effect of either of the agents alone; i.e., 1,25(OH)2D3 and RA synergistically induce the phagocytic capability of P388D1 cells. Dexamethasone and 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (TPA) did not induce a high-phagocytic phenotype in P388D1 cells and affected differentially the high-phagocytic phenotype induced by RA and 1,25(OH)2D3. Dexamethasone inhibited the phagocytic activity induced by RA (80% at 24 h), while it had small suppressive effects on that induced by 1,25(OH)2D3. TPA suppressed the phagocytic activity induced by RA (60% within 96 h) while potentiating the expression of the high-phagocytic phenotype induced by 1,25(OH)2D3 (50% increase with 96 h). The observed effects did not involve modulation of prostaglandin synthesis or intracellular cyclic adenosine 3':5'-monophosphate. Expression of transglutaminase activity in P388D1 cells was also modulated differentially by the four agents; 1,25(OH)2D3 treatment had no effect on enzyme level, RA and TPA suppressed it, and dexamethasone increased it. The data suggest that: 1,25(OH)2D3 and RA act via disparate mechanisms that can operate simultaneously; the elements induced in P388D1 cells by 1,25(OH)2D3 and RA, and which are responsible for the phagocytic activity, differ in their sensitivity to dexamethasone and TPA; and transglutaminase activity in P388D1 cells is readily manipulable, but there seems to be no straightforward correlation between the level of its activity and the phagocytic capability of the cells or their rate of proliferation.
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Aciltransferasas/metabolismo , Calcitriol/farmacología , Dexametasona/farmacología , Linfoma/fisiopatología , Fagocitosis/efectos de los fármacos , Forboles/toxicidad , Acetato de Tetradecanoilforbol/toxicidad , Tretinoina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Calcitriol/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Línea Celular , Sinergismo Farmacológico , Linfoma/enzimología , Ratones , Fenotipo , Prostaglandinas/farmacología , Transglutaminasas , Tretinoina/antagonistas & inhibidoresRESUMEN
1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1 alpha,25(OH)2D3, like phorbol ester tumor promoters, induces anchorage-independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1 alpha,25(OH)2D3 at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorage-independent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1 alpha,25(OH)2D3 in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1 alpha,25(OH)2D3: other derivatives of vitamin D3 also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1 alpha,25(OH)2D3. JB6 cells were found to contain a receptor specific for 1 alpha,25(OH)2D3 with a Kd of 55.7 pM and Nmax of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1 alpha,25(OH)2D3 also contained the receptor. DNA-cellulose chromatography showed that a 1 alpha,25(OH)2D3-receptor complex interacted with DNA. In contrast to 1 alpha,25(OH)2D3, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1 alpha,25(OH)2D3 when applied with it.