RESUMO
Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.
Assuntos
Pré-Eclâmpsia , Trofoblastos , Humanos , Gravidez , Feminino , Trofoblastos/metabolismo , Vitamina D/farmacologia , Pré-Eclâmpsia/genética , Calcitriol/metabolismo , Calcitriol/farmacologia , Linhagem Celular , Placenta , Hipóxia , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/farmacologia , Movimento Celular , Proteínas de Neoplasias/metabolismoRESUMO
Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.
Assuntos
Condrócitos , Solanum glaucophyllum , Ratos , Animais , Condrócitos/metabolismo , Animais Recém-Nascidos , Calcitriol/metabolismo , Ratos Wistar , Agrecanas/metabolismo , Fosfatase Alcalina , Cartilagem , Plantas , Vitamina D/metabolismo , Extratos Vegetais , Células CultivadasRESUMO
Cisplatin treatment is one of the most commonly used treatments for patients with cancer. However, thirty percent of patients treated with cisplatin develop acute kidney injury (AKI). Several studies have demonstrated the effect of bioactive vitamin D or calcitriol on the inflammatory process and endothelial injury, essential events that contribute to changes in renal function and structure caused by cisplatin (CP). This study explored the effects of calcitriol administration on proximal tubular injury, oxidative stress, inflammation and vascular injury observed in CP-induced AKI. Male Wistar Hannover rats were pretreated with calcitriol (6 ng/day) or vehicle (0.9% NaCl). The treatment started two weeks before i.p. administration of CP or saline and was maintained for another five days after the injections. On the fifth day after the injections, urine, plasma and renal tissue samples were collected to evaluate renal function and structure. The animals of the CP group had increased plasma levels of creatinine and of fractional sodium excretion and decreased glomerular filtration rates. These changes were associated with intense tubular injury, endothelial damage, reductions in antioxidant enzymes and an inflammatory process observed in the renal outer medulla of the animals from this group. These changes were attenuated by treatment with calcitriol, which reduced the inflammation and increased the expression of vascular regeneration markers and antioxidant enzymes.
Assuntos
Injúria Renal Aguda , Cisplatino , Ratos , Animais , Masculino , Cisplatino/farmacologia , Calcitriol/farmacologia , Calcitriol/metabolismo , Ratos Wistar , Antioxidantes/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Estresse Oxidativo , Inflamação/metabolismo , Rim/metabolismoRESUMO
BACKGROUND: Fetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals. METHODS: Female rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old. RESULTS: Vitamin D deficiency induced muscle atrophy in the male (M-VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast-twitch skeletal muscles [extensor digitorum longus (EDL)] from the M-VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross-sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M-CTRL; P < 0.05). M-VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF-1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser 473 Akt and Ser 21/9 GSK-3ß. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two-fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow-twitch skeletal muscles (soleus) from the M-VDD group as well as in both EDL and soleus muscles from the female offspring. CONCLUSIONS: These data show that maternal VDD selectively affects the development of type-II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast-twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.
Assuntos
Fibras Musculares de Contração Lenta , Deficiência de Vitamina D , Animais , Calcitriol/análise , Calcitriol/metabolismo , Calcitriol/farmacologia , Feminino , Glicogênio Sintase Quinase 3 beta/análise , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/farmacologia , Masculino , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/metabolismoRESUMO
The vitamin D receptor (VDR) constitutes a promising therapeutic target for the treatment of cancer. Unfortunately, its natural agonist calcitriol does not have clinical utility due to its potential to induce hypercalcemic effects at the concentrations required to display antitumoral activity. For this reason, the search for new calcitriol analogues with adequate therapeutic profiles has been actively pursued by the scientific community. We have previously reported the obtaining and the biological activity evaluation of new calcitriol analogues by modification of its sidechain, which exhibited relevant antiproliferative and selectivity profiles against tumoral and normal cells. In this work we conducted molecular modeling studies (i.e. molecular docking, molecular dynamics, constant pH molecular dynamics (CpHMD) and free energy of binding analysis) to elucidate at an atomistic level the molecular basis related to the potential of the new calcitriol analogues to achieve selectivity between tumoral and normal cells. Two histidine residues (His305 and His397) were found to exhibit a particular tautomeric configuration that produces the observed bioactivity. Also, different acid-based properties were observed for His305 and His307 with His305 showing an increased acidity (pKa 5.2) compared to His397 (pKa 6.8) and to the typical histidine residue. This behavior favored the pharmacodynamic interaction of the calcitriol analogues exhibiting selectivity for tumoral cells when VDR was modeled at the more acidic tumoral environment (pH â 6) compared to the case when VDR was modeled at pH 7.4 (normal cell environment). On the other hand, non-selective compounds, including calcitriol, exhibited a similar interaction pattern with VDR when the receptor was modeled at both pH conditions. The results presented constitute the first evidence on the properties of the VDR receptor in different physicochemical environments and thus represent a significant contribution to the in silico screening and design of new calcitriol analogues.
Assuntos
Modelos Moleculares , Receptores de Calcitriol/metabolismo , Calcitriol/metabolismo , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Receptores de Calcitriol/química , Microambiente TumoralRESUMO
The expression of vitamin D receptor (VDR) and 1,25-dihydroxyvitamin D3 [1,25(OH)D] levels exceed the values of controls in some but not all hypercalciuric stone formers (HSF). We aimed to evaluate serum 1,25(OH)D levels, the expression of VDR, CYP27B1, and CYP24A1 hydroxylases in HSF in comparison with normocalciuric stone formers (NSF) and healthy subjects (HS). Blood samples, 24-h urine collections and a 3-day dietary record were obtained from 30 participants from each of the groups. The expression of VDR, CYP27B1, and CYP24A1 was measured by flow cytometry. HSF presented significantly higher urinary volume, sodium, magnesium, oxalate, uric acid, and phosphorus than NSF and HS. Calcium intake was lower in HSF versus NSF and HS (442 ± 41 vs 594 ± 42 and 559 ± 41 mg/day, respectively, p = 0.027). Ionized calcium was significantly lower in HSF than NSF (1.29 ± 0.0 vs 1.31 ± 0.0 mmol/L, p < 0.01). Serum 1,25(OH)D was significantly higher in HSF and NSF than HS (22.5 ± 1.2; 22.2 ± 1.2 vs 17.4 ± 1.2 pg/ml, p = 0.007) but serum 25(OH)D, PTH, klotho and plasma FGF-23 did not differ between groups. VDR expression was higher in HSF and NSF than HS (80.8 ± 3.2; 78.7 ± 3.3 vs 68.6 ± 3.2%, p = 0.023). Although CYP27B1 and CYP24A1 expressions were similar among all groups, the ratio of 1,25(OH)D/CYP24A1 was higher in HSF and NSF than in HS (1.43 ± 0.25 and 0.56 ± 0.10 vs 0.34 ± 0.06, p = 0.00). Stone formers, regardless of urinary calcium excretion, had higher VDR expression and 1,25(OH)D levels than HS, even in ranges considered normal. Higher 1,25(OH)D/CYP24A1 ratio suggested a lower degradation of 1,25(OH)D by CYP24A1 in HSF and NSF.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcitriol/sangue , Hipercalciúria/patologia , Cálculos Renais/patologia , Receptores de Calcitriol/metabolismo , Vitamina D3 24-Hidroxilase/metabolismo , Adulto , Calcitriol/metabolismo , Cálcio/urina , Fator de Crescimento de Fibroblastos 23 , Voluntários Saudáveis , Humanos , Hipercalciúria/sangue , Hipercalciúria/complicações , Hipercalciúria/urina , Cálculos Renais/sangue , Cálculos Renais/etiologia , Cálculos Renais/urina , Leucócitos Mononucleares/metabolismo , MasculinoRESUMO
LCA and 1,25(OH)2D3 are vitamin D receptor ligands with different binding affinity. The secosteroid stimulates intestinal Ca2+ absorption. Whether LCA alters this process remains unknown. The aim of our work was to determine the effect of LCA on intestinal Ca2+ absorption in the absence or presence of NaDOC, bile acid that inhibits the cation transport. The data show that LCA by itself did not alter intestinal Ca2+ absorption, but prevented the inhibitory effect of NaDOC. The concomitant administration of LCA avoided the reduction of intestinal alkaline phosphatase activity caused by NaDOC. In addition, LCA blocked a decrease caused by NaDOC on gene and protein expression of molecules involved in the transcellular pathway of intestinal Ca2+ absorption. The oxidative stress and apoptosis triggered by NaDOC were abrogated by LCA co-treatment. In conclusion, LCA placed in the intestinal lumen protects intestinal Ca2+ absorption against the inhibitory effects caused by NaDOC. LCA avoids the reduction of the transcellular Ca2+ movement, apparently by blocking the oxidative stress and apoptosis triggered by NaDOC, normalizing the gene and protein expression of molecules involved in Ca2+ movement. Therefore, LCA might become a possible treatment to improve intestinal calcium absorption under oxidant conditions.
Assuntos
Cálcio/metabolismo , Ácido Desoxicólico/antagonistas & inibidores , Duodeno/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Ácido Litocólico/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Calcitriol/metabolismo , Galinhas , Ácido Desoxicólico/farmacologia , Duodeno/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Absorção Intestinal/fisiologia , Transporte de Íons/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
Factors affecting vitamin D metabolism may preclude anti-carcinogenic effects of its active metabolite calcitriol. Chronic ethanol consumption is an etiological factor for breast cancer that affects vitamin D metabolism; however, the mechanisms underlying this causal association have not been fully clarified. Using a murine model, we examined the effects of chronic moderate ethanol intake on tumoral and renal CYP27B1 and CYP24A1 gene expression, the enzymes involved in calcitriol synthesis and inactivation, respectively. Ethanol (5% w/v) was administered to 25-hydroxyvitamin D3-treated or control mice during one month. Afterwards, human breast cancer cells were xenografted and treatments continued another month. Ethanol intake decreased renal Cyp27b1 while increased tumoral CYP24A1 gene expression.Treatment with 25-hydroxyvitamin D3 significantly stimulated CYP27B1 in tumors of non-alcohol-drinking mice, while increased both renal and tumoral CYP24A1. Coadministration of ethanol and 25-hydroxyvitamin D3 reduced in 60% renal 25-hydroxyvitamin D3-dependent Cyp24a1 upregulation (P<0.05). We found 5 folds higher basal Cyp27b1 than Cyp24a1 gene expression in kidneys, whereas this relation was inverted in tumors, showing 5 folds more CYP24A1 than CYP27B1. Tumor expression of the calcitriol target cathelicidin increased only in 25-hydroxyvitamin D3-treated non-ethanol drinking animals (P<0.05). Mean final body weight was higher in 25-hydroxyvitamin D3 treated groups (P<0.001). Overall, these results suggest that moderate ethanol intake decreases renal and tumoral 25-hydroxyvitamin D3 bioconversion into calcitriol, while favors degradation of both vitamin D metabolites in breast cancer cells. The latter may partially explain why alcohol consumption is associated with vitamin D deficiency and increased breast cancer risk and progression.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Consumo de Bebidas Alcoólicas/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Etanol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vitamina D3 24-Hidroxilase/genética , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Neoplasias da Mama/complicações , Calcifediol/farmacologia , Calcitriol/metabolismo , Etanol/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Camundongos Nus , Vitaminas/farmacologiaRESUMO
Placenta is an important source and target of hormones that contribute to immunological tolerance and maintenance of pregnancy. In preeclampsia (PE), placental calcitriol synthesis is low; whereas pro-inflammatory cytokines levels are increased, threatening pregnancy outcome. Previously, we showed that calcitriol inhibits Th-1 cytokines under experimental inflammatory conditions in normal trophoblasts. However, a study of the regulation of inflammatory cytokines by calcitriol in trophoblasts from a natural inflammatory condition, such as PE, is still lacking. Therefore, the aim of the present study was to investigate calcitriol effects upon TNF-α, IFN-γ, IL-6 and IL-1ß in cultured placental cells from preeclamptic women by using qPCR and ELISA. Placentas were collected after cesarean section from preeclamptic women and enriched trophoblastic preparations were cultured in the absence or presence of different calcitriol concentrations during 24h. In these cell cultures, pro-inflammatory cytokines TNF-α and IL-6 secretion and mRNA expression were downregulated by calcitriol (P<0.05). No significant effects of calcitriol upon IFN-γ and IL-1ß were observed. In addition, basal expression of TNF-α, IL-6 and IL-1ß decreased as the cells formed syncytia. Our study supports an important autocrine/paracrine role of placental calcitriol in controlling adverse immunological responses at the feto-maternal interface, particularly in gestational pathologies associated with exacerbated inflammatory responses such as preeclampsia.
Assuntos
Calcitriol/farmacologia , Interleucina-6/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Calcitriol/metabolismo , Células Cultivadas , Regulação para Baixo , Feminino , Expressão Gênica/efeitos dos fármacos , Células Gigantes/metabolismo , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/metabolismo , Interleucina-6/biossíntese , Placenta/metabolismo , Gravidez , RNA Mensageiro/biossíntese , Trofoblastos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Environment may influence the development and prevention of cancer. Calcitriol has been associated with calcium homeostasis regulation. Many epidemiological, biochemical, and genetic studies have shown non-classic effects of vitamin D, such as its involvement in the progression of different cancers. Although vitamin D induces cellular arrest, triggers apoptotic pathways, inhibits angiogenesis, and alters cellular adhesion, the precise mechanisms of its action are still not completely established. This article will present a revision about the molecular aspects proposed to be involved in the anticancer action of calcitriol. Adequate levels of vitamin D to prevent cancer development will also be discussed.