1,25（OH）2D3通过STAT5抑制Th17细胞分化的调控作用研究*

目的 研究1.25二羟基维生素D₃[1,25（OH）₂D₃]抑制辅助性T细胞17（Th17）的分化与STAT5调控的关系。方法 通过分选出的CD4⁺T细胞,在1,25（OH）₂D₃和/或STAT5抑制剂的作用下,采用ELISA法检测抑制剂处理后细胞培养上清液Th17细胞因子（IL-17A、IL-22）水平的变化;采用细胞免疫荧光技术检测STAT5的磷酸化水平,采用Western blot技术检测STAT5蛋白表达水平。结果 1,25（OH）₂D₃组细胞培养上清IL-17A和IL-22水平（12.5±0.5 ng/ml,48.5±0.9 pg/ml）明显低于对照组（22.7±0.5 ng/ml,73.8±1.9 pg/ml）,而STAT5抑制剂组IL-17A和IL-22水平明显升高（33.5±0.7 ng/ml,89.1±1.4 pg/ml）,1,25（OH）₂D₃与STAT5抑制剂联合作用细胞IL-17A和IL-22水平（18.5±0.7 ng/ml,54.1±1.6 pg/ml）显著高于1,25（OH）₂D₃组,但低于STAT5抑制剂组,差异有统计学意义（P<0.01）;1,25（OH）₂D₃组细胞p-STAT5表达显著强于对照组,1,25（OH）₂D₃联合STAT5抑制剂组p-STAT5表达量低于对照组,而STAT5抑制剂组细胞p-STAT5表达量最低;1,25（OH）₂D₃组STAT5蛋白表达明显升高,而1,25（OH）₂D₃联合STAT5抑制剂组或STAT5抑制剂组STAT5蛋白表达明显降低,以STAT5抑制剂组细胞表达最弱,差异均具有统计学意义（P<0.01）。结论 1,25（OH）₂D₃通过STAT5信号通路能抑制Th17细胞分化。     

Effect of vitamin D deficiency and 1,25-dihydroxycholecalciferol treatment on epidermal calcium-binding protein (ECaBP) RNA activity

We have previously reported that the amount of epidermal calcium binding protein (ECaBP) in the skin decreases in the absence of vitamin D. Since vitamin D influences epidermal differentiation, and the synthesis of ECaBP may vary with cell differentiation, it was necessary to know whether vitamin D acts directly on the translational or post-translational level of ECaBP synthesis or indirectly by its action on epidermopoiesis. The cell-free translation technique was used to demonstrate the presence of mRNA coding for ECaBP. The activity of this mRNA has been evaluated in the skin of vitamin D-fed and in vitamin D-deficient rats with or without treatment with 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃). Vitamin D deficiency decreased the ECaBP mRNA activity. The latter was selectively increased in animals given a single dose of 1,25(OH)₂D₃. These results suggest that 1,25(OH)₂D₃ stimulates the production of ECaBP mRNA or stabilizes this mRNA.     

Evidence for the participation of protein kinase C and 3′,5′-cyclic AMP-dependent protein kinase in the stimulation of muscle cell proliferation by 1,25-dihydroxy-vitamin D3

Treatment with 1,25-dihydroxy-vitamin D₃ (1,25(OH)₂D₃) (1–12 h, 10⁻¹⁰ M) stimulates DNA synthesis in proliferating myoblasts, with an early response at 2–4 h of treatment followed by a maximal effect at 10 h. To investigate the mechanism involved in the mitogenic action of the hormone we studied the possible activation of intracellular messengers by 1,25(OH)₂D₃. The initial phase of stimulation of [³H]thymidine incorporation into DNA by the sterol was mimicked by the protein kinase C activator tetradecanoylphorbol acetate (TPA) in a manner which was dose dependent and specific as the inactive analog 4-phorbol was without effect. Maximal responses to TPA (100 nM) were obtained at 4 h. Staurosporine, a protein kinase C inhibitor, blocked the effect of 1,25(OH)₂D₃ on myoblast proliferation at 4 h. In addition, a fast (1–5 min) elevation of diacylglycerol levels and membrane-associated protein kinase C activity was observed in response to 1,25(OH)₂D₃. The adenylate cyclase activator forskolin (20 μM) and dibutyryl-cAMP (50 μM) increased DNA synthesis reproducing the second 1,25(OH)₂D₃-dependent stimulatory phase at 10 h. Inhibitors of protein kinase A blocked the increase in muscle cell DNA synthesis induced by 1,25(OH)₂D₃ at 10 h. Significant increases in cyclic AMP levels were detected in myoblasts treated with the sterol for 1–10 h. The calcium channel antagonist nifedipine (5–10 μM) abolished both the effects of 4-h treatment with 1,25(OH)₂D₃ or TPA and 10-h treatment with 1,25(OH)₂D₃ or dibutyryl-cAMP. Similar to the calcium channel agonist Bay K8644, 1,25(OH)₂D₃ stimulated myoblast ⁴⁵Ca uptake and its effects were blocked by nifedipine. Our results suggest that activation of calcium channels by phosphorylation via protein kinases C and A and is involved in the mitotic response of myoblasts to 1,25(OH)₂D₃.     

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)₂D₃ concentrations decreased with age in rats of both genders. Abundance of calbindin-D_28K and its mRNA in kidney and calbindin-D_9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)₂D₃ may be responsible for low plasma 1,25-(OH)₂D₃ concentrations observed in older animals. Plasma PTH and 1,25-(OH)₂D₃ concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D_9K and calbindin-D_9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)₂D₃ concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)₂D₃-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.     

Prostate cancer cell type-specific regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases the growth and enhances the osteolytic effects of prostate cancer cells, it is important to control the level of PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-calcemic analogue, EB1089, suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines PC-3 and LNCaP. The human PTHrP gene contains a sequence element homologous to the negative vitamin D response element within the parathyroid hormone gene. This DNA sequence (nVDRE(hPTHrP)) bound the vitamin D receptor (VDR) present in nuclear extracts from both PC-3 and LNCaP cells. However, when cloned upstream of the SV40 promoter and transiently transfected into PC-3 and LNCaP cells, nVDRE(hPTHrP) downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP, but not in PC-3, cells. These results may help to explain why some prostate cancers appear to be refractory to treatment with vitamin D analogues.     

Demonstration of a high affinity receptor for 1,25-dihydroxyvitamin D3 in rat pancreas

The existence of a high affinity receptor for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in rat pancreas was biochemically demonstrated in this study. In order to study the properties of this putative receptor, we took advantage of the analysis of low ionic strength chromatin-localized 1,25(OH)₂D₃ receptor. Using this method, the susceptibility of receptor protein to enzymatic degradation was so decreased, and the contamination by plasma vitamin D binding protein (DBP) component was so efficiently eliminated that a specific, saturable binding for 1,25(OH)₂D₃ could be demonstrated in the saturation analysis and the peak for the receptor was consistently apparent in the sucrose density gradient analysis. The equilibrium dissociation constant (Scatchard K_d) was found to be 3.7 ± 1.5 × 10^-10 (M), and the concentration of specific binding sites was calculated to be 1.22 ± 0.40 (fmol/mg protein). The number of specific binding sites in the rat pancreas was only 0.44% of that present in rat intestine (277 ± 19 (fmol/mg protein)) and 6.7% of that in rat kidney (18.1 ± 1.0 (fmol/mg)). However, when a correction is made for the 1,25(OH)₂D₃ receptor distribution in the tissues and expressed as the receptor concentration per receptor-containing cells, the rat pancreatic receptor level was calculated to be about 30% of the rat intestine. Sucrose density gradient sedimentation of this receptor yielded a value of 3.2 ± 0.1 (S) for the sedimentation coefficient and this peak was displaceable by a 100-fold excess of nonradioactive 1,25(OH)₂D₃. These data provide evidence for the presence of a specific 1,25(OH)₂D₃ receptor in mammalian pancreas, and we suggest, in conjunction with the facts that vitamin D₃ and its active metabolites play a physiological role on insulin secretion from rat pancreatic β-cells, that this receptor might be involved in the mechanisms of action of vitamin D₃ on insulin secretion from rat pancreas via a genomic effect.     

1,25（OH）2D3通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察

目的 体内体外观察1,25（OH）₂D₃通过调节miR-146a水平抑制大鼠肝纤维化的作用机制。方法 建立CCl₄诱导的大鼠肝纤维化模型,体外转染肝星状细胞（HSCs）miR-146a 模拟剂/抑制剂,观察1,25（OH）₂D₃处理对动物肝组织变化和HSC增殖和凋亡的影响。采用qPCR法检测肝组织miR-146a水平,采用CCK8法检测细胞增殖,使用流式细胞术检测HSC凋亡。结果 在干预8 w末,1,25（OH）₂D₃干预组大鼠肝纤维化程度明显减轻; 1,25（OH）₂D₃干预组大鼠肝组织miR-146a水平为（0.70± 0.03）,显著高于橄榄油组【（0.33±0.17,P<0.05）】; 1,25（OH）₂D₃组细胞增殖率为58.8%,较DMSO组下降了15.9%,转染miR-146a 模拟剂组大鼠HSC增殖率为46.5%,较对照组下降了53.3%,转染miR-146a 抑制剂组HSC增殖率为132.8%,较对照物组升高了32.8%（P<0.05）,1,25（OH）₂D₃干预组细胞凋亡率为12.6%,较DMSO组增加了5.2%,转染miR-146a 模拟剂组细胞凋亡率为16.8%,较对照组细胞凋亡率增加了8.2%,转染miR-146a 抑制剂组细胞凋亡率为6.3%,较对照组细胞凋亡率减少了2.2%（P<0.05）,提示1,25（OH）₂D₃具有抑制HSC增殖、促进凋亡作用。结论 1,25（OH）₂D₃可能通过调节miR-146a水平抑制HSC活化和抑制大鼠肝纤维化。     

丁酸钠和1，25-（OH）2D3对人结肠癌细胞增殖和hTERT表达的影响

背景：端粒酶在肿瘤细胞永生化过程中起重要作用．人端粒酶逆转录酶（hTERT）是调节端粒酶活性的关键因素。有研究发现生物活性制剂丁酸钠和1n,25-二羟维生素D3[1．25-（OH）,D3]具有潜在抗肿瘤效应。目的：观察丁酸钠和1,25-（OH）：D3对人结肠癌细胞增殖的影响及其可能机制。方法：以不同浓度丁酸钠（0．5～2．0mmol／L）、1,25-（0H）2D3（10^-6～10^-6mol/L）或两者联合[1．0mmol／L丁酸钠＋10^-7mol／L1,25-（OH）2D3]诱导人结肠癌细胞株HT29,MTT法检测细胞生长抑制率,流式细胞术检测细胞周期和细胞凋亡,RT—PCR检测hTERTmRNA表达。结果：丁酸钠和1,25-（OH）2D3能剂量和时间依赖性地抑制HT29细胞生长：1．0mmol／L丁酸钠和10^-7mol／L 1．25-（OH）2D3能诱导HT29细胞G0／G1期阻滞和细胞凋亡,抑制hTERTmRNA表达。联合用药组的上述作用较两者单用更为明显（P〈0．05）。结论：丁酸钠和1,25-（OH）2D3抑制人结肠癌细胞增殖的作用与其通过下调hTERT基因表达而抑制端粒酶活性、阻滞细胞周期、诱导细胞凋亡有关：两者联合可产生协同作用．     

Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.     

Epidermal growth factor and calcitriol synergistically induce osteoblast maturation

Calcitriol (1,25(OH)₂D₃) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.     

1,25-Dihydroxy-vitamin D3 (calcitriol)-dependent protein phosphorylation in rat duodenum: effects of ageing

We have examined the ability of 1,25(OH)₂-vitamin D₃ [1,25(OH)₂D₃; calcitriol], the hormonal form of vitamin D₃, to stimulate the phosphorylation of proteins in rat duodenum from young (3 months) and aged (22–24 months) rats. Brief (30 s) exposure of duodenum preincubated with ³²P-orthophosphate to the hormone increased the labeling of whole tissue proteins, an effect that was greatly diminished in aged animals. The response was dose-dependent, with maximal stimulation achieved at 1 nM calcitriol (+113% and +10% for young and aged rats, respectively). Phosphoproteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography. The hormone potentiated the phosphorylation predominantly on serine, threonine, and tyrosine residues of five acidic proteins of relative molecular masses of 66, 48, 45, 28, and 16 kDa. Moreover, the effects of calcitriol were exerted at the membrane level and varied as a function of exposure time. Direct treatment of purified basal lateral membranes for 30 s with the hormone (1 nM) stimulated the incorporation of ³²P of a 66kDa protein by 75% and of a 48 and 45kDa proteins by 60%. The effects of the hormone on basal lateral membrane protein phosphorylation were suppressed by the PKA, PKC, and tyrosine kinase inhibitors, Rp-cAMPS, bisindolylmaleimide, and genistein, respectively. In basal lateral membrane isolated from old animals, only minor changes in calcitriol–induced protein phosphorylation of the 66-kDa protein were observed. Taken together, these results suggest that calcitriol modulates duodenal membrane protein phosphorylation, at least in part through PKA, PKC, and tyrosine kinases, and that this mechanism is severely altered with ageing. The identity of the proteins whose phosphorylation was stimulated by calcitriol and their physiological role is currently under investigation.     

The regulation of aldosterone synthase expression

Vitamin D 1alpha-hydroxylase (1alpha(OH)ase), which converts the circulating prohormone 25-hydroxyvitamin-D(3) (25(OH)D(3)) to the active 1alpha-25-dihydroxyvitamin-D(3) (1,25(OH)(2)D(3)), is present in normal prostatic epithelium. However, prostate cancer cells, both primary cultured cells and cell lines, have greatly decreased activity of 1alpha(OH)ase and are therefore resistant to the tumor suppressor activity of circulating 25(OH)D(3). We quantitated 1alpha(OH)ase mRNA and protein levels to investigate mechanism(s) responsible for decreased 1alpha(OH)ase enzymatic activity in prostate cancer. Prostate cancer cell lines had low 1alpha(OH)ase mRNA levels. Primary prostate cell cultures derived from normal and cancer tissues had equivalent levels of 1alpha(OH)ase RNA and protein. Equivalent 1alpha(OH)ase protein levels were observed in prostate tissue sections containing normal and malignant cells. The protein levels of hsc70, whose homolog intracellular Vitamin D binding protein (IDBP-1) facilitates delivery of 25(OH)D(3) to 1alpha(OH)ase in monkey cells, were equivalent in the normal and cancer cells. Equivalent activity in normal and cancer cells of Vitamin D 24-hydroxylase, a mitochondrial enzyme that also uses 25(OH)D(3) as a substrate, further ruled out lack of access to substrate as a basis for low activity of 1alpha(OH)ase in cancer cells. We conclude that diminished 1alpha(OH)ase activity in prostate cancer cell lines is through decreased gene expression, whereas decreased activity in primary cultures and tissues is post-translational.     

Parathyroid hormone-related protein in hypercalcaemia associated with haematological malignancy

The incidence of hypercalcaemia and its association with humoral mechanisms involving parathyroid hormone-related protein (PTHrP), parathyroid hormone (PTH), or 1,25(OH)₂ vitamin D were assessed in a prospective study of patients admitted to a clinical haematology unit. Hypercalcaemia was detected in 18/165 patients, and was due to primary hyperparathyroidism in 3/17 patients in whom results of humoral mediator assessments were obtained. In the other patients, hypercalcaemia was associated in nine instances with myeloma, in five with B-cell non-Hodgkin's lymphoma (NHL), and in one with myeloid neoplasia. No evidence was obtained of a humoral mechanism involving 1,25(OH)₂ vitamin D, but elevated circulating levels of PTHrP, comparable with those in humoral hypercalcaemia of malignancy, were present in 2/4 patients with NHL, and in 3/9 with myeloma. The relationship between presence or absence of elevated circulating PTHrP, and presence or absence of hypercalcaemia during the course of treatment, indicated PTHrP was involved in the production of hypercalcaemia. Such an association raises the possibility that PTHrP released by neoplastic cells in these disorders acts in a paracrine manner to produce local bone resorption, and when produced in greater amounts causes elevated circulating levels which make an additional humorally-mediated contribution to the development of hypercalcaemia.     

Regulation of proliferation of prostate epithelial cells by 1,25-dihydroxyvitamin D3 is accompanied by an increase in insulin-like growth factor binding protein-3

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to regulate the proliferation of human prostate epithelial cell lines. Since the insulin-like growth factor (IGF) system is involved in the transformation process of epithelial cells, the following study was undertaken to determine if the IGF system, in particular IGF binding protein-3 (IGFBP-3), is altered by 1,25-(OH)2D3 in normal prostate epithelial cells as part of a mechanism for inhibition of transformation. Two cell systems were used in this study: (1) primary cultures of benign human prostate epithelial cells (PECs) and (2) an SV40-T immortalized prostate epithelial cell line (P153) that is non-tumorigenic. 1,25-(OH)2D3 was added to parallel sets of PECs and P153 cells in addition to the presence or absence of IGF-I or des(1-3)IGF-I. Treatment with 1,25-(OH)2D3 resulted in significant growth inhibition of both PECs and P153 cells. Furthermore, 1,25-(OH)2D3 inhibited IGF-induced proliferation, but this was partially reversed by high concentrations of IGF-I. Western ligand blots of condition media demonstrated a significant increase in IGFBP-3; likewise Northern blots demonstrated an increase in mRNA for IGFBP-3. Proliferation assays using an antibody designed to block the IGF-independent effects of IGFBP-3 failed to reverse the inhibitory effect of 1,25-(OH)2D3. Thus, IGFBP-3 acts in an IGF-dependent manner to inhibit cell growth of benign prostate epithelial cells.