密骨胶囊和健脾方对切卵大鼠小肠及腓肠肌中VDR mRNA表达的影响

目的：观察密骨胶囊、健脾方对大鼠小肠、腓肠肌中维生素D受体（vitamin D receptor,VDR）mRNA表达的影响,探讨补肾、健脾不同功效的中药复方,是否对机体不同组织器官VDR mRNA的表达存在一定选择性。方法：复制切卵大鼠骨质疏松模型,随机分为假切组、模型组、密骨胶囊组和健脾方组,术后12周开始灌胃,灌胃12周后取材。测定骨密度、子宫重量、腓肠肌重／体重,同时对大鼠小肠、腓肠肌中VDR mRNA进行半定量。结果：密骨胶囊能提高切卵大鼠骨密度（P〈0．05）,提高腓肠肌重／体重（P〈0．05）,上调小肠和腓肠肌中VDR mRNA的表达（P〈0．01）;健脾方能维持大鼠骨密度（P〉0．05）,上调腓肠肌中VDR mRNA的表达（P〈0．01）;密骨胶囊、健脾方均无增加子宫重量作用。结论：密骨胶囊和健脾方能通过上调切卵大鼠小肠和腓肠肌中VDR mRNA的表达,而起到治疗骨质疏松症的作用。     

1，25（OH）2D3对脂多糖作用下大鼠腹膜间皮细胞维生素D受体及肿瘤坏死因子α、转化生长因子β1表达的影响

目的 研究脂多糖（LPS）对大鼠腹膜间皮细胞（RPMC）维生素D受体(VDR)及肿瘤坏死因子α（TNF-α）、转化生长因子β1（TGF-β1）表达的影响,从而为1,25（OH）2D3在腹膜透析相关腹膜炎中的应用提供理论依据。 方法 胰蛋白酶消化法原代培养腹膜间皮细胞、传代、经鉴定后分组：(1)正常对照组;(2)脂多糖组：不同浓度的脂多糖（1、10、100 mg/L）分别作用6 h;10 mg/L脂多糖分别作用2、6、12 h;(3)1,25（OH）2D3作用组：10 mg/L脂多糖预孵育2 h后,加1,25（OH）2D3(10－8 mol/L、10－7 mol/L、10－6 mol/L)再作用6 h。RT-PCR法检测VDR mRNA的表达;Western印迹法检测VDR蛋白表达;ELISA法检测上清液TNF-α、TGF-β1的表达。 结果 与对照组相比,LPS组RPMC VDR mRNA和蛋白表达均显著下调（均P < 0.05）。与LPS组相比,1,25（OH）2D3组VDR mRNA和蛋白表达均显著上调（均P < 0.01）。LPS组上清液中TNF-α、TGF-β1浓度均显著高于对照组（均P < 0.01）;1,25（OH）2D3组上清液中TNF-α、TGF-β1浓度均显著低于LPS组（均P < 0.01）。 结论 LPS能下调RPMC VDR mRNA和蛋白的表达,上调TNF-α、TGF-β1表达。1,25（OH）2D3可逆转LPS的作用,上调RPMC VDR mRNA和蛋白的表达,并下调TNF-α、TGF-β1表达。VDR对腹膜透析相关腹膜炎具有一定的保护作用,并具有抑制腹膜纤维化的作用。     

维生素D分子机制及其与成纤维生长因子23、Klotho 相关性的新进展

维生素D（ VD）的经典作用为调节钙磷和骨代谢,同时还与免疫系统、细胞增殖和分化等有重要联系。1α,25－羟基维生素D（1,25（OH）D）配体结合维生素D受体（VDR）引发VDR与维甲酸X受体（RXR）紧密结合,且配体结合的VDR－RXR异二聚体识别VD调控基因序列中的维生素D应答元件（VDREs）。虽然1,25（OH）D－VDR可通过非基因机制快速发挥作用,但1,25（OH）D－VDR主要通过基因机制实现功能。1,25（OH）D－VDR可控制基因转录,VD配体、VDRE的DNA序列以及招募的共激活因子／共阻遏因子都能影响基因表达。1,25（OH）D－VDR调节基因的VDREs具有重要功能。通过推测RANKL基因染色质成环模型表明DNA成环和染色质的结构在VD调节基因表达的作用中发挥主要作用。1,25（OH）D－VDR调节基因表达可以延缓衰老和老年性疾病,比如癌症、2型糖尿病和心血管疾病。针对VD在磷酸盐代谢与衰老方面的关系已有了新的认识,认为1,25（OH）D－VDR诱导的骨骼中FGF23和肾脏中Klotho蛋白在该代谢途径中发挥重要作用,VD调节磷酸盐稳态可能是延缓衰老及相关慢性疾病的机制。     

健脾清化方对局灶节段硬化模型大鼠α平滑肌肌动蛋白和Ⅳ型胶原的影响

目的探讨健脾清化方对阿霉素致局灶节段硬化（focalsegmentalglomerularsclerosis,FSGS）模型大鼠肾功能的影响。方法左侧。肾切除加尾静脉注射阿霉素致大鼠肾脏FSGS,观察健脾清化方对模型大鼠0t平滑肌肌动蛋白（α-smoothmuscleactin,α-SMA）mRNA、lV型胶原（collagentypeIV,ColⅣ）mRNA的作用。结果与模型组比较,健脾清化方能显著降低α-SMAmRNA、ColⅣmRNA水平,且优于尿毒清组。结论健脾清化方对阿霉素肾病模型大鼠的肾纤维化有一定的改善作用,且疗效优于尿毒清。     

纳米钙补肾中药对骨质疏松症大鼠肠黏膜中 1，25(OH)2D3受体的mRNA和蛋白表达影响

目的研究骨质疏松症大鼠肠黏膜中维生素1,25(OH)2D3受体的mRNA和蛋白表达,揭示骨质疏松症的发病机制;同时,阐述纳米钙补肾中药的调节作用机制。方法切除大鼠双侧卵巢的方法复制骨质疏松症模型,采用纳米钙组及具有益肾填精、补钙壮骨作用的纳米钙补肾中药(大、中、小)剂量对实验大鼠治疗12周,以骨疏康颗粒剂、雌二醇(E2)及钙尔奇D600作为阳性对照组,正常大鼠和模型空白组为空白对照组;用PCR法、Western印迹法检测骨质疏松症大鼠肠黏膜中维生素1,25(OH)2D3受体的mRNA和蛋白表达。结果RT-PCR及Western方法检测,正常大鼠肠黏膜中含有维生素1,25(OH)2D3受体的mRNA和蛋白表达;模型空白组大鼠肠黏膜组织中的mRNA和蛋白表达水平有明显的下降。纳米钙、纳米钙补肾中药各剂量组、钙尔奇D600组、骨疏康和雌二醇E2组用药后12周,与模型空白组比较,可不同程度上调肠黏膜组织中的mRNA和蛋白表达水平。但其中各治疗用药组的效果有所不同,一般表现为纳米钙补肾中药剂量组作用较好。结论①正常大鼠肠黏膜组织可以在基因、蛋白水平表达维生素1,25(OH)2D3受体,而且在肠钙吸收中可能起到重要作用。②大鼠骨质疏松症的发生,与肠黏膜组织中维生素1,25(OH)2D3受体的mRNA和蛋白表达的变化有关,从而影响肠钙的吸收。③纳米钙补肾中药通过调控大鼠肠黏膜维生素1,25(OH)2D3受体的mRNA和蛋白表达,促进肠钙的吸收,对于骨质疏松症有一定的防治作用。     

25( OH) D3、维生素D受体与糖皮质激素性骨质疏松关系的研究

目的 观察地塞米松(Dex)不同作用时间对大鼠骨量、骨组织维生素D受体（VDR)和血清25羟维生素D3(25( OH) D3)的影响。方法 40只3.5月龄的sprague-dawley( SD)雌性大鼠,随机分为Dex组和对照组,每组20只,分别干预4周和9 周。Dex组肌肉注射Dex 2.5 mg/kg,每周2次,对照组注射等量生理盐水。干预4周和9周分别用双能X线骨密度仪测骨密度,逆转录聚合酶链反应和免疫组化法检测骨组织VDR mRNA和蛋白的表达水平,用ELISA法检测血清25( OH) D3的含量。 结果干预4周,Dex组大鼠体重下降(56. 67 ±24. 43) g,9周下降(85. 83 ±26. 35) g( P < 0. 05); Dex组体重低于对照组（P < 0.01)。干预9周,Dex组骨密度低于对照组(P < 0. 05)。干预4周,Dex组大鼠骨组织VDR mRNA表达高于对照组（1. 48 士 0. 32 vs 1. 15 ±0. 19)( P <0.05);9 周,Dex 组大鼠骨组织 VDR mRNA 表达低于对照组（1. 07 ± 0. 35 vs 1.38 ±0. 29) (P <0.05.。Dex组大鼠骨组织VDR蛋白表达在4周和9周与对照组均无差异(P >0.05)。干预4周,Dex组血清25( OH) D3含 量与对照组无差异（P >0.05);9周,Dex组血清25( OH) D3含量低于对照组（P <0.05)。结论 SD大鼠肌肉注射Dex 2.5mg/kg,每周2次,9周能成功建立糖皮质激素性骨质疏松模型。Dex可能通过减少VDR的转录和25( OH) D3的含量来引起骨密度下降,导致骨质疏松的发生。     

补肾方药对骨质疏松防治的实验研究

目的 探讨补肾方药对骨质疏松的防治作用及其机理。方法 本研究选用3种模型，即地塞米松和卵巢切除所诱发的骨质疏松模型，自然衰老的大鼠骨质疏松模型，并给予中药治疗。测定大鼠骨密度、骨代谢相关生化指标及骨形态计量学指标及基因表达分析。结果 补肾方药可显提高地塞米松所致骨质疏松大鼠的骨密度、血清骨钙素水平，降低尿钙排泄，同时，促进骨组织中I型胶原和LMP－1 mRNA表达，补肾方药还提高小肠黏膜CaBp-D9K基因表达，从而使小肠黏膜钙结合蛋白（CaBp)的合成增加，促进小肠对钙的吸收；补肾方药可提高卵巢切除所诱发的骨质疏松大鼠的骨密度，促进骨组织中I型胶原mRNA及小肠黏膜中VDR mRNA的表达，提高卵巢切除大鼠血清雌激素的水平，同时，提高骨组织中雌激素受体α和β（ERα，ERβ）的表达，增加雌激素对骨代谢的调节作用；补肾方药改善老年骨质疏松大鼠骨代谢，降低尿钙排泄，通过提高骨转换，主要是促进骨形成防止骨量丢失。结论 补肾方药通过多环节、多途径，调节骨质疏松大鼠的骨形成与骨吸收，使其达到骨形成与骨吸收相偶联，而防治骨质疏松。     

1,25-二羟基维生素D3对致死剂量脂多糖攻击的大鼠CD_4~＋CD_（25）~＋Treg细胞的调节作用

目的：观察不同剂量1,25-二羟基维生素D3（1,25-（OH）2D3）短期应用对大鼠受致死剂量脂多糖（LPS）攻击后的保护作用,并探讨此保护作用是否与1,25-（OH）2D3对CD4＋CD25＋Treg细胞的调节作用有关。方法：将大鼠分成3个剂量组,每组20只。分别给予1,25-（OH）2D3 0.125μg/只、0.25μg/只、1μg/只灌胃,3次/周,共2周。另设对照组给予赋形剂。给药后第15天腹腔注射致死剂量LPS（10mg/kg）,每组10只用于观察96 h内的死亡率;其余10只大鼠注射LPS 6 h后抽取外周血并留取脾脏标本。流式细胞仪检测大鼠外周血及脾脏CD4＋CD25＋Treg细胞数量,实时定量PCR检测脾脏Foxp3mRNA水平,ELISA检测外周血IL-10和TGF-β水平。结果：用1,25-（OH）2D3预处理的各组大鼠死亡率均显著低于对照组,用药各组外周血及脾脏CD4＋CD25＋Treg细胞数量、脾脏Foxp3mRNA表达水平、外周血IL-10及TGF-β水平也显著高于对照组。结论：1,25-（OH）2D3能够有效保护大鼠抵抗致死剂量LPS的攻击,这种保护作用可能与1,25-（OH）2D3上调CD4＋CD25＋Treg细胞的数量和功能有关。     

阿胶强骨口服液对去卵巢骨质疏松大鼠骨密度、生物力学、25-(OH)D3和1,25-(OH)2D3的影响

目的：研究阿胶强骨口服液对去卵巢骨质疏松大鼠骨密度（BMD）、生物力学、25-（OH）D3和1，25-（OH）2D3，的影响，探讨阿胶强骨口服液治疗原发性骨质疏松症的疗效机制。方法：4月龄健康雌性SD大鼠36只，随机分为3组，每组12只，分别为模型组，假手术组，阿胶强骨口服液治疗组。除假手术组外所有大鼠手术摘除双侧卵巢后导致雌激素缺失从而诱导骨质疏松症模型，分别在实验的第4、8、12周采用DEXA法分析股骨头及粗隆部的骨密度，生物力学技术分析股骨头生物力学参数，酶联免疫吸附方法检测25-（OH）D3和1,25-（OH）2D3的含量。结果：阿胶强骨口服液治疗组与模型组比较，股骨头及粗隆部骨密度明显提高（P〈0．05）；最大载荷（ML）及最大压应变（MS）等指标明显增强（P〈0．05）；血液、肝脏和肾脏组织中25-（OH）D3和1，25-（OH）2D3的含量明显提高，且组间比较差异有统计学意义（P〈0．05）。阿胶强骨口服液治疗组与假手术组比较差异无统计学意义（P〉0．05）。结论：阿胶强骨口服液在雌激素缺失早期即可在蛋白水平上调节25-（OH）D3和1，25-（OH）2D3的表达，激活骨代谢，提高骨密度，增强骨质量，起到预防骨质疏松的作用。     

成纤维细胞生长因子23在血液透析患者磷和维生素D代谢中的作用

目的 探讨成纤维细胞生长因子23（FGF-23）在维持性血液透析（MHD）患者磷和维生素D代谢中的作用及相关调控机制。 方法 采用酶联免疫分析法（ELISA）对59例MHD患者（血透组）及20例健康志愿者（对照组）进行血清全段FGF-23测定，同时应用放免法测定血清1，25-二羟维生素D（1,25(OH)2VitD）水平。血透组患者测定血清白蛋白（Alb）、血红蛋白（Hb）、血肌酐（Scr）、尿素氮（BUN）、钙（Ca）、磷（P）及全段甲状旁腺激素（iPTH）等指标。 结果 血透组血清FGF-23水平明显高于对照组[（215.23±123.55）比（28.72±11.49） ng/L，P < 0.01]，而血清1,25(OH)2VitD水平明显低于对照组[（13.25±8.73）比（42.24±12.45） μg/L，P < 0.01]。Pearson相关分析显示，血透组血清FGF-23水平与血清P、Scr、Ca、iPTH及透析疗程时间呈正相关（P < 0.05）；与血清1,25(OH)2VitD水平和年龄呈负相关（P < 0.05）；而与性别、血压、血清Alb、Hb、BUN等指标无相关。多元回归分析显示，血清P、Ca、Scr、iPTH和1,25(OH)2VitD是影响血清FGF-23的主要变量，5者组成的模型解释了总变异的约62%（R2=0.623，P < 0.01）。 结论 MHD患者血清全段FGF-23水平明显增高，而1,25(OH)2VitD水平明显降低。FGF-23的调控是由复杂的多种因素共同作用的结果，血清P、Ca、Scr、iPTH和1,25(OH)2VitD是影响血清FGF-23水平的主要调控因子。     

1 Alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3 have anabolic effects on cortical bone, but induce intracortical remodeling at toxic doses in ovariectomized rats

It is well established that vitamin D metabolites have anabolic properties on cancellous bone in rats. However, few data are available on cortical bone effects of vitamin D metabolites. In this study, we examined the effects of the synthetic vitamin D analogs 1alpha-hydroxyvitamin D2 (1alpha(OH)D2) and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) on cortical bone of the tibial shaft in ovariectomized (OVX) rats using bone histomorphometry. Six-month-old Fischer 344 rats were either OVX or sham-operated (SHAM). OVX rats received vehicle, 1alpha(OH)D2 or 1alpha(OH)D3 orally via the diet in a dose range from 0.025 to 0.2 microg/kg/day. All animals were killed 3 months postsurgery after in vivo fluorochrome labeling. Relative to SHAM rats, vehicle-treated OVX rats showed a reduction in cortical bone area (%) due to expansion of the marrow cavity. Treatment of OVX rats with either 1alpha(OH)D2 or 1alpha(OH)D3 dose-dependently decreased marrow area, and increased cortical area, periosteal perimeter, and periosteal and endocortical bone formation rate compared with OVX vehicle controls. Interestingly, OVX animals receiving the highest doses showed intracortical resorption cavities, a phenomenon only exceptionally observed in rats. The intracortical hole area was significantly lower in 1alpha(OH)D2-treated compared with 1alpha(OH)D3-treated rats. We conclude that 1alpha(OH)D2 and 1alpha(OH)D3 prevent cortical bone loss in OVX rats and have anabolic effects on cortical bone at higher doses. However, very high, toxic doses of both vitamin D analogs induce intracortical remodeling as an untoward side effect.     

Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin-D28k and the 1,25-dihydroxyvitamin D3 receptor.

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.     

Vitamin D metabolites prevent vertebral osteopenia in ovariectomized rats

Summary The present study investigated the prophylactic effects of vitamin D metabolites and vitamin D metabolite combinations on static and dynamic, tetracycline-based, histomorphometric parameters in the axial skeleton of ovariectomized rats. Forty-three Fischer-344 rats (10 weeks old, 130 g each body weight, BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated into the following groups: SHAM; OVX; OVX+7.5 ng 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]/rat/day; OVX +15 ng 1,24R,25-trihydroxyvitamin D₃ [1,24,25-(OH)₃D₃]/rat/day; OVX+75 ng 24R,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃]/rat/day; OVX+7.5 ng 1,25(OH)₂D₃/rat/ day+15 ng 1,24,25(OH)₃D₃/rat/day; OVX+7.5 ng 1,25(OH)₂D₃/rat/day+75 ng 24,25(OH)₂D₃/rat/day. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were collected 12 and 16 weeks postovariectomy, respectively. Sixteen weeks after surgery, all rats were sacrificed, and the first lumbar vertebrae were processed undecalcified for histomorphometric analysis. Ovariectomy induced a highly significant reduction (P<0.001) of cancellous bone mass in the secondary spongiosa of the lumbar vertebral body. The bone loss in OVX rats was accompanied by a distinct elevation of all histomorphometric parameters of bone formation and resorption. 1,25(OH)₂D₃ and both vitamin D metabolite combinations significantly raised serum calcium levels and prevented the bone loss by inhibiting the increased bone resorption in OVX rats. In the applied dosage, 1,24,25(OH)₃D₃ and 24,25(OH)₂D₃ alone were ineffective in preserving the cancellous bone of the lumbar vertebra in OVX rats. We conclude that the oral prophylactic application of low doses of active vitamin D metabolites can effectively prevent the osteopenia induced by ovariectomy in the axial skeleton of the rat.     

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.     

Increased vitamin D receptor level enhances 1,25-dihydroxyvitamin D3-mediated gene expression and calcium transport in Caco-2 cells.

Altered vitamin D receptor (VDR) level has been proposed to explain differences in intestinal responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We tested whether the enterocyte VDR level influences 1,25(OH)2D3-mediated gene expression and transepithelial calcium (Ca) transport in the human intestinal cell line Caco-2. Cells were stably transfected with a human metallothionein (hMT) IIA promoter-human VDR (hVDR) complementary DNA (cDNA) transgene that overexpressed hVDR in response to heavy metals. In MTVDR clones, induction of 25-hyroxyvitamin D3-24-hydroxylase (24-OHase) messenger RNA (mRNA) expression by 1,25(OH)2D3 (10(-9) M, 4 h) was correlated to metal-induced changes in nuclear VDR level (r2 = 0.99). In MTVDR clones, basal VDR level was 2-fold greater and 1,25(OH)2D3-mediated Ca transport (10(-7) M, 24 h) was 43% higher than in parental Caco-2 cells. Treatment of MTVDR clones with Cd (1 microM, 28 h) increased VDR level by 68%, significantly enhanced 1,25(OH)2D3-mediated Ca transport by 24%, and increased accumulation of calbindin D9K mRNA by 76% relative to 1,25(OH)2D3 alone. These observations support the hypothesis that the enterocyte VDR level is an important modulator of intestinal responsiveness to 1,25(OH)2D3.     

Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog,EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.     

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats.

The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats. INTRODUCTION: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known. MATERIALS AND METHODS: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots. RESULTS AND CONCLUSIONS: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.