1α,25-二羟维生素D_3对大鼠成骨细胞骨架、间隙连接通讯及[Ca~(2+)]_i的影响

目的研究不同浓度1α,25-二羟维生素D3[1,25-(OH)2·D3]0、10-9、10-8、10-7mol/L对体外培养3～4d龄SD大鼠成骨细胞(OB)骨架F-actin、间隙连接通讯(GJIC)及细胞内钙离子浓度([Ca2+]i)的影响。方法1,25-(OH)2·D3作用20min、24h后测定[Ca2+]i;作用24、48h后观察F-actin及GJIC。结果20min时,不同浓度1,25-(OH)2·D3组[Ca2+]i均显著高于对照组;24h时10-9mol/L组[Ca2+]i则显著低于对照组,其余各组间差异不显著。此时10-8、10-7mol/L组大部分细胞变得扁平,F-actin排列较对照组有序,形成应力纤维;48h时,对照组及10-9mol/L组F-actin表达减少,10-9mol/L组GJIC非常显著地弱于对照组,而10-8、10-7mol/L组大部分细胞F-actin表达完好,GJIC均非常显著地强于其余两组。结论高浓度1,25-(OH)2·D3能够维持OB形态,增强OB间通讯,而低浓度1,25-(OH)2·D3抑制细胞间通讯。     

1,25二羟维生素D3对肝癌HepG2细胞增殖侵袭影响

目的探讨1,25二羟维生素D3[1,25(OH)_2D_3]及PI3K/AKT信号通路抑制剂(LY294002)对人肝癌Hep G2细胞的增殖、侵袭影响及作用机制。方法实验设10~(-8)、10~(-7)、10~(-6)mol/L 1,25(OH)_2D_3组及2.5、5.0、10.0、20.0、40.0、80.0μmol/L LY294002组,10~(-7)mol/L 1,25(OH)_2D_3+5μmol/L LY294002联合组,噻唑蓝法检测人肝癌Hep G2细胞增殖抑制率;Compu Syn软件计算联合指数;Tanswell小室检测HepG2细胞侵袭数;Western blot检测Hep G2细胞增殖细胞核抗原(PCNA),细胞基质金属蛋白酶9(MMP-9)、磷酸化丝氨酸苏氨酸蛋白激酶(p-AKT)、10号染色体缺失且与张力蛋白同源物磷酸脂酶基因(PTEN)蛋白。结果 1,25(OH)_2D_3及LY294002对人肝癌HepG2细胞增殖抑制率呈时间-剂量依赖效应(P0.05);1,25(OH)2D3联合LY294002组细胞增殖抑制率明显高于二者单独处理组(P0.05),联合指数=0.728,2者具有协同效应;10-7mol/L 1,25(OH)2D3组、5μmol/L LY294002组以及联合组Hep G2细胞侵袭数[分别为(45.9±6.4)、(49.9±6.0)、(27.8±4.0)个]明显低于对照组[(64.6±8.0)个](P0.05)。与对照组比较,10-7mol/L 1,25(OH)2D3组及联合组HepG2细胞PTEN蛋白表达量增加,差异有统计学意义(P0.05),10-7mol/L 1,25(OH)_2D_3组、5μmol/L LY294002组及联合组HepG2细胞PCNA、MMP-9、p-AKT蛋白表达均降低,差异有统计学意义(P0.05),且联合组蛋白表达量低于二者单独处理组(P0.05)。结论 1,25(OH)_2D_3可抑制人肝癌HepG2细胞增殖、侵袭,其机制可能与上调PTEN表达,抑制PI3K/AKT信号通路活性,下调PCNA、M M P-9蛋白有关;与LY294002合用具有协同效应。     

1,25-(OH)_2D_3抑制人卵巢癌细胞SKOV-3增殖与调控microRNA-22的表达有关北大核心CSCD

目的通过观察1,25-(OH)2D3对SKOV-3细胞增殖和microRNA-22和microRNA-21表达的影响,探讨microRNAs在1,25-(OH)2D3抗肿瘤增殖中的作用。方法以不同浓度1,25-(OH)2D3(10-9、10-8、10-7mol/L)处理SKOV-3细胞,CCK-8法检测细胞增殖率,real time RT-PCR分析microRNA-22和microRNA-21的表达,流式细胞仪分析细胞周期,TUNEL法检测细胞凋亡。结果 1,25-(OH)2D3对SKOV-3细胞增殖有明显的抑制作用,并且存在着时间剂量依赖关系(P<0.01)。1,25-(OH)2D3可以降低microRNA-22的表达水平(P<0.05),但是不影响microRNA-21的表达。细胞周期分析显示,1,25-(OH)2D3可以诱导SKOV-3细胞阻滞于G0/G1,浓度越高阻滞越明显(P<0.05)。1,25-(OH)2D3促进SKOV-3细胞核内DNA断裂点增加,细胞凋亡增加(P<0.05)。结论在SKOV-3细胞中,1,25-(OH)2D3可能是通过抑制microRNA-22,促进细胞凋亡,调节细胞周期,抑制细胞增殖,从而到达抑制肿瘤的作用。[营养学报,2014,36(1):35-39]     

1,25-(OH)_2D_3抑制人卵巢癌细胞SKOV-3增殖与调控microRNA-22的表达有关

目的通过观察1,25-(OH)2D3对SKOV-3细胞增殖和microRNA-22和microRNA-21表达的影响,探讨microRNAs在1,25-(OH)2D3抗肿瘤增殖中的作用。方法以不同浓度1,25-(OH)2D3(10-9、10-8、10-7mol/L)处理SKOV-3细胞,CCK-8法检测细胞增殖率,real time RT-PCR分析microRNA-22和microRNA-21的表达,流式细胞仪分析细胞周期,TUNEL法检测细胞凋亡。结果 1,25-(OH)2D3对SKOV-3细胞增殖有明显的抑制作用,并且存在着时间剂量依赖关系(P<0.01)。1,25-(OH)2D3可以降低microRNA-22的表达水平(P<0.05),但是不影响microRNA-21的表达。细胞周期分析显示,1,25-(OH)2D3可以诱导SKOV-3细胞阻滞于G0/G1,浓度越高阻滞越明显(P<0.05)。1,25-(OH)2D3促进SKOV-3细胞核内DNA断裂点增加,细胞凋亡增加(P<0.05)。结论在SKOV-3细胞中,1,25-(OH)2D3可能是通过抑制microRNA-22,促进细胞凋亡,调节细胞周期,抑制细胞增殖,从而到达抑制肿瘤的作用。[营养学报,2014,36(1):35-39]     

活性维生素D_3对大鼠肾小球系膜细胞增殖影响

目的探讨活性维生素D3(1,25(OH)2D3)对大鼠系膜细胞增殖的影响。方法体外培养大鼠系膜细胞,随机分为正常对照组、表皮生长因子(EGF)(10 ng/m L)组、1,25(OH)2D3(10-8mol/L)组、EGF联合1,25(OH)2D3组,采用MTT法检测各组干预48 h后对大鼠系膜细胞增殖的影响,流式细胞术检测各组干预48 h后大鼠系膜细胞周期分布情况,免疫荧光法检测各组干预48 h后,大鼠系膜细胞中增生性细胞核抗原(PCNA)的表达情况。结果与正常对照组比较,EGF组能明显促进大鼠系膜细胞增殖,G0/G1期细胞减少,S期及G2/M期细胞增加,且PCNA表达增加,1,25(OH)2D3组大鼠系膜细胞增殖受抑制,G0/G1期细胞增加,S期及G2/M期细胞减少,且PCNA表达降低;与EGF组比较,EGF联合1,25(OH)2D3干预组系膜细胞增殖受抑制,G0/G1期细胞增多,S期及G2/M期细胞减少,且PCNA表达降低。结论 1,25(OH)2D3可阻滞大鼠系膜细胞的细胞周期并抑制大鼠系膜细胞的增殖和EGF对大鼠系膜细胞的促增殖作用。     

1,25-二羟维生素D_3对β淀粉样蛋白1-42诱导PC12细胞焦亡的保护作用

目的探讨1,25-二羟维生素D_3[1,25(OH)_2D_3]对β淀粉样蛋白1-42(Aβ_(1-42))诱导的PC12细胞焦亡的保护作用。方法 Aβ_(1-42)诱导PC12细胞构建体外阿尔茨海默病(Alzheimer’s disease, AD)细胞模型,设立对照组、模型组(20μmol/L Aβ_(1-42))及不同浓度1,25(OH)_2D_3预处理组[1、10、100 nmol/L 1,25(OH)_2D_3+20μmol/L Aβ_(1-42)]。CCK-8检测1,25(OH)_2D_3对Aβ_(1-42)诱导的PC12细胞活性,吖啶橙/溴乙锭(AO/EB)染色检测细胞膜通透性,比色法、酶联免疫吸附法检测乳酸脱氢酶(lactic dehydrogenase, LDH)和白细胞介素1β(interleukin-1β,IL-1β)水平,免疫蛋白印迹法检测NOD样受体家族蛋白(NOD-like receptor family protein 1, NLRP1)、含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1, caspase-1)和消皮素D(gasdermin D, GSDMD)蛋白表达。结果与对照组相比,模型组细胞活性显著降低(P0.01),细胞膜通透性、LDH、IL-1β、NLRP1、caspase-1和GSDMD蛋白显著升高(P0.01)。与模型组相比,3个1,25(OH)_2D_3预处理组的细胞活性均有所提高(P0.05),细胞膜破损减少。10和100 nmol/L 1,25(OH)_2D_3预处理组细胞LDH释放、IL-1β水平显著降低(P0.01)。1 nmol/L 1,25(OH)_2D_3组中NLRP1和GSDMD蛋白的表达量明显降低(P0.05),10和100 nmol/L 1,25(OH)_2D_3组降低更为显著(P0.01),10和100 nmol/L 1,25(OH)_2D_3组的caspase-1蛋白表达显著降低(P0.05,P0.01)。结论 1,25(OH)_2D_3对Aβ_(1-42)诱导的PC12细胞焦亡具有保护作用。     

1α,25-(OH)_2D_3对体外培养成骨细胞内钙离子浓度的影响

目的研究不同浓度1α,25-(OH)2D3(0、10-11、10-9、10-7 mol/L)对体外培养成骨细胞(osteoblasts,OB)内游离钙离子浓度([Ca2+]i)的影响及钙离子通道机制。方法在体外培养SD大鼠OB基础上,添加不同浓度的1α,25-(OH)2D3或/和10-8 mol/L硝苯地平(NIF)作用20 min,流式细胞仪测定[Ca2+]i。结果 10-11 mol/L 1α,25-(OH)2D3组[Ca2+]i显著低于对照组(P<0.05),10-9、10-7 mol/L组[Ca2+]i显著或极显著高于对照组(P<0.05或P<0.01);单独添加10-8 mol/L NIF组[Ca2+]i与对照组差异不显著(P>0.05);联合添加10-9 mol/L 1α,25-(OH)2D3和10-8 mol/L NIF组[Ca2+]i与对照组差异不显著(P>0.05),但显著低于单独添加10-9 mol/L 1α,25-(OH)2D3组(P<0.05)。结论 1α,25-(OH)2D3引起[Ca2+]i改变的过程涉及L-型钙离子通道。     

Ca~(2+)信号对1α,25-(OH)_2D_3调控破骨细胞形成及骨吸收活性的影响

目的探讨Ca2+信号对1α,25-二羟维生素D3(1α,25-(OH)2D3)调控破骨细胞(osteoclast,OC)形成及骨吸收活性的影响。方法在巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)及核因子-κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)联合诱导RAW264.7细胞分化为OC的基础上,添加10-8 mol/L 1α,25-(OH)2D3,并设Ca2+螯合剂1,2-双(2-氨基苯氧基)乙烷-N,N,N`,N`-四乙酸四乙酸甲酯(BAPTA-AM)干预组,通过抗酒石酸酸性磷酸酶(TRAP)染色鉴定OC的形成,环境扫描电镜观察牛皮质骨片吸收陷窝。结果添加10-8 mol/L 1α,25-(OH)2D3可显著增加OC数量,促进骨吸收活性。然而,与添加10-8 mol/L 1α,25-(OH)2D3比较,5μmol/L BAPTA-AM干预可显著降低OC数量,抑制骨吸收活性。结论 10-8 mol/L 1α,25-(OH)2D3能够促进OC形成和骨吸收活性,此过程涉及Ca2+信号机制。     

1,25二羟基维生素D3对大鼠脾调节性T细胞及其特异性转录因子表达的影响

目的:探讨用1,25二羟基维生素D3[1,25(OH)2VitD3]预干预对大鼠辅助性T细胞(Th1)免疫应答中脾CD4 CD25 调节性T细胞及其特异性转录因子Foxp3基因表达的影响. 方法:采用近交系大鼠,随机分为对照组、脂多糖(LPS)组和VitD3组.分别给予对照组和LPS组赋形剂,VitD3组用1,25(OH)2VitD3(0.25 μg/只·d-1),连续灌胃14 d.第15天LPS组和VitD3组腹腔注射致死剂量LPS(10 mg/kg).6 h后处死,用流式细胞仪检测脾CD4 CD25 调节性T细胞比例,用Realtime RT-PCR检测脾中Foxp3 mRNA的表达. 结果:1,25(OH)2VitD3明显上调大鼠Th1免疫应答中脾CD4 CD25 调节性T细胞的比例及其特异性转录因子Foxp3 mRNA的表达. 结论:1,25(OH)2VitD3预干预对大鼠Th1免疫应答中脾CD4 CD25 调节性T细胞及其特异性转录因子Foxp3的基因表达有显著影响.1,25(OH)2VitD3有可能通过促进CD4 CD25 调节性T细胞的发育,发挥其免疫调节作用.     

胰岛素对人成骨肉瘤MG-63细胞胰岛素受体底物-2基因表达的影响

目的观察胰岛素对人成骨肉瘤MG-63细胞胰岛素受体底物-2（IRS-2）基因mRNA表达的影响。方法用胰岛素干预人成骨肉瘤MG-63细胞，半定量RT-PCR检测细胞IRS-2基因mRNA的表达量。结果10^-10mol/L～10^-6mol/L胰岛素上调细胞IRS-2基因mRNA的表达（P＜0．05），且具有剂量依赖性。胰岛素的调节还存在时效依赖性，10^-8mol/L胰岛素干预12h可下调细胞IRS-2基因mRNA的表达（P＜0．05），干预24h～48h上调细胞IRS-2基因mRNA的表达（P＜0．05）。结论胰岛素可影响人成骨肉瘤MG-63细胞IRS-2基因mRNA的表达，胰岛素可能对1型糖尿病患者骨质疏松的发生和发展起着重要的作用。     

Effect of 17β-oestradiol on transepithelial calcium transport in human intestinal-like Caco-2 cells and its interactions with 1,25−dihydroxycholecalciferol and 9-cis retinoic acid

Background Oestrogen therapy helps prevent bone loss in postmenopausal women and corrects a decline in Ca absorption efficiency at the onset of menopause. However, the mechanism by which 17β-oestradiol (17β-E₂) stimulates Ca absorption is unclear. Oestrogen may exert its effect indirectly via increasing 1,25-dihydroxycholeciferol (1,25 (OH)₂D₃) or its receptor, or act more directly on the intestines via the oestrogen receptor (OR). Since oestrogen also increases retinol levels, this may influence Ca absorption. Aim To investigate the effect of 17β-E₂ alone and in combination with 1,25 (OH)₂D₃ on intestinal Ca uptake and absorption in Caco-2 cells cultured under deplete- and replete-9-cis retinoic acid (9-cis RA) conditions. Methods Twenty-one day-old Caco-2 cell monolayers (n 9 wells per treatment) were exposed to 9-cis RA-deplete and -replete media containing dimethyl sulfoxide (control), 10 nM-1,25 (OH)₂D₃, 10 nM-17β-E₂, or 10 nM-1,25 (OH)₂D₃ plus 10 nM-17β-E₂, for 48 h. Results 1,25 (OH)₂D₃ stimulated Ca uptake, total Ca transport, calbindin D_9K and CaT1 mRNA levels, while 17β-E₂ and 9-cis RA had no effect on Ca absorption or uptake. Nor did they augment the stimulatory effect of 1,25 (OH)₂D₃. Conclusion These in vitro findings suggest that oestrogen does not have a direct effect on intestinal Ca absorption.     

维生素D_3对报告载体荧光素酶表达的作用

目的：　研究１,２５－二羟维生素Ｄ３ 对结肠癌细胞系Ｃａｃｏ－２ 细胞中报告基因表达的作用,并探讨在报告载体ｐＧＬ２ 序列中存在潜在的抑制性维生素Ｄ应答元件（ＶＤＲＥ）的可能性。方法：　采用磷酸钙沉淀法将报告载体转染入Ｃａｃｏ－２ 细胞。Ｃａｃｏ－２细胞经不同浓度１,２５－二羟维生素Ｄ３ 处理后测定细胞裂解液中表达的荧光素酶活性。结果：　应用ｐＧＬ２ 报告载体时,当用ｐＳＧ５－ＶＤＲ表达载体共转染后,１,２５－二羟维生素Ｄ３显著地抑制Ｃａｃｏ－２ 细胞荧光素酶的表达（Ｐ＜ ０．０５）;而未使用该表达载体共转染则无抑制作用（Ｐ＞ ０．０５）。应用ｐＧＬ３ 报告载体时,不同浓度的１,２５－二羟维生素Ｄ３ 对ｐＬＧ３转染后Ｃａｃｏ－２ 细胞表达的荧光素酶活性均无显著抑制作用（Ｐ＞ ０．０５）,该作用不依赖是否存在有ｐＳＧ５－ＶＤＲ表达载体共转染。结论：１,２５－二羟维生素Ｄ３ 对报告载体ＰＧＬ２ 荧光素酶表达具有抑制作用,而对ｐＧＬ３ 则否;类似人类ＰＴＨ基因中的潜在抑制性ＶＤＲＥ存在于报告载体ｐＧＬ２,在ｐＧＬ３ 中该ＶＤＲＥ业已改变。     

1,25 dihydroxycholecalciferol-mediated calcium absorption and gene expression are higher in female than in male mice

Recent advances in bone and calcium (Ca) metabolism have relied upon genetically modified mice. However, although human studies have identified gender as an important modulator of Ca metabolism, its effect on Ca metabolism has not been examined in mice. Here we examined basal and vitamin D-regulated Ca absorption (in situ ligated loops) and mRNA levels for the apical membrane calcium channel, TRPV6, and the calcium binding protein, calbindin D(9k) (CaBP) mRNA levels (real-time PCR) in duodenum of female and male mice. At 2 mo of age, females fed a 5 g Ca/kg diet had higher Ca absorption (62.3 +/- 4.8 vs. 47 +/- 3.6%) and TRPV6 mRNA levels than males even though plasma 1,25 dihydroxyvitamin D [1,25(OH)(2) D] was not different. In mice fed high (20 g/kg), normal (5 g/kg), or low (0.2 g/kg) Ca diets for 7 d to alter plasma 1,25(OH)(2) D (91 +/- 12, 322 +/- 25, and 587 +/- 43 pmol/L, respectively), the relation between Ca absorption (slope = 0.116 vs. 0.084, P = 0.021) or duodenum TRPV6 mRNA (slope = 0.042 vs. 0.025, P = 0.034) and circulating 1,25(OH)(2) D was steeper in females. After a single 1,25(OH)(2) D injection (200 ng/100 g body weight), peak induction of TRPV6 mRNA was 2-fold greater (at 6 h) and CaBP mRNA was 20% higher in females (at 16 h). Duodenal vitamin D receptor mRNA levels did not differ between genders. Our data indicate that female mice are more sensitive to changes in serum 1,25(OH)(2) D levels than males and that this must be considered when using mice to study calcium and bone biology.     

Serum metabolite profiles and target tissue gene expression define the effect of cholecalciferol intake on calcium metabolism in rats and mice

We studied the effect of cholecalciferol (VD3) intake on VD3 status and markers of calcium (Ca) homeostasis in mice and rats. Serum 25 hydroxycholecalciferol (25OH-VD3) concentrations were increased in animals fed diets containing 400-20,000 international units (IU) VD3/kg (37 nmol.L(-1).1000 IU VD3(-1)), but body weight, serum Ca, and duodenal gene expression were not altered. High-VD3 intake decreased serum 1, 25-dihydroxycholecalciferol [1,25(OH)2-VD3] and renal 25 hydroxycholecalciferol-1alphahydroxylase (CYP27B1) mRNA, suggesting that rodents tolerate high-VD3 intake by suppressing the activity of the VD3 endocrine system. Serum 25OH-VD3 declined when animals were fed diets containing 1000 to 25 IU VD3/kg (9-11 wk, inflection at 200 IU/kg, 4-fold steeper slope below this). Neither body weight nor serum Ca were influenced by low-VD3 intake. However, mice fed the 25-IU/kg diet had lower serum 1,25(OH)2-VD3, duodenal calbindin D9k mRNA, bone mineral density, and renal 25 hydroxycholecalciferol-24 hydroxylase mRNA, whereas renal CYP27B1 mRNA was elevated when rodents were fed < 200 IU VD3/kg. These data reveal a stress on VD3 and Ca metabolism at low dietary VD3 intake. Dietary Ca restriction (0.25 vs. 0.5%, 9 wk) increased serum 1,25(OH)2-VD3 and was 30% greater in rats fed a 10,000-IU VD3/kg diet. High-VD3 intake did not prevent Ca restriction-induced bone loss. Our data show that modeling human VD3 status requires lower intake than the current NRC rodent requirement (1000-IU/kg diet). Also, although rodents are very tolerant of high-VD3 intake, it cannot compensate for moderate Ca restriction.     

Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA

To study the role of calbindin D(9k) (CaBP) and epithelial calcium channel ECaC2 in intestinal calcium (Ca) absorption, vitamin D receptor knockout (KO) and wild-type (WT) mice were fed either 0.5% Ca or a 2.0% Ca rescue diet starting at 21 d of age. Ca absorption and parameters involved in this process were measured at 60 or 90 d of age. Compared with WT, KO mice fed the 0.5% Ca diet had higher plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], and lower plasma Ca and insulin-like growth factor-I (IGF-I). Duodenal Ca absorption (% Ca absorbed) in KO mice was reduced 71% relative to WT mice and was associated with 55% lower CaBP mRNA, 47% lower CaBP protein and 95% lower ECaC2 mRNA levels. Compared with WT mice, the percentage of Ca absorbed in KO mice fed the 0.5% Ca diet was inappropriately low for the level of duodenal CaBP. The 2% Ca rescue diet normalized plasma Ca, prevented osteomalacia, increased growth and plasma IGF-I levels, but did not normalize plasma PTH or 1,25(OH)(2)D(3) in KO mice. In addition, the relationship between CaBP protein and the percentage of Ca absorbed was normalized, whereas ECaC2 mRNA fell to near zero. Our data demonstrate that higher CaBP levels do not ensure high rates of duodenal Ca absorption and that transcellular Ca absorption can occur even when ECaC2 gene expression is very low. In addition, our data suggest that the 2% Ca diet promotes a vitamin D receptor-independent anabolic effect on bone formation and calcium absorption, leading to improved calcium balance even in the presence of high PTH levels.     

老年妇女PTH分泌异常及1，25（OH）2D3的疗效观察

目的 研究老年妇女甲状旁腺激素的分泌变化以及维生素1，25(OH)2D3的疗效。方法测定20例年轻妇女(25～35岁)和20例老年妇女(70～78岁)血清PTH浓度，用RIA方法测定使用1，25(OH)2D3前后VFH的变化。使用RIA方法测定血清1，25(OH)2D和25(OH)D的血清浓度。结果 血清PTH浓度随增龄而升高，使用1，25(OH)2D3后，两组的PTH水平均下降。结论 1，25(OH)2D3能减少老年妇女PTH分泌的异常增加。     

1alpha,25-dihydroxycholecalciferol increases the expression of vascular endothelial growth factor in C3H10T1/2 mouse embryo fibroblasts

Evidence suggests that biologically active vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)], may inhibit carcinogenesis. Because angiogenesis is crucial to carcinogenesis, 1,25(OH)(2)D(3) regulation of proangiogenic vascular endothelial growth factor (VEGF) secretion was investigated in cellular models for multistage carcinogenesis. Conditioned media from 1,25(OH)(2)D(3)-treated C3H10T(1/2) mouse fibroblasts and their Harvey ras-oncogene transfected counterparts (rasneo11a cells) induced human umbilical vein endothelial cell (HUVEC) proliferation (1.3 and 0.3 times, respectively, P < 0.05), suggesting that 1,25(OH)(2)D(3) altered the angiogenic phenotype of the cells. Although rasneo11a cells secreted less VEGF than C3H10T(1/2) cells (97%, P < 0.005), 1,25(OH)(2)D(3) induced C3H10T(1/2) and rasneo11a cells to secrete 2 and 3 times, respectively, more VEGF than controls (P < 0.05). Similar effects on VEGF release occurred after 1,25(OH)(2)D(3) treatment of MCF10A and MCF10Aras cells, a human breast epithelial cell model for multistage carcinogenesis. In C3H10T(1/2) cells, 1,25(OH)(2)D(3) activated the VEGF promoter in a dose-dependent (5-100 nmol/L) manner (maximum 60%) and all doses induced VEGF secretion (P < 0.05). 1,25(OH)(2)D(3) induced VEGF mRNA expression ( approximately 50%) from 2 through 24 h; VEGF release was significantly increased at 8 h and sustained for 24 h. VEGF mRNA expression and release declined as C3H10T(1/2) cells grew more confluent, whereas the magnitude of 1,25(OH)(2)D(3)-stimulated changes in VEGF was greater in confluent (3.3 times RNA; 3.5 times release) than in subconfluent (50% RNA; 100% release) cultures (P < 0.05). Thus, 1,25(OH)(2)D(3) increases VEGF secretion, and in C3H10T(1/2) cells, this is likely through activation of the VEGF promoter and induction of gene expression. These data contribute to understanding the role 1,25(OH)(2)D(3) plays in regulation of angiogenesis in normal compared with disease states.