Differential effects of 19-nor-1,25-dihydroxyvitamin D(2) and 1,25-dihydroxyvitamin D(3) on intestinal calcium and phosphate transport

19-Nor-1,25-dihydroxyvitamin D(2) (19-norD(2)) a less calcemic and phosphatemic analog of 1,25-dihydroxyvitamin D (1,25[OH](2)D(3)), is approved for the treatment of secondary hyperparathyroidism in patients with kidney failure. We have previously demonstrated that 19-norD(2) is less active than 1,25(OH)(2)D(3) in stimulating bone resorption. In this study, we compared the potencies of 19-norD(2) and 1,25(OH)(2)D(3) in stimulating net calcium and phosphate absorption in the intestine. Mineral balance was assessed in normal rats during the last 4 days of a 14-day treatment with various daily doses of 19-norD(2) or 1,25(OH)(2)D(3). Calcium absorption increased from 16.5% +/- 7.8% in vehicle-treated rats to 27.5% +/- 7.2% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 21.6% +/- 3.9%, 26.2% +/- 5.5%, and 27.4% +/- 5.1% in rats treated with 10, 50, and 100 ng/day 19-norD(2), respectively. Thus comparable stimulation of calcium transport was attained with 10 ng 1,25(OH)(2)D(3) and 100 ng 19-norD(2). Similar results were obtained for phosphate absorption, with an increase from 28.2% +/- 5.5% in vehicle-treated rats to 40.2% +/- 4.7% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 32.9% +/- 2.2%, 36.2% +/- 4.5%, and 36.8% +/- 3.8% in rats given 10, 50, and 100 ng/day 19-norD(2), respectively. Vitamin D compounds are believed to increase calcium absorption by inducing a calcium channel (epithelial calcium transporter or calcium transporter-1 [CaT1]) on the luminal membrane, a calcium-binding protein (Calbindin D9k) in the cytosol, and a calcium pump (plasma membrane calcium adenosine triphosphatase-1 [PMCA1]) on the basolateral membrane. Northern-blot analysis of intestinal ribonucleic acid of vitamin D-deficient rats given seven daily injections of vehicle or 100 ng 1,25(OH)(2)D(3) or 19-norD(2) revealed that 19-norD(2) was less potent than 1,25(OH)(2)D(3) in stimulating expression of CaT1, Calbindin D9k and PMCA1. In summary, the reduced calcemic and phosphatemic activities of 19-norD(2) can be attributed to lower potency in stimulating intestinal calcium and phosphate absorption.     

Role of protein kinase C in parathyroid hormone stimulation of renal 1,25-dihydroxyvitamin D3 secretion.

PTH is a major regulator of renal proximal tubule 1,25(OH)2D3 biosynthesis. However, the intracellular pathways involved in PTH activation of the mitochondrial 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase) remain unknown. PTH can activate both the adenylate cyclase/protein kinase A (PKA) and the plasma membrane phospholipase C/protein kinase C (PKC) pathways. The present study was undertaken to determine whether PKC may mediate PTH activation of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase activity. Rat PTH 1-34 fragment in vitro translocated PKC activity from cytosolic to soluble membrane fraction from freshly prepared rat proximal tubules. Physiologic concentrations (10(-11)-10(-10) M) of rat PTH 1-34 fragment increased PKC translocation three- to fourfold while PKA activity ratio increased at PTH 10(-7) M. PTH stimulation of PKC and PKA was reduced in the presence of staurosporine (10 nM) by 41 and 29%, respectively. Sangivamycin (10 and 50 microM) also reduced PTH-stimulated PKC translocation, but did not alter PKA activity ratio. In vitro perifusion of renal proximal tubules with PTH (10(-11) M) increased 1,25(OH)2D3 steady-state secretion two- to fourfold. Sangivamycin at the same concentration that inhibited PKC translocation by 52% completely inhibited PTH-stimulated 1,25(OH)2D3 secretion. The present studies indicate that the phospholipase C/PKC pathway may mediate PTH stimulation of mammalian renal proximal tubule 1,25(OH)2D3 secretion.     

Effect of 1,25-dihydroxyvitamin D deficiency on the metabolic clearance rate of 1,25-dihydroxyvitamin D3: studies using pigs with vitamin D-dependent rickets type 1

We have used pigs with inherited vitamin D-dependent rickets type 1 to study the effect of 1,25-dihydroxyvitamin D deficiency on the metabolic clearance rate of 3H-1,25-dihydroxyvitamin D3 infused to steady-state levels in plasma. Plasma levels of 1,25-dihydroxyvitamin D were 24 +/- 1 (SEM) pmol/l in the hypocalcaemic, homozygous piglets and 196 +/- 27 pmol/l in their normocalcaemic, heterozygous siblings. The metabolic clearance rate of 1,25-dihydroxyvitamin D3 was the same in both normal heterozygous (0.90 +/- 0.02) and hypocalcaemic, homozygous piglets (0.90 +/- 0.01 ml-1 min-1 kg-1 metabolic body size). We conclude that a deficiency of circulating 1,25-dihydroxyvitamin D does not influence the clearance of 1,25-dihydroxyvitamin D3 from the circulation of pigs.     

Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25-dihydroxyvitamin D.

Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited (40-60%) by 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) at physiological concentrations (10(-10)-10(-9) M). The lymphocytes acquire specific receptors for 1,25(OH)2D3 upon activation by the lectins. This process precedes the inhibitory effect of 1,25(OH)2D3. We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1,25(OH)2D (the so-called vitamin D-dependent rickets type II). In five patients (three kindreds) peripheral blood mononuclear cells did not acquire receptors for 1,25(OH)2D3 upon phytohemagglutinin-induced activation. Moreover, in contrast to normal lymphocytes, the mitogenic stimulation of these patients' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1,25(OH)2D3. Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [3H]1,25(OH)2D3 but the hormone failed to inhibit the mitogenic stimulation. A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients. The conclusions from these findings are: (a) the inhibition of mitogenic stimulation by 1,25(OH)2D3 is mediated by specific functional receptors to the hormone; and (b) the receptors for 1,25(OH)2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone, such as intestine and kidney.     

1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex

1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D(3)-induced monocyte differentiation, independent of any effects on p21.     

慢性肾病患者血1,25-二羟基维生素D3水平变化

目的 研究慢性肾病(CKD)患者血1,25-二羟基维生素D3(维生素D3)水平异常与肾功能受累程度的关系.方法 研究对象包括150例健康对照者和101例CKD患者,检测其血维生素D3、甲状旁腺素、钙、磷、肌酐、清蛋白及尿素氮水平,计算肾小球滤过率(GFR).结果 与对照组相比,第4~5期患者的血维生素D3水平显著降低(P<0.05),第3~5期患者的血甲状旁腺素(PTH)、肌酐及尿素氮(BUN)水平显著升高(P<0.05),第4~5期患者的血磷水平显著升高(P<0.05),各期CKD患者的清蛋白水平均显著降低(P<0.05),仅第5期CKD患者的血钙浓度显著降低(P<0.05).血维生素D3水平与血PTH、尿素氮及肌酐水平呈负线性相关关系,与血钙、清蛋白及GFR水平呈正线性相关关系.结论 第4~5期CKD患者缺乏维生素D3,说明只有当肾功能受损严重时,CKD患者血维生素D3水平才会出现明显异常.此外,本研究还表明CKD患者血维生素D3水平与其他肾功能相关生化指标存在一定的相关性.     

1,25-二羟维生素D3对人结肠癌细胞凋亡和自噬的影响及机制研究

目的 观察1,25-二羟维生素D3[1,25-(OH)2D3]对人结肠癌HCT-116细胞凋亡和自噬的影响并初步探讨其机制.方法 体外培养人结肠癌细胞HCT-116,分成对照组、低剂量组、中剂量组、高剂量组和联合组[1,25-(OH)2D3高剂量联合腺苷单磷酸活化蛋白激酶(AMPK)抑制剂]共5组,观察并比较各组细胞凋...     

Therapy of 'high risk' myelodysplastic syndromes with an association of low-dose Ara-C, retinoic acid and 1,25-dihydroxyvitamin D3.

Forty-four patients with high risk primary myelodysplastic syndromes and an excess of marrow blasts were treated with a combination of low-dose Ara-C, retinoic acid and vitamin D3. Morphological subtypes were refractory anemia with excess of blasts (RAEB) in 16, RAEB in transformation (RAEB-T) in 20 and chronic myelomonocytic leukemia (CMML) in eight patients. The therapy was continued in responders until relapse or death. The results were compared to those of a matched control of 44 patients given a supportive therapy only. In the treated group the overall response rate was 50% (75% in RAEB, 50% in RAEB-T and 0% in CMML) and the survival was significantly better than in the control group (P < 0.025). Comparing separately each FAB subgroup gave statistical evidence that the treatment prolonged the survival in the RAEB-T subgroup only (P < 0.002). The median duration of response was 15 months and the survival in responders was statistically better than in non-responders (P < 0.0001). Myelosuppression has been the most important side effect, however, no death related to the treatment was observed. Our study suggests that patients with RAEB-T, who are not suitable candidates for aggressive chemotherapy, could benefit from our treatment schedule. The long duration of therapy seems to be of value for patients achieving a response in order to prolong the survival. The toxicity is acceptable and the therapy can be given on an outpatient basis.     

糖尿病肾病患者血清1，25-二羟维生素D3水平及临床意义

目的探讨血清1,25-二羟维生素D。水平在糖尿病肾病（diabeticnephropathy,DN）进展中的作用。方法131例2型糖尿病患者分为单纯糖尿病组（DM组）30例、早期DN组（EDN组）32例、临床DN组（CDN组）35例与DN终末期组（ESDN组）34例,选择体检健康者30例为对照组,各组采用ELISA法检测血清1,25-二羟维生素D3和25-羟维生素D。水平。结果糖尿病及DN各组血清1,25-二羟维生素D3及25-羟维生素D。水平均明显低于对照组（P〈0．01）,且随DN逐渐进展二者水平均逐渐降低（P〈0．05）;糖尿病患者血清1,25-二羟维生素D3水平与尿微量白蛋白排泄率呈负相关（r=0．452,P=0．034）,与肾小球滤过率呈正相关（r=0．390,P=0．006）。结论血清1,25-二羟维生素D3水平检测可能有利于DN的早期发现和病情判断。     

The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD184352 (CI-1040) selectively induces apoptosis in malignant schwannoma cell lines

Type 1 neurofibromatosis (NF1) is a common autosomal dominant disorder that results in neuroectodermal tumors. The NF1 tumor-suppressor gene encodes neurofibromin, which includes a GTPase-activating domain for Ras inactivation. Affinity purification showed N-Ras to be the predominant activated isoform of Ras in two independent neurofibrosarcoma cell lines from NF1 patients (lines ST88-14 and NF90-8). These NF1 cells also demonstrated increased constitutive activity of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) mitogen-activated protein (MAP) kinases compared with a sporadic malignant schwannoma cell line that maintains neurofibromin expression (STS-26T). Thus, MAP kinase kinase (MEK) inhibitors may be a rational approach to NF1 therapy. The MEK inhibitors PD98059 [2'-amino-3'-methoxyflavone], PD184352 (also called CI-1040) [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] all produced concentration-dependent suppression of the proliferation of the three cell lines. Individual MEK inhibitors had similar effects in all three cell lines. However, only the antiproliferative effects of PD184352 correlated closely with the elimination of ERK1,2 MAP kinase activities. PD98059 was primarily cytostatic, whereas U0126 and PD184352 were cytotoxic. Only PD184352 induced apoptosis in all three lines, as indicated by morphology, activation of DEVDase, procaspase-3 cleavage, and the appearance of populations having sub-G(0)/G(1) DNA contents. The differential effects of the MEK inhibitors on cell survival were not dependent on p53 status or effects on the ERK5 pathway. PD184352 was also proapoptotic to primary rat Schwann cells. Hence, although PD184352 effectively killed neurofibrosarcoma cells, its effects on normal Schwann cells may limit its usefulness in the clinic.     

ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo

IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13-induced inflammation and alveolar remodeling. There were associated decreases in IL-13-induced chemokines (MIP-1alpha/CCL-3, MIP-1beta/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of alpha1-antitrypsin. IL-13-induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13-induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13-induced diseases and disorders.     

运动对正常大鼠心肌JNK激活及MKP-1表达的影响

目的 探讨运动对大鼠心肌c-Jun氨基末端活化蛋白激酶(JNK)活性及丝裂原活化蛋白激酶磷酸酶-1(MKP-1)表达的影响，从JNK失活的角度探讨运动对心肌组织生长的影响。方法 14周龄雄性SD大鼠40只，随机分为对照组、急性运动组和运动训练1周组、2周组、3周组。采用运动平板进行运动训练，坡度为10％，速度为15m／min，每次运动30min。急性运动组仅运动30min；运动训练各组，每日1次。采用免疫沉淀法检测左室心肌JNK活性，westernblot检测MKP-1蛋白表达水平。结果 急性运动显著增加心肌：p46JNK、p54JNK活性，分别为对照组的2．2，2．6倍；MKP-1的表达水平与对照组相比有增加趋势，但无统计学意义。运动训练呈时间依赖性促进运动后心肌MKP-1表达水平、抑制JNK活化。结论 运动训练能增强正常大鼠心肌组织MKP-1的可诱导性，抑制JNK过度活化。     

1,25二羟维生素D3对哮喘小鼠的Rac1-IL33-ILC2通路的作用

目的观察1,25二羟维生素D31,25-(OH)2-D3对哮喘小鼠Ras相关的C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)、白细胞介素-33(IL-33)和2型固有淋巴细胞(type 2 innate lymphocytes,ILC2)的影响。方法将30只7周龄BALB/c雌性小鼠随机分为对照组、哮喘组、干预组,每组各10只小鼠,卵清蛋白混合液致敏并雾化吸入建立哮喘小鼠模型,干预组每次激发前腹腔注射1,25(OH)2-D3混合液0.08 ml,对照组和哮喘组以生理盐水代替。末次激发24 h后对小鼠肺组织中IL-33、Rac1、ILC2进行检测并比较。结果与对照组比较,哮喘组Rac1下降,IL-33水平增高,ILC2数目升高。且Rac1与IL-33、ILC2呈明显的负相关性(r=-0.954、r=-0.957,P<0.05)。干预组IL-33及ILC2数目较哮喘组显著下降,Rac1较哮喘组显著上升,差异有统计学意义(P<0.05)。结论哮喘小鼠存在Rac1下降,IL-33水平增高,ILC2数目升高的免疫变化;1,25-(OH)2-D3可能通过上调哮喘小鼠Rac1的表达,导致IL-33等细胞因子水平产生减少,进而抑制ILC2的活化、减轻哮喘的免疫炎症。     

Chronotherapy of high-dose 1,25-dihydroxyvitamin D3 in hemodialysis patients with secondary hyperparathyroidism: a single-dose study

BACKGROUND: A high-dose oral intermittent vitamin D (pulse therapy) is widely used for the treatment of secondary hyperparathyroidism associated with kidney failure. However, hypercalcemia by vitamin D sometimes interrupts this treatment. Because serum calcium concentration possesses a circadian rhythm, a chronopharmacologic approach of vitamin D may have merit for avoidance of adverse reactions. METHODS: Six female secondary hyperparathyroidism patients receiving maintenance hemodialysis received a single oral dose of 2 microg 1,25-dihydroxyvitamin D3 at either 8 AM or 8 PM in a crossover design. Serum concentrations of ionized and total calcium, phosphate, and vitamin D3 were determined for a 48-hour period after administration. We also measured serum intact parathyroid hormone before and 48 hours after dosing as an index for efficacy. RESULTS: A single oral administration of the drug caused an increase in concentration of ionized calcium, serum calcium, and phosphate. However, the area under concentration-time curve from zero to 48 hours [AUC(0-48)] and peak concentration of these variables were markedly lower after dosing at 8 PM. Pre-dose concentrations of these variables were lower at night. The AUC(0-48) of serum vitamin D3 of the morning and night trials did not differ significantly. Reduction of intact parathyroid hormone concentration was also similar between the two trials. CONCLUSION: The administration of vitamin D3 at night may reduce the occurrence of hypercalcemia and hyperphosphatemia in patients with secondary hyperparathyroidism, whereas the pharmacokinetics and intact parathyroid hormone-lowering effect of the drug does not vary with dosing time.     

Serum 1,25 dihydroxy vitamin D (1,25(OH)2D3), 25 hydroxy vitamin D (25(OH)D) and parathormone levels in diabetic retinopathy

OBJECTIVES: To investigate whether there is a relationship between serum 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], which is an inhibitor of angiogenesis, concentrations and severity of diabetic retinopathy (DR). DESIGN AND METHODS: Serum 1,25(OH)2D3, 25 hydroxy vitamin D [25(OH)D] and parathormone (PTH) concentrations were measured in diabetic patients (n = 66) and nondiabetic healthy subjects (n = 20). RESULTS: The mean serum 1,25(OH)2D3 concentration in diabetic patients was lower than that in nondiabetics (57.3+/-21.44 vs. 89.4+/-18.01 pmol/L, p<0.001); mean 1,25(OH)2D3 concentrations fell with increasing severity of DR [being 63.4+/-17.26 pmol/L for background DR (BDR), 47.7+/-13.27 pmol/L for preproliferative DR (pre-PDR), and 43.1+/-19.45 pmol/L for proliferative DR (PDR)]. Compared with the control group, serum 25(OH)D concentrations were found to be decreased in diabetic patients (p<0.001).There were negative correlations between 1,25(OH)2D3 and age (r = -0.331, p<0.01) and duration of diabetes (r = -0.255, p<0.05). CONCLUSION: From these findings, it was found that there was an inverse relationship between the severity of the retinopathy, i.e., neovascularization, and serum 1,25(OH)2D3 concentrations, being the lowest in PDR and the highest in diabetic patients without retinopathy (NDR) patients. The measurement of serum 1,25(OH)2D3 concentrations might be helpful to predict severity of DR in patients with diabetes mellitus.