Icariin attenuates high glucose‐induced type IV collagen and fibronectin accumulation in glomerular mesangial cells by inhibiting transforming growth factor‐β production and signalling through G protein‐coupled oestrogen receptor 1

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Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of 1,25-dihydroxyvitamin D3 and its 20-epi analogues (MC 1288, MC 1301, KH 1060), on clonal keratinocyte growth: evidence for differentiation of keratinocyte stem cells and analysis of the modulatory effects of cytokines

1. Keratinocytes are functionally divided into stem cells, transit amplifying cells and terminally differentiated cells. In a hyperproliferative skin disease, psoriasis, increased mitotic activity of the stem cells is chiefly responsible for epidermal hyperplasia. The effects of 1,25dihydroxyvitamin D₃ (1,25(OH)₂D₃) and potent vitamin D₃ analogues (MC 1288: 20-epi-1,25(OH)₂D₃, MC 1301: 20-epi-24a-homo-26,27-dimethyl-1,25(OH)₂D₃, KH 1060: 20-epi-22-oxa-24a-homo-26,27-dimethyl-1,25(OH)₂D₃) on the stem cells were investigated.
2. Stem cells were identified retrospectively as those giving rise to large keratinocyte colonies in culture (holoclones). 1,25(OH)₂D₃ (10⁻⁸–10⁻⁶ M) suppressed formation of holoclones by stimulating the progenitor cell differentiation into the phenotype expressing differentiation markers (keratins K1/K10 and involucrin).
3. 20-Epi vitamin D₃ analogues were more potent than 1,25(OH)₂D₃ in inhibiting the clonal keratinocyte growth. This activity correlated with the ability to induce cell differentiation (KH 1060>MC 1301>MC 1288>1,25(OH)₂D₃).
4. Cytokines modulated the effects of 1,25(OH)₂D₃ on clonal growth. One of the following cytokines (epidermal growth factor, transforming growth factor α, interleukin-1α, interleukin-1β, interleukin-6, interleukin-8) was required for 1,25(OH)₂D₃ to suppress clonal growth and induce cell differentiation. In contrast, keratinocyte growth factor and insulin-like growth factor I attenuated the effects of 1,25(OH)₂D₃.
5. In conclusion, 1,25(OH)₂D₃ and 20-epi vitamin D₃ analogues suppress clonal growth by directly inducing the differentiation of progenitor cells. It is conceivable that stimulation of stem cells differentiation is a major mechanism of action of vitamin D₃ compounds in psoriasis. Balance between different types of cytokines in psoriatic epidermis may be an important factor determining the clinical effect of vitamin D-based therapy.

     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

EFFECT OF SHORT COURSE OF 1,25-DIHYDROXYVITAMIN D3ON BIOCHEMICAL MARKERS OF BONE REMODELLING IN POSTMENOPAUSAL WOMEN

This study was designed to test the hypothesis that a short treatment course of 1,25(OH)₂D₃elicits a stimulation of osteoblast activity without any action on the osteoclast. To test this, oral daily doses of 0.5μg or 1μg of 1,25(OH)₂D₃were administered for 7 days to two groups (n=5 andn=7, respectively) of postmenopausal women with low bone mineral density. Markers of osteoblast activity, i.e. osteocalcin (BGP), total alkaline phosphatase activity (ALP) and bone alkaline phosphatase activity (BALP), and markers of osteoclast activity, i.e. hydroxylysyl-pyridinoline (Pyr), lysyl-pyridoline (D-Pyr), and galactosyl-hydroxylysine (GHyl) were measured in plasma and in fasting urinary samples, respectively, at sequential times during and after 1,25(OH)₂D₃administration. It resulted that short term 1μg 1,25(OH)₂D₃oral administration induced a significant (P<0.05) rise of BGP serum level without any associated increase ofD-Pyr and GHyl, the latter also expressed as GHyl to GGHyl ratio. Urinary Pyr increased significantly after 1μg daily doses of 1,25(OH)₂D₃. Thus, a short course of 1μg daily doses of 1,25(OH)₂D₃elicits a stimulation of osteoblast activity without any enhancement ofD-Pyr, the most specific marker of osteoclast activity. The enhancement of Pyr after 1μg daily doses of 1,25(OH)₂D₃might be due to the activation of extraosseous metabolic pathways rather than to the activation of osteoclast.     

1α,25-dihydroxyvitamin D3 on intestinal transporter function: studies with the rat everted intestinal sac

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) treatment (2.56 nmol/kg i.p. daily×4) increased PepT1, Mrp2, Mrp4, Asbt, but not Mdr1/P‐gp in the rat small intestine. In this study, the intestinal everted sac technique, together with various select probes: mannitol (paracellular transport), glycylsarcosine (PepT1), 5(and 6)‐carboxy‐2′,7′‐dichlorofluorescein (CDF) diacetate (precursor of CDF for Mrp2), adefovir dipivoxil (precursor of adefovir for Mrp4) and digoxin (P‐gp) was used to examine the functional changes of these transporters. After establishing identical permeabilities (P_app) of mannitol for the apical‐to‐basolateral (A‐to‐B) and basolateral‐to‐apical (B‐to‐A) directions at 20 min in 1,25(OH)₂D₃‐treated vs. vehicle‐treated duodenal, jejunal and ileal everted sacs, a significant enhancement of net A‐to‐B transport of glycylsarcosine in the duodenum, increased B‐to‐A transport of CDF and A‐to‐B and B‐to‐A transport of adefovir in the jejunum were observed with 1,25(OH)₂D₃ treatment. However, the A‐to‐B and B‐to‐A transport of digoxin in the ileum was unchanged. These changes in transporter function in the rat intestinal everted sac corresponded well to changes in proteins that were observed previously. This study confirms that the rat intestinal PepT1, Mrp2 and Mrp4, but not P‐gp are functionally induced by 1,25(OH)₂D₃ treatment via the vitamin D receptor (VDR). Copyright © 2011 John Wiley & Sons, Ltd.     

Alterations in gene expression in vitamin D‐deficiency: Down‐regulation of liver Cyp7a1 and renal Oat3 in mice

The vitamin D‐deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D‐deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α‐hydroxylase (Cyp7a1, rate‐limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up‐regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D‐deficiency. When vitamin D‐sufficient or D‐deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D‐deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D‐sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)₂D₃/cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D‐deficient mice, while 1,25(OH)₂D₃ treatment led to hypercalcemia. In conclusion, vitamin D‐deficiency resulted in down‐regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.     

Effects of 1α,25‐dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo

1α,25‐Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR‐rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill‐defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)₂D₃ treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)₂D₃ treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2‐ to 20‐fold) in 1,25(OH)₂D₃‐treated rats, and a 28‐fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal‐specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostα, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose‐dependent manner, and the ～50% concomitant reduction in FXR, SHP, HNF‐1α and HNF‐4α mRNA expression suggests the possibility of cross‐talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)₂D₃ changes are tissue‐specific, differing between the intestine and kidney which are VDR‐rich organs. Copyright © 2009 John Wiley & Sons, Ltd.     

1,25‑Dihydroxyvitamin D regulates macrophage polarization and ameliorates experimental inflammatory bowel disease by suppressing miR-125b

Macrophages are highly plastic cells. Depending on stimulation, macrophages rapidly polarize to functionally distinct phenotypes that are involved in the pathogenesis of inflammatory bowel disease (IBD). 1,25‑Dihydroxyvitamin D (1,25(OH)₂D₃) has immunomodulatory activity, and 1,25(OH)₂D₃ deficiency is correlated with autoimmune diseases, especially IBD. This study aimed to explore whether 1,25(OH)₂D₃ modulates macrophage polarization in inflammation. Peripheral blood mononuclear cells and colitis mice were treated with 1,25(OH)₂D₃. Macrophages were transfected with siRNA-vitamin D receptor (VDR) or miR-125b mimic or inhibitor, and 1,25(OH)₂D₃-pretreated colitis mice were injected with a miR-125b agomir. The distribution of macrophage subsets and macrophage subtype characteristics was analyzed. As expected, 1,25(OH)₂D₃ transformed lipopolysaccharide-induced M1 macrophages to the M2 subset, downregulated tumor necrosis factor-α and interleukin (IL)-6 expression and interferon regulatory factor 5 (IRF5) phosphorylation, and upregulated IL-10, arginase-1, VDR, and IRF4 expression. SiRNA-VDR and miR-125b mimic significantly impaired 1,25(OH)₂D₃ activity. In colitis mice, 1,25(OH)₂D₃ pretreatment ameliorated disease activity, converted M1 macrophages to the M2 subtype, suppressed IRF5 phosphorylation, and increased IRF4 expression in lamina propria mononuclear cells (LPMC). miR-125b agomir injections reversed 1,25(OH)₂D₃ action. Collectively, the results demonstrate that 1,25(OH)₂D₃ downregulates miR-125b expression and promotes M1 macrophage polarization to the M2 subtype. 1,25(OH)₂D₃ pretreatment ameliorated colitis by restoring the LPMC macrophage subtype balance.     

TGF‐β1 mediated Smad signaling pathway and EMT in hepatic fibrosis induced by Nano NiO in vivo and in vitro

Nickel oxide nanoparticles (Nano NiO) bears hepatotoxicity, while whether it leads to liver fibrosis remains unclear. The aim of this study was to establish the Nano NiO‐induced hepatic fibrosis model in vivo and investigate the roles of transforming growth factor β1 (TGF‐β1) in Smad pathway activation, epithelial‐mesenchymal transition (EMT) occurrence, and extracellular matrix (ECM) deposition in vitro. Male Wistar rats were exposed to 0.015, 0.06, and 0.24 mg/kg Nano NiO by intratracheal instilling twice a week for 9 weeks. HepG2 cells were treated with 100 μg/mL Nano NiO and TGF‐β1 inhibitor (SB431542) to explore the mechanism of collagen formation. Results of Masson staining as well as the elevated levels of type I collagen (Col‐I) and Col‐III suggested that Nano NiO resulted in hepatic fibrosis in rats. Furthermore, Nano NiO increased the protein expression of TGF‐β1, p‐Smad2, p‐Smad3, alpha‐smooth muscle actin (α‐SMA), matrix metalloproteinase9 (MMP9), and tissue inhibitors of metalloproteinase1 (TIMP1), while decreased the protein content of E‐cadherin and Smad7 in rat liver and HepG2 cells. Most importantly, Nano NiO‐triggered the abnormal expression of the abovementioned proteins were all alleviated by co‐treatment with SB431542, implying that TGF‐β1‐mediated Smad pathway, EMT and MMP9/TIMP1 imbalance were involved in overproduction of collagen in HepG2 cells. In conclusion, these findings indicated that Nano NiO induced hepatic fibrosis via TGF‐β1‐mediated Smad pathway activation, EMT occurrence, and ECM deposition.     

Modulatory role of 1,25 dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP pathway in the rat

1. Previous studies have shown that vitamin D₃ deficiency impairs the insulin response to glucose via an alteration of signal transduction pathways, such as Ca²⁺ handling and the phosphoinositide pathway. In the present study the adenylyl cyclase pathway was examined in islets from 3 independent groups: normal rats, 4 weeks-vitamin D₃ deficient rats and one week-1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) treated rats.
2. We found that the very low rate of insulin release observed in vitamin D₃ deficient rats could be restored in vitamin D₃ deficient islets only with high concentrations of dioctanoyl-cyclic AMP (DO-cyclic AMP), whereas 1,25(OH)₂D₃ improved the sensitivity of the islets to this exogenous cyclic AMP analogue.
3. The beneficial effect of 1,25(OH)₂D₃ observed with or without DO-cyclic AMP was protein kinase A-dependent, since the addition of N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H-89), a specific inhibitor of cyclic AMP-dependent protein kinases, decreased the insulin release of treated rats back to the level seen in vitamin D₃ deficient islets.
4. The low rate of insulin release could not be consistently related to an alteration in cyclic AMP content of the islets. Indeed, low insulin response to a barium + theophylline stimulus observed in vitamin D₃ deficient islets was paradoxically associated with a supranormal cyclic AMP content in the islets.
5. This paradoxical increase in cyclic AMP observed in these conditions could not be attributed to a lower total phosphodiesterase (PDE) activity, although the portion of Ca²⁺-calmodulin-independent PDE was predominant in islets from vitamin D₃ deficient rats.
6. On the other hand, the higher cyclic AMP content of vitamin D₃ deficient islets could be related to an increase in glucagon-induced cyclic AMP synthesis in relation to the hyperglucagonaemia previously observed in vitamin D₃ deficient rats. Since higher concentrations of exogenous glucagon and higher endogenous cyclic AMP concentrations were required in vitro to restore insulin release to normal values, the cyclic AMP-dependent pathways that usually potentiate insulin secretion appeared to be less efficient in relation to an alteration in the post cyclic AMP effector system.
7. 1,25(OH)₂D₃ exerted a stimulating effect on insulin release via protein kinase A activation but reduced the supranormal cyclic AMP synthesis, thus exerting a differential modulatory influence on biochemical disturbances in islets induced by vitamin D₃ deficiency.

     

Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)₂D₃- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)₂D₃ or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)₂D₃- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.     

1α,25‐dihydroxyvitamin D3 triggered vitamin D receptor and farnesoid X receptor‐like effects in rat intestine and liver in vivo

1α,25‐Dihydroxyvitamin D₃ (1,25(OH)₂D₃), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)₂D₃ (0–2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)₂D₃ treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the fibroblast growth factor 15 (FGF15), organic solute transporter (Ostα and Ostβ) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1±14.9 vs 41.9±7.8 µm , p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)₂D₃ treatment. Increased Bsep and Ostα mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid‐FXR‐SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF15, were found. Increased hepatic multidrug resistance‐associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P‐gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR‐SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF15, events that led to the activation of FXR. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of <Emphasis Type="Italic">C</Emphasis><Subscript>6</Subscript>-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)₂D₃ or ATRA and chemotherapy, adverse effects 1,25-(OH)₂D₃ or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C₆-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C₆-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6β-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)₂D₃ or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)₂D₃ or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.     

在人的类成骨细胞TE85中雌激素对活性维生素D作用的影响

目的　观察雌激素对活性维生素D ［1,25(OH)₂D₃］调节人的类成骨细胞TE85作用的影响。方法　以TE85为成骨细胞模型,³H-胸腺嘧啶参入法测细胞增殖,³H-脯氨酸参入法测细胞胶原合成,放射免疫法测骨钙素（BGP）含量,对硝基苯酚法测碱性磷酸酶（ALP）活性。结果　单独以0.5～5 nmol.L^-1 1,25(OH)₂D₃处理细胞96 h,³H-胸腺嘧啶参入被抑制,作用呈剂量依赖性。5 nmol.L^-1 1,25(OH)₂D₃可使细胞ALP活性增加29.7％。 雌二醇（E₂）可取消1,25(OH)₂D₃对细胞增殖的抑制作用。两药合用时能促进细胞胶原、骨钙素及ALP的合成。结论　1,25(OH)₂D₃有促进成骨细胞骨形成的作用。1,25(OH)₂D₃与E₂联合应用时,其促进成骨的作用增强。     

Interaction between calcium and cadmium in the 1,25-dihydroxyvitamin D3 stimulated rat duodenum

It has been suggested that calcium and cadmium compete for an intestinal transport system that is vitamin D dependent. To further test this hypothesis, the interaction between calcium and cadmium during transport in the duodenum of rat was investigated. Control rats maintained on a diet adequate in vitamin D, calcium, and phosphorus were compared to rats on the same diet administered 500 ng of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) 4 hr before transport measurement with everted sacs. Active transport of calcium was evident in control rats and was further stimulated by exogenous 1,25(OH)₂D₃. Added to mucosal bathing fluid 10 μm cadmium partially inhibited active calcium transport both in controls and in rats receiving 1,25(OH)₂D₃; 100 μm cadmium completely blocked active transport in both groups. Water transport was also inhibited by 10 and 100 μm cadmium. Cadmium uptake and transport were not affected by 1,25(OH)₂D₃. The accumulation of cadmium in mucosal tissue was significantly inhibited by 1 mm calcium, but there was no significant effect on uptake or transmural transport. The findings suggest that cadmium and calcium do not interact specifically at a 1,25(OH)₂D₃-dependent transport site. The interaction between calcium and cadmium in the duodenal mucosa could be related to the action of cadmium in blocking active transport processes.     

Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity

20-Hydroxyvitamin D₃ (20(OH)D₃), the major product of CYP11A1 action on vitamin D₃, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), to metabolize 20(OH)D₃. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)₂D₃ with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D₃ (25(OH)D₃). CYP24A1 hydroxylated 20(OH)D₃ to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D₃ (20,24(OH)₂D₃) and 20,25-dihydroxyvitamin D₃ (20,25(OH)₂D₃). The catalytic efficiency of CYP24A1 for 20(OH)D₃ metabolism was more than 10-fold lower than for either 25(OH)D₃ or 1,25(OH)₂D₃ and no secondary metabolites were produced. The two major products, 20,24(OH)₂D₃ and 20,25(OH)₂D₃, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)₂D₃ or the parent 20(OH)D₃, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D₃.     

Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro1α,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafl uoro-1α, 25-dihydroxyvitamin D₃ [F₆-1,25(OH)₂D₃] to 26,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D₃ [F₆-1,23,25(OH)₃D₃ and that of F₆-1,23,25(OH)₃D₃ to 26,27-hexafluoro-23-oxo-1α,25-dihydroxyvitamin D₃ [F₆-23-oxo-1,25(OH)₂D₃] are catalysed by 25-hydroxyvitamin D 24-hydroxylase (CYP24), ROS17}2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24–] and a corresponding blank plasmid [pSLVCYP24R(®)] were used. 2. Incubation of [1β-³H]-F-1,25(OH)₂D₃ for 2 and 5 days with ROS17}2.8 cells transfected with pSVL-CYP24– generated a metabolite that co-migrated with authentic F₆ -1,23,25(OH)₃D₃ in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1β-³H]-F₆ -1,23,25(OH)₃D₃ for 5 days with pSVL-CYP24– - transfected ROS 17}2.8 cells generated a metabolite that co-migrated with authentic F₆ -23-oxo-1,25(OH)₂D₃. In contrast, the metabolites F₆ -1,23,25(OH)₃D₃ or F₆ -23-oxo- 1,25(OH)₂D₃ were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F₆ -1,25(OH)₂D₃ to F₆ -1,23,25(OH)₃D₃ and that of F₆ -1,23,25(OH)₃D₃ to F₆ -23-oxo-1,25(OH)₂D₃.