Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase β (IKKβ) to block NF-κB activation. 1,25(OH)₂D₃ rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKβ-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKβ and that this interaction is enhanced by 1,25(OH)₂D₃. Protein mapping reveals that VDR-IKKβ interaction occurs between the C-terminal portions of the VDR and IKKβ proteins. Reconstitution of VDR^−/− cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKβ interaction disrupts the formation of the IKK complex and, thus, abrogates IKKβ phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)₂D₃-VDR inhibits NF-κB activation.     

Role of Vitamin D3 Receptor in the Synergistic Differentiation of WEHI-3B Leukemia Cells by Vitamin D3 and Retinoic Acid

WEHI-3B D⁻ cells differentiate in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)₂D₃ interact to produce synergistic differentiation of WEHI-3B D⁻ cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)₂D₃ receptor (VDR) and retinoid receptors, RARα and RXRα, was measured. No VDR was detected in untreated WEHI-3B D⁻ cells; however, RA and 1,25-(OH)₂D₃ when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARα and RXRα were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)₂D₃ produced synergistic differentiation of WEHI-3B D⁻ cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D⁺ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)₂D₃ in WEHI-3B D⁺ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)₂D₃ and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1,25-(OH)₂D₃.     

Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)₂D₃ and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)₂D₃ enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)₂D₃ treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)₂D₃-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)₂D₃ and did not change that by 1,25(OH)₂D₃ plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)₂D₃-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na⁺,K⁺-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.     

The Vitamin D Analogue ED71 but Not 1,25(OH)2D3 Targets HIF1α Protein in Osteoclasts

Although both an active form of the vitamin D metabolite, 1,25(OH)₂D₃, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)₂D₃ -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)₂D₃, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH)₂D₃ or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH)₂D₃ in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)₂D₃ in vitro, were both significantly higher following treatment with 1,25(OH)₂D₃ than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)₂D₃, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.     

Stabilisation of β-Catenin Downstream of T Cell Receptor Signalling

Background

The role of TCF/β-catenin signalling in T cell development is well established, but important roles in mature T cells have only recently come to light.

Methodology/Principal Findings

Here we have investigated the signalling pathways that are involved in the regulation of β-catenin in primary human T cells. We demonstrate that β-catenin expression is upregulated rapidly after T cell receptor (TCR) stimulation and that this involves protein stabilisation rather than an increase in mRNA levels. Similar to events in Wnt signalling, the increase in β-catenin coincides with an inhibition of GSK3, the kinase that is required for β-catenin degradation. β-catenin stabilisation in T cells can also be induced by the activation of PKC with phorbol esters and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PKC). Upon TCR signalling, β-catenin accumulates in the nucleus and, parallel to this, the ratio of TCF1 isoforms is shifted in favour of the longer β-catenin binding isoforms. However, phosphorylated β-catenin, which is believed to be inactive, can also be detected and the expression of Wnt target genes Axin2 and dickkopf is down regulated.

Conclusions/Significance

These data show that in mature human T cells, TCR signalling via PI3K and PKC can result in the stabilisation of β-catenin, allowing β-catenin to migrate to the nucleus. They further highlight important differences between β-catenin activities in TCR and Wnt signalling.     

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)₂D₃ inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D₃ to 1,25(OH)₂D₃, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)₂D₃, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)₂D₃ enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)₂D₃ was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)₂D₃ promoted maturation. 25(OH)D₃ also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)₂D₃. Consistent with this possibility, incubation of preadipocytes with 25(OH)D₃ led to 1,25(OH)₂D₃ accumulation in the media. 1,25(OH)₂D₃ also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.     

Components of the Canonical and Non-Canonical Wnt Pathways Are Not Mis-Expressed in Pituitary Tumors

Introduction

Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.

Objective

This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.

Materials and Methods

Genes of the Wnt canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.

Results

There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.

Conclusions

Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

A Role for Interleukin-1 Alpha in the 1,25 Dihydroxyvitamin D3 Response in Mammary Epithelial Cells

Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D₃ as a promising chemopreventative agent for breast cancer. Vitamin D₃ metabolites induce anti-proliferative effects in breast cancer cells in vitro and in vivo, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)₂D₃, the metabolically active form of vitamin D₃, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)₂D₃ in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)₂D₃ response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)₂D₃ in mammary epithelial cells. Furthermore, 1,25(OH)₂D₃ increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)₂D₃ in mammary cells. Together, these findings support the chemopreventative potential of vitamin D₃ in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)₂D₃.     

Aberrant Activation of the WNT/β-Catenin Signaling Pathway in Lupus Nephritis

Objective

The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.

Methods

The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.

Results

Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.

Conclusions

The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.