1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)₂D₃, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)₂D₃-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)₂D₃. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)₂D₃. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)₂D₃ effects. 1,25(OH)₂D₃ blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)₂D₃ down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide

1,25(OH)₂D₃, the active metabolite of vitamin D₃, its activity is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of its activity in the regulation of cell proliferation, cell differentiation and immunomodulation. Here we report lipopolysaccharide (LPS), a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, plus high glucose (HG) can significantly inhibit mesothelial cell viability while induce more apoptosis in rat peritoneal mesothelial cells (RPMC). Pretreatment with 1,25(OH)₂D₃ can reverse the above effect in a concentration dependent manner. HG plus LPS can down-regulate the levels of both mRNA and protein of VDR, and up-regulate the expression of TGF-β1 and TNF-α in RPMC, which can also be effectively reversed by pretreatment with 1,25(OH)₂D₃. The above results suggest that HG plus LPS may induce changes in RPMC’s viability and apoptosis, leading to peritoneal injury. 1,25(OH)₂D₃ can reverse the inhibition of cell viability, the increase of apoptotic rate and induction of fibrosis related cytokine TGF-β1 and TNF-α by HG plus LPS in RPMC, thus protect peritoneal membrane.     

The calcitonin/calcitonin gene related peptide-α gene is not required for 1α,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), can suppress disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Calcium appears to be a critical component of 1,25(OH)₂D₃-mediated suppression of EAE, as complete disease prevention only occurs with a concomitant increase in serum calcium levels. Calcitonin (CT) is a peptide hormone released in response to acute increases in serum calcium, which led us to explore its importance in 1,25(OH)₂D₃-mediated suppression of EAE. Previously, we discovered that co-administration of pharmacological doses of CT enhanced the suppressive effect of 1,25(OH)₂D₃ on EAE, suggesting CT may play a role in 1,25(OH)₂D₃-mediated suppression of EAE. To determine the importance of CT in EAE we have utilized a mouse strain in which the gene encoding CT and its alternative splice product, calcitonin gene related peptide-α (CGRP), have been deleted. Deletion of the CT/CGRP gene had no effect on EAE progression. Furthermore, treatment with 1,25(OH)₂D₃ suppressed EAE in CT/CGRP knock-out mice equal to that in wild type mice. Therefore, we conclude that CT is not necessary for 1,25(OH)₂D₃-mediated suppression of EAE.     

Effects of 1,25(OH)2D3 and vitamin D receptor on peripheral CD4+/CD8+ double‐positive T lymphocytes in a mouse model of systemic lupus erythematosus

This study aims to explore effects of 1,25(OH)₂D₃ and vitamin D receptor (VDR) on peripheral CD4⁺/CD8⁺ double‐positive (DP) T lymphocytes in systemic lupus erythematosus (SLE). MRL‐LPr/LPr mice with SLE (n = 20) and normal MRL mice (n = 20) were assigned into the control group (normal mice, without feeding with 1,25(OH)₂D₃), the 1,25(OH)₂D₃ group (SLE mice, feeding with 1,25(OH)₂D₃), the VDR‐knock‐in + 1,25(OH)₂D₃ group (SLE mice, VDR‐knock‐in, feeding with 1,25(OH)₂D₃) and the VDR‐knockout group (normal mice, VDR‐knockout, without feeding with 1,25(OH)₂D₃) (n = 10 per group). Levels of T lymphocytes were measured by flow cytometry. The mRNA and proteins expressions of inflammatory factors were measured by qRT‐PCR and ELISA. Extracellular signal‐regulated kinase‐1/2 (ERK1/2) expression was measured by Western blotting. Compared with normal mice, SLE mice showed reduced levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes. The levels of SLE‐related indicators all increased significantly, followed with severe skin ulcers and urinary system infection. With the increase in time, skin ulcers and urinary system infection were significantly improved, levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes increased, and levels of SLE‐related indicators all decreased in the 1,25(OH)₂D₃ group. There were no significant changes in bioindicators in the control and the VDR‐knock‐in + 1,25(OH)₂D₃ groups. The symptoms of SLE gradually occurred in the VDR‐knockout group. This study demonstrates that VDR and 1,25(OH)₂D₃ could elevate CD4⁺/CD8⁺ DP T lymphocytes and reduce expressions of inflammatory factors, thus inhibiting the development and progression of SLE.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)₂D₃ in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)₂D₃ on chondrocytes in OA remains obscure. Effect of 1,25-(OH)₂D₃ on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)₂D₃ activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)₂D₃-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)₂D₃ on MMP13 expression, transfection assays were used to show that 1,25-(OH)₂D₃ activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)₂D₃ in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)₂D₃ activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)₂D₃ to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.     

Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

1,25-Dihydroxyvitamin D₃ suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D? (1,25(OH)?D?), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)?D?, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)?D?-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at -299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)?D?. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)?D?. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)?D? effects. 1,25(OH)?D? blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)?D? down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

Activation of rapid signaling pathways does not contribute to 1α,25‐dihydroxyvitamin D3‐induced growth inhibition of mouse prostate epithelial progenitor cells

The active form of vitamin D, 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D) inhibits the growth of prostate epithelial cells, however the underlying mechanisms have not been clearly delineated. In the current study, the impact of 1,25(OH)₂D on the rapid activation of extracellular‐regulated kinase (ERK) 1/2 and protein kinase C α (PKCα), and the role of these pathways in growth inhibition was examined in immortalized mouse prostate epithelial cells, MPEC3, that exhibit stem/progenitor cell characteristics. 1,25(OH)₂D treatment suppressed the growth of MPEC3 in a dose and time dependent manner (e.g., 21% reduction at three days with 100 nM 1,25(OH)₂D treatment). However, ERK1/2 activity was not altered by 100 nM 1,25(OH)₂D treatment for time points from 1 min to 1 h in either serum‐containing or serum‐free medium. Similarly, PKCα activation (translocation onto the plasma membrane) was not regulated by short‐term treatment of 100 nM 1,25(OH)₂D. In conclusion, 1,25(OH)₂D did not mediate rapid activation of ERK1/2 or PKCα in MPEC3 and therefore the growth inhibitory effect of 1,25(OH)₂D is independent of rapid activation of these signaling pathways in this cell type. J. Cell. Biochem. 107: 1031–1036, 2009. © 2009 Wiley‐Liss, Inc. This article was published online 2 June 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 15 June 2009.     

1,25-dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine-stimulated adult human osteoblast-like cells

The steroid derivative 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a regulator of bone biology, and there is evidence that 1,25(OH)₂D₃ modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)₂D₃ decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-β₁ (TGF-β) or by tumor necrosis factor-α (TNF). The inhibition of the TGF-β-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)₂D₃ pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-β. Free arachidonic acid levels were decreased following 1,25(OH)₂D₃ pretreatment in the TNF stimulated samples. hOB cell phospholipase A₂ activity was measured in subcellular fractions, and this activity was decreased by 20–25% in the 1,25(OH)₂D₃ pretreated samples. The addition of the selective inhibitor AACOCF₃ to the phospholipase A₂ assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)₂D₃ pretreatment of the hOB cells. Thus, 1,25(OH)₂D₃ regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites. J. Cell. Biochem. 68:237–246, 1998. © 1998 Wiley-Liss, Inc.     

1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)₂D₃ and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)₂D₃ altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)₂D₃-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)₂D₃. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)₂D₃ in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.