Vitamin D3 Signaling Research Articles

Circadian regulation of calcium and phosphorus homeostasis during the oviposition cycle in laying hens

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (PiT-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens.

Stage of infection with Mycobacterium avium subsp. paratuberculosis impacts expression of Rab5, Rab7, and CYP27B1 in macrophages within the ileum of naturally infected cows.

Macrophages are the preferential target of Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of ruminant paratuberculosis. Uptake of pathogens by intestinal macrophages results in their trafficking through endosomal compartments, ultimately leading to fusion with an acidic lysosome to destroy the pathogen. MAP possesses virulence factors which disrupt these endosomal pathways. Additionally, levels of serum vitamin D3 have proven relevant to host immunity. Dynamics of endosomal trafficking and vitamin D3 metabolism have been largely unexplored in bovine paratuberculosis. This study aimed to characterize expression of early and late endosomal markers Rab5 and Rab7, respectively, within CD68+ macrophages in frozen mid-ileum sections harvested from cows at different stages of natural paratuberculosis infection. Additionally, factors of vitamin D3 signaling and metabolism were characterized through expression of vitamin D3 activating enzyme 1α-hydroxylase (CYP27B1), vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and vitamin D3 receptor (VDR) within CD68+ ileal macrophages. Cows with clinical paratuberculosis had significantly greater macrophage and MAP burden overall, as well as intracellular MAP. Total expression of Rab5 within macrophages was reduced in clinical cows; however, Rab5 and MAP colocalization was significantly greater in this group. Intracellular Rab7 colocalization with MAP was not detected in subclinical or Johne's Disease negative (JD-) control cows but was present in clinical cows. Additionally, macrophage CYP27B1 expression was significantly reduced in clinical cows. Taken together, the results from this study show disparate patterns of expression for key mediators in intracellular MAP trafficking and vitamin D metabolism for cows at different stages of paratuberculosis.

Research Highlights

Research Highlights

Do Interactions of Vitamin D3 and BMP Signaling Hold Implications in the Pathogenesis of Fibrodysplasia Ossificans Progressiva?

Fibrodysplasia ossificans progressiva (FOP) is a debilitating rare disease known for episodic endochondral heterotopic ossification (HO) caused by gain-of-function mutations in ACVR1/ALK2. However, disease severity varies among patients with identical mutations suggesting disease-modifying factors, including diet, may have therapeutic implications. The roles of vitamin D3 in calcium metabolism and chondrogenesis are known, but its effects on BMP signaling and chondrogenesis are less studied. This review attempts to assess the possibility of vitamin D's effects in FOP by exploring relevant intersections of VD3 with mechanisms of FOP flares. In vitro and in vivo studies suggest vitamin D suppresses inflammation, while clinical studies suggest that vitamin D3 protects against arteriosclerosis and inversely correlates with non-genetic intramuscular HO. However, the enhancement of chondrogenesis, BMP signaling, and possibly Activin A expression by vitamin D may be more relevant in FOP. There appears to be little potential for vitamin D to reduce HO in FOP, but testing the potential for excess vitamin D to promote HO may be warranted.

IL-1β Drives Production of FGF-23 at the Onset of Chronic Kidney Disease in Mice.

FGF-23 has arisen as an early biomarker of renal dysfunction, but at the onset of chronic kidney disease (CKD), data suggest that FGF-23 may be produced independently of the parathyroid hormone (PTH), 1,25(OH)2 -vitamin D3 signaling axis. Iron status is inversely correlated to the level of circulating FGF-23, and improvement in iron bioavailability within patients correlates with a decrease in FGF-23. Alternately, recent evidence also supports a regulatory role of inflammatory cytokines in the modulation of FGF-23 expression. To determine the identity of the signal from the kidney-inducing upregulation of osteocytic FGF-23 at the onset of CKD, we utilized a mouse model of congenital CKD that fails to properly mature the glomerular capillary tuft. We profiled the sequential presentation of indicators of renal dysfunction, phosphate imbalance, and iron bioavailability and transport to identify the events that initiate osteocytic production of FGF-23 during the onset of CKD. We report here that elevations in circulating intact-FGF-23 coincide with the earliest indicators of renal dysfunction (P14), and precede changes in serum phosphate or iron homeostasis. Serum PTH was also not changed within the first month. Instead, production of the inflammatory protein IL-1β from the kidney and systemic elevation of it in the circulation matched the induction of FGF-23. IL-1β's ability to induce FGF-23 was confirmed on bone chips in culture and within mice in vivo. Furthermore, neutralizing antibody to IL-1β blocked FGF-23 expression in both our congenital model of CKD and a second nephrotoxic serum-mediated model. We conclude that early CKD resembles a situation of primary FGF-23 excess mediated by inflammation. These findings do not preclude that altered mineral availability or anemia can later modulate FGF-23 levels but find that in early CKD they are not the driving stimulus for the initial upregulation of FGF-23. © 2020 American Society for Bone and Mineral Research.

Synergistic Effects of Interferon-γ and Vitamin D3 Signaling in Induction of ILT-3highPDL-1high Tolerogenic Dendritic Cells.

In the past, interferon (IFN)-γ and vitamin D3 (vit D3) have both been associated with induction of tolerogenic characteristics in human dendritic cells (DCs). Although there are only a few reports on interdependency of their actions, the interplay between IFN-γ and vit D3 has been clearly demonstrated in certain aspects of immune reactivity. Since both agents have been associated with regulation of immune responses, we set out to examine their functional and mechanistic interactions in context of principal regulators of immunity, the DCs. Combined treatment with vit D3 and IFN-γ caused an extensive expression of immunoglobulin-like transcript (ILT)-3 and programmed death ligand (PDL)-1 on γ/D3DCs, significantly greater than that caused by vit D3 alone. Such γ/D3DCs retained all general DC characteristics. After CD40 ligand-induced activation, they produced increased amounts of IL-10 with almost absent production of IL-12p70. On the other hand, the co-stimulatory potential of γ/D3DCs was weak, with cells possessing the capacity to inhibit CD4+ T cell, CD8+ T cell, as well as memory T cell responses. Naive CD4+ T cells stimulated with γ/D3DCs produced increased amounts of IL-10 with concomitantly low IFN-γ production, upon T cell receptor activation. Additionally, γ/D3DCs completely inhibited granzyme B expression by CD8+ T cells. The percentage of FoxP3-positive cells in co-cultures with naive CD4+ T cells was significantly higher where γ/D3DCs were used as stimulators compared to DCs treated with vit D3 alone and it could be partially reversed by PDL-1 blockade. Interestingly, γ/D3DCs were inefficient at suppressing mDC-induced CD4+ T cell proliferation, but were twice as effective as D3DCs at suppressing mDC-induced CD8+ T cell proliferation. Blockade of indoleamine-2,3-dioxygenase did not reduce the tolerogenic phenotype induced by IFN-γ and vit D3 treatment. Examination of signaling pathways activation revealed a tendency toward increased ERK and Akt phosphorylation in γ/D3DCs. Inhibition of MEK/ERK and PI3K/mTOR pathways significantly reduced the expression of ILT-3 and PDL-1 on γ/D3DCs. In summary, we present the first evidence for existing synergy between IFN-γ and vit D3 in shaping a unique tolerogenic DC activation state.

IL-6 Impairs the Activity of Vitamin D3 in the Regulation of Epithelial-Mesenchymal Transition in Triple Negative Breast Cancer.

Objective:The present study aimed to investigate the possible role of IL-6 and 1α,25-dihydroxyvitamin D3 (1,25D) signaling in epithelial-mesenchymal transition (EMT) and stemness in triple-negative breast cancer (TNBC) cell line.Methods:TNBC cell line, HCC 1806, was treated with IL-6 and 1,25D for three and six days. Also, the role of vitamin D receptor (VDR) was studied by transfection of TNBC cell line with VDR gene and transfection efficiency was assessed using Human VDR enzyme-linked immunosorbent assay (ELISA). Changes in E-cadherin gene expression were analyzed by quantitative real-time PCR (qRT-PCR). Also, changes in CD44+ cells were analyzed by flow cytometry. Finally, morphological changes were investigated by light microscopy after 6 days. Results:Treatment of HCC1806 cells with IL-6 has no significant effect either on E-cadherin gene expression or CD44+ cells, (p > 0.05). However, E-cadherin gene expression was significantly up-regulated after treatment with 1,25D for 6 days, (p < 0.05). Also, CD44+ cells were significantly reduced after treatment with 1,25D either for 3 or 6 days, (p < 0.05). Transfection of TNBC cell line with VDR gene significantly up-regulated VDR protein expression, (p < 0.05). In addition, overexpression of VDR in TNBC cells and treatment with 1,25D significantly up-regulated E-cadherin gene expression, (p < 0.05) and reduced CD44+ cells, (p < 0.05). Moreover, transfection with VDR and treatment with a combination of 1,25D and IL-6 significantly down-regulated E-cadherin gene expression and increased CD44+ cells compared with transfected cells with VDR treated with 1,25D alone, (p < 0.05). No significant morphological changes were observed in treated cells, 6 days post-treatment. Conclusion:The presence of IL-6 in the breast tumor microenvironment may impair the activity of vitamin D3 signaling, limiting its anti-tumor effects in TNBC.

MiR-99b-3p is induced by vitamin D3 and contributes to its antiproliferative effects in gastric cancer cells by targeting HoxD3.

Vitamin D3 is known to have anticancer actions by affecting tumorigenesis including the cell cycle and cell apoptosis in gastric cancer (GC) cells; the genes including microRNAs (miRNAs) regulated by vitamin D3 signaling remain discovered. miR-99b-3p, the tumor suppressor gene, is not only decreased in GC tissues, but is also induced by vitamin D3 through the vitamin D receptor (VDR) binding on the promoter domain of miR-99b. Further study indicates that miR-99b-3p inhibits cell viability and induces cell arrest in the S-phase in GC cells, the direct target gene of miR-99b-3p is verified to be HoxD3, which is also overexpressed in GC cell lines. Overall, our results show that miR-99b-3p mediates the antiproliferative of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

Abstract 2976: A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat

Abstract Background: Vitamin D3 (VD) and its analogue, Paricalcitol (Pcal), have previously shown anti-tumorigenic activities against colorectal cancer (CRC). Hence, this study compared the therapeutic efficacy of both VD types with or without chemotherapy (5-FU) in azoxymethane (AOM)-induced CRC in rat. Methods: seventy male Wistar rats were equally allocated into 7 groups: Control, AOM, VD, Pcal, 5-FU, 5-FU/VD and 5-FU/Pcal. AOM was subcutaneously injected for 2 weeks (15mg/kg/week). The study duration was 25 weeks. VD (500 IU/rat/day; 3 days/week) and Pcal (2.5 μg/kg/day; 3 days/week) were injected subcutaneously from week-15 till the end of the study. 5-FU intraperitoneal injections started in week-21 (12 mg/kg/day; 4 successive days) and continued in week-22 (6 mg/kg/4 doses every other day). The colons were collected for gross and histopathological examination. The expression of VD signalling system, Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, smad4, p21 and p27 proteins was measured by immune-histochemistry/fluorescence. The mRNA expression of Wnt, β-catenin, DKK-1 and COX-2 genes was measured by Quantitative RT-PCR. ELISA was used to measure the concentrations of TGF-β1, VEGF and COX-2 proteins in tissues. Results: The AOM and 5-FU groups showed marked inhibitions in the VD receptor and binding protein compared with controls (P &amp;lt; 0.05). VD and Pcal monotherapies significantly increased the expression of the VD molecules and the VD group showed the utmost elevations. Moreover, the tumour numbers and sizes were equally and significantly reduced in the 5-FU, VD and Pcal groups compared with the AOM group (P &amp;lt; 0.05). Interestingly, the 5-FU/VD and 5-FU/Pcal resulted in significantly lower numbers of tumours compared with the remaining study groups and the highest significant reduction was observed in the former group. At the molecular level, the 5-FU/VD markedly inhibited the pro-oncogenic molecules (Wnt, β-catenin, NF-κB, COX-2, iNOS &amp; VEGF) as well as significantly upregulated the anti-tumorigenic molecules (DKK-1, TGF-β1, smad4, p21 and p27) compared with the other study groups (P &amp;lt; 0.05). Conclusions: Vitamin D3 showed a better synergism than Pcal with 5-FU. The enhanced anti-tumorigenic actions of 5-FU/VD involved the modulation of the Wnt/β-catenin and TGF-β1/smad4 pathways, angiogenesis and cell cycle arrest. Further studies are required to elucidate the therapeutic significance of VD in clinical settings Citation Format: Mohammed Abbas Baghdadi, Mohammed Aslam, Jawwad Ahmad, Shakir Idris, Bassem Refaat. A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2976.

The role of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation

Abstract Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (Pdia3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. Pdia3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking Pdia3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E2, which is released during Pdia3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE2, was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.

MiR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 by directly targeting E2F3 in gastric cancer cells.

VitaminD3 signaling is involved in inhibiting the development and progression of gastric cancer (GC), while the active vitamin D metabolite 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated gene regulatory mechanisms in GC remain unclear. We found that miR-145 is induced by 1,25(OH)2D3 in a dose- and vitamin D receptor (VDR)-dependent manner in GC cells. Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3. Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining. Overexpression of miR-145 inhibited colony formation, cell viability and induced cell arrest in S-phase in GC cells by targeting E2F3 and CDK6. miR-145 inhibition consistently abrogates the 1,25(OH)2D3-mediated suppression of E2F3, CDK6, CDK2 and CCNA2 genes. Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

Vitamin D receptor signaling enhances locomotive ability in mice.

Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of wild-type (WT) mice by 1.6-fold, whereas exercise did not enhance locomotive ability of VDR knockout (KO) mice. Compared with WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED-71, ELD), an analog of 1,25(OH)2 D3 , administered to rotarod-trained C57BL/6 mice enhanced locomotor performance compared with vehicle-treated nontrained mice. The area of AChR cluster on the extensor digitorum longus was greater in ELD-treated mice than in vehicle-treated mice. ELD and 1,25(OH)2 D3 enhanced expression of IGF-1, myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability after physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.

Characterization of Vitamin D Production by Human Ocular Barrier Cells

Vitamin D3 is a secosteroid mainly synthesized from the conversion of the skin precursor 7-dehydrocholesterol (7DHC) to vitamin D3 by ultraviolet (UV) B sunlight. Extrarenal synthesis of vitamin D3 has been reported in many tissues and cells, including barrier sites. This study characterizes the expression of components of vitamin D3 signaling in human ocular barrier cells. Primary human scleral fibroblasts (HSF), human corneal endothelial (HCEC-12), nonpigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines were analyzed for the expression of vitamin D receptor (VDR), the vitamin D3 activating enzymes 1α-hydroxylase (CYP27B1), 25-hydroxylases (CYP27A1 and CYP2R1), the vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and the endocytic receptors cubilin and megalin using a combination of RT-PCR, immunocytochemistry, and enzyme immunoassay (EIA). The HSF, HCEC-12, ODM-2, and ARPE-19 express mRNA and protein for all vitamin D3 synthesizing and metabolizing components. The cell types tested, except HSF, are able to convert inactive 25-hydroxyvitamin D3 (25[OH]D3) into active 1,25-hydroxyvitamin D3 (1,25[OH]2D3). This novel study demonstrated that ocular barrier epithelial cells express the machinery for vitamin D3 and can produce 1,25(OH)2D3. We suggest that vitamin D3 might have a role in immune regulation and barrier function in ocular barrier epithelial cells.

αSMA+ Macrophages Skewed From Hematopoietic Stem Cells By Vitamin D3 Initiate Myelofibrosis and Subsequent Osteosclerosis

Myelofibrosis (MF) is characterized as the proliferation of fibroblasts resulting in the replacement of marrow space by collagenous connective tissue fibers and is also known to be frequently complicated with osteosclerosis. However, the pathogenesis of this phenomenon is largely unknown. Allogeneic stem cell transplantation is the therapeutic choice in clinic with complete resolution of the disorder despite the recognition as microenvironment problem by the hematologists. Here, we establish a novel inducible murine MF model and propose a new paradigm in the pathophysiology of MF. Vitamin D receptor-deficient (VDR-/-) mice display rickets type II, which can be restored by high calcium diet, resulting in their usefulness as transplant recipients. We transplanted wild-type (WT) bone marrow (BM) cells into lethally irradiated VDR-/- mice and found that the vast majority died due to BM failure (n=25, median survival 66 days), though for the time being hematopoiesis was reconstituted during the first month after transplantation. The homing of long-term repopulating hematopoietic stem cells (HSCs) into marrow space was normal at 3 hrs, but the HSCs selectively disappeared at 3 weeks after transplantation as assessed by competitive reconstitution. Since VDR-/- mice showed normal hematopoiesis in steady-state, we transplanted VDR-/- BM into lethally irradiated VDR-/- recipients, which resulted in survival with no BM failure. Thus, engraftment failure of the WT HSCs in VDR-/- recipients did not originate from radiation-induced irreversible niche destruction, but it was likely intrinsic to donor HSC behavior. Histological analysis of femurs at 1-2 months after transplantation of WT BM into VDR-/- recipients revealed that the BM cavity was occupied by spindle-shaped cells and silver fibers. There was also prominently increased trabecular bones only in the metaphysis; whereas, normal hematopoietic appearance was observed in the diaphysis. This was initiated by hematopoietic cells since CD45+lin-c-kit+ cells isolated from WT CAG-EGFP transgenic mice as donor source induced the same MF and osteosclerosis in VDR-/- recipients. Metaphysial BM was replaced by monotonous fibroblastic cells in this particular setting; however, these cells were composed of two distinct populations with mutual distribution, 1) GFP+F4/80+ donor-derived macrophages and 2) GFP-osterix+ (or runx2+) host-derived preosteoblasts. These two distinct cells were tangled around each other equally in the fibrotic tissue area, and preosteoblasts were dominant in the osteoscrelotic area. Both populations were positive for αSMA. Since VDR-/- donor cells did not induce MF and it was reported that the level of 1,25(OH)2D3 (vitamin D3) is extremely high in VDR-/- mice, we hypothesized that WT HSCs exposed to high vitamin D3 might differentiate into αSMA+ macrophages and proliferate in vivo. Furthermore, since it is widely known that macrophages are strong supporter of osteoblasts, these cells might drive osteoblast-lineage cells. In the culture of hematopoietic stem/progenitor cell line FDCP-mix, vitamin D3 induced strong F4/80 upregulation together with partial αSMA induction, and MCP1 secretion in the culture supernatant was highly induced depending on vitamin D3 concentration. Strikingly, a diet low in vitamin D3 prevented the development of MF with osteosclerosis in VDR-/- recipients transplanted with WT BM. Thus, in our novel MF model, the true pathogenesis is likely that αSMA+ macrophages as MF-initiating cells perhaps directly differentiated from HSCs through vitamin D3 stimulation, drive the activity of preosteoblasts as a major producer of collagen fibers, and initiate osteosclerosis. We next examined whether this new paradigm could be applied for JAK2 V617F transgenic mice, which display MF with osteosclerosis, and human MF patients (n=3 including two cases with JAK2 V617F mutation). As we expected, marrow fibroblastic cells of both mouse genetic model and human patients were similarly composed of αSMA+CD169+ (or CD163+) macrophages and αSMA+ osteoblastic lineage cells with osterix or runx2 expression. Our study may explain why BMT is useful in clinic because MF is likely initiated by hematopoietic cells. We propose the modulation of vitamin D3 signaling or macrophage-targeted strategies as novel therapeutic choices for MF. Disclosures:No relevant conflicts of interest to declare.

Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.

Vitamin D3 Modulates Definitive Hematopoiesis by Two Distinct Mechanisms in the Developing Zebrafish Embryo

Abstract 763Studies of the vitamin D3 signaling pathway have revealed a broad role for this hormone in tissue homeostasis and as a result there is great interest in exploring the therapeutic potential of vitamin D3 for the treatment of various diseases including cancer. Studies investigating the role of vitamin D3 in hematological malignancies have shown that in vitro vitamin D3 has anti-proliferative and pro-differentiation effects resulting in differentiation of leukemic cell lines towards the monocytic lineage. Surprisingly, the role of vitamin D3 in HSC homeostasis and leukemia progression in vivo is not well understood. To elucidate the mechanisms of action of vitamin D3 in vivo during HSC self-renewal and differentiation, we utilized the zebrafish (Danio rerio) as vertebrate model due to their evolutionary conserved blood system and their amenability for genetic and chemical manipulation. Vitamin D3 was identified in a chemical screen performed in our laboratory as a regulator of runx1 and c-myb expression in the aorta-gonad mesonephros (AGM), the first site of definitive hematopoiesis. Treatments of zebrafish embryos with active vitamin D3 (1,25OH D3) between 12–36 hours post fertilization (hpf) resulted in increased expression of HSC markers (runx1, c-myb, CD41) as determined by whole mount in situ hybridization (WISH). In contrast, treatment with the non-hydroxylated vitamin D3 precursor cholecalciferol (D3) resulted in decreased runx1 and c-myb expression. D3 treatment during HSC induction and expansion from 24–36 hpf did not affect runx1 and c-myb expression, suggesting that D3 is acting early during the establishment of the vascular niche. To quantify the difference in HSC progenitors observed by WISH, FACS analysis was performed on double positive Tg(Lmo2:dsRed), Tg(c-myb:gfp) embryos. 1,25OH D3 treated embryos had a 20% increase in the double positive cell population corresponding to HSCs, while treatment with D3 resulted in a 25% decrease in the number of HSCs. The differential effect of 1,25OH D3 and D3 suggest that these two compounds act via two distinct mechanisms. To determine if the HSC enhancement observed by 1,25OH D3 was acting through the canonical vitamin D3 receptor (VDR), loss of function experiments were performed by injecting morpholinos targeting the vitamin D receptors. Morpholino knockdown of VDRA and VDRB (zebrafish VDR orthologs) resulted in decreased expression of runx1 c-myb, and CD41 via WISH. The decrease in HSC positive cells was confirmed by fluorescence microscopy using the Tg(Runx1P2:gfp) and Tg(-6.0itga:gfp) reporter lines, further supporting our hypothesis. To address the decrease in HSCs by D3, we postulated that D3 was inhibiting hedgehog signaling, as vitamin D3 has been previously shown to act as a negative regulator of the hedgehog pathway. In support of our hypothesis, treatment with D3 resulted in decreased ptch2 mRNA expression a downstream target of hedgehog signaling. In addition, FACS analysis using the hedgehog reporter Tg(6xGli:mCherry) line showed a 15% reduction in the number of mCherry positive cells in D3 treated embryos compared to controls. Co-treatments of zebrafish embryos with the hedgehog antagonist cyclopamine and D3 resulted in additive loss of reporter activity (45%) and loss of runx1 positive cells in the AGM, revealing synergy between these two compounds. In addition, co-exposure with the hedgehog agonist SAG rescued the HSC defect in D3-treated embryos. Hedgehog signaling is known to regulate vein and artery specification through the activation of notch signaling. Consistent with our hypothesis that D3 reduces hedgehog signaling, an expansion of the venous marker flt4 and a reduction in the expression of the arterial marker ephrinb2a was observed by WISH on D3-treated embryos. Furthermore, treatment of the notch reporter line Tg(Notch:gfp) with D3 resulted in lowered notch activity. In summary, these studies reveal that the active 1,25OH D3 positively regulates HSC progenitors in vivo during the onset of definitive hematopoiesis. In contrast, the non-hydroxylated vitamin D3 precursor acts as negative regulator of hematopoiesis by inhibiting hedgehog signaling and affecting vascular niche formation. Based on our studies, the differential effect between D3 and 1,25OH D3 should be considered when investigating the therapeutic potential of vitamin D3 in the context of hematological malignancies and other cancers. Disclosures:Goessling:Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy.

1,25-Dihydroxyvitamin D3 Promotes a Sustained LPS-Induced NF-κB-Dependent Expression of CD55 in Human Monocytic THP-1 Cells

The vitamin D3 system imposes immunosuppressive effects on monocytic cells, in part, by inhibiting NF-κB-dependent expression of proinflammatory mediators. CD55, a cell surface complement regulatory protein that promotes protective and anti-inflammatory properties, is reportedly an NF-κB target gene transiently induced in monocytic cells by the bacterial endotoxin LPS. CD55 is elevated on white cells in women experiencing preterm labor (a pathophysiology commonly associated with bacterial infection) and failure to maintain CD55 was associated with subsequent preterm delivery. We examined the influence of vitamin D3 signaling on LPS-induced expression of CD55 in human monocytic THP-1 cells using quantitative PCR, immunoblot, immunohistochemistry, and NF-κB activation pathway inhibitors. Non-NF-κB targets CD14 and CD11b, which modulate bacterial surveillance and eradication, respectively, were also examined. LPS produced a rapid transient 1.6-fold increase in CD55 mRNA. 1,25-D3 alone did not affect CD55 mRNA expression within the first 48 h. However, in 1,25-D3 pretreated cells, LPS produced a >4-fold immediate and sustained increase in CD55 mRNA and protein expression, which was blocked by NF-κB inhibitors. Our results unexpectedly suggest that vitamin D3 signaling may promote an anti-inflammatory response through an NF-κB-dependent increase in CD55 expression. As expected, LPS or 1,25-D3 alone led to sustained increases in CD14 and CD11b expression. In 1,25-D3 pretreated cells, LPS differentially regulated protein expression - CD14 (21-fold increase) and CD11b (a transient 2-fold decrease) - principally at the posttranscriptional level. The coordinated temporal expression of CD55, CD14 and CD11b would contribute to an anti-inflammatory response by providing protection against complement-mediated cell lysis during pathogen recognition and eradication. Overall, the vitamin D3 system may play a role coordinating an anti-inflammatory response pattern of the host complement immune system. This may be particularly important when considering the high rates of preterm births in blacks, a population that exhibits reduced circulating vitamin D3 levels.

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor‐mediated vitamin D3 metabolism

Cortisol was reported to downregulate body‐fluid Ca2+ levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca2+ from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca2+ uptake functions to cope with fluctuating Ca2+ levels in aquatic environments. Cortisol was previously found to regulate Ca2+ uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low‐Ca2+ fresh water stimulated Ca2+ influx and expression of epithelial calcium channel (ecac), 11β‐hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca2+influx and the expressions of ecac and hydroxysteroid 11‐beta dehydrogenase 2 (hsd11b2), but downregulated 11β‐hydroxylase and the gr with no effects on other Ca2+ transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca2+ uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca2+ uptake, the involvement of vitamin D3 was analyzed. Cortisol stimulated expressions of vitamin D‐25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α‐OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D3 in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca2+ uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D3 signaling to stimulate Ca2+ uptake in zebrafish.

Abstract B43: The Vitamin D receptor and retinoid X receptor-alpha expression related to DNMT levels in a murine model of colitis

Abstract Vitamin D3, whose functionality is controlled by signaling through the vitamin D receptor (VDR), plays a regulatory role in inflammation. To properly signal, VDR must heterodimerize with its signaling partner, Retinoid X Receptor (RXRα). When the signaling capability of Vitamin D3 goes awry, this mechanism of inflammation regulation is unchecked and cells are free to proliferate and release inflammatory signaling molecules. Inflammation and dysregulated Vitamin D3 signaling have been implicated in allowing the progression of colitis to cancer, but the mechanism by which this happens is poorly understood. Previous studies in our laboratory revealed that expression of RXRα is decreased in high-grade dysplasia in a mouse model of colon cancer. Whether RXRα or VDR are downregulated in human colitis remains to be clarified. The dextran sodium sulfate (DSS) model is a well-accepted animal model for human colitis, based on its histological similarity to human disease. Addition of azoxymethane (AOM), a carcinogen, is often utilized to encourage colon cancer development in mice with DSS-induced colitis. As a first step, we examined the expression profiles of RXR and VDR in the DSS induced and the DSS/AOM induced mouse models of colitis. To achieve colitis with the DSS model, ten C57BL/6J mice were given 2% DSS for one week followed by a two week recovery period with water and five control mice received only water. This was repeated two times to simulate the remission and relapses evidenced in human colitis. Mice were sacrificed after the third cycle. For the AOM/DSS model, ten C57BL/6J mice were intraperitoneally (IP) injected with 10mg/kg of AOM (control mice were IP with saline) and allowed to recover for one week with water before being given the same DSS model as described above. Mice were killed after one cycle for the acute model or after three cycles for the chronic model. At sacrifice, colons were either swiss-rolled for IHC analysis of VDR, RXRα and PCNA or the mucosal layer was scraped for determination of VDR, RXRα and DNA methyltranferase (DNMT) expression and protein levels. We found that in both mouse colitis protocols VDR protein levels were decreased with altered levels of RXRα protein. In accord with these primary effects, we also found DNMT expression to be altered. We conclude that downregulation of key transcription factors by epigenetic mechanisms could play a role in murine colitis models. We are also investigating whether this find translates to one's risk for colon cancer Citation Information: Cancer Prev Res 2011;4(10 Suppl):B43.

Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D3 in Prostate Progenitor/Stem Cells

Vitamin D(3) is a promising preventative and therapeutic agent for prostate cancer, but its implementation is hampered by a lack of understanding about its mechanism of action. Upon treatment with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3), vitamin D(3)], the metabolically active form of vitamin D(3), adult prostate progenitor/stem cells (PrP/SC) undergo cell-cycle arrest, senescence, and differentiation to an androgen receptor-positive luminal epithelial cell fate. Microarray analyses of control- and vitamin D(3)-treated PrP/SCs revealed global gene expression signatures consistent with induction of differentiation. Interestingly, one of the most highly upregulated genes by vitamin D(3) was the proinflammatory cytokine interleukin-1α (IL-1α). Systems biology analyses supported a central role for IL-1α in the vitamin D(3) response in PrP/SCs. siRNA-mediated knockdown of IL-1α abrogated vitamin D(3)-induced growth suppression, establishing a requirement for IL-1α in the antiproliferative effects of vitamin D(3) in PrP/SCs. These studies establish a system to study the molecular profile of PrP/SC differentiation, proliferation, and senescence, and they point to an important new role for IL-1α in vitamin D(3) signaling in PrP/SCs.