Vitamin D Receptor Agonists Research Articles

Abstract RAOS15-03: Pancreatic tumor stroma as a therapeutic target

Abstract Pancreatic ductal adenocarcinoma (PDAC) occurs in the context of an exuberant desmoplastic response that produces a ‘living chimera’ of tumor and activated stromal components. A prominent feature of the stromal response is the transformation of the Vitamin A storing pancreatic stellate cell (PSC) to activated myofibroblasts. We hypothesized that restoration of the quiescent PSC phenotype could disrupt tumor-stroma crosstalk and thus improve response to chemotherapy. Recently, we have shown that the vitamin D receptor (VDR) is expressed at high levels in activated human pancreatic tumor stroma and, in response to ligand, acts as a master transcriptional regulator of PSCs to reprise the quiescent state. In vivo, a synthetic VDR agonist (Cal) induced signal-dependent transcriptional remodeling of tumor stroma, enhancing the anti-tumor effects of gemcitabine by increasing intra-tumoral concentration 5-fold to reduce tumor growth. A survival study in KPC mice shows that addition of CAL results in a 57% increase in lifespan relative to Gemcitabine treatment alone. An approved IND enables the exploration of CAL in the neo-adjuvant setting in patients with resectable PDAC. This Phase I trial is designed to establish whether CAL (Zemplar) is able to reach the tumor and alter genes and other measurable responses linked to stromal reprogramming. Citation Format: Ronald M. Evans. Pancreatic tumor stroma as a therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr RAOS15-03. doi:10.1158/1538-7445.AM2015-RAOS15-03

Epigenetic Modulation of Human Podocyte Vitamin D Receptor in HIV Milieu

HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression.

Abstract IA9: Vitamin D: Shining light on pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) occurs in the context of an exuberant desmoplastic response that produces a ‘living chimera’ of tumor and activated stromal components. A prominent feature of the stromal response is the transformation of the Vitamin A storing pancreatic stellate cell (PSC) to activated myofibroblasts. We hypothesized that restoration of the quiescent PSC phenotype could disrupt tumor-stroma crosstalk and thus improve response to chemotherapy. Recently, we have shown that the vitamin D receptor (VDR) is expressed at high levels in activated human pancreatic tumor stroma and, in response to ligand, acts as a master transcriptional regulator of PSCs to reprise the quiescent state. In vivo, a synthetic VDR agonist (Cal) induced signal-dependent transcriptional remodeling of tumor stroma, enhancing the anti-tumor effects of gemcitabine by increasing intratumoral concentration to reduce tumor growth. Secreted factors are key drivers of the communication between tumor and stroma. To explore the impact of PSC secreted factors on gene expression in pancreatic cancer, PDAC cell lines were grown in the presence of stromal conditioned media (StCM). Addition of StCM was sufficient to induce expression of thousands of PDAC genes linked to epigenetic reprograming including genome wide histone hyperacetylation. The gene expression profile induced by stromal factors overlap with those previously identified following oncogenic Kras, suggesting functional complementarity between cell-autonomous and micro-environmental cues. As epigenome modification results in changes in gene expression through histone modifications, we tested a small molecule inhibitors epigenetic regulators for the capacity to inhibit gene expression changes downstream of stromal factors. Paracrine induction of pro-growth and inflammatory genes was inhibited by this small molecule, suggesting that acetyl-lysine readers serve as key transducers of stromal inputs to drive alterations in gene expression in pancreatic cancer cells. This work describes a molecular strategy through which transcriptional reprogramming of tumors and tumor stroma by Vitamin D or epigenetic regulators may serve as a therapeutic portal in the treatment of pancreatic cancer. Citation Format: Mara H. Sherman, Ning Ding, Eric A. Collisson, Michael Downes, Ronald M. Evans. Vitamin D: Shining light on pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA9.

AT1R blockade in adverse milieus: role of SMRT and corepressor complexes.

ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.

Optimization of histone deacetylase inhibitor activity in non-secosteroidal vitamin D-receptor agonist hybrids

The combination of (1α,25)-dihydroxyvitamin D3 (1,25D) and histone deacetylase inhibitor (HDACi) trichostatin A is highly antiproliferative in numerous cancer cell lines. We have previously prepared novel non-secosteroidal hybrid molecules which simultaneously act as both vitamin D receptor (VDR) agonists and HDACi. These molecules function as cytostatic and cytotoxic agents in 1,25D-resistant SCC4 squamous carcinoma cells. Here we have extended the scope of the hybrids by making several modifications to the diarylpentane core and to the aliphatic spacer unit to develop molecules with increased potency towards HDACs while maintaining VDR agonist activity. Notably, hybrid DK-366 (33a), a direct analog of first-generation hybrids but lacking a methyl group on one aryl ring possesses low micromolar potency for HDAC3 and HDAC6 and is a highly effective antiproliferative agent in SCC4 cells. Chain extended hybrids such as DK-367 (33c) possess even greater HDAC potency and are also highly antiproliferative. These results show that we can optimize HDACi potency in hybrid molecules without sacrificing VDR agonism.

FP016PARICALCITOL INHIBITED ALDOSTERONE-INDUCED PROINFLAMMATORY FACTORS BY MODULATING EPIDERMAL GROWTH FACTOR RECEPTOR PATHWAY IN CULTURED TUBULAR EPITHELIAL CELLS

Chronic kidney disease is characterized by Vitamin D deficiency and activation of the renin-angiotensin-aldosterone system. Increasing data show that vitamin D receptor agonists (VDRAs) exert beneficial effects in renal disease and possess anti-inflammatory properties, but the underlying mechanism remains unknown. Emerging evidence suggests that “a disintegrin and metalloproteinase” (ADAM)/epidermal growth factor receptor (EGFR) signalling axis contributes to renal damage. Aldosterone induces EGFR transactivation regulating several processes including cell proliferation and fibrosis. However, data on tubular epithelial cells is scarce. We have found that, in cultured tubular epithelial cells, aldosterone induced EGFR transactivation via TGF-α/ADAM17. Blockade of the TGF-α/ADAM17/EGFR pathway inhibited aldosterone-induced proinflammatory gene upregulation. Moreover, among the potential downstream mechanisms, we found that TGF-α/ADAM17/EGFR inhibition blocked ERK and STAT-1 activation in response to aldosterone. Next, we investigated the involvement of TGF-α/ADAM17/EGFR axis in VDRA anti-inflammatory effects. Preincubation with the VDRA paricalcitol inhibited aldosterone-induced EGFR transactivation, TGF-α/ADAM-17 gene upregulation, and downstream mechanisms, including proinflammatory factors overexpression. In conclusion, our data suggest that the anti-inflammatory actions of paricalcitol in tubular cells could depend on the inhibition of TGF-α/ADAM17/EGFR pathway in response to aldosterone, showing an important mechanism of VDRAs action.

Vitamin D receptor activation protects against myocardial reperfusion injury through inhibition of apoptosis and modulation of autophagy.

To determine the roles of vitamin D receptor (VDR) in ischemia/reperfusion-induced myocardial injury and to investigate the underlying mechanisms involved. The endogenous VDR expression was detected in the mouse heart, and myocardial ischemia/reperfusion (MI/R) upregulated VDR expression. Activation of VDR by natural and synthetic agonists reduced myocardial infarct size and improved cardiac function. Mechanistically, VDR activation inhibited endoplasmic reticulum (ER) stress (determined by the reduction of CCAAT/enhancer-binding protein homologous protein expression and caspase-12 activation), attenuated mitochondrial impairment (determined by the decrease of mitochondrial cytochrome c release and caspase-9 activation), and reduced cardiomyocyte apoptosis. Furthermore, VDR activation significantly inhibited MI/R-induced autophagy dysfunction (determined by the inhibition of Beclin 1 over-activation, the reduction of autophagosomes, the LC3-II/LC3-I ratio, p62 protein abundance, and the restoration of autophagy flux). Moreover, VDR activation inhibited MI/R-induced oxidative stress through a metallothionein-dependent mechanism. The cardioprotective effects of VDR agonists mentioned earlier were impaired in the setting of cardiac-specific VDR silencing. In contrast, adenovirus-mediated cardiac VDR overexpression decreased myocardial infarct size and improved cardiac function through attenuating oxidative stress, and inhibiting apoptosis and autophagy dysfunction. Our data demonstrate that VDR is a novel endogenous self-defensive and cardioprotective receptor against MI/R injury, via mechanisms (at least in part) reducing oxidative stress, and inhibiting apoptosis and autophagy dysfunction-mediated cell death.

Paricalcitol Inhibits Aldosterone-Induced Proinflammatory Factors by Modulating Epidermal Growth Factor Receptor Pathway in Cultured Tubular Epithelial Cells.

Chronic kidney disease is characterized by Vitamin D deficiency and activation of the renin-angiotensin-aldosterone system. Increasing data show that vitamin D receptor agonists (VDRAs) exert beneficial effects in renal disease and possess anti-inflammatory properties, but the underlying mechanism remains unknown. Emerging evidence suggests that “a disintegrin and metalloproteinase” (ADAM)/epidermal growth factor receptor (EGFR) signalling axis contributes to renal damage. Aldosterone induces EGFR transactivation regulating several processes including cell proliferation and fibrosis. However, data on tubular epithelial cells is scarce. We have found that, in cultured tubular epithelial cells, aldosterone induced EGFR transactivation via TGF-α/ADAM17. Blockade of the TGF-α/ADAM17/EGFR pathway inhibited aldosterone-induced proinflammatory gene upregulation. Moreover, among the potential downstream mechanisms, we found that TGF-α/ADAM17/EGFR inhibition blocked ERK and STAT-1 activation in response to aldosterone. Next, we investigated the involvement of TGF-α/ADAM17/EGFR axis in VDRA anti-inflammatory effects. Preincubation with the VDRA paricalcitol inhibited aldosterone-induced EGFR transactivation, TGF-α/ADAM-17 gene upregulation, and downstream mechanisms, including proinflammatory factors overexpression. In conclusion, our data suggest that the anti-inflammatory actions of paricalcitol in tubular cells could depend on the inhibition of TGF-α/ADAM17/EGFR pathway in response to aldosterone, showing an important mechanism of VDRAs action.

Hyperglycemia enhances kidney cell injury in HIVAN through down-regulation of vitamin D receptors

In the present study, we evaluated the effect of short term hyperglycemia on renal lesions in a mouse model (Tg26) of HIV-associated nephropathy (HIVAN). Control and Tg26 mice in groups (n=6) were administered either normal saline (FVBN or Tg) or streptozotocin (FVBN+STZ or Tg26+STZ). After two weeks, biomarkers were collected and kidneys were harvested. FVBN+ STZ and Tg26+STZ displayed elevated serum glucose levels when compared to FVBN and Tg26 respectively. Tg26+STZ displayed elevated (P<0.05) blood urea nitrogen (BUN) levels (P<0.05) and enhanced (P<0.01) proteinuria when compared to Tg26. Tg26+STZ displayed enhanced (P<0.001) number of sclerotic glomeruli and microcysts vs. Tg26. Renal tissues of Tg26 displayed down regulation of vitamin D receptor (VDR) expression and enhanced Ang II production when compared to FVBN mice. Hyperglycemia exacerbated down regulation of VDR and production of Ang II in FVBN and Tg mice. Hyperglycemia increased kidney cell reactive oxygen species (ROS) production and oxidative DNA damage in both FVBN and Tg26 mice. In in vitro studies, HIV down regulated podocyte VDR expression and also enhanced renin angiotensin system activation. In addition, both glucose and HIV stimulated kidney cell ROS generation and DNA damage and compromised DNA repair; however, tempol (superoxide dismutase mimetic), losartan (Ang II blocker) and EB1089 (VDR agonist) provided protection against DNA damaging effects of glucose and HIV. These findings indicated that glucose activated the RAS and inflicted oxidative stress-mediated DNA damage via down regulation of kidney cell VDR expression in HIV milieu both in vivo and in vitro.

Vitamin D receptor agonist VS-105 improves cardiac function in the presence of enalapril in 5/6 nephrectomized rats.

Vitamin D receptor (VDR) agonists (VDRAs) are commonly used to manage hyperparathyroidism secondary to chronic kidney disease (CKD). Patients with CKD experience extremely high risks of cardiovascular morbidity and mortality. Clinical observations show that VDRA therapy may be associated with cardio-renal protective and survival benefits in patients with CKD. The 5/6 nephrectomized (NX) Sprague-Dawley rat with established uremia exhibits elevated serum parathyroid hormone (PTH), hypertension, and abnormal cardiac function. Treatment of 5/6 NX rats with VS-105, a novel VDRA (0.05 and 0.5 μg/kg po by gavage), once daily for 8 wk in the presence or absence of enalapril (30 mg/kg po via drinking water) effectively suppressed serum PTH without raising serum calcium. VS-105 alone reduced systolic blood pressure (from 174 ± 6 to 145 ± 9 mmHg, P < 0.05) as effectively as enalapril (from 174 ± 6 to 144 ± 7 mmHg, P < 0.05). VS-105 improved cardiac functional parameters such as E/A ratio, ejection fraction, and fractional shortening with or without enalapril. Enalapril or VS-105 alone significantly reduced left ventricular hypertrophy (LVH); VS-105 plus enalapril did not further reduce LVH. VS-105 significantly reduced both cardiac and renal fibrosis. The lack of hypercalcemic toxicity of VS-105 is due to its lack of effects on stimulating intestinal calcium transport and inducing the expression of intestinal calcium transporter genes such as Calb3 and TRPV6. These studies demonstrate that VS-105 is a novel VDRA that may provide cardiovascular benefits via VDR activation. Clinical studies are required to confirm the cardiovascular benefits of VS-105 in CKD.

Mesenchymal stem cells and a vitamin D receptor agonist additively suppress T helper 17 cells and the related inflammatory response in the kidney.

Mesenchymal stem cells (MSCs) suppress T helper (Th)17 cell differentiation and are being clinically pursued for conditions associated with aberrant Th17 responses. Whether such immunomodulatory effects are enhanced by coadministration of MSCs with other agents is not well known. In the present study, individual and combined effects of MSCs and the vitamin D receptor (VDR) agonist paricalcitol on Th17 induction were investigated in vitro and in a mouse model of sterile kidney inflammation (unilateral ureteral obstruction). In vitro, MSCs and paricalcitol additively suppressed Th17 differentiation, although only MSCs suppressed expression of Th17-associated transcriptions factors. Combined administration of MSCs and paricalcitol resulted in an early (day 3) reduction of intrarenal CD4(+) and CD8(+) T cells, CD11b(+)/lymphocyte antigen 6G(+) neutrophils, and inflammatory (lymphocyte antigen 6C(hi)) monocytes as well as reduced transcript for IL-17 compared with untreated animals. Later (day 8), obstructed kidneys of MSC/paricalcitol double-treated mice, but not mice treated with either intervention alone, had reduced tubular injury and interstitial fibrosis as well as lower numbers of neutrophils and inflammatory monocytes and an increase in the ratio between M2 (CD206(+)) and M1 (CD206(-)) macrophages compared with control mice. Adjunctive therapy with VDR agonists may enhance the immunosuppressive properties of MSCs in the setting of pathogenic Th17-type immune responses and related inflammatory responses.

Structural development of p-carborane-based potent non-secosteroidal vitamin D analogs

Non-secosteroidal vitamin D receptor (VDR) ligands are promising candidates for many clinical applications. We recently developed novel non-secosteroidal VDR agonists based on p-carborane (an icosahedral carbon-containing boron cluster) as a hydrophobic core structure. Here, we report the design, synthesis and biological evaluation of carborane-based vitamin D analogs bearing various substituents at the diol moiety. Among the synthesized compounds, methylene derivative 31 exhibited the most potent vitamin D activity, which was comparable to that of the natural hormone, 1α,25(OH)2D3. This compound is one of the most potent non-secosteroidal VDR agonists reported to date, and is a promising lead for development of novel drug candidates.

Elimination of vitamin D receptor in vascular endothelial cells alters vascular function.

Vitamin D deficiency has been associated with cardiovascular dysfunction. We evaluated the role of the vitamin D receptor (VDR) in vascular endothelial function, a marker of cardiovascular health, at baseline and in the presence of angiotensin II, using an endothelial-specific knockout of the murine VDR gene. In the absence of endothelial VDR, acetylcholine-induced aortic relaxation was significantly impaired (maximal relaxation, endothelial-specific VDR knockout=58% versus control=73%; P<0.05). This was accompanied by a reduction in endothelial NO synthase expression and phospho-vasodilator-stimulated phosphoprotein levels in aortae from the endothelial-specific VDR knockout versus control mice. Although blood pressure levels at baseline were comparable at 12 and 24 weeks of age, the endothelial VDR knockout mice demonstrated increased sensitivity to the hypertensive effects of angiotensin II compared with control mice (after 1-week infusion: knockout=155±15 mm Hg versus control=133±7 mm Hg; P<0.01; after 2-week infusion: knockout=164±9 mm Hg versus control=152±13 mm Hg; P<0.05). By the end of 2 weeks, angiotensin II infusion-induced, hypertrophy-sensitive myocardial gene expression was higher in endothelial-specific VDR knockout mice (fold change compared with saline-infused control mice, type-A natriuretic peptide: knockout mice=3.12 versus control=1.7; P<0.05; type-B natriuretic peptide: knockout mice=4.72 versus control=2.68; P<0.05). These results suggest that endothelial VDR plays an important role in endothelial cell function and blood pressure control and imply a potential role for VDR agonists in the management of cardiovascular disease associated with endothelial dysfunction.

Vitamin D in pediatric rheumatology

One century after its discovery vitamin D is shown to be, in fact, a pleiotropic steroid hormone which, beside regulation of calcium homeostasis and bone turnover, has anti-proliferative, pro-differentiation, anti-bacterial, immunomodulatory and anti-inflammatory properties in various cells and tissues. The D hormone [1α,25(OH)2D], regulated in endocrine, autocrine and paracrine manner, must be bound to the specific nuclear vitamin D receptor (VDR) to exert epigenetic and genetic effects influencing more than 2000 genes in all tissues and immune cells, essential for proliferation, differentiation and immunoregulation. VDR agonists inhibit in myeloid DCs, but not in plazmacytoid DCs, expression of surface co-stimulatory molecules such as MHC class II, CD40, CD80 and CD86. In T cells, 1,25(OH)2D decreases the production of IL-2, IL-17 and interferon-γ (IFNγ) and attenuates the cytotoxic activity and proliferation of CD4+ and CD8+ T cells, and promote the development of FoxP3+ regulatory T (TReg) cells and IL-10-producing T regulatory type 1 cells. In contrast to glucocorticoids which are non-selective immunosuppressive compaunds, 1α,25(OH)2D induces monocyte proliferation and the expression of interleukin-1 (IL-1) and cathelicidin (an antimicrobial peptide) by macrophages, thereby contributing to innate immune responses to some bacteria. Additionally it was shown that 1α,25(OH)2D can override steroid resistance and antagonize its negative bone turnover influence. The discovery of the immunomodulatory and anti-tumor properties of D-hormone prompted researchers to investigate possibility of its use as a preventive and therapeutic agent for different autoimmune and malignant diseases. Several publications in last years have found a high prevalence of vitamin D deficiency in JIA, SLE and other chronic inflammatory diseases in childhood, with the mean values of 25(OH)D levels at the lower end of ‘acceptable’ range. Measurement of 25(OH)D level (as the only standardized toll to estimate vitamin D status) is actually only the reflection of the balance between food and/or supplement vitamin D diet intake and its utilization in the local tissues into the active D hormone (especially immune cells in the state of chronic inflammation). Recent recommendations and clinical guidelines have suggested vitamin D supplementation of up to 2000 IU/d to be safe and well tolerated in children with chronic diseases. New recommendations and guidelines for vitamin D supplementation, as adjunct treatment option in JIA and other chronic inflammatory rheumatic diseases, is an appealing need due to its pleiotropic effects. It can both minimize bone fragility and contribute improvement of impaired immunity regulation in rheumatic diseases in children, especially those with necessity for long term steroid treatment.

Vitamin D receptor agonists' anti-inflammatory properties.

One century after its discovery, vitamin D has been shown to be, in fact, a pleiotropic steroid hormone, which, besides regulation of calcium homeostasis and bone turnover, has antiproliferative, prodifferentiation, antibacterial, immunomodulatory, and anti-inflammatory properties in various cells and tissues. D hormone (1α,25(OH)2 D), regulated in an endocrine, autocrine, and paracrine manner, must be bound to the specific nuclear vitamin D receptor (VDR) to exert epigenetic and genetic effects, acting as a connection between extracellular stimuli and genomic responses of the cells. Since only high doses of hormone, provoking hypercalcemia, can achieve immunomodulatory effects, more than 3000 VDR agonists have been synthesized. Numerous experimental trials have been performed in animal models, evidencing the preventive and therapeutic potential of VDR agonists for chronic inflammatory diseases and cancer. Considering the selective anti-inflammatory effects of VDR agonists compared to glucocorticoids, sparing microbicidal functions, the fear of hypercalcemia as their only frequent side effect becomes a questionable reason for the lack of clinical studies.

Ginkgo biloba extract potentiates human vitamin D receptor‐mediated increase in CYP2B6 reporter activity by 1α,25‐dihydroxyvitamin D3 (1141.8)

Vitamin D receptor (VDR) regulates the expression of a broad array of genes, including those involved in drug biotransformation (e.g. CYP2B6). Ginkgo biloba extract induces CYP2B6, but it is not known whether it activates VDR. Therefore, we characterized the effect of G. biloba extract on human VDR (hVDR) activity. As assessed in a cell‐based dual‐luciferase reporter gene assay, G. biloba extract did not activate hVDR in cells transfected with a hVDR expression plasmid and a CYP2B6‐PBREM/XREM reporter plasmid. In contrast, 1α,25‐dihydroxyvitamin D3, which is a hVDR agonist, increased it by 16‐fold compared to the vehicle‐treated group. The combination of G. biloba extract and 1α,25‐dihydroxyvitamin D3 further increased hVDR activity by another 14‐fold (i.e. 225‐fold increase compared to the vehicle‐treated group). This potentiation effect was evident in multiple lots of G. biloba extract and with other VDR agonists (i.e. lithocholic acid and 9‐cis retinoic acid), and it increased as the concentration of the extract or 1α,25‐dihydroxyvitamin D3 increased. In contrast, the potentiation did not occur when cells were transfected with a plasmid expressing human constitutive androstane receptor, human pregnane X receptor, or mouse VDR. Overall, the findings indicate that G. biloba extract potentiates hVDR‐mediated CYP2B6 reporter activity.Grant Funding Source: Supported by the Canadian Institutes of Health Research and Michael Smith Fdn. for Health Res.

LncRNA: a new player in 1α, 25(OH)2 vitamin D3/VDR protection against skin cancer formation

Sunlight, vitamin D and skin cancer form a controversial brew. While too much sunlight exposure causes skin cancer, it is the major source of vitamin D from skin. We propose that these processes can be balanced. Vitamin D signalling (VDS) protects against skin cancer as demonstrated by the susceptibility of the skin to tumor formation in VDR null mice and protection from UVB-induced mutations when VDR agonists are administered. The question is how is protection afforded. Previously, we have focused on the Wnt/β-catenin/hedgehog and DNA damage repair (DDR) pathways. As VDR regulates hundreds of genes with thousands of VDR response elements (VDRE) throughout the genome, and many VDREs are in non-coding regions, we decided to explore long non-coding RNAs (lncRNA). LncRNAs are mRNA-like transcripts ranging from 200 bases ~100 kb lacking significant open reading frames. They are aberrantly expressed in human cancers and involved in a spectrum of tumorigenic/metastatic processes (cell proliferation/apoptosis/angiogenesis). We discovered that VDS regulated the expression of certain lncRNAs in a manner consistent with VDS protection against skin cancer. Given the huge variation in genes actively regulated by 1,25(OH)2 D from different cell types, it is conceivable that our results could apply to personalized medicine based on the distinctive lncRNA profiles. These lncRNAs could also serve as skin cancer biomarkers secreted into the blood or urine via exosomes as demonstrated in other cancer types (breast, prostate). Modulation of lncRNA profile by VDS may also provide insight into regulating pathways such as Wnt/ß-catenin and hedgehog.

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

Vitamin D receptor (VDR) agonists supporting human osteoblast (hOB) differentiation in the absence of bone resorption are attractive agents in a bone regenerative setting. One potential candidate fulfilling these roles is 24,25-dihydroxy vitamin D3 (24,25D). Over forty years ago it was reported that supraphysiological levels of 24,25D could stimulate intestinal calcium uptake and aid bone repair without causing bone calcium mobilisation. VDR agonists co-operate with certain growth factors to enhance hOB differentiation but whether 24,25D might act similarly in promoting cellular maturation has not been described. Given our discovery that lysophosphatidic acid (LPA) co-operated with VDR agonists to enhance hOB maturation, we co-treated MG63 hOBs with 24,25D and a phosphatase-resistant LPA analog. In isolation 24,25D inhibited proliferation and stimulated osteocalcin expression. When co-administered with the LPA analog there were synergistic increases in alkaline phosphatase (ALP). These are encouraging findings which may help realise the future application of 24,25D in promoting osseous repair.

Synthesis and structure–activity relationship of p-carborane-based non-secosteroidal vitamin D analogs

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3: 1] is a specific modulator of nuclear vitamin D receptor (VDR), and novel vitamin D analogs are therapeutic candidates for multiple clinical applications. We recently developed non-secosteroidal VDR agonists bearing a p-carborane cage (a carbon-containing boron cluster) as a hydrophobic core structure. These carborane derivatives are structurally quite different from classical secosteroidal vitamin D analogs. Here, we report systematic synthesis and activity evaluation of carborane-based non-secosteroidal vitamin D analogs. The structure–activity relationships of carborane derivatives are different from those of secosteroidal vitamin D derivatives, and in particular, the length and the substituent position of the dihydroxylated side chain are rather flexible in carborane derivatives. The structure–activity relationships presented here should be helpful in development of non-secosteroidal vitamin D analogs for clinical applications.

Vitamin D receptor agonists: suitable candidates as novel therapeutic options in autoimmune inflammatory myopathy.

The primary aim in the treatment of autoimmune inflammatory myopathies (IMs) is to recover muscle function. The presence of immune/inflammatory cell infiltrates within muscle tissues represents the common feature of different IM subtypes, albeit a correlation between muscular damage extent and inflammation degree is often lacking. Treatments for IMs are based on life-long immunosuppressive therapy, with the well known adverse effects; recovery is incomplete for many patients. More effective therapies, with reduced side-effects, are highly desirable. Vitamin D receptor (VDR) agonists emerge to retain pleiotropic anti-inflammatory properties, since they regulate innate and adaptive immunity by switching the immune response from proinflammatory T helper 1 (Th1) type to tolerogenic T helper 2 (Th2) type dominance. In skeletal muscle cells less hypercalcemic VDR ligands target powerful mediators of inflammation, such as TNFα and TNFα driven paths, without affecting immune or muscle cells viability, retaining the potentiality to counteract Th1 driven overreactivity established by the self-enhancing inflammatory loop between immune and skeletal muscle cells. This review summarizes those features of VDR agonists as candidates in future treatment of IM.