Wnt signalling is required for hair follicle development and for the growth phase (anagen) of postnatal follicles. When the pathway is activated at high levels in adult mouse epidermis, ectopic follicles form from existing follicles, interfollicular epidermis (IFE) and sebaceous glands, revealing a remarkable ability of the tissue to be reprogrammed. To compare the competence of different epidermal cell populations to form ectopic follicles, we expressed a 4-hydroxy-tamoxifen (4OHT) inducible, stabilised beta-catenin transgene (DeltaNbeta-cateninER) under the control of two different promoters. We targeted the reservoir of stem cells in the hair follicle bulge via the keratin 15 (K15) promoter and targeted the sebaceous glands and base of the follicle (bulb) with a truncated K5 promoter (DeltaK5). No ectopic follicles formed in the IFE in either model, establishing the autonomy of the IFE stem cell compartment in undamaged epidermis. Activation of beta-catenin in the bulge stimulated proliferation and bulge expansion. Existing hair follicles entered anagen, but no ectopic follicles formed. DeltaK5DeltaNbeta-cateninER expressing hair follicles also entered anagen on 4OHT treatment. In addition, a subpopulation of cells at the base of the sebaceous gland readily formed ectopic follicles, resulting in complete and reversible conversion of sebaceous glands into hair follicles. Combined activation of beta-catenin and the vitamin D receptor enhanced differentiation of sebaceous gland-derived hair follicles and stimulated ectopic follicle formation in the hair follicle bulb, but not in the bulge. Our results suggest that the bulge and sebaceous gland are, respectively, non-permissive and permissive niches for Wnt induced hair follicle differentiation.

Active vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH)2D3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER+) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER+ (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER+ BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER+ BC both in vitro and in vivo.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+ CD25high CD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+ CD25high CD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+ CD25high CD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.

The measurement of 1α,25(OH)2D3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere.

The detection of cis-regulatory modules (CRMs) that mediate transcriptional responses in eukaryotes remains a key challenge in the postgenomic era. A CRM is characterized by a set of co-occurring transcription factor binding sites (TFBS). In silico methods have been developed to search for CRMs by determining the combination of TFBS that are statistically overrepresented in a certain geneset. Most of these methods solve this combinatorial problem by relying on computational intensive optimization methods. As a result their usage is limited to finding CRMs in small datasets (containing a few genes only) and using binding sites for a restricted number of transcription factors (TFs) out of which the optimal module will be selected.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5'-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

In chronic obstructive pulmonary disease (COPD), the bronchial epithelium is the first immune barrier that is triggered by cigarette smoke. Although vitamin D (vitD) has proven anti-inflammatory and antimicrobial effects in alveolar macrophages, little is known about the direct role of vitD on cigarette smoke-exposed bronchial epithelial cells. We examined the effects of vitD on a human bronchial epithelial cell line (16HBE) and on air-liquid culture of primary bronchial epithelial cells (PBEC) of COPD patients and controls exposed for 24 h to cigarette smoke extract (CSE). VitD decreased CSE-induced IL-8 secretion by 16HBE cells, but not by PBEC. VitD significantly increased the expression of the antimicrobial peptide cathelicidin in 16HBE and PBEC of both COPD subjects and controls. VitD did not affect epithelial to mesenchymal transition or epithelial MMP-9 expression and was not able to restore impaired wound healing by CSE in 16HBE cells. VitD increased the expression of its own catabolic enzyme CYP24A1 thereby maintaining its negative feedback. In conclusion, vitD supplementation may potentially reduce infectious exacerbations in COPD by the upregulation of cathelicidin in the bronchial epithelium.

Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts.

Three analogues of 1a,25-dihydroxyvitamin D(3) (calcitriol), featuring a trans-fused decalin C,D-core with local S(2)-symmetry, and possessing identical side-chain and seco-B,A-ring structures, have been synthesized starting from readily available (4aR,8aS)-octahydronaphthalene-1,5-dione (7). The very short sequences involve the simultaneous introduction of the side-chain and seco-B,A-ring fragments via Suzuki and Sonogashira coupling reactions. The analogues are devoid of relevant biological activity.

We have previously shown that 1α,25(OH)2-Vitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 inhibit the proliferation of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR) and this could be partially explained by the inhibition of the NF-κB pathway. In this work, we further explored the mechanism of action of both vitamin D compounds in Kaposi sarcoma. We investigated whether the cell cycle arrest and subsequent apoptosis of endothelial cells (SVEC) and SVEC transformed by vGPCR (SVEC-vGPCR) elicited by 1α,25(OH)2D3 and TX 527 were mediated by the vitamin D receptor (VDR). Cell cycle analysis of SVEC and SVEC-vGPCR treated with 1α,25(OH)2D3 (10nM, 48h) revealed that 1α,25(OH)2D3 increased the percentage of cells in the G0/G1 phase and diminished the percentage of cells in the S phase of the cell cycle. Moreover, the number of cells in the S phase was higher in SVEC-vGPCR than in SVEC due to vGPCR expression. TX 527 exerted similar effects on growth arrest in SVEC-vGPCR cells. The cell cycle changes were suppressed when the expression of the VDR was blocked by a stable transfection of shRNA against VDR. Annexin V-PI staining demonstrated apoptosis in both SVEC and SVEC-vGPCR after 1α,25(OH)2D3 and TX 527 treatment (10nM, 24h). Cleavage of caspase-3 detected by Western blot analysis was increased to a greater extent in SVEC than in SVEC-vGPCR cells, and this effect was also blocked in VDR knockdown cells. Altogether, these results suggest that 1α,25(OH)2D3 and TX 527 inhibit the proliferation of SVEC and SVEC-vGPCR and induce apoptosis by a mechanism that involves the VDR.

Inflammatory bowel diseases are gastrointestinal diseases that include Crohn disease and ulcerative colitis. The chronic inflammation is thought to result from an excessive inflammatory response to environmental factors such as luminal bacteria in genetically predisposed individuals. Studies have revealed that mice with impaired vitamin D signaling are more susceptible to experimental colitis. To better understand the contribution of vitamin D signaling in different cells of the gut to this disease, we investigated the effects of intestinal-specific or myeloid vitamin D receptor deletion. Our study addressed the importance of vitamin D receptor expression in intestinal epithelial cells using intestine-specific vitamin D receptor null mice and the contribution of vitamin D receptor expression in macrophages and granulocytes using myeloid-specific vitamin D receptor null mice in a dextran sodium sulfate model for experimental colitis. Loss of intestinal vitamin D receptor expression had no substantial effect on the clinical parameters of colitis and did not manifestly change mucosal cytokine expression. Inactivation of the vitamin D receptor in macrophages and granulocytes marginally affected colitis-associated symptoms but resulted in increased proinflammatory cytokine and increased β-defensin-1 expression in the colon descendens of mice with colitis. Intestinal deletion of the vitamin D receptor did not aggravate symptoms of chemically induced colitis. Loss of the vitamin D receptor in macrophages and granulocytes mildly affected colitis-associated symptoms but greatly increased proinflammatory cytokine expression in the inflamed colon, suggesting a prominent role for innate immune cell vitamin D signaling in controlling gut inflammation.

Besides its actions on minerals and bone, the bioactive vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has important immunomodulatory properties. Within the immune system, dendritic cells represent key targets for this hormone and 1,25(OH)2D3-induced changes in their phenotype and function ultimately affects T lymphocytes. However, the presence of vitamin D receptors (VDR) in activated T cells proposes additional mechanisms for 1,25(OH)2D3 to directly regulate T cell responses. Here, we investigated the expression and kinetics of vitamin D-related genes in human activated T lymphocytes. Different activation stimuli elicited increased VDR- and 1-alpha-hydroxylase expression, with a highly similar kinetic pattern. Addition of 1,25(OH)2D3 effectively triggered VDR signaling, as evidenced by 24-hydroxylase induction, but only when introduced to T lymphocytes expressing high levels of VDR. This enhanced degree of VDR signaling correlated with a stronger inhibition of cytokines (IFN-gamma, IL-10) and modulation of homing receptor expression (CCR10, CLA) in long-term T cell cultures. Importantly, chronic 1,25(OH)2D3-exposure further amplified VDR signaling and the concomitant T cell modulating effects. In conclusion, we validate T cells as direct targets for 1,25(OH)2D3 and provide this optimized in vitro model to improve our understanding of the role of vitamin D as a direct regulator of T cell responses.

The Vitamin D receptor (VDR) plays a key role in calcium homeostasis, as well as in cell proliferation and differentiation. Among the large number of VDR ligands that have been developed, we have previously shown that BXL-62 and Gemini-72, two C-20-modified vitamin D analogs are highly potent VDR agonists. In this study, we show that both VDR ligands restore the transcriptional activities of VDR variants unresponsive to the natural ligand and identified in patients with rickets. The elucidated mechanisms of action underlying the activities of these C-20-modified analogs emphasize the mutual adaptation of the ligand and the VDR ligand-binding pocket.