Vitamin D Receptor Knockout Mice Research Articles

VDR restores the expression of PINK1 and BNIP3 in TECs of streptozotocin-induced diabetic mice.

Defective mitophagy in renal tubular epithelial cells is one of the main drivers of renal fibrosis in diabetic kidney disease. Our gene sequencing data showed the expression of PINK1 and BNIP3, two key molecules of mitophagy, was decreased in renal tissues of VDR-knockout mice. Herein, streptozotocin (STZ) was used to induce renal interstitial fibrosis in mice. VDR deficiency exacerbated STZ-induced renal impairment and defective mitophagy. Paricalcitol (pari, a VDR agonist) and the tubular epithelial cell-specific overexpression of VDR restored the expression of PINK1 and BNIP3 in the renal cortex and attenuated STZ-induced kidney fibrosis and mitochondrial dysfunction. In HK-2 cells under high glucose conditions, an increased level of α-SMA, COL1, and FN and a decreased expression of PINK1 and BNIP3 with severe mitochondrial damage were observed, and these alterations could be largely reversed by pari treatment. ChIP-qPCR and luciferase reporter assays showed VDR could positively regulate the transcription of Pink1 and Bnip3 genes. These findings reveal that VDR could restore mitophagy defects and attenuate STZ-induced fibrosis in diabetic mice through regulation of PINK1 and BNIP3.

The role of vitamin D receptor in predentin mineralization and dental repair after injury.

Dentin is a permeable and complex tubular composite formed by the mineralization of predentin that mineralization and repair are of considerable clinical interest during dentin homeostasis. The role of Vdr, a receptor of vitamin D, in dentin homeostasis remains unexplored. The aim of the present study was to assess the impact of Vdr on predentin mineralization and dental repair. Vdr-knockout (Vdr-/-) mice models were constructed; histology and immunohistochemistry analyses were conducted for both WT and Vdr-/- mice. The finding revealed a thicker predentin in Vdr-/- mice, characterized by higher expression of biglycan and decorin. A dental injury model was employed to observe tertiary dentin formation in Vdr-/- mice with dental injuries. Results showed that tertiary dentin was harder to form in Vdr-/- mice with dental injury. Over time, heightened pulp invasion was observed at the injury site in Vdr-/- mice. Expression of biglycan and decorin was reduced in the predentin at the injury site in the Vdr-/- mice by immunohistochemistry. Taken together, our results imply that Vdr plays a regulatory role in predentin mineralization and tertiary dentin formation during dentin homeostasis.

VDR is an essential regulator of hair follicle regression through the progression of cell death.

Vitamin D receptor (VDR) is essential for hair follicle homeostasis as its deficiency induces hair loss, although the mechanism involved remains unknown. Our research shows that, in Vdr-knockout mice, the hair cycle is halted during the catagen stage, preceding alopecia. In addition, in Vdr-knockout hair follicles, epithelial strands that normally regress during the catagen phase persist as "surviving epithelial strands." Single-cell RNA sequencing analysis suggests that these surviving epithelial strands are formed by cells in the lower part of the hair follicle. These findings emphasize the importance of the regression phase in hair follicle regeneration and establish VDR as a regulator of the catagen stage.

Myeloid vitamin D receptor regulates Paneth cells and microbial homeostasis.

Cross talk between immune cells and the intestinal crypt is critical in maintaining intestinal homeostasis. Recent studies highlight the direct impact of vitamin D receptor (VDR) signaling on intestinal and microbial homeostasis. However, the tissue-specific role of immune VDR signaling is not fully understood. Here, we generated a myeloid-specific VDR knockout (VDRΔLyz ) mouse model and used a macrophage/enteroids coculture system to examine tissue-specific VDR signaling in intestinal homeostasis. VDRΔLyz mice exhibited small intestine elongation and impaired Paneth cell in maturation and localization. Coculture of enteroids with VDR-/- macrophages increased the delocalization of Paneth cells. VDRΔLyz mice exhibited significant changes in the microbiota taxonomic and functional files, and susceptibility to Salmonella infection. Interestingly, loss of myeloid VDR impaired Wnt secretion in macrophages, thus inhibiting crypt β-catenin signaling and disrupting Paneth cell differentiation in the epithelium. Taken together, our data have demonstrated that myeloid cells regulate crypt differentiation and the microbiota in a VDR-dependent mechanism. Dysregulation of myeloid VDR led to high risks of colitis-associated diseases. Our study provided insight into the mechanism of immune/Paneth cell cross talk in regulating intestinal homeostasis.

Intestinal vitamin D receptor protects against extraintestinal breast cancer tumorigenesis

ABSTRACT The microbiota plays critical roles in regulating the function and health of the intestine and extraintestinal organs. A fundamental question is whether an intestinal-microbiome-breast axis exists during the development of breast cancer. If so, what are the roles of host factors? Vitamin D receptor (VDR) involves host factors and the human microbiome. Vdr gene variation shapes the human microbiome, and VDR deficiency leads to dysbiosis. We hypothesized that intestinal VDR protects hosts against tumorigenesis in the breast. We examined a 7,12-dimethylbenzanthracene (DMBA)-induced breast cancer model in intestinal epithelial VDR knockout (VDRΔIEC) mice with dysbiosis. We reported that VDRΔIEC mice with dysbiosis are more susceptible to breast cancer induced by DMBA. Intestinal and breast microbiota analysis showed that VDR deficiency leads to a bacterial profile shift from normal to susceptible to carcinogenesis. We found enhanced bacterial staining within breast tumors. At the molecular and cellular levels, we identified the mechanisms by which intestinal epithelial VDR deficiency led to increased gut permeability, disrupted tight junctions, microbial translocation, and enhanced inflammation, thus increasing tumor size and number in the breast. Furthermore, treatment with the beneficial bacterial metabolite butyrate or the probiotic Lactobacillus plantarum reduced breast tumors, enhanced tight junctions, inhibited inflammation, increased butyryl-CoA transferase, and decreased levels of breast Streptococcus bacteria in VDRΔIEC mice. The gut microbiome contributes to the pathogenesis of diseases not only in the intestine but also in the breast. Our study provides insights into the mechanism by which intestinal VDR dysfunction and gut dysbiosis lead to a high risk of extraintestinal tumorigenesis. Gut-tumor-microbiome interactions represent a new target in the prevention and treatment of breast cancer.

Vitamin D-VDR (vitamin D receptor) alleviates glucose metabolism reprogramming in lipopolysaccharide-induced acute kidney injury.

Background: Our previous study showed that vitamin D (VD)-vitamin D receptor (VDR) plays a nephroprotective role in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Recently, glucose metabolism reprogramming was reported to be involved in the pathogenesis of AKI. Objective: To investigate the role of VD-VDR in glucose metabolism reprogramming in LPS-induced AKI. Methods: We established a model of LPS-induced AKI in VDR knockout (VDR-KO) mice, renal proximal tubular-specific VDR-overexpressing (VDR-OE) mice and wild-type C57BL/6 mice. In vitro, human proximal tubular epithelial cells (HK-2 cells), VDR knockout and VDR overexpression HK-2 cell lines were used. Results: Paricalcitol (an active vitamin D analog) or VDR-OE reduced lactate concentration, hexokinase activity and PDHA1 phosphorylation (a key step in inhibiting aerobic oxidation) and simultaneously ameliorated renal inflammation, apoptosis and kidney injury in LPS-induced AKI mice, which were more severe in VDR-KO mice. In in vitro experiments, glucose metabolism reprogramming, inflammation and apoptosis induced by LPS were alleviated by treatment with paricalcitol or dichloroacetate (DCA, an inhibitor of p-PDHA1). Moreover, paricalcitol activated the phosphorylation of AMP-activated protein kinase (AMPK), and an AMPK inhibitor partially abolished the protective effect of paricalcitol in LPS-treated HK-2 cells. Conclusion: VD-VDR alleviated LPS-induced metabolic reprogramming in the kidneys of AKI mice, which may be attributed to the inactivation of PDHA1 phosphorylation via the AMPK pathway.

1,25-Dihydroxyvitamin D Deficiency Accelerates Aging-related Osteoarthritis via Downregulation of Sirt1 in Mice.

Emerging observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, the relationship between vitamin D level and OA and the role of vitamin D supplementation in the prevention of knee OA are controversial. To address these issues, we analyzed the articular cartilage phenotype of 6- and 12-month-old wild-type and 1α(OH)ase-/- mice and found that 1,25(OH)2D deficiency accelerated the development of age-related spontaneous knee OA, including cartilage surface destruction, cartilage erosion, proteoglycan loss and cytopenia, increased OARSI score, collagen X and Mmp13 positive chondrocytes, and increased chondrocyte senescence with senescence-associated secretory phenotype (SASP). 1,25(OH)2D3 supplementation rescued all knee OA phenotypes of 1α(OH)ase-/- mice in vivo, and 1,25(OH)2D3 rescued IL-1β-induced chondrocyte OA phenotypes in vitro, including decreased chondrocyte proliferation and cartilage matrix protein synthesis, and increased oxidative stress and cell senescence. We also demonstrated that VDR was expressed in mouse articular chondrocytes, and that VDR knockout mice exhibited knee OA phenotypes. Furthermore, we demonstrated that the down-regulation of Sirt1 in articular chondrocytes of 1α(OH)ase-/- mice was corrected by supplementing 1,25(OH)2D3 or overexpression of Sirt1 in mesenchymal stem cells (MSCs) and 1,25(OH)2D3 up-regulated Sirt1 through VDR mediated transcription. Finally, we demonstrated that overexpression of Sirt1 in MSCs rescued knee OA phenotypes in 1α(OH)ase-/- mice. Thus, we conclude that 1,25(OH)2D3, via VDR-mediated gene transcription, plays a key role in preventing the onset of aging-related knee OA in mouse models by up-regulating Sirt1, an aging-related gene that promotes articular chondrocyte proliferation and extracellular matrix protein synthesis, and inhibits senescence and SASP.

VDR activation attenuates osteoblastic ferroptosis and senescence by stimulating the Nrf2/GPX4 pathway in age-related osteoporosis.

Ferroptosis plays an essential role in the pathology of osteoporosis. This study investigated whether vitamin D receptor (VDR) activation could protect against age-related osteoporosis through an anti-ferroptosis mechanism. d-galactose (D-gal)-induced mice and VDR-knockout mice were used in the in-vivo study. The VDR activator (1,25(OH)2D3) attenuated senescence and ferroptosis in the D-gal-induced bone, as illustrated by downregulated senescence-associated secretory phenotype genes, improved mitochondrial morphology, elevated glutathione, and decreased lipid peroxidation markers (malondialdehyde and 4-hydroxynonenal). The pre-osteoblast MC3T3-E1 cells and primary rat osteoblasts were applied in the in-vitro studies. 1,25(OH)2D3 or ferroptosis inhibitor (ferrostatin-1) treatment downregulated the cellular senescence markers in D-gal-induced osteoblasts. Mechanistically, 1,25(OH)2D3 activated the VDR and its downstream nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway, resulting in the downregulation of lipid peroxidation. Nrf2 knockdown or addition of GPX4 inhibitor (RSL-3) blocked the protective effect of 1,25(OH)2D3 against D-gal-induced ferroptosis and senescence. VDR knockdown impeded the 1,25(OH)2D3-induced activation of Nrf2/GPX4 pathway in osteoblasts. Proteomics and immunofluorescence analysis confirmed that ferroptosis and suppression of the Nrf2/GPX4 pathway occurred in VDR-knockout mice. Our data demonstrated that ferroptosis played an essential role in age-related osteoporosis. The VDR activation attenuated osteoblast ferroptosis via stimulating the Nrf2/GPX4 signaling pathway.

Vitamin D receptor enhances NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasomes plays a critical role in the inflammatory response against intracellular bacterial infection. The NLR family apoptosis inhibitory proteins (NAIPs) detect Flagellin or type III secretion system (T3SS) microbial components to activate NLRC4 inflammasome. However, the underlying mechanism of NLRC4 inflammasome activation is not completely understood. Here, we show that the vitamin D receptor (VDR) is an essential immunological regulator of the NLRC4 inflammasome. Conditional VDR knockout mice (VDRflox/flox lyz2-Cre) exhibited impaired clearance of pathogens after acute Salmonella Typhimurium infection leading to poor survival. In macrophages, VDR deficiency reduced caspase-1 activation and IL-1β secretion upon S. Typhimurium infection. For NAIPs act as upstream sensors for NLRC4 inflammasome assembly, the further study demonstrated that VDR promoted the NAIP-NLRC4 association and triggered NAIP-NLRC4 inflammasome activation, not NLRP3 activation. Moreover, Lys123 residue of VDR is identified as the critical amino acid for VDR-NLRC4 interaction, and the mutant VDR (K123A) effectively attenuates the NLRC4 inflammasome activation. Together, our findings suggest that VDR is a critical regulator of NAIPs-NLRC4 inflammasome activation, mediating innate immunity against bacterial infection.

Vitamin D Receptor affects male mouse fertility via regulation of lipid metabolism and testosterone biosynthesis in testis

Vitamin D and vitamin D receptor (VD/VDR) plays a vital role in the development of spermatozoa, which is largely determined by the testosterone level in serum. Testosterone biosynthesis is closely related to lipid metabolism in gonadal adipose around testes. VDR could regulate lipid metabolism in adipocytes as well. However, it still remains unknown how VDR regulates lipid metabolism to impact testosterone biosynthesis in testis. Hereby, various parameters of male fertility were compared between wildtype (WT) and Vdr knockout (Vdr-KO) male mouse. For Vdr-KO mice, the size of testis and gonadal adipose was smaller than that of WT, and the sperm quality and testosterone level were lower than WT. Subsequently, testis proteome data between Vdr-KO and WT mice indicated that dysregulation of lipid metabolism was closely associated with decreased testosterone biosynthesis in Vdr-deficient mouse. And further evaluation of VDR functions in Leydig cells verified that VDR impacted lipid metabolism and regulated the expression of a range of genes involved in testosterone biosynthesis. Knockdown VDR could significantly decrease testosterone synthesis and secretion in Leydig cells. Meanwhile, expression of genes involved in androgen synthesis was decreased but genes related to lipolysis were up-regulated. Collectively, the present study unveiled the relationship between lipid metabolism and testosterone biosynthesis mediated by VDR in mouse testis and its effect on male fertility. These findings will greatly enhance our current understanding of VDR intermediate in lipid metabolism and androgen synthesis.

Deletion of the Vitamin D Receptor in Skeletal Muscle is Associated with Improved Glucose Tolerance and Reduced Muscle Function

Skeletal muscle insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus. Epidemiological studies have demonstrated that vitamin D deficiency is associated with the presence of type 2 diabetes in humans. To probe the role of the vitamin D receptor (VDR), an intracellular and nuclear protein that mediates the bioactivity of the active vitamin D ligand, 1,25‐dihydroxyvitamin D, in skeletal muscle mediated glucose disposal, we deleted the skeletal muscle VDR in adult mice. We assessed glucose disposal, insulin concentrations and sensitivity, and skeletal muscle gene expression in Vdr knockout and wild‐type Vdr mice. Following the induction of a skeletal muscle‐specific Crerecombinase with tamoxifen in adult male Vdrfl/flmice, efficient deletion of the Vdrwas demonstrated. In skeletal muscle‐specific Vdrknockout mice, basal glucose concentrations were similar to those in wild‐type Vdrmice. In Vdrknockout mice, blood glucose concentrations decreased more rapidly following the administration of intraperitoneal glucose than in control mice expressing wild‐type Vdr (Figure 1A and Figure 1B). The AUC for glucose concentrations in Vdrfl/fl.actin iCre mice treated with tamoxifen was reduced by 20% (p=0.015) compared to the AUC of blood glucose concentrations prior to tamoxifen treatment. Pre‐and post‐treatment AUC’s for blood glucose in Vdrfl/fl.actin iCre mice treated with vehicle and in Vdrfl/flmice treated with tamoxifen were unchanged (p=0.97,0.23). Plasma insulin concentrations were identical in knockout and wild‐type Vdr mice following a bolus of glucose, demonstrating that deletion of the Vdr in skeletal muscle did not influence the release of insulin in response to glucose (one‐way ANOVA, p=0.45). Insulin sensitivity was similar in Vdrknockout and wild‐type Vdrmice. Gene expression analysis of skeletal muscle mRNA derived from Vdrknockout mice and wild‐type mice demonstrated an increase in the expression of genes associated with oxidative phosphorylation, fatty acid oxidation and adipogenesis pathways that could potentially alter glucose utilization. We conclude that selective deletion of the Vdr in the skeletal muscle of adult mice results in more efficient glucose disposal than in wild‐type Vdrmice; basal and glucose‐stimulated insulin serum concentrations are identical in knock‐out and wild type VDR mice with no changes in insulin sensitivity. The data suggest that modulation of the vitamin‐D endocrine system is unlikely to improve glucose utilization.

Inhibition of Nrf2 degradation alleviates age-related osteoporosis induced by 1,25-Dihydroxyvitamin D deficiency

Previous studies have shown that 1,25(OH)2D plays an anti-osteoporosis role by an anti-aging mechanism. Oxidative stress is a key mediator of aging and bone loss; however, whether 1,25(OH)2D can exert its anti-osteoporosis effect by inhibiting oxidative stress is unclear. In this study, osteoporosis and the bone aging phenotype induced by 1,25(OH)2D deficiency in male mice were significantly rescued in vivo upon the supplementation of oltipraz, an inhibitor of Nrf2 degradation. Increased oxidative stress, cellular senescence and reduced osteogenesis of BM-MSCs from VDR knockout mice were also significantly rescued when the cells were pre-treated with oltipraz. We found that 1,25(OH)2D3 promoted Nrf2 accumulation by inhibiting its ubiquitin-proteasome degradation, thus facilitating Nrf2 activation of its transcriptional targets. Mechanistically, 1,25(OH)2D3 enhances VDR-mediated recruitment of Ezh2 and facilitation of H3K27me3 action at the promoter region of Keap1, thus transcriptionally repressing Keap1. To further validate that the Nrf2-Keap1 pathway serves as the key mediator in the anabolic effect of 1,25(OH)2D3 on bone, Nrf2−/− mice, or hBM-MSCs with shRNA-mediated Nrf2-knockdown, were treated with 1,25(OH)2D3; we found that Nrf2 knockout largely blocked the bone anabolic effect of 1,25(OH)2D3 in vivo and ex vivo, and Nrf2 knockdown in hBM-MSCs markedly blocked the role of 1,25(OH)2D3 in inhibiting oxidative stress and promoting osteogenic differentiation and bone formation. This study provides insight into the mechanism whereby 1,25(OH)2D3 postpones age-related osteoporosis via VDR-mediated activation of Nrf2-antioxidant signaling and inhibition of oxidative stress, and thus provides evidence for oltipraz as a potential reagent for clinical prevention and treatment of age-related osteoporosis.

Vitamin D receptor alleviates hepatic ischemia and reperfucion injury by mediating endoplasmic reticulum stress through autophagy.

Hepatic ischemia and reperfusion (IR) injury is a common complication in clinical practice. Endoplasmic reticulum (ER) stress and autophagy are the key factors in the process of hepatic IR injury. The vitamin D receptor (VDR) can mediate ER stress and autophagy; however, it can also mitigate IR injury. The relationship between VDR, ER stress, and autophagy in hepatic IR injury is unknown. VDR knockout mice and wild-type littermates underwent 70% liver ischemia (90 min) and reperfusion (6 h). To observe the effect of autophagy in the relationship with VDR and ER stress in hepatic IR injury, the autophagy agonist rapamycin and its inhibitor chloroquine were used in the study. Meanwhile, RAW264.7 cells were studied in vitro to verify the relationship between VDR, autophagy, and ER stress. VDR was involved in hepatic IR injury and its activation reduced liver injury and inhibited an inflammatory response. ER stress took part in the liver injury during the process of IR. Meanwhile, VDR activation was found to inhibit inflammation by ER stress, and vice versa. Furthermore, autophagy was the connection between VDR and ER stress. After treatment with rapamycin or chloroquine, the effect of VDR activation or VDR silencing in ER stress was partially reversed. The same tendency was observed in vitro. ER stress and autophagy are important mechanisms of hepatic IR injury. VDR can regulate ER stress through autophagy and then protect the liver from IR injury.

Vitamin D-VDR (vitamin D receptor) regulates defective autophagy in renal tubular epithelial cell in streptozotocin-induced diabetic mice via the AMPK pathway

ABSTRACT Diabetic nephropathy (DN) has become a major cause of end-stage renal disease, and autophagy disorder is implicated in the pathogenesis of DN. Our previous studies found that vitamin D (VD) and VDR (vitamin D receptor) played a renoprotective role by inhibiting inflammation and fibrosis. However, whether VD-VDR regulates autophagy disorders in DN remains unclear. In this study, we established a streptozotocin (STZ)-induced diabetic model in vdr knockout (vdr-KO) mice and VDR specifically overexpressed in renal proximal tubular epithelial cells (Vdr-OE) mice. Our results showed that paricalcitol (an activated vitamin D analog) or Vdr-OE could alleviate STZ-induced ALB (albumin) excretion, renal tubule injury and inflammation, while these were worsened in vdr-KO mice. Defective autophagy was observed in the kidneys of STZ mice, which was more pronounced in vdr-KO mice and could be partially restored by paricalcitol or Vdr-OE. In high glucose-induced HK-2 cells, defective autophagy and decreased PRKAA1/AMPK phosphorylation was observed, which could be partially restored by paricalcitol in a VDR-dependent manner. AMPK inhibitor abolished paricalcitol-induced autophagy activation, and AMPK activator restored the defective autophagy in high glucose-induced HK-2 cells. Furthermore, paricalcitol-mediated AMPK activation was abrogated by CAMKK2/CaMKKβ inhibition, but not by STK11/LKB1 knockout. Meanwhile, paricalcitol rescued the decreased Ca2+ concentration induced by high glucose. In conclusion, VD-VDR can restore defective autophagy in the kidney of STZ-induced diabetic mice, which could be attributed to the activation of the Ca2+-CAMKK2-AMPK pathway in renal tubular epithelial cells. Abbreviations: ACTB/β-actin: actin beta;AGE: advanced glycation end-products;AMPK: AMP-activated protein kinase;CAMKK2/CaMKKβ: calcium-calmodulin dependent protein kinase kinase 2;CQ: chloroquine;DN: diabetic nephropathy;HG: high levels of glucose;KO: knockout;LG: low levels of glucose;MAP1LC3/LC3: microtubule associated protein 1 light chain 3;NOD2: nucleotide binding oligomerization domain containing 2;OE: overexpression;PAS: periodic acid Schiff; Pari: paricalcitol;PTECs: proximal renal tubule epithelial cells;RT: room temperature;SQSTM1/p62: sequestosome 1;STK11/LKB1: serine/threonine kinase 11;STZ: streptozotocin;TEM: transmission electron microscopy;VD: vitamin D;VDR: vitamin D receptor;WT: wild-type

Bone mass protective potential mediated by bovine milk basic protein requires normal calcium homeostasis in mice

ObjectivesMilk provide protective effects against bone loss caused by an impaired calcium balance. Although the effects of some elements have previously been confirmed, the involvement of milk basic protein (MBP) in bone mineral metabolism remains poorly characterized. Moreover, the importance of mineral nutrition sufficiency to establish the effect of MBP must be evaluated. MethodsFirst, to evaluate the physiological conditions required for MBP activity, we examined the bone and mineral phenotypes of mice that suffer from insufficient calcium absorption due to a lack of intestinal vitamin D signaling. Second, to determine whether vitamin D signaling affects the effect of MBP on bone resorption, in vitro osteoclastogenesis were assessed using bone marrow cells. ResultsIn mice with systemic vitamin D receptor (Vdr) inactivation, dietary MBP supplementation was unable to normalize hypercalcemia and hyperparathyroidism and failed to rescue bone mineralization impairments. In contrast, calcium and bone homeostasis responded to MBP supplementation when Vdr inactivation was restricted to the intestines. Hyperparathyroidism in intestine-specific Vdr knockout mice was also improved by MBP supplementation, along with a decrease in bone resorption in response to the level of serum tartrate-resistant acid phosphatase 5b. These results corresponded with a reduction in tartrate-resistant acid phosphatase-stained osteoclast numbers and the eroded surface on the tibia. MBP treatment dose-dependently suppressed osteoclastogenesis in cultured bone marrow macrophages regardless of vitamin D activity. These effects of MBP were blunted when parathyroid hormone was added to the culture medium, which is in line with the in vivo phenotype observed with systemic Vdr inactivation and suggests that severe hyperparathyroidism limits MBP activity in the bone. ConclusionsTherefore, adaptive calcium homeostasis is an essential requirement when MBP exerts protective effects through the inhibition of bone resorption.

Downregulation of VDR in benign paroxysmal positional vertigo patients inhibits otolith‑associated protein expression levels.

Benign paroxysmal positional vertigo (BPPV) is the most common peripheral vertigo‑associated disease. Vitamin D (VD) helps maintain normal otolith function and may be associated with BPPV. VD exerts its biological functions primarily via the VD receptor (VDR). The present study demonstrated that serum VD levels were significantly decreased in patients with BPPV compared with in controls. VDR, otolith‑associated protein otoconin‑90 (OC90) and NADPH oxidase 3 (NOX3) expression levels were also significantly decreased in patients with BPPV compared with in controls. Furthermore, a positive correlation was observed between VD levels and VDR expression. Receiver operating characteristic curve analysis identified VDR expression levels as a potential diagnostic marker for BPPV. OC90 and NOX3 expression levels were notably lower in the inner ear tissue of VDR knockout mice compared with in those of wild‑type mice. In mice overexpressing VDR, OC90 and NOX3 were also overexpressed. Following intravenous injection of VD in VDR knockout mice, expression levels of OC90 and NOX3 were not significantly different from those in VDR knockout mice injected with saline. This indicated that VDR may be underexpressed in patients with BPPV and was associated with the expression levels of otolith‑associated proteins. Moreover, VDR mediated VD activation, leading to otolith protein formation. The present study provided a novel theoretical basis for BPPV onset that may facilitate the development of more effective diagnostic and treatment options.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

Imbalance of the intestinal virome and altered viral-bacterial interactions caused by a conditional deletion of the vitamin D receptor

ABSTRACT Vitamin D receptor (VDR) deficiency is associated with cancer, infection, and chronic inflammation. Prior research has demonstrated VDR regulation of bacteria; however, little is known regarding VDR and viruses. We hypothesize that VDR deficiency impacts on the intestinal virome and viral-bacterial interactions. We specifically deleted VDR from intestinal epithelial cells (VDRΔIEC), Paneth cells (VDRΔPC), and myeloid cells (VDRΔLyz) in mice. Feces were collected for shotgun metagenomic sequencing and metabolite profiling. To test the functional changes, we evaluated pattern recognition receptors (PRRs) and analyzed microbial metabolites. Vibrio phages, Lactobacillus phages, and Escherichia coli typing phages were significantly enriched in all three conditional VDR-knockout mice. In the VDRΔLyz mice, the levels of eight more virus species (2 enriched, 6 depleted) were significantly changed. Altered virus species were primarily observed in female VDRΔLyz (2 enriched, 3 depleted) versus male VDRΔLyz (1 enriched, 1 depleted). Altered alpha and beta diversity (family to species) were found in VDRΔLyz. In VDRΔIEC mice, bovine viral diarrhea virus 1 was significantly enriched. A significant correlation between viral and bacterial alterations was found in conditional VDR knockout mice. There was a positive correlation between Vibrio phage JSF5 and Cutibacterium acnes in VDRΔPC and VDRΔLyz mice. Also, there were more altered viral species in female conditional VDR knockout mice. Notably, there were significant changes in PRRs: upregulated TLR3, TLR7, and NOD2 in VDRΔLyz mice and increased CLEC4L expression in VDRΔIEC and VDRΔPC mice. Furthermore, we identified metabolites related to virus infection: decreased glucose in VDRΔIEC mice, increased ribulose/xylulose and xylose in VDRΔLyz mice, and increased long-chain fatty acids in VDRΔIEC and VDRΔLyz female mice. Tissue-specific deletion of VDR changes the virome and functionally changes viral receptors, which leads to dysbiosis, metabolic dysfunction, and infection risk. This study helps to elucidate VDR regulating the virome in a tissue-specific and sex-specific manner.

Paneth Cell Alertness to Pathogens Maintained by Vitamin D Receptors

Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown. We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn's disease. We generated Paneth cell-specific VDR knockout (VDRΔPC) mice to investigate the molecular mechanisms. Lysozymes in the Paneth cells were significantly decreased in the VDRΔPC mice. Isolated VDRΔPC Paneth cells exhibited weakened inhibition of pathogenic bacterial growth and displayed reduced autophagic responses. VDRΔPC mice had significantly higher inflammation after Salmonella infections. VDRΔPC mice also showed high susceptibility to small intestinal injury induced by indomethacin, a nonsteroidal anti-inflammatory drug. Co-housing of VDRΔPC and VDRlox mice made the VDRΔPC less vulnerable to dextran sulfate sodium colitis, suggesting the transmission of protective bacterial from the VDRlox mice. Thus, a lack of VDR in Paneth cells leads to impaired antibacterial activities and consequently increased inflammatory responses. Genetically and environmentally regulated VDRs in the Paneth cells may set the threshold for the development of chronic inflammation, as observed in inflammatory bowel diseases. We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.

Maxacalcitol (22-Oxacalcitriol (OCT)) Retards Progression of Left Ventricular Hypertrophy with Renal Dysfunction Through Inhibition of Calcineurin-NFAT Activity

Left ventricular hypertrophy (LVH) is a cardiovascular complication highly prevalent in patients with chronic kidney disease (CKD). Previous studies analyzing 1α-hydroxylase or vitamin D receptor (Vdr) knockout mice revealed active vitamin D as a promising agent inhibiting LVH progression. Paricalcitol, an active vitamin D analog, failed to suppress the progression of LV mass index (LVMI) in pre-dialysis patients with CKD. As target genes of activated VDR differ depending on its agonists, we examined the effects of maxacalcitol (22-oxacalcitriol: OCT), a less calcemic active vitamin D analog, on LVH in hemodialysis patients and animal LVH models with renal insufficiency. In retrospective cohort study, patients treated with OCT who underwent hemodialysis were enrolled. Using cardiac echocardiography, LV mass was evaluated by the area-length method. In animal study, angiotensin II (Ang II)-infused Wister rats with heminephrectomy or Ang II-stimulated neonatal rat ventricular myocytes (NRVM) were treated with OCT. OCT significantly inhibited the progression of LVMI in hemodialysis patients. In Ang II-infused heminephrectomized rats, OCT suppressed the progression of LVH in a blood pressure-independent manner. OCT also suppressed the activity of calcineurin in the left ventricle of model rats. Specifically, OCT reduced the protein levels of calcineurin A, but not the mRNA levels of Ppp3ca (calcineurin Aα). Luciferase assays showed that OCT increased the promoter activity of Fbxo32 (atrogin1), an E3 ubiquitin ligase targeting calcineurin A. Finally, OCT promoted ubiquitination and degradation of calcineurin A. Our works indicated that OCT retards progression of LVH through calcineurin-NFAT pathway, which reveal a novel aspect of OCT in attenuating pathological LVH.