[The role of calcium sensitive and vitamin D receptors in the pathogenesis of sporadic multiple parathyroid gland disease].

BACKGROUND: Sporadic multiple parathyroid gland disease is » cases of primary hyperparathyroidism (PHPT). However, a single tactic for diagnosing and operating volume in patients with this variant of PHPT has not yet been developed. One of the possible directions in the search for pathogenetically substantiated methods of diagnosis and treatment is the study of the molecular genetic features of the disease and associated clinical and laboratory factors. AIM: To study the features of the expression of calcium sensitive (CaSR) and vitamin D (VDR) receptors on the surface of parathyroid cells in primary hyperparathyroidism with solitary and multiple lesions of the parathyroid glands, as well as its changes under the influence of a decrease in the filtration function of the kidneys. MATERIALS AND METHODS: In a single center observational prospective study with retrospective data collection, there were patients who during 2019-2021. operated on for PHPT, secondary hyperparathyroidism (SHPT) and all cases of tertiary hyperparathyroidism (THPT) operated during 2014-2021. The expression of CaSR, VDR and its relationship with the main laboratory parameters, the clinical variant of hyperparathyroidism, and the morphological substrate were studied. RESULTS: The study included 69 patients: 19 with multiple and 25 with solitary PTG near PHPT, 15 with SHPT, 10 with THPT. A statistically significant decrease in the frequency of detection of normal expression of CaSR and VDR receptors occurs in any morphological variant of hyperparathyroidism and is observed in 93-60% of drugs. A decrease in the normal expression of CaSR in hyperplasia is detected statistically significantly less frequently than in adenoma (p≤0.01). The median expression intensity in adenoma was 2.5 (2:3), in hyperplasia 3.5 (3-4) (p≤0.01). The difference in the molecular mechanisms of the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma (PHPT with solitary adenoma) or hyperplasia (SHPT and PHPT with multiple PTG lesions) is realized in the frequency of maintaining normal CaSR expression in the PTG tissue. These mechanisms are implemented at the local level, their variability does not change under the influence of RRT. A common molecular genetic mechanism for the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma or hyperplasia has been found to reduce the frequency of maintaining normal VDR expression in PTG (up to 7-13%), p<0.01. This mechanism is implemented at the local level, its variability changes under the influence of RRT, reaching statistically significant differences in patients with THPT. CONCLUSION: The study demonstrates the features of changes in the expression of CaSR and VDR in PHPT with multiple lesions of the parathyroid glands. The relationship between the expression of these receptors and the clinical variant of hyperparathyroidism, the morphological substrate, the main laboratory parameters, and renal function was shown.

Evaluation of BMP-2, VEGF, and Vitamin D Receptor Levels in the Ligamentum Flavum of Patients with Lumbar Spinal Stenosis and Disc Herniation.

AIM: To histopathologically evaluate and compare bone morphogenetic protein (BMP)-2, vascular endothelial growth factor (VEGF), and vitamin D receptor (VDR) levels in the ligamentum flavum (LF) of patients with lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). MATERIAL AND METHODS: Surgical specimens of the LF in 25 patients who underwent surgery for LDH and 25 patients who underwent surgery for LSS were examined histopathologically. The prevalence and severity of BMP-2, VEGF, and VDR immunoreactivity were evaluated to create histoscores (prevalence × severity), which were compared between groups. RESULTS: The mean BMP-2 histoscore was similar in both groups. In the LSS group, the mean VEGF histoscore was significantly higher and the mean VDR histoscore was significantly lower. CONCLUSION: Elevated VEGF and decreased VDR levels in the LF in LSS are associated with more intense inflammation and chronic process of the disease. The prominent expression of BMP-2 in the LF in both diseases suggests that BMP-2 might be affected by inflammation regardless of chronic pressure and degeneration.

Local NF-κB Activation Promotes Parathyroid Hormone Synthesis and Secretion in Uremic Patients.

Secondary hyperparathyroidism (SHPT) in uremic patients is characterized by parathyroid gland (PTG) hyperplasia and parathyroid hormone (PTH) elevation. Previously, we demonstrated that NF-κB activation contributed to parathyroid cell proliferation in rats with chronic kidney disease. Although vitamin D inhibits inflammation and ameliorates SHPT, the contribution of vitamin D deficiency to SHPT via local NF-κB activation remains to be clarified. PTGs collected from 10 uremic patients with advanced SHPT were used to test the expressions of vitamin D receptor (VDR), NF-κB, and proliferating cell nuclear antigen (PCNA). Freshly excised PTG tissues were incubated for 24 hours in vitro with VDR activator (VDRA) calcitriol or NF-κB inhibitor pyrrolidine thiocarbamate (PDTC). Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to investigate the regulation of PTH transcription by NF-κB. We found higher levels of activated NF-κB and lower expression of VDR in nodular hyperplastic PTGs than in diffuse hyperplasia. In cultured PTG tissues, treatment with VDRA or PDTC inhibited NF-κB activation and PCNA expression, and downregulated preproPTH mRNA and intact PTH levels. ChIP assays demonstrated the presence of NF-κB binding sites in PTH promoter. Furthermore, in luciferase reporter assays, addition of exogenous p65 significantly increased PTH luciferase activity by 2.4-fold (P < 0.01), while mutation of NF-κB binding site at position -908 of the PTH promoter suppressed p65-induced PTH reporter activity (P < 0.01). In summary, local NF-κB activation contributes to SHPT and mediates the transcriptional activation of PTH directly in uremic patients. Vitamin D deficiency may be involved in SHPT via the activation of NF-κB pathway.

Defining vitamin D receptor expression in the brain using a novel VDRCre mouse.

Vitamin D action has been linked to several diseases regulated by the brain including obesity, diabetes, autism, and Parkinson's. However, the location of the vitamin D receptor (VDR) in the brain is not clear due to conflicting reports. We found that two antibodies previously published as specific in peripheral tissues are not specific in the brain. We thus created a new knockin mouse with cre recombinase expression under the control of the endogenous VDR promoter (VDRCre ). We demonstrated that the cre activity in the VDRCre mouse brain (as reported by a cre-dependent tdTomato expression) is highly overlapping with endogenous VDR mRNAs. These VDR-expressing cells were enriched in multiple brain regions including the cortex, amygdala, caudate putamen, and hypothalamus among others. In the hypothalamus, VDR partially colocalized with vasopressin, oxytocin, estrogen receptor-α, and ß-endorphin to various degrees. We further functionally validated our model by demonstrating that the endogenous VDR agonist 1,25-dihydroxyvitamin D activated all tested tdTomato+ neurons in the paraventricular hypothalamus but had no effect on neurons without tdTomato fluorescence. Thus, we have generated a new mouse tool that allows us to visualize VDR-expressing cells and to characterize their functions.

Preparation and Use of Turn-on Fluorescent Probe for Detection and Live Cell Imaging of Vitamin D Receptor as a Target Protein.

Turn-on fluorescent probe mediated by conjugate addition and cyclization (TCC probe) is a small molecule that reacts with a protein of interest in cells. TCC probe is applicable to various types of proteins by exchanging the ligand unit for target proteins. TCC probes are a potent tool for molecular imaging and chemical proteomics. This protocol describes the synthesis of a TCC probe via unstable intermediate and how to use this probe to visualize vitamin D receptor as a target protein. For complete details on the use and execution of this protocol, please refer to Kojima et al. (2020).

The role of TRPM7 in vascular calcification: Comparison between phosphate and uremic toxin.

AIMS: Vascular calcification is a common complication in patients with chronic kidney disease and associated with increased morbidity and mortality. The role of TRPM7 in vascular smooth muscle cell (VSMC) transformation during vascular calcification is not clear. We aim to investigate the effects of phosphate and indoxyl sulphate on the expression of TRPM7 and calcification-related molecules in VSMC. MAIN METHODS: Human aortic smooth muscle cells (HASMC) were treated with phosphate (3.3 mM) or indoxyl sulphate (500 µM and 1000 µM). 2-APB, a channel blocker of TRPM7 was added simultaneously in blocking experiment. Cells were then examined grossly and alizarin red solution was employed for calcification assessment. Lastly, cells were harvested for gene expression and protein abundance analysis. KEY FINDINGS: Phosphate treatment induced significant increase in BMP2, RUNX2, BMP7, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and TRPM7, but 1-alpha hydroxylase, klotho, DKK1 and sclerostin were not changed. The addition of 2-APB prevented increase of BMP2, RUNX2, BMP7, VDR, CaSR and TRPM7. Indoxyl sulphate treatment was associated with decrease in TRPM7 and DKK1, but increase in RUNX2, BMP2 and VDR were noted. There were no significant alterations in BMP7, CaSR, klotho,1-alpha hydroxylase and sclerostin. Co-treatment with 2-APB reversed the increase in VDR. SIGNIFICANCE: Both phosphate and indoxyl sulphate induced calcification in VSMC but it was more prominent in phosphate. TRPM7 was upregulated by phosphate but downregulated in indoxyl sulphate treatment. Vascular calcification was reduced by blocking TRPM7 with 2-APB and there was partial anti-calcification effect in indoxyl sulphate.

Modified expression of vitamin D receptor and CYP27B1 in denervation-induced muscle atrophy.

The vitamin D pathway is related to the mass and function of skeletal muscles. Several studies have demonstrated the role of vitamin D receptor (VDR) and CYP27B1 in skeletal muscles, suggesting that these proteins may regulate skeletal muscles and their function. However, it remains unclear whether the expression of VDR and CYP27B1 is modified in skeletal muscle atrophy. We investigated whether denervation-induced muscle atrophy is associated with altered expression of VDR and CYP27B1 in murine skeletal muscles. Skeletal muscles were excised from C57BL/6J mice, 3 and 7 days after the mice underwent denervation surgery. Denervation induced muscle atrophy and enhanced the expression of MuRF1 and Atrogin-1 in the gastrocnemius and soleus. The protein expression of VDR was increased in the denervated gastrocnemius; in contrast, denervation decreased the protein expression of CYP27B1 in the gastrocnemius and soleus. These results suggest that denervation-induced muscle atrophy is associated with changes in the expression of vitamin D-related proteins in murine skeletal muscles.

Immunohistochemical expression of vitamin D receptor and forkhead box P3 in classic Hodgkin lymphoma: correlation with clinical and pathologic findings.

BACKGROUND: Expression of forkhead box P3 (FOXP3), a key regulator of T-cell function, in the tumor immune microenvironment is related to survival in classic Hodgkin lymphoma (CHL). Vitamin D receptor (VDR), a transcription factor agonists have been shown to induce FOXP3 expression in T-cells and enhance recruitment of these cells to the inflammatory sites. VDR expression is CHL has been described. However, there is no data on expression of VDR in context of quantity of FOXP3 positive cells in CHL. METHODS: We examined and correlated immunohistochemical expression of VDR and FOXP3 along with clinical and pathology findings in 29 cases of CHL. RESULTS: VDR was expressed in Hodgkin Reed-Sternberg (HRS) cells and background lymphocytes and FOXP3 was expressed in background lymphocytes. 82% of CHL cases, regardless of the subtype, expressed VDR and in majority of the cases, VDR expression was directly proportional to the quantity of FOXP3 expressing lymphocytes in the tumor microenvironment. In cases with higher clinical stage (III/IV), only 28.5% of cases diffusely expressed VDR and FOXP3 compared to 71.4% showing focal positivity. Whereas in cases with lower clinical stages (I/II), the expression pattern of VDR and FOXP3 was almost similar (41.6% diffuse versus 33.3% focal). Interestingly, focal VDR and FOXP3 expression pattern was significantly higher among males. Mixed cellularity cases showed predilection for focal VDR and FOXP3 expression (80% cases); whereas nodular sclerosis subtype had focal and diffuse VDR and FOXP3 expression patterns in similar proportion. Cases with diffuse VDR and FOXP3 expression were less likely to have bone marrow involvement. Epstein Barr virus- encoded small RNA (EBER) positive cases were predominantly focally positive (80%) for VDR and FOXP3. CONCLUSIONS: In summary, quantity of FOXP3 positive T-cells in CHL microenvironment seems to correlate with VDR expression. Clinical stage show a trend of inverse correlation with expression of VDR and quantity of FOXP3 positive T-cells. These findings suggest that VDR could be a possible prognostic and therapeutic target in CHL.

Cytoplasmic VDR expression as an independent risk factor for ovarian cancer.

The vitamin D receptor (VDR), primarily known as a crucial mediator of calcium homeostasis and metabolism, has been shown to play a significant role in various cancer entities. Previous studies have focused on vitamin D and its receptor in gynecological cancers, noting that the receptor is upregulated in epithelial ovarian cancer (EOC). The aim of this study is to analyze the prognostic impact of VDR and its functional significance in ovarian cancer. Through immunohistochemistry, VDR staining was examined in 156 ovarian cancer samples. Evaluation of VDR staining was conducted in the nucleus and the cytoplasm using the semi-quantitative immunoreactive score, and the scores were classified into high- and low-level expressions. Expression levels were correlated with clinical and pathological parameters as well as with overall survival to assess for prognostic impact. Differences in cytoplasmic VDR expression were identified between the histological subtypes (p = 0.001). Serous, clear cell, and endometrioid subtypes showed the highest staining, while the mucinous subtype showed the lowest. Cytoplasmic VDR correlated with higher FIGO stage (p = 0.013; Cc = 0.203), positive lymph node status (p = 0.023; Cc = 0.236), high-grade serous histology (p = 0.000; Cc = 0.298) and grading from the distinct histological subtypes (p = 0.006; Cc = - 0.225). Nuclear VDR did not correlate with clinicopathological data. High cytoplasmic expression of VDR was associated with impaired overall survival (HR 2.218, 32.5 months vs. median not reached; p < 0.001) and was confirmed as a statistically independent prognostic factor in the Cox regression multivariate analysis. Additional knowledge of VDR as a biomarker and its interactions within the mitogen-activated protein kinase (MAPK) signaling pathway could potentially improve the prognosis of therapeutic approaches for specific subgroups in EOC.

Lymphocyte and monocyte vitamin D receptor expression during paricalcitol or calcitriol treatments in patients with stage 5 chronic kidney disease.

PURPOSE: In this study, we aimed to investigate the effect of paricalcitol and calcitriol usage on vitamin D receptor (VDR) contents of CD8+ , CD4+ lymphocytes and monocytes in stage 5d chronic kidney disease (CKD) patients. METHODS: Thirty-six hemodialysis patients older than 18 years of age and 19 healthy controls (group HC) without any known acute or chronic diseases were included in the study. The group of patients undergoing scheduled hemodialysis comprised three subgroups: group CL: patients on calcitriol (n: 10), group PC: patients on paricalcitol (n: 13), and group NT: patients not taking any vitamin D or VDR activating medications (n: 13). CD8+/VDR, CD4+/VDR and MONO/VDR values were representing the ratio of VDR representing cells among related cell group. On the other hand, values of CD8+/MFI, CD4+/MFI and MONO/MFI have shown the total amount of cellular VDR content per cell which has been given as of mean fluorescence intensity in the flow cytometric process. Main CKD mineral bone disorder parameters such as a hemogram, serum BUN, creatinine, albumin, Ca, iP, iPTH, 25(OH)D3 levels were also measured. RESULTS: Average VDR contents in CD8+, CD4+ and monocytes were not different among three patient groups on hemodialysis. But in all hemodialysis subgroups, CD8+/VDR, CD4+/VDR, MONO/VDR, CD8+/MFI, CD4+/MFI and MONO/MFI levels were found to be higher compared with the healthy control subjects (p < 0.001). Among hemodialysis groups, no significant CD8+/VDR, CD4+/VDR, and MONO/VDR content differences were found with regard to the type of VDR activator agent used. There was no difference in serum levels of 25(OH)D3 and CRP among groups participating in the study. CONCLUSION: There was no difference between CD8+/VDR, CD4+/VDR, and MONO/VDR levels in hemodialysis patients using calcitriol or paricalcitol, suggesting that both treatment agents may have a similar effect on VDR contents in lymphocytes and monocytes in that patient population. But in all hemodialysis subgroups, CD8+/VDR, CD4+/VDR, and MONO/VDR levels were found to be higher compared with the healthy control subjects, suggesting an overexpression of VDR through a non CRP and/or 25(OH)D3 dependent mechanism.

Propionate promotes vitamin D receptor expression via yes-associated protein in rats with short bowel syndrome.

Vitamin D deficiency and refractory osteoporosis are common complications in patients with short bowel syndrome (SBS). The symptom of bone loss is not effectively alleviated, even after the oral administration of vitamin D in SBS patients who had been weaned off parenteral nutrition. In this study, we aimed to investigate the effect of propionate on the expression of the vitamin D receptor (VDR) in the small intestine of rats with SBS. Firstly, IEC-6 (intestinal epithelioid cell line No. 6) cells were incubated in vitro with 1 mM sodium propionate for 24 h. This resulted in a significant increase in the expression of VDR and yes-associated protein (YAP) compared with that in the control group. Transfection of IEC-6 cells with YAP siRNA significantly down-regulated the expression of VDR. By contrast, after incubating IEC-6 cells with lysophosphatidic acid, an agonist of YAP, upregulation of VDR and YAP was observed. Next, we investigated whether this effect occurs in vivo. Five-week-old male Sprague-Dawley rats underwent 80% small bowel resection to establish an SBS model. Rats treated with 1% w/v sodium propionate had high levels of VDR and YAP expression in the intestine and intestinal adaptation was clearly observed compared to the control group. However, these effects were blocked by intraperitoneal injection of verteporfin. Thus, this study showed that propionate promoted VDR expression in the intestine via the activity of YAP, both in vitro and in vivo. Moreover, propionate was shown to play an active role in postoperative intestinal adaptation in SBS rats.

1,25-(OH)2D3/Vitamin D receptor alleviates systemic lupus erythematosus by downregulating Skp2 and upregulating p27.

BACKGROUND: Recent evidence has suggested that the 1,25(OH)2D3/Vitamin D receptor (VDR) acts to suppress the immune response associated with systemic lupus erythematosus (SLE), a serious multisystem autoimmune disease. Hence, the aim of the current study was to investigate the mechanism by which 1,25-(OH)2D3/VDR influences SLE through regulating the Skp2/p27 signaling pathway. METHODS: Initially, the levels of 1,25(OH)2D3, VDR, Skp2, and p27 were measured in collected renal tissues and peripheral blood. Meanwhile, the levels of inflammatory factors, biochemical indicators (BUN, Cr, anti-nRNP IgG, anti-dsDNA IgG) and urinary protein levels were assayed in in VDRinsert and VDR-knockout mice in response to 1,25(OH)2D3 supplement. In addition, the distribution of splenic immune cells was observed in these mice. RESULTS: Among the SLE patients, the levels of 1,25(OH)2D3, VDR and p27 were reduced, while the levels of Skp2 were elevated. In addition, the levels of anti-nRNP IgG and anti-dsDNA IgG were increased, suggesting induction of inflammatory responses. Notably, 1,25(OH)2D3/VDR mice had lower concentrations of BUN and Cr, urinary protein levels, precipitation intensity of the immune complex and complement, as well as the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice. Additionally, 1,25(OH)2D3 or VDR reduced the degree of the inflammatory response while acting to regulate the distribution of splenic immune cells. CONCLUSION: This study indicated that 1,25-(OH)2D3/VDR facilitated the recovery of SLE by downregulating Skp2 and upregulating p27 expression, suggesting the potential of 1,25-(OH)2D3/VDR as a promising target for SLE treatment.

Lower Vitamin D Levels, but Not VDR Polymorphisms, Influence Type 2 Diabetes Mellitus in Brazilian Population Independently of Obesity.

Background and Objectives: Vitamin D levels have been associated with a diversity of diseases, including obesity. Vitamin D presents a pleiotropic action, and can regulate insulin secretion and inflammatory responses. Vitamin D receptor (VDR) gene polymorphisms are involved in the gene expression regulation and have been associated with type 2 diabetes mellitus (T2DM). This study aimed to evaluate the association between the polymorphisms ApaI (rs7975232), BsmI (rs1544410), FokI (rs10735810), and TaqI (rs731236) in the VDR gene in people diagnosed with T2DM, and plasma 25-hydroxivitamin D levels [25(OH)D]. Materials and Methods: A total of 101 T2DM patients and 62 gender, age, and body mass index (BMI) matched non-diabetic controls were included in this study. Molecular analyzes were performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The plasma 25(OH)D levels were measured by high performance liquid chromatography. Results: The plasma 25(OH)D levels were lower in T2DM patients (17.2 (16.6) ng/mL) when compared with the control subjects (30.8 (16.2) ng/mL, p < 0.0001), independently of obesity status. We found no difference between genotypic and allelic frequencies of the VDR polymorphisms when comparing the T2DM group and control group (p > 0.05 for all), and did not show any association with plasma 25(OH)D levels. Conclusions: These results suggest that T2DM is associated with lower plasma 25(OH)D levels, which are not related to BMI and VDR gene polymorphisms.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Neuroprotective effects of vitamin D on high fat diet- and palmitic acid-induced enteric neuronal loss in mice.

BACKGROUND: The role of vitamin D in obesity and diabetes is debated. Obese and/or diabetic patients have elevated levels of free fatty acids, increased susceptibility to gastrointestinal symptoms and are suggested to have altered vitamin D balance. The enteric nervous system is pivotal in regulating gastrointestinal activity and high fat diet (HFD) has been shown to cause loss of enteric neurons in ileum and colon. This study investigates the effect of vitamin D on HFD- and palmitic acid-induced enteric neuronal loss in vivo and in vitro. METHODS: Mice were fed either a normal diet (ND) or HFD supplemented with varying levels of vitamin D (from 0x to 20x normal vitamin D level) for 19 weeks. Ileum and colon were analyzed for neuronal numbers and remodeling. Primary cultures of myenteric neurons from mouse small intestine were treated with palmitic acid (4x10-4M) and/or 1α,25-hydroxy-vitamin D3 (VD, 10-11- 10-7M) with or without modulators of lipid metabolism and VD pathways. Cultures were analyzed by immunocyto- and histochemical methods. RESULTS: Vitamin D supplementation had no effect on enteric neuronal survival in the ND group. HFD caused substantial loss of myenteric neurons in ileum and colon. Vitamin D supplementation between 0-2x normal had no effect on HFD-induced neuronal loss. Supplementation with 20x normal, prevented the HFD-induced neuronal loss. In vitro supplementation of VD prevented the palmitic acid-induced neuronal loss. The VD receptor (VDR) was not identified in enteric neurons. Enteric glia expressed the alternative VD receptor, protein disulphide isomerase family A member 3 (PDIA3), but PDIA3 was not found to mediate the VD response in vitro. Inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) and immune neutralization of isocitrate lyase prevented the VD mediated neuroprotection to palmitic acid exposure. CONCLUSIONS: Results show that VD protect enteric neurons against HFD and palmitic acid induced neuronal loss. The mechanism behind is suggested to be through activation of PPARγ leading to improved neuronal peroxisome function and metabolism of neuronal lipid intermediates.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. OBJECTIVE: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. MATERIAL AND METHODS: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. RESULTS: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. CONCLUSIONS: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Vitamin D-induced vitamin D receptor expression induces tamoxifen sensitivity in MCF-7 stem cells via suppression of Wnt/ß-catenin signaling.

Objective: Cancer stem cells (CSCs) are responsible for the drug resistance of breast cancers. Vitamin D deficiency promotes tumor resistance. The present study examined the effect of vitamin D and vitamin D receptor (VDR) expression on the tamoxifen resistance of CSCs. Methods: MCF-7 cells were treated with 1,25(OH)2D3 and their levels of VDR expression, viability, and apoptosis were detected. CD133+ MCF-7 stem cells were identified and transfected with a VDR-overexpression plasmid. The tamoxifen concentration that reduced MCF-7 cell viability by 50% (IC50) was determined. The activation of Wnt/ß-catenin signaling was also investigated. Results: Vitamin D reduced the viability of MCF-7 cells and promoted their apoptosis. Vitamin D enhanced VDR expression and induced DNA damage. When CD133+ stem cells were separated from MCF-7 cells, the IC50 of tamoxifen for stem cells was significantly higher than that of parental MCF-7 cells, suggesting a higher tamoxifen resistance in MCF-7 stem cells. Levels of VDR expression and Wnt/ß-catenin signaling in CD133+ cells were markedly lower and higher than those in CD133- cells, respectively. Stem cells transfected with VDR overexpression plasmids showed decreased tamoxifen IC50 values, viability, spheroid formation, and expression of Wnt and ß-catenin proteins when compared with control cells. Cell apoptosis was increased by transfection with a VDR overexpression plasmid. Finally, the inhibitory effects induced by VDR overexpression could be reversed by the VDR inhibitor, calcifediol. Conclusion: Stem cells contributed to the tamoxifen resistance of MCF-7 cells. Vitamin D-induced VDR expression increased the sensitivity of MCF-7 stem cells to tamoxifen by inhibiting Wnt/ß-catenin signaling.

Protective effect of calcitriol on podocytes in spontaneously hypertensive rat.

BACKGROUND: Hypertension is a major global public health issue. Uncontrolled hypertension leads to organ damage, especially renal damage. Calcitriol is used to treat osteoporosis, promote bone formation, and increase bone mass. Previous studies have demonstrated that 1,25(OH)2D3, in addition to its classic role, also has multiple immune regulation and renoprotective functions and inhibits the activity of the renin-angiotensin-aldosterone system (RASS). The aim of the current study was to investigate the renoprotective effects of calcitriol in a spontaneously hypertensive rat (SHR) model. METHODS: A total of 18 SHRs and 8 age-matched normal Wistar rats were enrolled. SHRs were randomly divided into a hypertensive nephropathy group (H), a hypertensive nephropathy treated with calcitriol group (D) and a control group (NS). The rats were sacrificed after 16 weeks of treatment. The blood pressure (BP) of rats were measured one time every 4 weeks. The levels of serum albumin, serum creatinine, blood calcium, serum Vitamin D and 24-h urinary protein were measured after 16 weeks treatment. The protein level of WT1, nephrin and vitamin D receptor (VDR) was examined by Western blotting and immunohistochemical staining. RESULTS: There were no notable changes in blood pressure or serum creatinine in group H and D compared with group NS. The albumin, calcium and vitamin D serum levels in group H were significantly decreased compared with group NS and significantly increased in group D compared with group H. The level of 24-h urine protein significantly increased in group H compared with group NS and significantly decreased in group D compared with group H. The expression of VDR, WT1 and nephrin in the kidney were all significantly decreased in group H compared with group NS and significantly increased in group D compared with group H. CONCLUSION: The present results indicated that there was injury of podocytes in hypertensive nephropathy, which can be ameliorated by calcitriol in SHR, but there was no significant anti-hypertensive effect. Vitamin D/VDR decreased proteinuria perhaps by increasing expression of nephrin and WT1 protein in podocyte of SHRs.

Stromal expression of JNK1 and VDR is associated with the prognosis of esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide, and its outcome is poor. The purpose of this study was to determine the association between JNK1 and vitamin D receptor (VDR) expression and the prognosis of ESCC. METHODS: Immunohistochemical staining was conducted on ESCC tissue microarrays (362 pairs of ESCC and normal esophagus tissues). The epithelial and stromal expression levels of c-jun NH2-terminal kinase 1 (JNK1) and VDR were scored and correlated with the ESCC characteristics. Laser-capture-based quantitative RT-PCR was performed on ESCC tissues. The effects of JNK1 and VDR on ESCC cell proliferation and migration were analyzed in vitro by transient transfection, and protein changes were evaluated by immunoblotting. RESULTS: Both JNK1 and VDR were reduced in ESCC epithelial cells in comparison with the normal esophagus, but the expression of JNK1 and VDR in ESCC stromal tissues, not epithelial cells, was strongly associated with the survival time of ESCC patients. Functional studies showed that increased JNK1 suppressed cancer cell proliferation, mobility, and migration, which were linked to the alterations of VDR and metastasis-associated proteins. CONCLUSION: JNK1 and VDR act as tumor suppressors, and their stromal expression levels are associated with prognosis in esophageal squamous cell carcinoma.