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Objectives: Myocardial inflammation underlies a broad spectrum of conditions that cause damage to the myocardium and lead to structural and functional defects. Homocysteine (Hcy) is closely related to the occurrence and development of cardiovascular diseases. We investigated the mechanism underlying the effects of vitamin D as a prophylactic treatment for Hcy-induced cardiac inflammation., Methods: The levels of 25(OH)D 3 and Hcy were assessed using ELISA kits. Expression levels of the vitamin D receptor (VDR), NFE2 like bZIP transcription factor 2 (NFE2L2), methylenetetrahydrofolate reductase (MTHFR) and inflammatory factors were examined by Western blotting, immunohistochemistry and real time polymerase chain reaction. NFE2L2/MTHFR-knockdown HL-1 cells and NFE2L2 +/- mouse were used to test the effects of vitamin D., Results: We found the levels of Hcy in the serum and myocardial tissue of mice in the Hcy + CCE group were lower than in the Hcy groups, which was opposed to the trend exhibited by the serum 25(OH)D 3 level of mice. The mRNA and protein expression levels of the inflammatory factors in cardiac tissues and cardiomyocytes were strongly decreased by the Hcy treatment, compared to the Hcy + CCE/Hcy + 1,25(OH) 2 D 3 groups. Moreover, the results revealed that the level of nuclear NFE2L2 in Hcy + CCE/Hcy + 1,25(OH) 2 D 3 group was increased compared to Hcy group with a reciprocal decrease in the level of cytosolic NFE2L2 in vivo and in vitro. Similarly, the MTHFR mRNA and protein expression in the Hcy + CCE group was higher than the Hcy group. We determined that NFE2L2 promoted the expression of MTHFR. However, based on Hcy treatment, the combination of 1,25(OH)2D3 and MTHFR -/- reversed the decline in IL-6 and TNFα expression caused by 1,25(OH)2D3 alone. Chromatin immunoprecipitation and luciferase reporter assays showed the up-regulation effect of VDR on NFE2L2 and NFE2L2 on MTHFR., Conclusions: Our findings indicate that vitamin D/VDR could improve Hcy-induced myocardial inflammation through activation of NFE2L2 mediated MTHFR., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Homocysteine (Hcy) is a synthetic amino acid containing sulfhydryl group, which is an intermediate product of the deep metabolic pathway of methionine and cysteine. The abnormal increase in fasting plasma total Hcy concentration caused by various factors is called hyperhomocysteine (HHcy). HHcy is closely relevant to the occurrence and progression of diverse cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension and diabetes, etc. Vitamin D/vitamin D receptor (VDR) pathway is pointed out that prevent cardiovascular disease by reducing serum homocysteine levels. Our research is designed to explore the potential mechanism of vitamin D in the prevention and treatment of HHcy., Methods and Results: The Hcy and 25(OH)D 3 levels in mouse myocardial tissue, serum or myocardial cells were detected using ELISA kits. The expression levels of VDR, Nrf2 and methionine synthase (MTR) were observed using Western blotting, immunohistochemistry and real time polymerase chain reaction (PCR). General information of the mice, including diet, water intake and body weight, was recorded. Vitamin D up-regulated the mRNA and protein expression of Nrf2 and MTR in mouse myocardial tissue and cells. CHIP assay determined that the combination of Nrf2 binding to the S1 site of the MTR promoter in cardiomyocytes using traditional PCR and real time PCR. Dual Luciferase Assay was applied to detect the transcriptional control of Nrf2 on MTR. The up-regulation effect of Nrf2 on MTR was verified by Nrf2 knockout and overexpression in cardiomyocytes. The role of Nrf2 in vitamin D inhibition of Hcy was revealed using Nrf2-knockdown HL-1 cells and Nrf2 heterozygous mice. Western blotting, real time PCR, IHC staining and ELISA showed that Nrf2 deficiency could restrain the increase in MTR expression and the decrease in Hcy level induced by vitamin D. The transcriptional activities of Nrf2/MTR were activated by vitamin D/VDR with a decrease in Hcy., Conclusion: Vitamin D/VDR upregulates MTR in an Nrf2-dependent manner, thereby reducing the risk of HHcy., Competing Interests: Conflict of interest All authors state that no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Some epidemiological studies have revealed that vitamin D (VD) deficiency is closely linked with the prevalence of obesity, however, the role of VD in energy homeostasis is yet to be investigated, especially in central nervous system. Given that VD negatively regulates renin in adipose tissue, we hypothesized that central VD might play a potential role in energy homeostasis., Objectives: The present study aims to investigate the potential role of VD in energy homeostasis in the CNS and elaborate its underlying mechanisms., Methods: This study was conducted in Cyp27b1 -/- mice, VD-treated and wild-type mice. After the intraventricular injection of renin or its inhibitors, the changes of renin-angiotensin system (RAS) and its down-stream pathway as well as their effects on metabolic rate were examined., Results: The RAS activity was enhanced in Cyp27b1 -/- mice, exhibiting a increased metabolic rate. Additionally, corticotropin-releasing hormone (CRH), a RAS-mediated protein regulating energy metabolism in the hypothalamus, increased significantly in Cyp27b1 -/- mice. While in VD-treated group, the RAS and sympathetic nerve activities were slightly inhibited, hence the reduced metabolic rate., Conclusion: Collectively, the present study demonstrates that the VD/vitamin D receptor (VDR) has a significant impact on energy homeostasis through the modulation of RAS activity in the hypothalamus, subsequently altering CRH expression and sympathetic nervous activity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Liver fibrosis may progress toward cirrhosis and cancer without effective therapy. Here, we investigated the underlying mechanism of Vitamin D as a therapeutic approach., Methods: Carbon tetrachloride (CCL4)-induced mice model and transforming growth factor-β1 (TGF-β1) induced human hepatic stellate cell line LX-2 were used in vivo and in vitro. The fibrotic profiles, degree of liver injury and HRC expression were assessed by histology, Western blot, immunohistochemistry and Real-Time PCR. The proliferation of cells transfected with HRC +/+ and HRC -/- plasmids was detected by MTS and cell cycle methods., Results: Vitamin D significantly suppressed the expression of HRC in liver fibrosis model both in vivo and in vitro (P < 0.01). The cell with overexpression of HRC significantly increased TGF-β1/Smad3 expressions and the percentage of the S peak in cell cycle (P < 0.05). However, Vitamin D can significantly reverse the levels of TGF-β1, Smad3 and p-smad3 caused by HRC in vitro. Furthermore, the overexpression of HRC in cell lines can attenuate the function of Vitamin D, suggesting that VD played a role by regulating HRC. Mechanically, HRC as the target of VDR is detected by CHIP method., Conclusions: Vitamin D can delay hepatic fibrosis by reducing activation of hepatic stellate cells and TGF-β/Smad signaling through negative regulation of HRC. The findings revealed the important regulatory effect of Vitamin D in hepatic stellate cells and provided new insights into the therapeutic function of Vitamin D on liver fibrosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Intrinsic vitamin D affects the proliferation, apoptosis, invasion, metastasis, and tumorigenesis of lung cancer by regulating tumor signaling pathways. Histidine-rich calcium-binding protein (HRC) maintains Ca 2+ homeostasis, which plays crucial roles in the occurrence and development of cancer., Objectives: Our study aims to investigate the ability of vitamin D in the regulation of HRC and the role of HRC playing in lung cancer., Methods: We investigated the effects of vitamin D on lung cancer and the underlying mechanisms, by measuring HRC and vitamin D receptor (VDR) expression in lung cancer, paracancer, and normal tissues from patients using immunohistochemistry, western blotting, and real time RT-PCR. We transfected H460 lung cancer cells (supplemented or not with vitamin D) with PX458-HRC and pcDNA3.1-HRC plasmids and injected mice with lung cancer cells harboring pcDNA3.1-vector or pcDNA3.1-HRC plasmids., Results: Vitamin D inhibited HRC expression and H460 cell migration and proliferation, and promoted apoptosis compared with controls. The expression of HRC and VDR was significantly upregulated and downregulated, respectively, in lung cancer versus paracancer or normal tissues. Cell proliferation and migration were reduced, apoptotic cells were more and tumors were smaller in mice treated with vitamin D/cholecalciferol cholesterol emulsion (CCE) than in vitamin D/CCE+HRC +/+ mice., Conclusion: Vitamin D inhibited lung cancer tumor growth, migration, and proliferation by downregulating HRC., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Aim: Vitamin D plays an important role in water and salt homeostasis. The aim of our study was to investigate the underlying relationship of Vitamin D and Aquaporins (AQP)., Methods: The behaviors of 1 α (OH)-ase knockout mice and wild type mice were observed before analysis. The ICR mice were treated with vehicle or paricalcitol, a vitamin D analogue, followed by animals receiving a standard diet and free access to drinking water either with aliskiren (renin blocker; 37.5 mg aliskiren in 100 ml water), or telmisartan (a angiotensin II type I receptor blocker; 40 mg telmisartan in 100 ml water) a week before study. The expressions of AQP-1, AQP-4, and renin in mice kidneys were detected by western bolting, immunohistochemistry, and immunofluorescence., Results: Diuresis and polydipsia were observed in 1 α (OH)-ase knockout mice, and a decreased water intake and urine output in ICR mice was observed after paricalcitol treatment. Compared with wild type, the AQP-1 expressions were increased in renal papilla and AQP-4 expressions were decreased in renal proximal tubule of 1 α (OH) ase knockout mice. In addition, AQP-1 was decreased in renal papilla and AQP-4 expressions were increased in proximal tubule by suppressing renin activity or supplement of Vitamin D analogue. After injecting renin into the lateral ventricle of the 1 α (OH)ase knockout mice, the renin expression level was decreased in the kidney, followed by the decrease of AQP-1 in renal papilla and increase of AQP-4 in proximal tubule., Conclusions: Overall, Vitamin D and renin inhibitors have synergistic effects in regulating water channels in mice kidneys.

Objective: To explore the effect of vitamin D analogs regulated mice urinary output on depressed expression of renin-angiotensin in paraventricular nucleus., Methods: A total of 51 C57BL/6 mice were randomly divided into control (n = 36) and vitamin D receptor knockout (VDR, n = 15) groups. Then the tests of water deprivation, vitamin D analogue injection and 0.25% captopril water drinking were performed. Renin levels in paraventricular nucleus and plasma and aquaporin in kidney were determined by immunofluorescence, real-time polymerase chain reaction (PCR) and Western blot., Results: The urinary output of VDR knockout mice decreased markedly after 48 h water deprivation [(129 ± 45 vs 1 354 ± 224) ml/24 h, P < 0.05] while there was little change of urinary and plasma osmolarity. The expression levels of AQP-4 and rennin in VDR knockout mice were higher than those of wild type. Renin and angiotensin II in periventricular nucleus of VDR knockout mice were higher than those of wild type and was reduced by injecting vitamin D analogs into wild type. Rennin activity: (139 ± 31 vs 185 ± 56) mcU/ml; angiotensin II: (33 ± 32 vs 72 ± 27) pg/ml, (both P < 0.05). There was statistically significant differences. The expression of c-fos in periventricular nucleus in VDR knockout mice was higher and it could be inhibited after an injection of vitamin D analog., Conclusions: Thirsty in central nervous system is inhibited by vitamin D analogs through reduced renin-angiotensin activity in paraventricular nucleus. Polydipsia and polyuria may be caused by a lack of vitamin D. Captopril lowers urinary output.

The negative feedback mechanism is essential to maintain effective immunity and tissue homeostasis. 1,25-dihydroxyvitamin D (1,25[OH]2D3) modulates innate immune response, but the mechanism remains poorly understood. In this article, we report that vitamin D receptor signaling attenuates TLR-mediated inflammation by enhancing the negative feedback inhibition. Vitamin D receptor inactivation leads to hyperinflammatory response in mice and macrophage cultures when challenged with LPS, because of microRNA-155 (miR-155) overproduction that excessively suppresses suppressor of cytokine signaling 1, a key regulator that enhances the negative feedback loop. Deletion of miR-155 attenuates vitamin D suppression of LPS-induced inflammation, confirming that 1,25(OH)2D3 stimulates suppressor of cytokine signaling 1 by downregulating miR-155. 1,25(OH)2D3 downregulates bic transcription by inhibiting NF-κB activation, which is mediated by a κB cis-DNA element located within the first intron of the bic gene. Together, these data identify a novel regulatory mechanism for vitamin D to control innate immunity.

Leptin is an adipose tissue-derived hormone that plays a critical role in energy homeostasis. Vitamin D has been shown to regulate energy metabolism, but the relationship between vitamin D and leptin is unclear. Leptin expression and secretion was reduced in vitamin D receptor (VDR)-null mice and increased in transgenic (Tg) mice overexpressing the VDR in adipocytes; however, as leptin is mainly determined by fat mass, it is unclear whether the vitamin D hormone directly regulates leptin expression. To address this question, we determined the effect of vitamin D on leptin expression in vivo and ex vivo. One-week treatment of WT mice with the vitamin D analog RO-27-5646 led to a significant increase in adipose leptin mRNA transcript and serum leptin levels. Moreover, in adipose tissue cultures, 1,25-dihydroxyvitamin D markedly stimulated mRNA expression and secretion of leptin, but not resistin, in adipose tissues obtained from WT mice, but not from VDR-null mice, and leptin upregulation induced by 1,25-dihydroxyvitamin D was more robust in adipose tissues obtained from VDR Tg mice compared with WT mice. These data demonstrate that 1,25-dihydroxyvitamin D stimulates adipose leptin production in a VDR-dependent manner, suggesting that vitamin D may affect energy homeostasis through direct regulation of leptin expression.

Vitamin D inhibits renin expression and blocks the compensatory induction of renin associated with the use of renin-angiotensin system inhibitors. Here we test the therapeutic effects of two commonly used vitamin D analogs and their combination with losartan on the development of left ventricular hypertrophy. One-month-old male spontaneously hypertensive rats were treated with vehicle, losartan, paricalcitol, doxercalciferol, a combination of losartan and paricalcitol, or a combination of losartan and doxercalciferol for 2 months. Blood pressure was markedly reduced by losartan, but not by paricalcitol or doxercalciferol alone. Echocardiograpy demonstrated a 65 to 80% reduction in left ventricular wall thickness with losartan, paricalcitol, or doxercalciferol monotherapy and almost complete prevention of left ventricular hypertrophy with the combination therapies. Attenuation of cardiac and cardiomyocyte hypertrophy, and suppression of atrial and brain natriuretic peptides, were most marked in the combination therapy groups. These changes were well correlated with left ventricular gene and microRNA expression profiles in the different treatment groups. Renal and cardiac renin expression was markedly increased in losartan-treated animals, but nearly normalized with combination therapy. The same vitamin D analogs suppressed plasma renin activity in patients receiving chronic hemodialysis. These data demonstrate that vitamin D analogs have potent antihypertrophic activity in part via suppression of renin in the kidney and heart, and combination of these analogs with losartan achieves much better therapeutic effects because of the blockade of the compensatory renin increase.

The renin-angiotensin system (RAS) plays a critical role in the development of diabetic nephropathy, and blockade of the RAS is currently used for treatment of diabetic nephropathy. One major problem for the current RAS inhibitors is the compensatory renin increase, which reduces the efficacy of RAS inhibition. We have shown that vitamin D exerts renoprotective actions by transcriptionally suppressing renin. Here we demonstrated that combination therapy with an AT1 receptor blocker and a vitamin D analog markedly ameliorated renal injury in the streptozotocin (STZ)-induced diabetes model due to the blockade of the compensatory renin rise by the vitamin D analog, leading to more effective RAS inhibition. STZ-treated diabetic DBA/2J mice developed progressive albuminuria and glomerulosclerosis within 13 weeks, accompanied by increased intrarenal production of angiotensin (Ang) II, fibronection, TGF-beta, and MCP-1 and decreased expression of slit diaphragm proteins. Treatment of the diabetic mice with losartan or paricalcitol (19-nor-1,25-dihydroxyvitamin D(2), an activated vitamin D analog) alone moderately ameliorated kidney injury; however, combined treatment with losartan and paricalcitol completely prevented albuminuria, restored glomerular filtration barrier structure, and markedly reduced glomerulosclerosis. The combined treatment suppressed the induction of fibronection, TGF-beta, and MCP-1 and reversed the decline of slit diaphragm proteins nephrin, Neph-1, ZO-1, and alpha-actinin-4. These were accompanied by blockade of intrarenal renin and Ang II accumulation induced by hyperglycemia and losartan. These data demonstrate that inhibition of the RAS with combination of vitamin D analogs and RAS inhibitors effectively prevents renal injury in diabetic nephropathy.

Emerging evidence supports a pathological link between vitamin D deficiency and the risk of inflammatory bowel disease (IBD). To explore the mechanism we used the dextran sulfate sodium (DSS)-induced colitis model to investigate the role of the vitamin D receptor (VDR) in mucosal barrier homeostasis. While VDR(+/+) mice were mostly resistant to 2.5% DSS, VDR(-/-) mice developed severe diarrhea, rectal bleeding, and marked body weight loss, leading to death in 2 wk. Histological examination revealed extensive ulceration and impaired wound healing in the colonic epithelium of DSS-treated VDR(-/-) mice. Severe ulceration in VDR(-/-) mice was preceded by a greater loss of intestinal transepithelial electric resistance (TER) compared with VDR(+/+) mice. Confocal and electron microscopy (EM) revealed severe disruption in epithelial junctions in VDR(-/-) mice after 3-day DSS treatment. Therefore, VDR(-/-) mice were much more susceptible to DSS-induced mucosal injury than VDR(+/+) mice. In cell cultures, 1,25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] markedly enhanced tight junctions formed by Caco-2 monolayers by increasing junction protein expression and TER and preserved the structural integrity of tight junctions in the presence of DSS. VDR knockdown with small interfering (si)RNA reduced the junction proteins and TER in Caco-2 monolayers. 1,25(OH)(2)D(3) can also stimulate epithelial cell migration in vitro. These observations suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD.

1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-kappaB activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR-/- mice, the basal level of kappaB inhibitor (IkappaB) alpha protein was markedly decreased compared with VDR+/- MEFs; however, degradation of IkappaBalpha and its phosphorylation in response to TNF-alpha treatment or Salmonella infection were not altered in VDR-/- cells, neither were the levels of IkappaB kinase-alpha and IkappaB kinase-beta proteins. Consistent with IkappaBalpha reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR-/- cells. In addition, the physical interaction between VDR and p65 was absent in VDR-/- MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-kappaB transcriptional activity; consistently, induction of IL-6 by TNF-alpha or IL-1beta was much more robust in VDR-/- than in VDR+/- cells, indicating that VDR-/- cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-kappaB activity. The reduction of IkappaBalpha in VDR-/- MEFs may be partially explained by the lack of VDR-mediated stabilization of IkappaBalpha by 1,25(OH)2D3. This is supported by the observation that IkappaBalpha degradation induced by TNF-alpha was inhibited by 1,25(OH)2D3 in VDR+/- cells, but not in VDR-/- cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-kappaB activation.

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.

The renin-angiotensin system (RAS) plays a central role in the regulation of blood pressure, volume and electrolyte homeostasis. Inappropriate activation of the RAS may lead to hypertension. Clinical and epidemiological studies have suggested a correlation between Vitamin D-deficiency and high blood pressure. Our recent studies demonstrate that Vitamin D is a potent endocrine suppressor of renin biosynthesis to regulate the RAS. Mice lacking the Vitamin D receptor (VDR) have elevated production of renin and angiotensin (Ang) II, leading to hypertension, cardiac hypertrophy and increased water intake. These abnormalities can be prevented by treatment with an ACE inhibitor or AT(1) receptor antagonist. Vitamin D repression of renin expression is independent of calcium metabolism, the volume- and salt-sensing mechanisms and the Ang II feedback regulation. In normal mice, Vitamin D-deficiency stimulates renin expression, whereas injection of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] reduces renin synthesis. In cell cultures, 1,25(OH)(2)D(3) directly suppresses renin gene transcription by a VDR-dependent mechanism. Furthermore, we have found that Gemini compounds have more potent renin-suppressing activity than 1,25(OH)(2)D(3). Collectively, our studies reveal a critical role of the Vitamin D endocrine system in the regulation of blood pressure and volume homeostasis, and suggest that low calcemic Vitamin D analogs may potentially be developed into a new class of anti-hypertensive agents to control renin production and blood pressure.

Background: With environmental and lifestyle changes, recent epidemiological studies have shown that the prevalence of Ulcerative Colitis (UC) is on the rise, while treatment options are limited. There is an urgent need to explore the underlying mechanisms of vitamin D (VD) as an effective treatment. Methods: Dextran sulfate sodium-induced mice and lipopolysaccharide-induced HCT116 cells were used to establish the classic UC models in vivo and in vitro, respectively. Typical symbols of inflammation (IL-6, COX-2), oxidative stress (MDA, MPO, GSH), and ferroptosis (ACSL4, GPX4, SLC7A11, and Iron) were analyzed by Western blot, Immunohistochemistry, RT-PCR, and relative assay kits. The inflammation factors and oxidative stress injury of cells transfected with ACSL4+/+ plasmids were tested by Western blot, MDA, and MPO methods. Results: Vitamin D attenuated the levels of COX-2, IL-6, Iron, MDA, and MPO and improved SOD1 and GSH contents in DSS + VD and LPS + VD groups, compared with model groups. Ferrostatin-1 (Fer-1) could relieve the levels of COX-2, IL-6, Iron, MDA, and MPO while increasing the contents of SOD1 and GSH in DSS + Fer-1 and LPS + Fer-1 compared to model groups. VD downregulated the expression of ACSL4 and upregulated GPX4 in tissues and cells. After transfected with ACSL4+/+ plasmids, we found VD's role of downregulating inflammation and oxidative stress was relieved. Conclusions: Vitamin D can relieve UC by inhibiting ferroptosis both in mice and in cells through the negative regulation of ACSL4, providing new insight into the therapeutic function of VD on UC. [ABSTRACT FROM AUTHOR]

[Display omitted] Intrinsic vitamin D affects the proliferation, apoptosis, invasion, metastasis, and tumorigenesis of lung cancer by regulating tumor signaling pathways. Histidine-rich calcium-binding protein (HRC) maintains Ca2+ homeostasis, which plays crucial roles in the occurrence and development of cancer. Our study aims to investigate the ability of vitamin D in the regulation of HRC and the role of HRC playing in lung cancer. We investigated the effects of vitamin D on lung cancer and the underlying mechanisms, by measuring HRC and vitamin D receptor (VDR) expression in lung cancer, paracancer, and normal tissues from patients using immunohistochemistry, western blotting, and real time RT-PCR. We transfected H460 lung cancer cells (supplemented or not with vitamin D) with PX458-HRC and pcDNA3.1-HRC plasmids and injected mice with lung cancer cells harboring pcDNA3.1-vector or pcDNA3.1-HRC plasmids. Vitamin D inhibited HRC expression and H460 cell migration and proliferation, and promoted apoptosis compared with controls. The expression of HRC and VDR was significantly upregulated and downregulated, respectively, in lung cancer versus paracancer or normal tissues. Cell proliferation and migration were reduced, apoptotic cells were more and tumors were smaller in mice treated with vitamin D/cholecalciferol cholesterol emulsion (CCE) than in vitamin D/CCE+HRC+/+ mice. Vitamin D inhibited lung cancer tumor growth, migration, and proliferation by downregulating HRC. [ABSTRACT FROM AUTHOR]

Introduction. Vitamin D plays a key role in maintaining calcium homeostasis and skeletal health. The liver is critically involved in vitamin D metabolism, as 25-hydroxyvitamin D3 (25(OH)D3) is synthesized in the liver. Therefore liver dysfunction may lead to vitamin D deficiency and bone problems. The aim of this study was to examine vitamin D status and bone turnover markers in hepatitis B patients from northeastern China. Methods. We recruited 39 patients with hepatitis B (23 noncirrhotic and 16 cirrhotic) and 48 healthy controls in Shenyang, a metropolitan city in northeastern China, and measured serum 25(OH)D3 levels and serum and urinary bone turnover markers in these subjects. Results. Serum 25(OH)D3 levels in the patients with or without cirrhosis were markedly lower compared to the nonhepatitis controls (19.2 ± 1.2 and 18.5 ± 1.3 vs. 31.6 ± 1.3 nmol/L control), whereas serum and urinary bone turnover markers (alkaline phosphatase, C-terminal telopeptide of type I collagen, and pyridinoline) were significantly higher in these patients than in the controls. Moreover, serum levels of osteoprotegerin, a bone mass-regulating protein, were substantially reduced in the patients, with the lowest seen in patients with cirrhosis (2.7 ± 1.1 and 1.4 ± 0.4 vs. 3.4 ± 0.7 pg/mL control). Serum 25(OH)D3 levels below 30 nmol/L were positively correlated with serum osteoprotegerin levels in this cohort. Conclusions. Severe vitamin D deficiency is very common in hepatitis B patients in northeastern China, which negatively impacts their bone health. These data strongly suggest a need to treat these patients with vitamin D supplementation to protect their bone health. [ABSTRACT FROM AUTHOR]

Abstract: Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D. [Copyright &y& Elsevier]

The intrarenal renin-angiotensin system (RAS) plays a key role in the development of diabetic nephropathy. Recently, we showed that combination therapy with an AT1 receptor blocker (ARB) and an activated vitamin D analog produced excellent synergistic effects against diabetic nephropathy, as a result of blockade of the ARB-induced compensatory renin increase. Given the diversity of vitamin D analogs, here we used a pro-drug vitamin D analog, doxercalciferol (1α-hydroxyvitamin D2), to further test the efficacy of the combination strategy in long-term treatment. Streptozotocin-induced diabetic DBA/2J mice were treated with vehicle, losartan, doxercalciferol (0.4 and 0.6 μg/kg), or losartan and doxercalciferol combinations for 20 wk. Vehicle-treated diabetic mice developed progressive albuminuria and glomeruloscierosis. Losartan alone moderately ameliorated kidney injury, with renin being drastically upregulated. A similar therapeutic effect was seen with doxercalciferol alone, which markedly suppressed renin and angiotensinogen expression. The losartan and doxercalciferol combination most effectively prevented albuminuria, restored glomerular filtration bartier structure, and dramatically reduced glomerulosclerosis in a dose-dependent manner. These effects were accompanied by blockade of intrarenal renin upregulation and ANG II accumulation. These data demonstrate an excellent therapeutic potential for doxercalciferol in diabetic renal disease and confirm the concept that blockade of the compensatory renin increase enhances the efficacy of RAS inhibition and produces synergistic therapeutic effects in combination therapy. [ABSTRACT FROM AUTHOR]

Previously, we showed that vitamin D receptor gene knockout leads to hyperreninemia independent of calcium metabolism; however, the contribution of parathyroid hormone to renin upregulation remained unclear. Here we separated the role of vitamin D and parathyroid hormone in the regulation of renin expression in vivo by generating transgenic mice that overexpressed the human vitamin D receptor in renin-producing cells using the 4.1 kb Ren-1c gene promoter. Targeting of human vitamin D receptor to the juxtaglomerular cells of the mice was confirmed by immunohistochemistry. Renal renin mRNA levels and plasma renin activity were decreased in these transgenic mice by about 50% and 30%, respectively, with no significant change in blood pressure, calcium, or parathyroid hormone levels. Moreover using vitamin D receptor knockout mice, we found that expression of the human receptor in their juxtaglomerular cells reduced renin expression in these mice without affecting calcium or parathyroid hormone status. Our study shows that suppression of renin expression by 1,25-dihydroxyvitamin D in vivo is independent of parathyroid hormone and calcium.Kidney International (2008) 74, 1577–1581; doi:10.1038/ki.2008.452; published online 1 October 2008 [ABSTRACT FROM AUTHOR]

1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-κB activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR-/- mice, the basal level of κB inhibitor (1κB) α protein was markedly decreased compared with VDR+/- MEFs; however, degradation of IκBα and its phosphorylation in response to TNF-α treatment or Salmonella infection were not altered in VDR-/- cells, neither were the levels of IκB kinase-α and IκB kinase-β proteins. Consistent with IκBα reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR-/- cells. In addition, the physical interaction between VDR and p65 was absent in VDR-/- MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-κB transcriptional activity; consistently, induction of IL-6 by TNF-α or IL-1β was much more robust in VDR-/- than in VDR+/- cells, indicating that VDR-/- cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-κB activity. The reduction of IκBα in VDR-/- MEFs may be partially explained by the lack of VDR-mediated stabilization of IκBα by 1,25(OH)2D3. This is supported by the observation that IκBα degradation induced by TNF-α was inhibited by 1,25(OH)2D3 in VDR+/- cells, but not in VDR-/- cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-κB activation. [ABSTRACT FROM AUTHOR]

Abstract: The renin-angiotensin system (RAS) plays a central role in the pathogenesis of hypertension. Recently, we discovered that 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] functions as a negative endocrine regulator of renin biosynthesis, which provides a molecular basis to explore the potential of Vitamin D analogs as renin inhibitors to control the RAS. To search for renin-inhibiting Vitamin D analogs, we screened 20 Vitamin D analog compounds using As4.1-hVDR cell (a juxtaglomerular cell line) culture by Northern blot and luciferase reporter assays. We found that the Gemini compounds, which have two side-chains at carbon-20 position, were particularly active in suppressing renin expression. Eight Gemini compounds were identified that were equally or 10- to 100-times more potent than 1,25(OH)2D3 in renin inhibition. These Gemini compounds also potently stimulate 25-hydroxyvitamin D 24-hydroxylase expression in As4.1-hVDR cells. Administration of compound RO-27-5646 [1,25-dihydroxy-21-(3-methyl-3-hydroxy-butyl)-19-nor-cholecalciferol] in mice caused a marked reduction in renal renin mRNA expression without invoking severe hypercalcemia as seen in 1,25(OH)2D3 treatment. These data establish in principle that Vitamin D analogs can indeed inhibit renin expression in vitro and in vivo, and support the notion that low calcemic Vitamin D analogs can potentially be used as therapeutic agents to control the RAS. [Copyright &y& Elsevier]

Our recent studies suggest that 1,25-dihydroxyvitamin D3 functions as an endocrine suppressor of renin biosynthesis. Genetic disruption of the vitamin D receptor (VDR) results in overstimulation of the rennin-angiotensin system (RAS), leading to high blood pressure and cardiac hypertrophy. Consistent with the higher heart-to-body weight ratio, the size of left ventricular cardiomyocytes in VDR knockout (KO) mice was markedly increased compared with wild-type (WT) mice. As expected, levels of atrial natriuretic peptide (ANP) mRNA and circulating ANP were also increased in VDRKO mice. Treatment of VDRKO mice with captopril reduced cardiac hypertrophy and normalized ANP expression. To investigate the role of the cardiac RAS in the development of cardiac hypertrophy, the expression of renin, angiotensinogen, and AT-1a receptor in the heart was examined by real-time RT-PCR and immunostaining. In VDRKO mice, the cardiac renin mRNA level was significantly increased, and this increase was further amplified by captopril treatment. Consistently, intense immunostaining was detected in the left ventricle of captopril-treated WT and VDRKO mice by use of an anti-renin antibody. Levels of cardiac angintensinogen and AT-1a receptor mRNAs were unchanged in the mutant mice. These data suggest that the cardiac hypertrophy seen in VDRKO mice is a consequence of activation of both the systemic and cardiac RAS and support the notion that 1,25-dihydroxyvitamin D3 regulates cardiac functions, at least in part, through the RAS. [ABSTRACT FROM AUTHOR]

Bolt, Merry J. G., Wenhua Liu, Guilin Qiao, Juan Kong, Wei Zheng, Thomas Krausz, Gabriella Cs-Szabo, Michael D. Sitrin, and Yan Chun Li. Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3. Am J Physiol Endocrinol Metab 287: E744-E749, 2004. First published May 27, 2004; 10.1152/ajpendo.00151.2004.—As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal. [ABSTRACT FROM AUTHOR]

Alopecia is a predominant feature of vitamin D receptor inactivation in mice and humans. To determine the role of vitamin D receptor in the regulation of hair growth directly, we used the human keratin 14 promoter to target human vitamin D receptor expression to the skin of transgenic mice, and generated vitamin D receptor null mice that express the human vitamin D receptor transgene. Parallel studies were carried out in littermates of wild-type, vitamin D receptor null, transgenic, and human vitamin D receptor-expressing null mice in two transgenic lines. The transgenic mice were grossly normal. The vitamin D receptor null and vitamin D receptor null/human vitamin D receptor mice were growth retarded and developed hypocalcemia, secondary hyperparathyroidism, and rickets. In contrast to the vitamin D receptor null mice that developed alopecia, however, the vitamin D receptor null/human vitamin D receptor mice displayed a normal hair coat, and their hair shaft and skin histology were indistinguishable from those of the wild-type mice. Immunohistochemical analyses revealed that the human vitamin D receptor was highly expressed in the basal layer of the epidermis and outer root sheath of the hair follicle. During follicular morphogenesis, no major histologic differences were seen in the skin of wild-type, vitamin D receptor null, transgenic, and vitamin D receptor null/human vitamin D receptor littermates. When anagen was induced by hair depilation at day 20 after birth, the vitamin D receptor null mice failed to initiate the hair cycle, whereas the vitamin D receptor null/human vitamin D receptor mice displayed the same pattern of anagen follicle formation as the wild-type mice. Interestingly, the transgenic mice initiated the follicular cycle earlier than the wild-type and vitamin D receptor null/human vitamin D receptor mice in a gene concentration-dependent manner. Taken together, these data provide direct evidence that vitamin D receptor is required for the... [ABSTRACT FROM AUTHOR]

As the prevalence of obesity and metabolic syndrome escalates, it has become increasingly important to understand the physiology of adipocytes. 1,25-dihydroxyvitamin D (1,25(OH)[sub 2]D[sub 3]) is a steroid hormone that is traditionally known to be a regulator of mineral homeostasis. 1,25(OH)[sub 2]D[sub 3] exerts its effects through the vitamin D receptor (VDR), a nuclear receptor that regulates gene transcription. Our recent studies suggest that VDR plays an integral role in adipocyte differentiation. VDR expression is tightly regulated during adipocyte differentiation and treatment of preadipocytic cells with 1,25(OH)[sub 2]D[sub 3] suppresses differentiation. However, the in vivo role of VDR in lipid metabolism has not been explored. We, therefore, evaluated the body fat composition and lipid parameters of VDR(-/-) mice. On a regular diet, VDR (-/-) mice weighed less and contained less body fat than wild type (WT) mice. On the high calcium-high lactose diet that rescues the hypocalcemia, the difference between VDR(-/-) and WT mice appeared sex dependent. In males, VDR(-/-) mice weighed less and had a lower body fat than WT mice; however no significant difference was observed in females. On a high fat diet, VDR (-/-) mice gained weight at a much lower rate than WT mice regardless of the sex. The gene profile of adipocytes from VDR(-/-) mice was also found to be altered. Levels of leptin and adiponectin were reduced in VDR(-/-) mice compared to WT mice, whereas resistin levels were increased in VDR(-/-) mice. Plasma cholesterol and triglyceride levels were significantly lower in VDR (-/-) mice than WT mice. These observations indicate that VDR plays a role in the regulation of adipose mass and lipid metabolism in vivo, which is independent of its role in calcium metabolism. [ABSTRACT FROM AUTHOR]

We have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2725 to -2647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)2D3 inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)2D3-induced suppression. EMSA revealed that 1,25(OH)2D3 markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)2D3 suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex. [ABSTRACT FROM AUTHOR]

Abstract: We have used mouse embryonic fibroblasts (MEFs) derived from VDR(+/−) and VDR(−/−) mice to determine whether the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-κB activation. We found that the basal IκBα protein level was markedly decreased in VDR(−/−) MEFs compared to VDR(+/−) MEFs; however, degradation of IκBα and its phosphorylation were not altered in VDR(−/−) cells, neither were the levels of IKKα and IKKβ proteins. Consistently, p65 nuclear translocation was increased in unstimulated VDR(−/−) cells. The physical interaction between VDR and p65 was absent in VDR(−/−) MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in NF-κB transcriptional activity. Consistently, induction of IL-6 by TNFα or IL-1β was much more robust in VDR(−/−) than in VDR(+/−) cells, indicating that VDR(−/−) cells are more susceptible to inflammatory stimulation. Therefore, fibroblasts lacking VDR appear to be more pro-inflammatory due to the intrinsic high NF-κB activity. The reduction of IκBα in VDR(−/−) MEFs may be partially explained by the lack of VDR-mediated stabilization of IκBα by 1,25(OH)2D3. These data suggest that VDR plays an inhibitory role in the regulation of NF-κB activation. [Copyright &y& Elsevier]

Abstract Vitamin D plays an important role in maintaining skeletal development and bone homeostasis. Although vitamin D has been extensively researched, the direct effect of 1,25(OH) 2 D 3 on osteoblasts is unclear. To explore the 1,25(OH) 2 D 3 action on murine osteoblasts, we performed tandem mass tag experiments on MC3T3-E1 cells treated with and without 1,25(OH) 2 D 3. Three up-regulated proteins (MRC2, WWTR1 and RASSF2) related to bone metabolism were confirmed in this study. 1,25(OH) 2 D 3 up-regulated the expression of MRC2 through vitamin D receptor. MRC2 affects collagen metabolism in osteoblasts. Combined with bioinformatics and parallel reaction monitoring analysis, we inhibited the expression of MRC2 to explore the relationship between MRC2 and collagens. Then we found MRC2 down-regulated COL5A2 and up-regulated MMP13. This study provides a protein profile of 1,25(OH) 2 D 3 -treated murine osteoblasts, reveals a newly discovered signaling axis (1,25(OH) 2 D 3 /VDR/MRC2/COL5A2 and MMP13), and explains the effect of 1,25(OH) 2 D 3 on bone metabolism from a new perspective. Graphical abstract Image 1 Highlights • This study provides a protein profile of 1,25(OH) 2 D 3 -treated murine osteoblasts. • 1,25(OH) 2 D 3 regulates some proteins related to collagen metabolism. • Vitamin D may play a role in negative regulation of bone ECM. [ABSTRACT FROM AUTHOR]

Bolt, Merry J. G., Wenhua Liu, Guilin Qiao, Juan Kong, Wei Zheng, Thomas Krausz, Gabriella Cs-Szabo, Michael D. Sitrin, and Yan Chun Li. Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3. Am J Physiol Endocrinol Metab 287: E744-E749, 2004. First published May 27, 2004; 10.1152/ajpendo.00151.2004.—As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal. [ABSTRACT FROM AUTHOR]

As a critical regulator of the cell cycle, cyclin-dependent kinase (CDK) inhibitor p21 or p21 is involved in the development of cardiac hypertrophy and heart failure. Calcitriol, or 1,25(OH) 2 D 3, the bioactive form of vitamin D (VD), can activate p21 expression and attenuate cardiac hypertrophy. To simulate cardiac hypertrophy in vitro and ex vivo, respectively, mice and cardiomyocytes were treated with isoproterenol (ISO). Moreover, the p21 signaling pathway was examined in ISO + VD and ISO + VD p21 inhibitor-treated cardiomyocytes. We found that calcitriol treatment led to a significant decrease in cardiac size and the mRNA levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in ISO-treated mice. Furthermore, the surface area of cardiomyocytes and the expression of ANP and BNP were decreased, and the expression of p21 was increased in the ISO + VD group compared with those in the ISO group. Furthermore, the surface area of cardiomyocytes and the expression of ANP and BNP were markedly upregulated in the ISO + VD p21 inhibitor group relative to the ISO + VD group, whereas the difference was not statistically significant compared with those of the ISO p21 inhibitor group. Therefore, our findings indicate that 1,25(OH) 2 D 3 protects against cardiac hypertrophy in mice through upregulating p21 expression. [ABSTRACT FROM AUTHOR]