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.

On the relationship between VDR, RORα and RORγ receptors expression and HIF1-α levels in human melanomas.

We analysed the correlation between the expression of HIF-1α (hypoxia-inducible factor 1 alpha), the nuclear receptors: VDR (vitamin D receptor), RORα (retinoic acid receptor-related orphan receptor alpha), and RORγ and CYP24A1 (cytochrome P450 family 24 subfamily A member 1) and CYP27B1 (cytochrome P450 family 27 subfamily B member 1), enzymes involved in vitamin D metabolism. In primary and metastatic melanomas, VDR negatively correlated with nuclear HIF-1α expression (r = -.2273, P = .0302; r = -.5081, P = .0011). Furthermore, the highest HIF-1α expression was observed in pT3-pT4 VDR-negative melanomas. A comparative analysis of immunostained HIF-1α and CYP27B1 and CYP24A1 showed lack of correlation between these parameters both in primary tumors and melanoma metastases. In contrast, RORα expression correlated positively with nuclear HIF-1α expression in primary and metastatic lesions (r = .2438, P = .0175; r = .3662, P = .0166). Comparable levels of HIF-1α expression pattern was observed in localized and advanced melanomas. RORγ in primary melanomas correlated also positively with nuclear HIF-1α expression (r = .2743, P = .0129). HIF-1α expression was the lowest in localized RORγ-negative melanomas. In addition, HIF-1α expression correlated with RORγ-positive lymphocytes in melanoma metastases. We further found that in metastatic lymph nodes FoxP3 immunostaining correlated positively with HIF-1α and RORγ expression in melanoma cells (r = .3667; P = .0327; r = .4208, P = .0129). In summary, our study indicates that the expression of VDR, RORα and RORγ in melanomas is related to hypoxia and/or HIF1-α activity, which also affects FoxP3 expression in metastatic melanoma. Therefore, the hypoxia can affect tumor biology by changing nuclear receptors expression and molecular pathways regulated by nuclear receptors and immune responses.

Women with recurrent spontaneous abortion have decreased 25(OH) vitamin D and VDR at the fetal-maternal interface

Immunological mechanisms have been proposed to underlie the pathogenesis of recurrent spontaneous abortion (RSA). Vitamin D has a potent immunomodulatory effect, which may affect pregnancy outcome. The objective of this study was to investigate 25-hydroxyvitamin D [25(OH) D] concentration and vitamin D receptor (VDR) expression in the decidual tissues of RSA patients. Thirty women with RSA (RSA group) and thirty women undergoing elective abortion (control group) were recruited during 2016 from gynecology outpatient clinics. We measured 25(OH) D, interleukin (IL)-17, IL-23, transforming growth factor β (TGF-β), VDR and 1-α-hydroxylase (CYP27B1) in decidual tissues collected during the abortion procedure. In the RSA group, 25(OH) D and TGF-β were significantly decreased while IL-17 and IL-23 were significantly increased compared with the control group. VDR expression was significantly decreased in the RSA group compared with the control group. Logistic regression analysis showed a significant negative correlation between 25(OH) D in decidual tissues and RSA. These results indicated that vitamin D concentrations in the decidua are associated with inflammatory cytokine production, suggesting that vitamin D and VDR may play a role in the etiology of RSA.

25-Hydroxyvitamin D deficiency is associated with inflammation-linked vascular endothelial dysfunction in middle-aged and older adults.

We tested the hypothesis that vascular endothelial function, assessed by endothelium-dependent dilation, is related to serum vitamin D status among middle-aged and older adults without clinical disease, and that this is linked to inflammation. Brachial artery flow-mediated dilation, a measure of endothelium-dependent dilation, was lower (P<0.01) in vitamin D-insufficient (3.7 ± 0.2%; serum 25-hydroxyvitamin D [25(OH)D]: 20 to 29 ng/mL; 62 ± 1 years of age; n = 31; mean± SE) and vitamin D-deficient (3.2 ± 0.3%; 25(OH)D: <20 ng/mL; 63 ± 2 years of age; n = 22) versus vitamin D-sufficient (4.6 ± 0.4%; 25(OH)D: >29 ng/mL; 61 ± 1 years of age; n = 22) subjects, whereas endothelium-independent dilation (brachial dilation to sublingual nitroglycerine) did not differ (P = 0.45). Among all subjects, brachial flow-mediated dilation was positively related to serum 25(OH)D (%Δ: r = 0.35; P<0.01) but not 1,25-dihydroxyvitamin D (r = -0.06; P = 0.61), the active form of vitamin D. Vascular endothelial cell expression of the proinflammatory transcription factor nuclear factor κB was greater in deficient versus sufficient subjects (0.59 ± 0.07 versus 0.44 ± 0.05; P<0.05), and inhibition of nuclear factor κB (4 days oral salsalate) improved flow-mediated dilation to a greater extent in subjects with lower versus higher 25(OH)D (+3.7 ± 0.6 versus +2.0 ± 0.2%; P<0.05). Endothelial cell expression of the downstream proinflammatory cytokine interleukin-6 also was higher in deficient versus sufficient subjects (0.67 ± 0.08 versus 0.47 ± 0.05; P<0.01) and inversely related to serum 25(OH)D level (r = -0.62; P<0.01), whereas vitamin D receptor and 1-α hydroxylase, the 25(OH)D to 1,25-dihydroxyvitamin D converting enzyme, were lower (P<0.05). Inadequate serum 25(OH)D is associated with vascular endothelial dysfunction among healthy middle-aged/older adults, and this is mediated in part by nuclear factor κB-related inflammation. Reduced vitamin D receptor and 1-α hydroxylase may be molecular mechanisms linking vitamin D insufficiency to endothelial dysfunction.

Mammary epithelial cell transformation is associated with deregulation of the vitamin D pathway.

The vitamin D endocrine system mediates anti-proliferative and pro-differentiating signaling in multiple epithelial tissues, including mammary gland and breast tumors. The vitamin D metabolite 1alpha,25(OH)2D3 mediates growth inhibitory signaling via activation of the vitamin D receptor (VDR), a ligand dependent transcription factor. 1alpha,25(OH)(2)D3 is synthesized from 25(OH)D3 (the major circulating form of the vitamin) by the mitochondrial enzyme CYP27b1 in renal and other tissues. Human mammary epithelial (HME) cells express VDR and CYP27b1 and undergo growth inhibition when exposed to physiological concentrations of 25(OH)D3, suggesting that autocrine or paracrine vitamin D signaling contributes to maintenance of differentiation and quiescence in the mammary epithelium. In the current studies we tested the hypothesis that cancer cells would exhibit reduced sensitivity to vitamin D mediated negative growth regulation. We used a series of progressively transformed HME cell lines expressing known oncogenic manipulations to study the effects of transformation per se on the vitamin D pathway. We report that mRNA and protein levels of VDR and CYP27b1 were reduced greater than 70% upon stable introduction of known oncogenes (SV40 T antigens and H-rasV12) into HME cells. Oncogenic transformation was also associated with reduced 1alpha,25(OH)2D3 synthesis, and cellular sensitivity to growth inhibition by 1alpha,25(OH)2D3 and 25(OH)D3 was decreased approximately 100-fold in transformed cells. These studies provide evidence that disruption of the vitamin D signaling pathway occurs early in the cancer development process.

Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts.

BACKGROUND: In pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi. METHODS: Quantification of hCG in culture media was performed by immunoassay. Expression studies were carried out by real time PCR. Analysis of CYP27B1 and VDR localization in placental slides were performed by immunohistochemistry. Statistical significance was established by one way ANOVA using Tukey test for comparisons. RESULTS: Calcitriol regulated hCG in a time-dependent manner: at 6 h the secosteroid stimulated hCG, whereas longer incubations (24 h) showed opposite effects. Interestingly, calcitriol stimulatory effects on hCG were accompanied by an increase in intracellular cAMP content and were abolished by pre-incubation of the cells with a selective protein kinase A inhibitor. Immunohistochemical techniques showed differential VDR localization in the syncytiotrophoblast layer or in the vascular smooth muscle cells depending on the epitope to which the antibodies were raised (specific for the carboxy- or amino-terminal regions, respectively). CYP27B1 was immunolocalized in the syncytiotrophoblast layer of placental villi. CONCLUSION: The presence and location of the vitamin D activating enzyme CYP27B1 as well as the specific receptor for vitamin D were shown in placental sections. The latter, together with findings demonstrating specific effects of calcitriol acting through the VDR and the cAMP/PKA signaling pathway upon hCG expression and secretion, indicate that there is a functional vitamin D endocrine system in the placenta, and recognize calcitriol as an autocrine regulator of hCG.

Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.

OBJECTIVES: Many patients with Crohn's disease (CD) have low bone mineral density (BMD) that may not be solely attributable to glucocorticoid use. We hypothesised that low BMD in patients with CD is associated with elevated circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D). We further hypothesised that this was secondary to increased synthesis of 1,25(OH)(2)D by inflammatory cells in the intestine. The aim of this study was to examine the relationship between 1,25(OH)(2)D levels and BMD in patients with CD. METHODS: An IRB approved retrospective review of medical records from patients with CD (n = 138) or ulcerative colitis (UC, n = 29). Measurements of vitamin D metabolites and immunoreactive parathyroid hormone (iPTH) were carried out. BMD results were available for 88 CD and 20 UC patients. Immunohistochemistry or real time reverse transcription-polymerase chain reaction (RT-PCR) for the enzyme 1alpha-hydroxylase was performed on colonic biopsies from patients with CD (14) or UC (12) and normal colons (4). RESULTS: Inappropriately high levels of serum 1,25(OH)(2)D (>60 pg/ml) were observed in 42% of patients with CD compared with only 7% in UC, despite no differences in mean iPTH. Serum 1,25(OH)(2)D levels were higher in CD (57 pg/ml) versus UC (41 pg/ml) (p = 0.0001). In patients with CD, there was a negative correlation between 1,25(OH)(2)D levels and lumbar BMD (r = -0.301, p = 0.005) independent of therapeutic glucocorticoid use. 1,25(OH)(2)D levels also correlated with CD activity. Lastly, immunohistochemistry and RT-PCR demonstrated increased expression of intestinal 1alpha-hydroxylase in patients with CD. CONCLUSIONS: These data demonstrate that elevated 1,25(OH)(2)D is more common in CD than previously appreciated and is independently associated with low bone mineral density. The source of the active vitamin D may be the inflamed intestine. Treatment of the underlying inflammation may improve metabolic bone disease in this subgroup of patients.

Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.

The mitochondrial enzyme 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D(3), in the kidney. However, enzyme activity assays indicate that 1 alpha-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1 alpha-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1 alpha-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1 alpha-hydroxylase in both normal and diseased tissues. Specific staining for 1 alpha-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Further studies using psoriatic skin highlighted overexpression of 1 alpha-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1alpha-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin from these patients 1 alpha-hydroxylase expression was observed in cells positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1 alpha-hydroxylase in several extrarenal tissues, such as skin, placenta, and lymph nodes. The function of this enzyme at novel extrarenal sites, such as adrenal medulla, brain, pancreas, and colon, remains to be determined. However, the discrete patterns of staining in these tissues emphasizes a possible role for 1 alpha-hydroxylase as an intracrine modulator of vitamin D function in peripheral tissues.

Vitamin D hydroxylases and their regulation in a naturally vitamin D-deficient subterranean mammal, the naked mole rat (Heterocephalus glaber).

The vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is generated by a series of hydroxylation steps in the liver and kidneys. We investigated whether naturally vitamin D-deficient subterranean mammals (naked mole rats, Heterocephalus glaber) employ the same enzymatic pathways, and whether these are regulated in a similar manner to that established for other mammals. Vitamin D3-25-hydroxylase in the liver and both 25-hydroxyvitamin D3-1-hydroxylase and 25-hydroxyvitamin D3-24 hydroxylase (1-OHase and 24-OHase) in the kidney were detectable in mole rats. As expected for vitamin D-deficient mammals, the 1-OHase activity predominated over the 24-OHase. After mole rats received a supraphysiological supplement of vitamin D3, 1-OHase activity was suppressed and 24-OHase activity was enhanced. Irrespective of vitamin D status, forskolin (a protein kinase A activator) and dibutyryl cyclic AMP did not alter the activity of either 1-OHase or 24-OHase. These findings suggest that the response of renal hydroxylases to parathyroid hormone was blunted. Phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) (protein kinase C activators), suppressed 1-OHase activity. 24-OHase activity was induced by TPA but not by OAG. These effects were similar to those illicited by vitamin D3 supplementation but were additive in that they increased the responses shown in vitamin D-replete mole rats. These data confirm that naturally vitamin D-deficient mole rats can convert vitamin D3 to the hormone, 1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of diabetes mellitus on parathyroid hormone-stimulated protein kinase activity, ferredoxin phosphorylation, and renal 1,25-dihydroxyvitamin D production.

Impairment in the stimulation of renal production of 1,25-dihydroxyvitamin D[1,25 (OH)2D] by parathyroid hormone (PTH) occurs in diabetes. Renal response to PTH in terms of 25-hydroxyvitamin D-1-hydroxylase (1-OHase) stimulation involves increased cyclic adenosine monophosphate (cAMP) production, increased cAMP-dependent protein kinase activity, and dephosphorylation of renal ferredoxin (renoredoxin). To identify the step where diabetes might impair PTH stimulation of 1-OHase, we studied the effects of PTH on 1,25(OH)2D production, cAMP content, cAMP-dependent protein kinase activity, and the phosphorylation state of renoredoxin by using renal slices from diabetic and nondiabetic rats. PTH and forskolin significantly stimulated 1,25(OH)2D production in renal slices from nondiabetic animals but not from diabetic animals. PTH-stimulated cAMP production and cAMP-dependent protein kinase activity in renal slices were not altered by diabetes. However, diabetes significantly impaired the capacity of PTH to dephosphorylate renoredoxin and to increase the activity of the 1-OHase enzyme complex. These results suggest that the decreased capacity of PTH to stimulate 1-OHase activity in diabetic animals may reflect the decreased capacity of PTH to alter the phosphorylation state of renoredoxin in these animals.

Parathyroid hormone secretion and target organ response in experimental acute pancreatitis.

To determine changes in parathyroid hormone secretion and target organ response caused by acute pancreatitis before the development of systemic toxic conditions, experimental acute pancreatitis was induced in rats with a choline-deficient, ethionine-supplemented diet. After 7 days, the rats were weighed and bled, and one kidney was assayed for 25-hydroxyvitamin D1 hydroxylase activity. Several manifestations of pancreatitis were observed in rats given the diet: weight loss (from 29.6 to 26.3 g vs that for control rats, from 29 to 52.8 g) and lower dietary intake (15.5 vs 47 g per rat per 7 days). Serum amylase levels fell from 1794 to 350 U/L in rats given the choline-deficient, ethionine-supplemented diet compared with levels of 1800 to 2100 U/L in control rats. The pancreases of rats given the choline-deficient, ethionine-supplemented diet showed degeneration, necrosis, and hemorrhaging. Serum levels of calcium, phosphorus, chloride, and parathyroid hormone did not change significantly throughout the experiment. Renal 25-hydroxyvitamin D1 hydroxylase activity was higher than in control rats (8.9 +/- 0.8 vs 7.6 +/- 0.6 fmol/mg of kidney per minute). Acute pancreatitis in this experimental animal model does not alter serum levels of calcium and parathyroid hormone or reduce target organ responsiveness to the hormone.

Vitamin D metabolism in nephrotic rats.

It is well known that patients with nephrotic syndrome and normal renal function have hypocalcemia in spite of high PTH concentration, caused by the low serum concentration of the active vitamin D metabolite, 1,25(OH)2D, presumably due to its loss in urine. However, it has been uncertain whether the conversion of 25(OH)D into 1,25(OH)2D in the kidney is impaired. In this study, we examined the responsibility of 1,25(OH)2D in PAN-induced nephrotic rats. Sprague-Dawley rats weighing 250 g were given subcutaneous injections 1.5 mg/100 g PAN for 12 days prior to use. Some of these rats were given intraperitoneal injection of 100 IU of 25(OH)D3 for 3 days prior to use and of 10 IU of PTH. We measured Ca2+ in plasma, vitamin D metabolites and mid-molecule PTH in serum, renal 25(OH)D-1-hydroxylase activity in vitro, and response of nephrogenous cyclic AMP to exogenous PTH administration. In nephrotic rats, plasma Ca2+, serum 25(OH)D and 1,25(OH)2D were lower than in control rats, and the serum PTH level was higher than in controls. In 25(OH)D3-injected nephrotic rats, Ca2+ and 1,25(OH)2D were higher than in nephrotic rats, indicating that the decreased level of 1,25(OH)2D in nephrotic rats was partially due to the low serum level of 25(OH)D. Despite the elevation of the serum level of PTH, the Vmax of renal 25(OH)D-1-hydroxylase in nephrotic rats was lower than in controls. Response of nephrogenous cyclic AMP to PTH in nephrotic rats was lower than in controls. Although nephrotic rats had higher PTH levels than control rats, Vmax of renal 25(OH)D-1-hydroxylase and response of cyclic AMP to exogenous PTH administration in nephrotic rats were lower than in controls, suggesting that abnormalities of calcium metabolism in patients with nephrotic syndrome might be partially attributed to the impaired renal response to PTH.

Inhibition of 25-hydroxyvitamin D 1 alpha-hydroxylase by renal mitochondrial protein kinase-catalyzed phosphorylation.

Partially purified chick kidney mitochondrial Type II protein kinase catalyzes the phosphorylation of 1 alpha-hydroxylase cytochrome P-450 without affecting the rate of product formation in vitro when 1 alpha-hydroxylase activity is reconstituted by the addition of [ferredoxin] and [ferredoxin reductase] to the phosphorylated cytochrome. The cytochrome's effective concentration, or its general spectral properties did not change upon phosphorylation. However, when the cytochrome and its ferredoxin were present simultaneously during the phosphorylation reaction, reconstitution of 1 alpha-hydroxylase activity by the addition of ferredoxin reductase failed to catalyze product formation. Although a several fold increase in the kinase activity could be demonstrated in the presence of cAMP, the above phosphorylation effects appear to be cAMP-independent.