Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10⁻⁷ M, 1,25(OH)₂D₃ for 24 h resulted in a 20–30% decrease in PKI mRNA. 1,25(OH)₂D₃ treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)₂D₃ directly and tissue-specifically downregulates PKI mRNA in the chick kidney.     

1α,25-Dihydroxyvitamin D(3) down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells

Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding neurite-promoting factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10⁻⁸ m 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)₂D₃. These effects were observed in a dose-dependent manner in a dose range between 10⁻¹¹ m to 10⁻⁸ m. This effect was specific to 1,25(OH)₂D₃, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)₂D₃ down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)₂D₃, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.     

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10⁻¹⁰–10⁻⁸ M 1,25-(OH)₂D₃ or 10⁻⁹–10⁻⁷ M 24,25-(OH₂)D₃, and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [³⁵S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)₂D₃ caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)₂D₃ responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)₂D₃; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)₂D₃. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.     

1,25-Dihydroxycholecalciferol Stimulates ICAM-1 Expression of Human Alveolar Macrophages in Healthy Controls and Patients with Sarcoidosis

Synthesis and release of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₂) by alveolar macrophages (AM) have been shown to be increased in granulomatous lung disease. ICAM-1 plays a major part in leukocyte homing to sites of chronic inflammation, which is a crucial step during the inflammatory response. Whether 1,25-(OH)₂D₂ alters the ICAM-1 expression of AM in humans has not been studied. Bronchoalveolar lavage (BAL) was performed in 12 healthy volunteers, in 13 patients with sarcoidosis (active disease n= 8, inactive disease n= 5), and in 9 patients with chronic bronchitis. AM were incubated with different concentrations of 1,25-(OH)₂D₂ (10⁻¹¹ to 10⁻⁶ M) with and without priming with interferon-γ (IFN-γ) and with and without preincubation with 10⁻⁸ M dexamethasone. In addition, the metabolites of vitamin D, 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol, were used. The AM expression of ICAM-1 (cELISA) and the release of tumor necrosis factor-α (TNF-α) (bioassay) by AM were determined. In healthy volunteers the ICAM-1 expression on AM was significantly and dose-dependently increased by 1,25-(OH)₂D₂, but not by 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol. Priming with IFN-γ resulted in an additive effect. Preincubation with dexamethasone inhibited ICAM-1 expression. Addition of 1,25-(OH)₂D₂ after inhibition by dexamethasone increased ICAM-1 expression significantly. TNF-α secretion of AM from healthy volunteers was significantly reduced by 1,25-(OH)₂D₂. In sarcoidosis patients ICAM-1 expression was significantly higher compared with healthy volunteers. Incubation with 1,25-(OH)₂D₂ resulted in a further significant increase of ICAM-1 expression. TNF-α secretion of AM was increased compared with healthy volunteers. 1,25-(OH)₂D₂ reduced TNF-α secretion; however, this difference was not significant. 1,25-(OH)₂D₂ has an immunomodulating effect on human AM both in healthy volunteers and in sarcoidosis patients with enhanced expression of ICAM-1. It may serve as an autocrine mediator in inflammatory lung disease. Accepted for publication: 5 November 1998     

Vitamin D treatment of senescence accelerated mice (SAM-P/6) induces several regulators of stromal cell plasticity

In an attempt to understand the regulation of bone marrow multipotential cells plasticity in vivo, we treated 4-month-old SAM-P/6 mice with a constant infusion of either 18 pmol/24 h of 1,25(OH)₂D₃ or vehicle alone for 6 weeks. In vehicle treated animals 78% ± 4 adipose volume vs. total volume was stained positive with oil red O as compared to only 32 ± 3% in 1,25(OH)₂D₃treated animals (P < 0.001). Furthermore, we aimed to identify the changes in gene expression induced by 1,25(OH)₂D₃in bone marrow cells by analyzing a set of 5440 genes in the NIA 15K Mouse cDNA microarray. Overall, a coordinated regulation of genes which both stimulate osteoblastogenesis and inhibit adipogenesis was observed in 1,25(OH)₂D₃-treated mice when compared to vehicle treated mice. In summary, this study illustrates the anti-adipogenic effect of 1,25(OH)₂D₃in bone cells and identifies some of the possible key signals involved in bone cell plasticity.     

VITAMIN D METABOLISM IN HYPERTHYROIDISM

The serum concentrations of 25-hydroxycholecalciferol (25 OH D₃), 24,25-dihydroxycholecalciferol [24,25(OH)₂D₃] and 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] were measured in twenty-one patients with untreated hyperthyroidism. Compared with control subjects, 25 OH D₃ concentrations were not altered, 24,25(OH)₂D₃ concentrations were increased, although not significantly and 1,25(OH)₂D₃ concentrations were decreased (P= 0.01). Following oral carbimazole therapy, 24,25(OH)₂D₃ concentrations fell (P<0.01), 1,25(OH)₂D₃ concentrations increased (P<0.01) and 25 OH D₃ concentrations were unchanged. The altered 1,25(OH)₂D₃ and 24,25(OH)₂D₃ concentrations found in hyperthyroidism are probably due to the effects of thyroid hormone on bone and mineral metabolism. Increased serum calcium and phosphate concentrations with secondary hypoparathyroidism result in stimulation of the renal 24-hydroxylase and suppression of the 1-hydroxylase enzymes. In addition, serum 24,25(OH)₂D₃ concentrations were significantly correlated with serum triiodothyronine levels (T3) (r= 0.66, P<0.002) before treatment. This may indicate a direct stimulatory effect of T3 on 24-hydroxylase activity. No relationship was found between serum 1,25(OH)₂D₃ concentrations before therapy and serum T3.     

Regulation of vitamin D-1alpha-hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney

We investigated the effects of dexamethasone on vitamin D-1alpha-hydroxylase and -24-hydroxylase expression and on vitamin D receptor (VDR) content in the kidneys of mice fed either a normal (NCD) diet or a calcium- and vitamin D-deficient (LCD) diet for 2 weeks. For the last 5 days mice received either vehicle or dexamethasone (2 mg/kg per day s.c.). Dexamethasone significantly increased plasma calcium concentrations without changing plasma concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in both NCD and LCD groups. Northern blot and enzyme activity analyses in NCD mice revealed that dexamethasone increased renal VDR mRNA expression modestly and greatly increased 24-hydroxylase mRNA abundance and enzyme activity, but did not affect 1alpha-hydroxylase mRNA abundance and enzyme activity. In mice fed an LCD diet, dexamethasone increased renal VDR mRNA expression 1.5-fold, decreased 1alpha-hydroxylase mRNA abundance (52%) and activity (34%), and markedly increased 24-hydroxylase mRNA abundance (16-fold) and enzyme activity (9-fold). Dexamethasone treatment did not alter functional VDR number (B(max) 125-141 fmol/mg protein) or ligand affinity (K(d) 0.13-0.10 nM) in LCD mice. Subcutaneous injections of 1,25(OH)(2)D(3) (0.24 nmol/kg per day for 5 days) into NCD mice strongly increased renal 24-hydroxylase mRNA abundance and enzyme activity, while there was no effect of dexamethasone on renal 24-hydroxylase expression in these mice. This may be due to overwhelming induction of 24-hydroxylase by 1,25(OH)(2)D(3). These findings suggest that glucocorticoid-induced osteoporosis is caused by direct action of the steroids on bone, and the regulatory effect of glucocorticoids on renal 25-hydroxyvitamin D(3) metabolism may be less implicated in the initiation and progression of the disease.     

Klotho protein activates the PKC pathway in the kidney and testis and suppresses 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression

Homozygous Klotho mutant (kl ^−/−) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl ^−/− mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D₃ 1α-hydroxylase in the kidney. We studied 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D₃ 1α-hydroxylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D₃ 1α-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.     

Effects of analogs of 1,25(OH)2 Vitamin D3 on the proliferation and differentiation of the human chronic myelogenous leukemia cell line,RWLeu-4

Summary We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)₂D₃ (ED₅₀-3-10 nM). Side-chain-fluorinated analogs were more potent (ED₅₀=0.7–2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)₂D₃. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferatiive activity [1,25(OH)₂-16,23-diene D₃ (ED₅₀=2.7 nM) and 1,25(OH)₂-16-ene-23-yne D₃ (ED₅₀=0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)₂D₃, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent.Abbreviations 1,25(OH)₂D₃ 1,25-(OH)₂ vitamin D₃ - CML chronic myelogenous leukemia - NBT nitroblue tetrazolium - ED₅₀ 50% effective dose - IC₅₀ 50% inhibitory concentration     

Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystal-lographic structure of the glucocorticold and oeltrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations. DESIGN Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25dlhydroxyvltamln D₃ (1,25(OH)₂D₃) and functional responses to this hormone. These cells were also used to Isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patlents was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)₂D₃. Computer analysis of mutations in the VDR gene was carried out using the glucocorticold and oestrogen receptors as model systems. PATIENTS Two patients with HVDRR from unrelated families. MEASUREMENTS Cytosollc binding and nuclear association of 1,25(OH)₂D₃ were determined in control and affected patients, and functional response to 1,25(OH)₂D₃ was assessed by measurement of 2bhydroxyvltamln D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)₂D₃ following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)₂D₃. Sequencing of cDNA revealed single mutations, in patient I (Phe44 → IIe) and in patient II (Lys42 → Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR. CONCLUSIONS Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.     

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)₂D₃ and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intronlexon boundarles sequenced. PATIENT A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)₂D₃. MEASUREMENTS Nuclear association of 1,25(OH)₂D₃ was determined in patient and control cells and the functional response to 1,25(OH)₂D₃ was assessed by measurement of 25-hydroxyvitamln D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH), D₃. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5’donor splice site of intron 4. CONCLUSION In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the Characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the + 5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.     

Stathmin levels in growth plate chondrocytes are modulated by vitamin D3 metabolites and transforming growth factor-beta1 and are associated with proliferation

Stathmin is a highly conserved, phosphorylated cytosolic protein that is found at decreased levels in all cells as they become more terminally differentiated, or when they decrease in their rate of proliferation. This study examined the hypothesis that stathmin levels in growth plate chondrocytes decreases as endochondral maturation increases. To test this hypothesis, we used a costochondral growth plate chondrocyte cell culture model. Cells derived from the resting zone (RC) express twice as much stathmin mRNA in culture and have twice as much stathmin protein as cells derived from the post proliferative growth zone ([GC];prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiation, RC and GC chondrocytes were cultured in the presence of 10⁻¹⁰ to 10⁻⁸ M 1α,25-(OH)₂D₃, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10⁻⁹ to 10⁻⁷ M 24R,25-(OH)₂D₃, which regulates differentiation and maturation of RC cells, but decreases proliferation of GC cells. In addition, RC cells were treated with 0.44 or 0.88 ng/mL of recombinant human transforming growth factor β1 (rhTGF-β1), which stimulates proliferation of RC cells and regulates proteoglycan production, but not alkaline phosphatase activity. Stathmin protein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are associated with proliferation. Proliferating chondrocytes in vivo exhibited higher levels of immunoreactive stathmin than either RC or GC cells in the growth plate. In culture, 1α,25-(OH)₂D₃ caused a dose-dependent de-crease in stathmin in RC and GC cells within 24 h. 24 R, 25-(OH)₂D₃ also reduced stathmin levels in GC cells within 24 h but only affected RC cells after prolonged exposures (96 h), at which time RC cells express a GC-like phenotype. rhTGF-β1 caused an increase in stathmin levels in RC cells. Stathmin levels are sensitive to protein kinase C (PKC) in other cells. Inhibition of PKC with chelerythrine had no effect on the response of RC cells to 1α,25-(OH)₂D₃ but it blocked the effect of rhTGF-β1, indicating that decreases in stathmin by vitamin D₃ metabolites may not be modulated by PKC, whereas increases in stathmin via rhTGF-β1 may be regulated via a PKC-dependent mechanism. These results support the hypothesis that constitutively expressed levels of stathmin are related to cell maturation state and that they are modulated by factors that regulate proliferation.     

In vitro metabolism of 25-hydroxyvitamin D3 by isolated rat kidney cells

Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with ³H-labeled 25-hydroxyvitamin D₃ (25-OH-D₃) several polar metabolites, including 1,25-(OH)₂[³H]D₃ and 24,25-(OH)₂[³H]D₃ were produced. The specific activities of the 25-OH-D₃:1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ were linear over a wide range in cell densities (0.65-5.0 × 10⁶ cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)₂[³H]D₃ from 25-OH-[³H]D₃ by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)₂[³H]D₃ was linear for over 90 min. The specific activity of the 25-OH-D₃:1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10⁶ cells) as well as calcium-deficient rats (8.1 fmol/min per 10⁶ cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10⁶ cells). Also, the specific activity of the 25-OH-D₃:24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10⁶ cells) and calcium-deficient rats (5.1 fmol/min per 10⁶ cells) compared to the controls (15.2 fmol/min per 10⁶ cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D₃ by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.     

Adrenergic agents,but not triiodo-L-thyronine induce c-fos and c-myc expression in the rat heart

Summary We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 g/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2h. The response of both cellular oncogenes to norepinephrine was reduced significantly by a blockade but (3 blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2x10^–7 M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.     

Decreased expression of vitamin D receptor may contribute to the hyperimmune status of patients with acquired aplastic anemia

Acquired aplastic anemia (AA) is an immune‐mediated bone marrow failure syndrome. 1α,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). Previous studies have established that 1,25(OH)₂D₃ and VDR were involved in the pathogenesis of some autoimmune diseases. In this study, we evaluated the involvement of 1,25(OH)₂D₃ and VDR on T‐cell responses in AA. Plasma 25(OH)D₃ levels were comparable between patients with AA and healthy controls. Surprisingly, VDR mRNA was significantly lower in untreated patients with AA than in healthy controls. Subsequent in vitro experiments revealed that 1,25(OH)₂D₃ treatment suppressed the proliferation of lymphocytes and inhibited the secretion of interferon‐γ, tumor necrosis factor‐α, and interleukin‐17A, meanwhile promoting the production of transforming growth factor‐β1 in patients with AA. Moreover, 1,25(OH)₂D₃ inhibited the differentiation of type 1 and Th17 cells but induced the differentiation of type 2 and regulatory T cells. Interestingly, VDR mRNA was elevated in healthy controls after 1,25(OH)₂D₃ treatment, but not in patients with AA. In conclusion, decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in patients with AA.     

Development of a vitamin D-responsive organ culture system for adult and old rat intestine

The purpose of this study was to develop an organ culture system for adult and old rat small intestine that is responsive to vitamin D. Explants from F344 rats were cultured on Millipore tissue culture inserts placed in 6-well dishes at a temperature of 28°C and in the presence of 95% oxygen. Explants from young (2 months old), adult (12 months old), and old (22 months old) rats were viable for up to 12 hr as determined by constant rates of DNA and protein synthesis. Hormonal responsiveness was characterized by measuring the capacity of 1,25-dihydroxyvitamin D [1,25(OH)₂D], the hormonal form of vitamin D, to increase mRNA levels of the intestinal 24-hydroxylase cytochrome P-450 (CYP24). Jejunal explants from young rats increased CYP24 mRNA levels in a linear fashion with an EC₅₀ of 3 nM in response to 1,25(OH)₂D. There was no change with age in the magnitude of the jejunal response with regard to time (0–12 hr) or dose (0.1–100 nM). However, in the duodenum, 1,25(OH)₂D increased CYP24 mRNA to significantly higher levels in the adult compared to the young. Since the 24-hydroxylase is the first step in the degradative pathway for 1,25(OH)₂D in the intestine, increased duodenal expression of the 24-hydroxylase may contribute to the decreased action of 1,25(OH)₂D on the adult duodenum.     

The gastrointestinal electrical mapping suite (GEMS): software for analyzing and visualizing high-resolution (multi-electrode) recordings in spatiotemporal detail

Background

Intestinal hyper-permeability plays a critical role in the etiopathogenesis of inflammatory bowel disease (IBD) by affecting the penetration of pathogens, toxic compounds and macromolecules. 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active form of vitamin D, has been shown to be an important regulator of IBD and recent epidemiology suggests that patients with IBD have an impaired vitamin D status. The purpose of this study is to investigate the possible protective effects of 1,25(OH)₂D₃ on mucosal injury and epithelial barrier disruption on dextran sulfate sodium (DSS)-induced acute colitis model.

Methods

We used DSS-induced acute colitis model to investigate the protective effects of 1,25(OH)₂D₃ on mucosal injury and epithelial barrier integrity. Severity of colitis was evaluated by disease activity index (DAI), body weight (BW) change, colon length, histology, myeloperoxidase (MPO) activity, and proinflammatory cytokine production including tumor necrosis factor-?? (TNF-??) and interferon-?? (IFN-??). In vitro the protective role of 1,25(OH)₂D₃ was assessed by incubating Caco-2 cells with or without DSS and measuring transepithelial electrical resistance (TEER) and fluorescein isothiocyanate dextran (FITC-D). The intestinal permeability was analyzed by FITC-D, bacterial translocation and measurement of lipopolysaccharide (LPS). Ultrastructural features of the colon tissue and Caco-2 cell monolayer were observed by electron microscopy. Expressions of tight junction (TJ) proteins in the colon mucosa and Caco-2 cells were detected by immunohistochemistry, immunofluorescence, Western blot and real-time fluorescent quantitative PCR, respectively.

Results

DSS-induced acute colitis model was characterized by a reduced BW, AUC of BW, serum calcium, higher DAI, AUC of DAI, shortened colon length, elevated MPO activity, worsened histologic inflammation, increased mononuclear cell numbers in mesenteric lymph nodes (MLNs) and colonic lamina propria (LP), and enhanced proteins and mRNA levels of TNF-?? and IFN-??. 1,25(OH)₂D₃ markedly increased expressions of TJ proteins and mRNA and decreased the FITC-D permeability and the level of LPS. Furthermore, 1,25(OH)₂D₃ abrogated bacterial translocation to MLNs and ameliorated ultrastructural features of the colon epithelium by scanning electron microscopy (SEM). In vitro, 1,25(OH)₂D₃ increased TEER, TJ proteins and mRNA expressions, decreased the FITC-D permeability, and preserved structural integrity of the TJ in Caco-2 cells.

Conclusions

1,25(OH)₂D₃ may play a protective role in mucosal barrier homeostasis by maintaining the integrity of junction complexes and in healing capacity of the colon epithelium. 1,25(OH)₂D₃ may represent an attractive and novel therapeutic agent for the adjuvant therapy of IBD.     

25-Hydroxyvitamin D-1α Hydroxylase: Studies in Mouse Models and Implications for Human Disease

Genetic mouse models with targeted deletion (“knockout”) of the 25-hydroxyvitamin D-1alpha-hydroxylase gene [1α(OH)ase^−/−], as well as with targeted deletion of the VDR gene, when exposed to different dietary regimens, have provided considerable insight into the molecular regulation of skeletal physiology by the 1,25(OH)₂D/VDR system. These regimens induced different phenotypic changes and demonstrated that parathyroid gland size and the development of the cartilaginous growth plate were each co-ordinately regulated by calcium and by 1,25(OH)₂D, and that parathyroid hormone (PTH) secretion and mineralization of bone reflected ambient calcium (and phosphorus) levels rather than the direct actions of the 1,25(OH)₂D/VDR system. In contrast, increased calcium absorption, optimal osteoblastogenesis, and baseline bone formation were observed to be modulated by 1,25(OH)₂D/VDR signaling. These bone anabolic effects of endogenous 1,25(OH)₂D were evident in neonatal mice as well as in older animals, and exogenous 1,25(OH)₂D₃ was also found to stimulate trabecular and cortical bone formation in neonatal double homozygous 1α(OH)ase^−/−PTH^−/− mice. Furthermore, the anabolic effect of exogenously administered PTH appeared to be partly dependent on the stimulation of endogenous 1,25(OH)₂D. Genetic mouse models have also been employed to study extra-skeletal actions modulated by the 1,25(OH)₂D/VDR system. For example, increased blood pressure, activation of the renin/angiotensin system, myocardial hypertrophy, and cardiac dysfunction were observed in 1αOHase^−/− mice, and these alterations could be prevented by treatment with 1,25(OH)₂D₃. These models allow controlled examination of the regulation of both skeletal and extra-skeletal pathophysiology associated with 1,25(OH)₂D deficiency which appear to be relevant to humans, and facilitate studies to prevent and treat these disorders by active vitamin D forms.