Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation

1,25-Dihydroxyvitamin D³ [1,25(OH)₂D₃] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that at physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)₂D₃. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)₂D₃ and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 μM) of 1,25(OH)₂D₃. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)₂D₃ to initiate a complete growth-inhibitory response as measured using [³H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)₂D₃ are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)₂D₃ and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)₂D₃ receptor (VDR). © 1995 Wiley-Liss, Inc.     

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.     

Relative resistance to 1,25-dihydroxyvitamin D3 in a keratinocyte model of tumor progression.

We have examined the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on mitogen-stimulated growth and on c-myc proto-oncogene expression in a keratinocyte model of tumor progression. A dose-dependent inhibition of cell growth by 1,25-(OH)2D3 was demonstrated in both established (HPK1A) and malignant (HPK1A-ras) cells. However, this inhibition was observed with the addition of 1,25-(OH)2D3 at a higher concentration in HPK1A-ras cells than in HPK1A cells. Cell cycle analysis revealed a blockage of the normal progression of the cell cycle from G0 to S phase in the presence of 1,25-(OH)2D3. A higher concentration of 1,25-(OH)2D3 was required in HPK1A-ras cells to overcome the mitogen-stimulated progression into S phase, when compared with HPK1A cells. Analysis of c-myc messenger RNA revealed a strong inhibition of its expression at early time points with higher concentrations of 1,25-(OH)2D3 being required to obtain an inhibition in HPK1A-ras cells similar to that obtained in HPK1A cells. 1,25-(OH)2D3 receptor characterization by sucrose gradient analysis and equilibrium binding demonstrated the presence of a single 3.7 S protein with similar receptor numbers and affinity in both cell lines. These observations therefore demonstrate that an alteration of the growth inhibitory response to 1,25-(OH)2D3 occurs when keratinocytes acquire the malignant phenotype and suggest that the alteration lies beyond the interaction of the ligand with its receptor. In addition, relative resistance to 1,25-(OH)2D3 was also observed in the expression of the cell-cycle associated oncogene c-myc. These studies may therefore have important implications in vivo in the development and growth of epithelial cell cancers.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.