1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte

The steroid hormone 1,25(OH)₂-vitamin D₃ [1,25D] has been shown to affect the growth and proliferation of primary cultures of ventricular myocytes isolated from neonatal rat hearts. The research presented here shows that the vitamin D receptor [VDR] is present in murine cardiac myocytes (HL-1 cells), and that 1,25D affects the growth, proliferation and morphology of these cells. In addition we show that 1,25D effects expression of ANP, myotrophin, and c-myc. Furthermore, 1,25D effects expression and localization of the VDR within the cell. Murine HL-1 cardiac myocytes were grown and treated with 1,25D in culture, and growth and morphology were assessed with microscopic analysis. Cells were counted and protein levels were evaluated through Western blot analysis. Subcellular localization of the VDR was determined using immunofluorescence and confocal microscopy. 1,25D was found to decrease proliferation and alter cellular morphology of the HL-1 cells. Treatment with 1,25D increased expression of myotrophin while decreasing expression of atrial natriuretic peptide [ANP] and c-myc. 1,25D treatment also increased expression and nuclear localization of the VDR in these cardiac myocytes. Thus 1,25D is an important hormone involved in modulating and maintaining heart cell structure and function.     

Differentiation-related regulation of 1,25 dihydroxy vitamin D3 receptor mRNA in human leukaemia cells HL-60

Vitamin D receptor (VDR) is a nuclear protein which mediates the physiological actions of its hormone ligand, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). While it appears that the receptor-hormone complex regulates the expression of hormone-dependent genes involved in mineral homeostasis, its role in induction of differentiation of leukaemic cells is less clear. We have studied the expression of the VDR gene in several sublines of HL-60 leukaemic cells with varying responsiveness to 1,25(OH)2D3. Sublines which rapidly differentiated to monocytic forms were shown to contain elevated steady-state levels of VDR mRNA within 1 h of exposure to high concentration of 1,25(OH)2D3. This up-regulation of the expression of VDR was not apparent in sublines in which monocytic differentiation occurred after a delay of several days. Beginning at approximately 3 h after exposure to 1,25(OH)2D3 in most cases, there was a gradual decline in VDR mRNA levels. Measurement of steady-state levels of mRNA for c-myc and c-fos showed that in sublines of HL-60 cells which respond rapidly to 1,25(OH)2D3, elevation of VDR mRNA is evident prior to the changes in proto-oncogene expression. These data are consistent with the hypothesis that a change in VDR gene expression is one of the steps that promote monocytic differentiation.     

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.     

Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the growth of normal human keratinocytes cultured in serum-free medium was investigated. 1,25(OH)2D3 inhibited the cell growth at 10(-7) M by 75.3% and at 10(-6) M almost completely. The growth inhibition was accompanied by changes related to proliferation: (1) remarkable inhibition of DNA synthesis, (2) the decrease in the number of high-affinity receptors for epidermal growth factor, with almost no change in total receptor number, (3) the rapid decrease in c-myc mRNA level. The inhibition of DNA synthesis and the decrease of c-myc mRNA expression occurred at 3 h after the addition of 1,25(OH)2D3. These results suggest that decrease of c-myc mRNA expression is one of the primary effects of 1,25(OH)2D3 in the growth inhibition of human keratinocytes.     

Nuclear co-translocation of myotrophin and p65 stimulates myocyte growth. Regulation by myotrophin hairpin loops

Myotrophin, a 12-kDa ankyrin repeat protein, stimulates protein synthesis and cardiomyocyte growth to initiate cardiac hypertrophy by activating the NF-kappaB signaling cascade. We found that, after internalization into myocytes, myotrophin cotranslocates into the nucleus with p65 to stimulate myocyte growth. We used structure-based mutations on the hairpin loops of myotrophin to determine the effect of the loops on myotrophin and p65 localization, induction of protein synthesis, and cardiac hypertrophy. Loop mutants, most prominently glutamic acid 33-->alanine (E33A), stimulated protein synthesis much less than wild type. Myotrophin-E33A internalized into myocytes but did not translocate into the nucleus and failed to promote nuclear translocation of p65. In addition, two cardiac hypertrophy marker genes, atrial natriuretic factor and beta-myosin heavy chain, were not up-regulated in E33A-treated cells. Myotrophin-induced myocyte growth and initiation of hypertrophy thus require nuclear co-translocation of myotrophin and p65, in a manner that depends crucially on the myotrophin hairpin loops.     

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Membrane localization,Caveolin-3 association and rapid actions of vitamin D receptor in cardiac myocytes

The active form of vitamin D, 1alpha, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), mediates both genomic and rapid non-genomic actions in heart cells. We have previously shown that the vitamin D receptor (VDR) is located in the t-tubular structure of cardiomyocytes. Here we show that VDR specifically interacts with Caveolin-3 in the t-tubules and sarcolemma of adult rat cardiac myocytes. Co-immunoprecipitation studies using VDR antibodies revealed that Caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with Caveolin-3 antibody. Confocal immuno-fluorescence microscopy analysis also showed co-localization of VDR and Caveolin-3 in t-tubules and sarcolemma. The non-genomic effects of the functional VDR were studied in electrically stimulated myocytes isolated from adult rat hearts. Sarcomere shortening and re-lengthening were measured in 1,25(OH)₂D₃ treated cardiac myocytes. A 1 nM treatment decreased peak shortening within minutes, suggesting a rapid effect through the membrane-bound VDR. This novel finding of the interaction between VDR and Caveolin-3 is fundamentally important in understanding 1,25(OH)₂D₃ signal transduction in heart cells and provides further evidence that VDR plays a role in regulation of heart structure and function.     

Myotrophin-kappaB DNA interaction in the initiation process of cardiac hypertrophy

To investigate how cardiac hypertrophy and heart failure develop, we isolated and characterized a candidate initiator, the soluble 12-kDa protein myotrophin, from rat and human hearts. Myotrophin stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes. Recombinant myotrophin from the cloned gene showed structural/functional motifs, including ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and casein kinase II. One repeat, homologous with I kappaB, interacts with rel/NF-kappaB in vitro. We analyzed the interaction of recombinant myotrophin and nuclear extracts prepared from neonatal and adult cardiomyocytes; gel mobility shift assay showed that myotrophin bound to kappaB DNA. To define PKC's role in myotrophin-induced myocyte growth, we incubated neonatal rat myocytes (normal and stretch) with specific inhibitors and found that myotrophin inhibits [3H]leucine incorporation into myocytes and different hypertrophic gene expression in neonatal myocytes. Using confocal microscopy, we observed that a basal level of myotrophin was present in both cytoplasm and nucleus under normal conditions, but under cyclic stretch, myotrophin levels became elevated in the nucleus. Myotrophin gene levels were upregulated when myocytes underwent cyclic stretch or were treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta and also when excised beating hearts were exposed to high pressure. Our data showed that the myotrophin-kappaB interaction was increased with age in spontaneously hypertensive rats (SHRs) only. Our data provide evidence that myotrophin-kappaB DNA interaction may be an important step in initiating cardiac hypertrophy.     

Impact of the Vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) interacts with the Vitamin D₃ receptor (VDR) to modulate proliferation and apoptosis in a variety of cell types, including breast cancer cells. In this review, we discuss three issues related to the role of the VDR in growth control: first, whether mammary glands lacking VDR exhibit abnormal growth; second, whether the VDR is essential for induction of apoptosis by 1,25(OH)₂D₃; and third, whether VDR up-regulation can sensitize cells to 1,25(OH)₂D₃. Studies from our laboratory have demonstrated that mammary glands from VDR knockout (VDR KO) mice exhibit accelerated growth and branching during puberty, pregnancy and lactation as compared to wild-type (WT) mice. In addition, involution after weaning, a process driven by epithelial cell apoptosis, proceeds at a slower rate in VDR KO mice compared to WT mice. Using cells isolated from VDR KO and WT mice, we report that both normal and transformed mammary cells derived from WT mice are growth inhibited by 1,25(OH)₂D₃, however, cells derived from VDR KO mice are completely unresponsive to 1,25(OH)₂D₃. In human breast cancer cells, we have identified a variety of agents, including steroid hormones, phytoestrogens and growth factors, that up-regulate VDR expression and enhance sensitivity to 1,25(OH)₂D₃-mediated growth inhibition. Collectively, these studies support a role for 1,25(OH)₂D₃ and the VDR in negative growth regulation of both normal mammary gland and breast cancer cells.