Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3

Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.     

A-ring analogs of 1,25-dihydroxyvitamin D(3)

The growing interest in1α,25(OH)(2)D(3), the hormonally active form of vitamin D(3), has prompted numerous efforts to synthesize vitamin D analogs as potential therapeutic agents, and some of these are already on the market and in clinical development. Although most vitamin D preparations developed thus far have focused on side-chain modifications, providing many useful analogues with high potency and selectivity, in recent years, modifications of the A-ring has attracted much attention because it can afford useful analogues exhibiting unique activity profiles as well. In this review we will focus on the current understanding of the relationship between selected modifications in the A-ring of the 1α,25(OH)(2)D(3) molecule, such as epimerization and/or substitution at C-1 and C-3, substitution at C-2, and removal of the 10,19-exocyclic methylene group, and their effect on biological potency and selectivity. Finally, suggestions for the structure-based design of therapeutically valuable A-ring vitamin D analogs will conclude the review.     

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.     

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.     

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions

Summary 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)₂-D₃ using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)₂-D₃. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)₂-D₃. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)₂-D₃. The antiproliferative effect of 1,25-(OH)₂-D₃ was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)₂-D₃ is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)₂-D₃ to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)₂-D₃, but can be used to assay other agents that modulate keratinocyte proliferation. Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology (J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342; 1988).     

Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system

In addition to its role in calcium and skeletal homeostasis, there is increasing evidence that the hormonal form of vitamin D, 1, 25-dihydroxyvitamin D(3), appears to serve as a modulator of the immune system. We have determined the level of the 1, 25-dihydroxyvitamin D(3) receptor (VDR) in resting and activated lymphocytes by immuno- and ligand-binding assays. As expected from previous work, the total T lymphocyte population contains VDR whose levels are increased when activated and treated with 1, 25-dihydroxyvitamin D(3). Surprisingly, the highest concentrations of VDR are found in CD8 lymphocytes, although significant amounts are also present in CD4 lymphocytes. Furthermore, B lymphocytes do not contain detectable amounts of VDR. Cells of the monocyte/macrophage lineage possess small amounts of VDR that are not affected by activation but are increased by treatment with 1, 25-dihydroxyvitamin D(3). These results suggest that CD8 lymphocytes may be a major site of 1,25-dihydroxyvitamin D(3) action, while B lymphocytes are likely not directly regulated by 1, 25-dihydroxyvitamin D(3).     

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.     

Molecular pathways involved in the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 in primary human keratinocytes

We previously reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against ultraviolet (UV)B-induced apoptosis. Here, we confirmed the anti-apoptotic effect of 1,25(OH)2D3 in keratinocytes, using cisplatin and doxorubicin as apoptotic triggers. We further showed that 1,25(OH)2D3 activates two survival pathways in keratinocytes: the MEK/extracellular signal regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. Activation of ERK and Akt by 1,25(OH)2D3 was transient, required a minimal dose of 10(-9) M and could be blocked by actinomycin D and cycloheximide. Moreover, inhibition of Akt or ERK activity with respectively a PI-3K inhibitor (LY294002) or MEK inhibitors (PD98059, UO126), partially or totally suppressed the anti-apoptotic capacity of 1,25(OH)2D3. Finally, 1,25(OH)2D3 changed the expression of different apoptosis regulators belonging to the Bcl-2 family. Indeed, 1,25(OH)2D3 treatment increased levels of the anti-apoptotic protein Bcl-2 and decreased levels of the pro-apoptotic proteins Bax and Bad in a time- and dose-dependent way. Induction of Bcl-2 by 1,25(OH)2D3 was further shown to be mediated by ERK and, to a lesser extent, by Akt. In conclusion, 1,25(OH)2D3 clearly protects keratinocytes against apoptosis (1) by activating the MEK/ERK and the PI-3K/Akt survival pathways and (2) by increasing the Bcl-2 to Bax and Bad ratio.     

The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents

The active form of vitamin D, 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)], is a secosteroid hormone that regulates calcium and bone metabolism, controls cell proliferation and differentiation, and exerts immunoregulatory activities. This range of functions has been exploited clinically to treat a variety of conditions, from secondary hyperparathyroidism to osteoporosis, to autoimmune diseases such as psoriasis. Recent advances in understanding 1,25(OH)(2)D(3) functions and novel insights into the mechanisms of its immunomodulatory properties suggest a wider applicability of this hormone in the treatment of autoimmune diseases and allograft rejection.     

Regulation of the procine 1,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 and parathyroid hormone in AOK-B50 cells

The 24-hydroxylase is the enzyme responsible for the first step in the catabolism of 1,25-dihydroxyvitamin D3, the active form of vitamin D. This enzyme was shown to be upregulated by 1,25-dihydroxyvitamin D3 itself and downregulated by parathyroid hormone (PTH). Upregulation of 24-hydroxylase by 1,25-dihydroxyvitamin D3 has been characterized; however, the mechanism by which PTH acts to downregulate 24-hydroxylase expression remains unknown. Here we report the cloning of the porcine 24-hydroxylase, and show that 1,25-dihydroxyvitamin D3-stimulated 24-hydroxylase mRNA and activity are repressed by PTH in AOK-B50 cells, a porcine kidney proximal tubule cell line with stably transfected opossum PTH receptors. Forskolin mimicked the effects of PTH consistent with in vivo data, and suppression by PTH was not due to changes in VDR levels. The first 1400 bp of the 24-hydroxylase promoter were not able to mediate the effects of PTH on a reporter gene. In view of the above findings we concluded that AOK-B50 cells are a suitable model for further studying the mechanism of action of PTH on 24-hydroxylase mRNA.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

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.     

The MRNA expression of the human 1,25-dihydroxyvitamin D3 receptor and the c-myc protooncogene in cultured human keratinocytes

Summary The human vitamin D receptor mRNA expression in preconfluent human cultured keratinocytes was upregulated by treatment of these cells with 10^−8 M 1,25(OH)₂D₃ for 24 hours. Additionally, human c-myc mRNA expression was decreased in a dose dependent manner by 1,25(OH)₂D₃ in both preconfluent and confluent cultured human keratinocytes.     

Immunomodulatory role of 1,25-dihydroxyvitamin D3.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25-D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. At a molecular level, 1,25-D3 inhibits the accumulation of mRNA for IL-2, IFN-gamma, and GM-CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th-clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone. When given in vivo, 1,25-D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25-D3-receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.