Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal

We measured the serum concentration of 25-hydroxyvitamin D₃ (25-OH-D₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂-D₃) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)₂-D₃ was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)₂-D₃ is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)₂-D₃ in target tissues for the hormone.     

Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

The structural requirements for the interaction of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with an anti-1,25(OH)₂D₃ antiserum and with the natural cytosolic receptor for 1,25(OH)₂D₃ isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)₂D₃-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)₂D₃ were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)₂D₃ is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)₂D₃ with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)₂D₃ decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)₂D₃, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K_D value of 1.7 × 10^?10m for the antiserum and 2.3 × 10^?10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)₂D₃ at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10⁷ M^?1 min^?1 for the antiserum and 0.55 × 10⁷ M^?1 min^?1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)₂D₃ to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10^?5 min^?1 versus 21.0 × 10^?5 min^?1).     

Evidence for multiple molecular weight forms of the chick intestinal 1,25-dihydroxyvitamin D3 receptor

Studies from many laboratories have reported apparent molecular weights for the chick intestinal 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] receptor varying from 47,000 to 67,000 daltons. We report here that in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.3 mM) and in the presence or absence of ligand, the apparent molecular weight of the receptor is 99,700 ± 9,400 (SD) daltons (as determined by gel filtration). In the absence of PMSF, however, the unoccupied receptor migrates with an apparent molecular weight of 51,400 ± 5,700 (SD) daltons. This smaller form of the 1,25(OH)₂D₃ receptor, upon incubation with [³H]-1,25(OH)₂D₃ in the presence of PMSF, then migrates with an apparent molecular weight of 95,900 ± 7,300 (SD) daltons. These results suggest the presence of heretofore unappreciated multiple molecular forms of the chick intestinal 1,25(OH)₂D₃ receptor.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Studies on calciferol metabolism: Metabolism of 25-hydroxy-Vitamin D3 by the chicken embryo

It is known that after birth of a vertebrate there is a requirement for the metabolism of Vitamin D₃ (cholecalciferol) to 1,25-(OH)₂-Vitamin D₃ to produce the hormonally active form essential for calcium homeostasis. However it is not known whether the enzymatic capability to produce 1,25-(OH)₂-D₃ only appears after birth or whether it is generated in the embryo. Presented in this paper are results of studies designed to measure the production and localization of 1,25-(OH)₂-D₃ in the embryo. It was found that the renal enzyme, 25-OH-cholecalciferol-1-hydroxylase, which is capable of producing 1,25-(OH)₂-D₃, is present as early as day 9 of incubation (12 days before hatch) in White Leghorn chicks. Further, the enzyme activity increases 6-fold to a maximal level which occurs on the day of hatching. 1,25-(OH)₂-D₃ was shown to be produced in vivo at day 17 and was found then in low levels in the embryonic intestine and kidney. Thus we have shown that 1,25-(OH)₂-D₃ is made by embryonic chick kidneys and is found in low levels in embryonic chick intestine and kidney significantly before hatch.     

Cytosol Preparations are Inadequate for Quantitating Unoccupied Receptors for 1,25-Dihydroxyvitamin D3

Abstract

Recent studies in this laboratory have indicated that 90% of the unoccupied receptors for 1,25-dihydroxyvitamin D₃ [1,25(OH)₂-D₃] are associated with nuclear components when chick intestinal mucosa is homogenized in low salt buffer (TED: 10 mM Tris, 1.5 mM EDTA, 1.0 mM dithiothreitol, pH 7.4). This observation suggested that previously reported cytosol 1,25(OH)₂D₃ receptors could result instead from salt extraction of nuclear receptors. The studies herein indicate that tissue 1,25(OH)₂D₃ receptor recovery is 30–50% lower in cytosol prepared from KTED (0.3 M KC1 + TED) or STKM (0.25 M sucrose, 50 mM Tris, 25 mM KC1, 5 mM MgCl₂, pH 7.4) than in TED-prepared chromatin. Thus tissue concentrations of unoccupied 1,25(OH)₂D₃ receptors can be closely estimated in TED-chromatin; full quantitation can be achieved by summing the number of receptors in TED-chromatin plus TED-cytosol. Incubation at different temperatures for varying times yielded maximal receptor recovery (6.1 pmol/g mucosal wet weight) at 4°C for 4–24 h or at 23° for 30 min. Scatchard analyses confirmed that only a single class of high affinity (K_d 0.4 nM) binding sites was present under all incubation conditions. Dithiothreitol significantly improved receptor recovery both in cytosol and in chromatin preparations. Conversely, inclusion of 20% glycerol caused an artificial increase in specific H-1,25(OH)₂D₃ binding due to a second class of chromatin binding sites with ten-fold higher K_d (8.1 nM) and a greater number of binding sites than the 1,25(OF)₂D₃ receptor. In conclusion, the TED-chromatin assay procedure provides better quantitation of the tissue content of unoccupied 1,25(OH)₂D₃ receptors than do previously described techniques. The presence of unoccupied nuclear-associated 1,25(OH)₂D₃ receptors in other target tissues emphasizes the potential for erroneous physiological conclusions if these chromatin-associated receptors are overlooked.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Induction of calcium-binding protein in organ-cultured chick intestine by fluoro analogs of vitamin D3

Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D₃ (D₃) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D₃) or in both the 1α and 25 positions (1α,25-F₂-D₃) were markedly more potent than vitamin D₃ itself although 1α,25-F₂-D₃ was only $\text{1}\text{7}\text{th}$ as potent as 1α-F-D₃. The 25-fluoro analog (25-F-D₃) was a very weak inducer; only $\text{1}\text{45}\text{th}$ as potent as vitamin D₃. The 25-fluoro analog of 1α-hydroxyvitamin D₃ (1α-OH-25-F-D₃) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24R-OH-25-F-D₃ was about 15 times more potent than 24R,25-(OH)₂-D₃. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F₂-25-OH-D₃ and 24,24-F₂-1α,25-(OH)₂-D₃ were about four times as potent as their nonfluorinated counterparts. The 24,24-F₂-1α,25-(OH)₂-D₃ is, therefore, the most potent vitamin D₃ analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D₃ metabolite, 1α,25-(OH)₂-D₃.     

The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding

The structural features of 1,25-dihydroxyvitamin D₃ that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D₃ molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D₂) did not alter the binding of 1,25-dihydroxyvitamin D₃, but significantly diminished the binding of 25-hydroxyvitamin D₃. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: Existence of a paracrine system for 1,25 dihydroxycholecalciferol

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecal-ciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)₂D₃ synthesis was studied by measuring the conversion of [³H] 25(OH)D₃ to [³H] 1,25(OH)₂D₃. The purified product was tested for its ability to compete with synthetic [³H] 1,25(OH)₂D₃ for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogeneous 1,25(OH)₂D₃ were evaluated by the increase in 25(OH)D₃-24 hydroxylase activity. Furthermore the expression of 1,25(OH)₂D₃ receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)₂D₃. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D₃-24 hydroxylase response to 1,25(OH)₂D₃ were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)₂D₃ and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)₂D₃ produced locally by neighboring immature ones. © 1994 wiley-Liss, Inc.     

Early effect of 25-hydroxycholecalciferol (25-OH-D3) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on the ability of parathyroid hormone (PTH) to elevate cyclic AMP of intact bone cells.

25-OH-D₃ and 1,25-(OH)₂-D₃ had no effects by themselves on the cyclic AMP levels of isolated bone cells but enhanced the stimulation seen following an exposure with submaximal concentrations of PTH for as little as 2 minutes. Preincubation with the 25-OH-D₃ or 1,25-(OH)₂-D₃ resulted in a time dependent decrease in the enhancement of PTH response over a 1 hr period. It is, therefore, suggested that cyclic AMP may be involved in some aspects of the action of vitamin D₃ derivatives on bone cells.     

The Establishment of a HeLa Cell Demonstrating Rapid Mitogen-Activated Protein Kinase Phosphorylation in Response to 1α,25-Dihydroxyvitamin D3 by Stable Transfection of a Chick Skeletal Muscle cDNA Library

1α,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] phosphorylates the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, within 30 sec in primary cultured chick skeletal muscle cells. MAPK of HeLa cell lines, which had been stably transfected with a cDNA library derived from mRNA of chick skeletal muscle cells, was also rapidly phosphorylated by 1,25-(OH)₂D₃. These cell lines have the potential to be a good tool for further investigation of rapid non-genomic mechanism activated by 1,25-(OH)₂D₃.     

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).     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D₃, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D₃], and that 1,25(OH)₂D₃ endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)₂D₃ is produced from 25(OH)D₃ by skin keratinocytes, 25(OH)[³H]D₃ was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)₂D₃ was 1,25(OH)₂D₃. Most of the 1,25(OH)₂D₃ endogenously produced from 25(OH)D₃ remained within the cells. To examine the time course of 1,25(OH)₂D₃ production, the amount of 1,25(OH)[³H]D₃ was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D₃. These data indicate that keratinocytes rapidly convert 25(OH)D₃ to 1,25(OH)₂D₃ and that 1,25(OH)₂D₃ is not released into the medium. To determine whether endogenously produced 1,25(OH)₂D₃ is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)₂D₃ and 25(OH)D₃ on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10^?8 M and 10^?7 M 1,25(OH)₂D₃ and to 80.5% and 23.9% by 10^?8 M and 10^?7 M 25(OH)D₃, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10^?7 M 1,25(OH)₂D₃, and to 11.8% by 2.5·10^?7 M 25(OH)D₃. These results clearly indicate that 25(OH)D₃ is as effective on human keratinocytes as 1,25(OH)₂D₃ in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)₂D₃. The possibility that the effect of 25(OH)D₃ is mediated through binding to the 1,25(OH)₂D₃ receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D₃ for the 1,25(OH)₂D₃ receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)₂D₃. Thus, these results suggest that 1,25(OH)₂D₃ endogenously produced in keratinocytes from 25(OH)D₃ is involved in the regulation of their growth and differentiation in vitro.     

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

The water soluble calcinogenic factor present in the plant $\text{Solanum}$$\text{malacoxylon}$ is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, $\text{Charonia}$$\text{lampus}$. Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH)₂D₃ for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH)₂D₃. The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH)₂D₃ and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH)₂D₃ hormone. We therefore conclude that the vitamin D-like principle in $\text{Solanum}$$\text{malacoxylon}$ is a sterol-glycoside which contains the 1,25-(OH)₂D₃ molecule as its active sterol component.