1 alpha,25-dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] at 10(-8)-10(-7) M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)2D3 increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)2D3 and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)2D3 is an active metabolite which may be involved in supporting cartilage growth.     

Effect of 1,25-dihydroxyvitamin D3 on cyclic AMP responses to hormones in clonal osteogenic sarcoma cells.

The effect of 1,25-dihydroxyvitamin D3 on adenylate cyclase responsiveness was studied in the clonal osteogenic sarcoma cell line, UMR 106-06, which responds to several bone active hormones. 1,25-dihydroxyvitamin D3 treatment had no consistent effect on basal formation of cyclic AMP in intact cells, but the responses to parathyroid hormone, isoproterenol, prostaglandin E2, salmon calcitonin and the plant diterpene, forskolin, were all attenuated, by up to 90%. The effect of 1,25-dihydroxyvitamin D3 was dose-dependent, with half-maximal effectiveness at 0.1 nM, and required 48 h treatment of cells before it became apparent. The relative potencies of other vitamin D3 compounds correlated closely with their relative affinities for the 1,25-dihydroxyvitamin D3 receptor and their biological activities in other systems. 1,25-dihydroxyvitamin D3 treatment had no effect on the kinetics of labelled calcitonin binding to UMR 106-06 cells. Furthermore, the fact that such a range of hormones was affected made a receptor mediated mechanism unlikely. Nucleotide stimulatory (Ns) unit activity was assayed after 1,25-dihydroxyvitamin D3 treatment and found to be unchanged. Islet activating protein, an inhibitor of nucleotide inhibitory unit (Ni) activity, failed to modify the 1,25-dihydroxyvitamin D3 effect. Thus the effect of 1,25-dihydroxyvitamin D3 appeared to be exerted beyond hormone receptor and nucleotide regulatory components of the adenylate cyclase complex. It is concluded that 1,25-dihydroxyvitamin D3 attenuates adenylate cyclase response to hormones by a direct or indirect action on the catalytic component of adenylate cyclase.     

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+-uptake by cultured vascular smooth muscle cells derived from rat aorta

We have recently shown the presence of receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 stimulates Ca-ATPase in vascular smooth muscle cells presumably via receptor mediated mechanism. These data suggest that the sterol may directly be involved in the regulation of cellular calcium homeostasis. To further define action of vitamin D in smooth muscle cells, we studied effect of the sterol on cellular uptake of calcium. 1,25-dihydroxyvitamin D3 stimulated 45Ca2+ uptake by cultured cells, A7r5, derived from fetal rat aorta, when the cells were incubated with the sterol for 18 hr. The effect was dose-dependent at 10(-10) to 10(-9) M, and three orders of magnitude higher concentration of 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 was needed to obtain similar effects. Furthermore, the effect of 1,25-dihydroxyvitamin D3 was abolished by cycloheximide (10(-5) M), a protein synthesis inhibitor. These data clearly suggest that 1,25-dihydroxyvitamin D3 may directly regulate cellular calcium homeostasis in vascular smooth muscle cells presumably via receptor mediated mechanism.     

The effect of vitamin D3 metabolites on normal and leukemic bone marrow cells in vitro

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.     

Sodium butyrate (SB) augments the effects of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) on neoplastic and osteoblastic phenotype in clonal rat osteosarcoma cells

1,25 Dihydroxyvitamin D3 suppressed colony formation in soft agar and increased alkaline phosphatase activity in clonal rat osteosarcoma cells. Sodium butyrate enhanced these effects of the hormone partly through a mechanism involving an alteration of nuclear binding of the hormone. It is suggested that 1,25 dihydroxyvitamin D3 in conjunction with sodium butyrate might be able to regulate differentiation and proliferation of neoplastic cells.     

Progesterone and 1,25-dihydroxyvitamin D₃ inhibit endometrial cancer cell growth by upregulating semaphorin 3B and semaphorin 3F

Class 3 semaphorins (SEMA), SEMA3B and SEMA3F, are secreted proteins that regulate angiogenesis, tumor growth, and metastasis by binding to their transmembrane receptor complex consisting of plexins and neuropilins (NP). Expression of SEMAs and their receptors was assessed in tissue microarrays by immunohistochemistry. SEMA3B, SEMA3F, and plexin A3 were expressed strongly in normal endometrial tissues, whereas grade-dependent decreases were found in endometrial carcinomas. No change was observed in the expression of plexin A1, NP1, and NP2 in normal versus endometrial cancer tissues. Endometrial cancer cells showed decreased expression of SEMA3B, SEMA3F, and plexin A3 compared with their normal counterparts. Treatment of cancer cells with progesterone (P4) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for a period of 72 hours induced a significant upregulation of SEMA3B and SEMA3F as well as inhibited growth of cancer cells by increasing caspase-3 activity. Cotreatment of cell lines with P4 or 1,25(OH)(2)D(3) and their respective antagonists confirmed the specificity of their actions. Transfection of siRNA-targeting SEMA3B and SEMA3F in endometrial cancer cells attenuated P4 or 1,25(OH)(2)D(3)-induced growth inhibition. Restoration of SEMA3B or SEMA3F expression in cancer cells caused growth inhibition, reduced soft agar colony formation, and cell invasiveness by inhibiting expression of matrix metalloproteinase-2 (MMP-2), MMP-9, integrin αvβ3, and proangiogenic genes and by upregulating antiangiogenic genes. Thus, we have identified two new P4 and 1,25(OH)(2)D(3)-regulated antitumor genes for endometrial cancer. These results suggest that the loss of SEMAs contribute to the malignant phenotype of endometrial cancer cells and that reexpression of SEMAs by ectopic expression or with anticancer agents P4 or 1,25(OH)(2)D(3) can be a promising therapeutic treatment against endometrial cancer.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

24-homologated 1,25-dihydroxyvitamin D3 compounds: separation of calcium and cell differentiation activities

A series of 24-homologated 1,25-dihydroxyvitamin D3 compounds have been chemically synthesized and studied with regard to their activity in inducing differentiation of human promyelocyte HL-60 cells to monocytes and in calcium mobilizing activity in vitamin D deficient rats. Homologation of 1,25-dihydroxyvitamin D3 or its delta 22 analogue by one or two carbons increases by 10-fold and three-carbon homologation reduces by half the activity in causing differentiation of HL-60. On the other hand, homologation causes a substantial decrease in in vivo calcium mobilization activity. The addition of each carbon at the 24-position decreases binding to the HL-60 receptor or rat intestinal receptor by 5-10-fold so that binding affinity of the trihomo compound for the receptors is 130 times less that of 1,25-dihydroxyvitamin D3. Thus, binding affinity for the receptor cannot account for the preferential activity of the 24-homologated compounds in inducing cell differentiation.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.     

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.     

1,25-Dihydroxyvitamin D receptors in an established bone cell line. Correlation with biochemical responses

A stable cell line derived from mouse bone (cell line MMB-1) has been used for studies of the cellular receptor for 1,25-dihydroxyvitamin D3 in osteoblasts. Previous studies have demonstrated that collagen synthesis in the MMB-1 cell line is specifically inhibited by 1,25-dihydroxyvitamin D3 as well as by other bone-regulating hormones. Incubation of cell homogenates with [3H]1,25-dihydroxyvitamin D3 indicated the presence of a specific receptor which was located primarily in the chromatin fraction. Optimum conditions for the receptor assay required the inclusion of 500 kallikrein-inactivating units of Trasylol/ml and 10 mM NaMoO4. Under these conditions the receptors were stable for 2 h at 23 degrees C and for 24 h at 4 degrees C. Cellular content of receptors was dependent upon the state of confluency of the cells: fully confluent cells contained minimal concentrations of receptors. In cultures of 70-80% confluency, the 1,25-dihydroxyvitamin D3 receptors demonstrated linear Scatchard plots with Kd = 0.4 nM. Peak receptor activity was found at 3.7 S in linear sucrose gradient fractions of cell homogenates. The synthesis of collagen by MMB-1 cells was inhibited by 1,25-dihydroxyvitamin D3 in direct proportion to the concentration of cellular receptors at varying levels of culture confluence. The data indicate that MMB-1 cells contain cytoplasmic/nuclear receptors for 1,25-dihydroxyvitamin D3 which are similar to the receptors found in other target tissues for this hormone and suggest that these receptors are mediators of the effects of 1,25-dihydroxyvitamin D3 on collagen synthesis.     

1,25-Dihydroxyvitamin D3 binds specifically to rat vascular smooth muscle cells and stimulates their proliferation in vitro

Cultured vascular smooth muscle cells (VSMC) derived from rat aorta were found to contain a specific receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Its Kd (5.0 x 10(-11) M) and capacity (22.9 fmol/mg of cytosol protein) for 1,25-(OH)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D3 metabolites [1,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than vitamin D3] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(OH)2D3 receptor in other tissues. 1,25-(OH)2D3 at concentrations of more than 10(-10) M caused dose-dependent enhancement of the proliferation of VSMC in DMEM with 10% FCS. 25-Hydroxyvitamin D3 stimulated the proliferation of VSMC only at its highest concentration tested (10(-6) M). These data show that 1,25-(OH)2D3 stimulates the proliferation of VSMC after its binding to a cytoplasmic receptor of the cells in vitro, and support the possibility that VSMC are target cells of the hormone.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.     

1α,25-Dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃]at 10⁻⁸-10⁻⁷ M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)₂D₃ increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)₂D₃ and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)₂D₃ is an active metabolite which may be involved in supporting cartilage growth.     

Regulation of the vitamin D receptor and cornifin beta expression in vaginal epithelium of the rats through vitamin D3

The aim of the present study was to determine the respective role of 1,25-dihydroxyvitamin D3 on vaginal epithelium and 1,25-dihydroxyvitamin D3 receptor expression in ovariectomized rats and vitamin D3 treated rats. Bilateral ovariectomies were performed in 20 mature, non-pregnant Wistar female rats. All the animals were divided into 2 groups consisting of 10 rats each. Group I served as control. In group II, animals were injected intramuscularly with vitamin D3 (50, 00 IU/kg). Two weeks after the injections, vaginas of animals in group I and group II were removed removed and processed for immunohistochemistry. Epithelial differentiation, 1,25-dihydroxyvitamin D3 receptor and cornifin beta expression were investigated in vaginal epithelium of control group (ovariectomized) and vitamin D3 treated rats. Vaginal epithelial cells from vitamin D3 treated animals changed into highly- stratified keratinizing layers. 1,25-dihydroxyvitamin D3 receptor and cornifin beta as a marker of squamous differentiation were present in ovariectomized rats treated with 1,25-dihydroxyvitamin D3. In contrast, cornifin beta and 1,25-dihydroxyvitamin D3 receptor were absent in all layers of vaginal epithelium in control group. We demonstrated for the first time that 1,25-dihydroxyvitamin D3 induced proliferation of vaginal epithelium consistent with the cornifin beta expression and 1,25-dihydroxyvitamin D3 up-regulated 1,25-dihydroxyvitamin D3 receptor expression in vaginal epithelium.     

Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.

1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.     

1,25-Dihydroxyvitamin D3 increases epidermal growth factor receptors and transforming growth factor beta-like activity in a bone-derived cell line

To investigate possible mechanisms through which 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) affects cell proliferation and differentiation, we have studied the effects of 1,25-(OH)2D3 on the binding and mitogenic activity of epidermal growth factor (EGF) in RCJ 1.20 cells, an established, non-tumorigenic cell line derived from 21-day-old fetal rat calvaria. 1,25-(OH)2D3 caused a dose- and time-dependent 2- to 3-fold increase in the number of receptors for EGF. The 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 metabolites of vitamin D3 were ineffective in eliciting changes in EGF binding. Saturation and Scatchard analyses indicated that an increase in available unoccupied high affinity EGF binding sites was responsible for the 1,25-(OH)2D3-induced EGF binding. In addition, 1,25-(OH)2D3 enhanced EGF-dependent growth of RCJ 1.20 cells in soft agar. The potentiation of EGF effects on RCJ 1.20 cell growth by 1,25-(OH)2D3 may be related to the 1,25-(OH)2D3 regulation of EGF binding. However, the induction of anchorage-independent growth by 1,25-(OH)2D3 appears to be due to the stimulation of transforming growth factor beta-like activity. These results provide a possible explanation for the mechanism whereby the effects of 1,25-(OH)2D3 on cell proliferation and bone metabolism may be mediated.