Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

The effect of vitamin D analogs and of vitamin D-binding protein on lymphocyte proliferation

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.     

Effects of 1,25-dihydroxyvitamin D3 on cell-cycle kinetics of T 47D human breast cancer cells

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25-(OH)2D3, which inhibit cellular replication, on the cell-cycle kinetics of a 1,25-(OH)2D3-responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25-(OH)2D3 in the range 10(-9) M to 10(-6) M caused a time- and concentration-dependent decrease in cell numbers. Treatment of cells growing in charcoal-treated fetal calf serum with 10(-8) M 1,25-(OH)2D3 for 6 days reduced cell numbers to 49% +/- 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% +/- 0.5% (n = 11) to 19.6% +/- 2.3% (n = 9), significant by paired analysis (P less than 0.002). At higher concentrations of 1,25-(OH)2D3 (10(-7)-10(-6) M), there was a concentration-dependent decline in S phase and increases in both G0/G1 and G2 + M phase cells. Detailed analysis of the temporal changes in cell-cycle phase distribution following treatment with 2.5 X 10(-8) and 10(-7) M 1,25-(OH)2D3 showed an initial accumulation of cells in G0/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in G0/G1 phase and an increase in G2 + M. In contrast cells treated with 10(-7) M 1,25-(OH)2D3 had significantly elevated % G0/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from G0/G1 following 4 days of exposure to 10(-7) M 1,25-(OH)2D3. This accumulation of cells in G0/G1 was accompanied by a fall in % S phase cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

1,25-Dihydroxyvitamin D3 increases bone alkaline phosphatase isoenzyme levels in human osteogenic sarcoma cells

The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines, SAOS and TE85, after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3). Enzyme activity increased when the cells were incubated with concentrations of 1,25(OH)2D3 between 10(-9) and 10(-7) M and cell growth was not inhibited at these concentrations. The specific activity of alkaline phosphatase was 4- to 7-fold higher than that in the control cells after 5 to 7 days of continuous exposure to 1,25(OH)2D3. Immunochemical studies demonstrated that the enzyme from both control and 1,25(OH)2D3-treated cultures cross-reacted with antisera specific for the phosphatase isoenzyme produced by normal human bone, and did not cross-react with antisera specific for the placental alkaline phosphatase isoenzyme. The increased enzyme activity in cultures induced with 1,25(OH)2D3 correlated with an absolute increase in the number of bone-specific phosphatase molecules, as determined by radioimmunoassay. No effect on alkaline phosphatase activity was observed when the cells were treated with other vitamin D metabolites or with 5-bromo-2'-deoxyuridine. Comparative studies demonstrated that hydrocortisone, another steroid hormone, increased the phosphatase activity with a different time course than did 1,25(OH)2D3. High affinity cytoplasmic receptors for 1,25(OH)2D3 and hydrocortisone were found in the SAOS and TE85 cells.     

Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells

Dihydroxy-cholecalciferol [1,25(OH)2D3] has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC) based on species and culture conditions. We have examined the effects of 1,25(OH)2D3 on the differentiation of porcine MSC under culture conditions designed to promote proliferation in order to attempt to mimic the conditions in young, rapidly growing animals. The MSC were isolated from bone marrow of a young pig and grown in basal media (BM) containing DMEM+10% fetal bovine serum and antibiotics. Cells received either BM, BM+10(-8) M 1,25(OH)2D3 or BM+10(-7) M 1,25(OH)2D3 with complete media changes every 3 days for a total of 12 days of culture. On days 3, 6, 9 and 12, viable cell numbers were determined, and samples were collected for gene expression analysis and cytochemical staining. There was a treatment-based reduction in cell numbers on 6, 9 and 12 days (P<.05). The concentrations of mRNAs encoding peroxisome proliferator-activated receptor gamma, lipoprotein lipase, and adipocyte-binding protein 2 were increased (P<.05) in a manner indicative of adipocytic differentiation by treatment with 1,25(OH)2D3 in a dose-dependent manner. However, the mRNA levels of osteocalcin, a late stage marker of osteoblastic differentiation, was also increased (P<.05) by treatment with 1,25(OH)2D3. An increased percentage of lipid filling, based on Oil Red O staining, and decreased alkaline phosphatase activity, was also seen with 1,25(OH)2D3 treatment. These data suggest that 1,25(OH)(2)D(3) stimulates the differentiation of porcine MSC towards an adipocytic phenotype.     

Effects of 1,25-dihydroxyvitamin D3 and its analogs on butyrate-induced differentiation of HT-29 human colonic carcinoma cells and on the reversal of the differentiated phenotype

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) greatly enhances sodium butyrate (NaB)-induced enterocyte differentiation of HT-29 human colonic carcinoma cells while 1,25-(OH)2D3 alone induces growth restriction without associated differentiation. In the present study, the efficacies of various analogs of 1,25-(OH)2D3 to enhance NaB-induced HT-29 differentiation and to prolong the reversal of the differentiated phenotype under NaB-free growth conditions were subsequently examined. Extent of HT-29 differentiation was assessed by measurement of alkaline phosphatase (AP) activity, appearance of mucin-producing cells, changes in morphological characteristics, and expression of differentiation-associated cytokeratin proteins. Among active analogs of 1,25-(OH)2D3, 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 (F6-1,25-(OH)2D3), 24,24-difluoro-24-homo-1,25-(OH)2D3, and 26,27-dimethyl-1,25-(OH)2D3 were 100-, 10-, and 5-fold, respectively, more effective than 1,25-(OH)2D3 in enhancing NaB-induced mucin production. Combined use of NaB and F6-1,25-(OH)2D3 (10(-9) M) also induced HT-29 cells to form highly differentiated goblet-like enterocytes, and increased both cellular AP enzymatic activity and tissue-type cytokeratin content. This differentiated state was qualitatively more advanced than that achieved by a combination of NaB and 10(-7) M 1,25-(OH)2D3. NaB-mediated HT-29 differentiation (in short-term inductions) was found to be reversible following a return to NaB-free medium. HT-29 cells differentiated by combined use of NaB and 1,25-(OH)2D3 or its analogs exhibited a significant prolonged reversal time relative to cells differentiated with NaB alone. The most prominent effect was achieved using cells differentiated with NaB and 10(-9) M F6-1,25-(OH)2D3 which exhibited a 7-fold prolonged reversal time over colonocytes differentiated by NaB alone. Our data suggest that a combined use of NaB and 1,25-(OH)2D3 or its derivatives may provide a convenient in vitro model system to probe molecular events associated with steroid-target tissue interactions in a differentiating cell system as commonly occurs in vivo. Such an analysis might lend itself to design of a rational combination differentiation-based therapy for the clinical management of colon cancer.     

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)D3 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 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone

A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin. A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA. Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell. In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3. In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined. However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity. Incubation of viable cell suspensions with 25-hydroxy[26,27-3H]vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2[3H]D3 formation. Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively. The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ. In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity. Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity. Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.     

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.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae

The synthesis of osteocalcin, the major non-collagenous protein of adult bone, was examined in cultures of 21-day fetal rat calvariae. Osteocalcin was measured by a sensitive and specific radioimmunoassay. Osteocalcin concentration in unincubated calvariae was 14.5 +/- 0.5 ng/calvaria. After incubation, there was a continuous increase in bone and medium osteocalcin, and by 96 h the values were about 100% higher than in unincubated calvariae. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) at 10(-11) to 10(-8)M increased osteocalcin synthesis. The effect appeared as early as 6 h after treatment and was primarily observed in the culture medium, and 1,25-(OH)2D3 stimulated osteocalcin up to 9-fold by 96 h. Concomitant with the effect on osteocalcin synthesis, 1,25-(OH)2D3 inhibited collagen synthesis. Cycloheximide markedly decreased osteocalcin concentrations in control and 1,25-(OH)2D3-treated calvariae. The stimulatory effect on osteocalcin synthesis was specific to 1,25-(OH)2D3 since 24,25-dihydroxyvitamin D3, parathyroid hormone, epidermal growth factor, and prostaglandin E2 did not stimulate osteocalcin synthesis, and parathyroid hormone and epidermal growth factor opposed the 1,25-(OH)2D3 stimulatory effect. Insulin did not alter osteocalcin concentration by itself but enhanced the effect of 1,25-(OH)2D3. In conclusion, 1,25-(OH)2D3 stimulates osteocalcin synthesis in cultures of normal calvariae, but this effect is not shared by other hormones known to affect bone metabolism.     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.     

1,25(OH)2D3 and calcipotriol (MC903) have similar effects on the induction of osteoclast-like cell formation in human bone marrow cultures

MC903 is a novel analogue of 1,25(OH)2D3 which exhibits similar inhibitory effects on cell proliferation and like, 1,25(OH)2D3, stimulates synthesis of osteoblast specific proteins by osteoblast-like cells in vitro. It is less active than 1,25(OH)2D3 in causing hypercalcemia in vivo. Since 1,25(OH)2D3 is known to stimulate bone resorption and increase the number of osteoclasts in several systems (in vivo and in vitro) we examined the effects of MC903 on the formation of osteoclast-like cells in vitro. As reported previously 1,25(OH)2D3 promoted the formation of multinucleated cells with phenotypic and functional characteristics of osteoclasts from adult human bone-marrow cultures at concentrations between 10(-8)M to 10(-12)M. Higher doses consistently suppressed multinucleated cell formation to values seen in the absence of 1,25(OH)2D3. Cells cultured in the presence of MC903 or for three weeks consistently induced the formation of multinucleated cells at concentrations 10(-8)M to 10(-12)M. As seen with 1,25(OH)2D3, MC903 also inhibited multinucleated cell formation at very high concentrations (10(-6)M). In two separate experiments MC903 appeared to be more potent than 1,25(OH)2D3 at lower concentrations (10(-10)M - 10(-12)M). From this study we conclude that MC903 is at least as potent as 1,25(OH)2D3 in inducing the formation human osteoclast-like cells in vitro. The decreased ability of MC903 to induce hypercalcemia in vivo is not therefore a result of a less marked effect than 1,25(OH)2D3 on the regulation of osteoclast formation.     

Vitamin D metabolites stimulate phosphatidylcholine transfer to renal brush-border membranes

The phosphatidylcholine content of both the intestinal and renal brush-border membranes and ion transport are affected by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). To investigate the mechanism of this effect, liposomes were prepared containing self-quenching concentrations of fluorescent phospholipid derivatives. When these liposomes were incubated with rat renal brush-border membrane vesicles, an immediate increase in the relative fluorescence of N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylcholine (NBD-PC) was detected, indicating transfer of NBD-PC into a non-quenched membrane. Addition of 1,25(OH)2D3 to the liposomes produced a dose-dependent stimulation of NBD-PC transfer to the acceptor brush-border membrane vesicles. Peripheral fluorescence was visible when the brush-border membrane vesicles were viewed with a fluorescent microscope. Using brush-border membrane vesicles from kidneys of vitamin D-deficient animals, quantitation of lipid transfer revealed a 1,25(OH)2D3 (10(-7) M) stimulation of NBD-PC transfer from 1.38 +/- 0.27 to 2.07 +/- 0.26 micrograms/h, and of PC transfer, assessed by vesicle phosphatidylcholine content, from 49.7 +/- 12 to 57.3 +/- 12 micrograms/mg protein per h (P less than 0.05). There was no significant transfer of N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In the absence of hormone, the amount of NBD-PC transferred to brush-border membrane vesicles prepared from normal rats was significantly greater than that transferred to brush-border membrane vesicles prepared from vitamin D-deficient animals (2.12 +/- 0.02 vs. 1.39 +/- 0.27 micrograms of NBD-PC/h, P less than 0.05). Both physiologic and pharmacologic concentrations of 1,25(OH)2D3 stimulated NBD-PC transfer with maximum response at 10(-14) M (2.98 +/- 0.15 micrograms/h). 24,25-Dihydroxycholecalciferol and 25-hydroxycholecalciferol (25(OH)D3) also stimulated transfer, although dose-response curves were less effective than for 1,25(OH)2D3. Cortisol and vitamin D-3 did not stimulate transfer. 1,25(OH)2D3 did not stimulate NBD-PC transfer between liposome populations.     

1,25-Dihydroxyvitamin D(3) and 9-cis-retinoids are synergistic regulators of 24-hydroxylase activity in the rat and 1, 25-dihydroxyvitamin D(3) alters retinoic acid metabolism in vivo.

The RXR forms a heterodimer with the VDR to activate genes that are regulated by 1,25(OH)(2)D(3). In the absence of RXR's ligand, 9-cis-RA, RXR appears to be a silent partner to VDR. The effect of 9-cis-RA on VDR/RXR heterodimer formation and 1, 25(OH)(2)D(3)-mediated gene expression in vivo remains unclear. We examined the effect of exogenous 9-cis-RA or 9-cis-RA precursors, 9, 13-di-cis-RA and 9-cis-RCHO, on 1,25(OH)(2)D(3)-mediated induction rat renal 24-hydroxylase. The rats were treated as follows: (1) vehicle; (2) 1,25(OH)(2)D(3); (3) 1,25(OH)(2)D(3) + 9-cis-RA; (4) 1, 25(OH)(2)D(3) + 9,13-di-cis-RA; (5) 1,25(OH)(2)D(3) + 9-cis-RCHO; (6) 9-cis-RA; (7) 9,13-di-cis-RA; and (8) 9-cis-RCHO. 1, 25(OH)(2)D(3) was administered IP 18 h prior to sacrifice. The retinoids were administered every 4 h, starting 28 h prior to sacrifice. The last retinoid dose was administered 4 h prior to sacrifice. Treatment with 1,25(OH)(2)D(3) alone increased 24-hydroxylase from 35 +/- 6 (controls) to 258 +/- 44 pmol/min/g tissue. When 1,25(OH)(2)D(3) was administered with 9-cis-RA, 9, 13-di-cis-RA, or 9-cis-RCHO, 24-hydroxylases were 568 +/- 56, 524 +/- 56, and 463 +/- 62 pmol/min/g tissue, respectively. Furthermore, codosing of 1,25(OH)(2)D(3) and 9-cis-retinoids resulted in higher circulating concentrations of 9-cis-RA and 9,13-di-cis-RA when compared to rats dosed with 9-cis-retinoids alone. This was shown to be due to 1,25(OH)(2)D(3) increasing the half-life of 9,13-di-cis-RA by three to four times. These results show that 9-cis-RA can act synergistically with 1,25(OH)(2)D(3) in the regulation of 24-hydroxylase in vivo. Additionally, 1,25(OH)(2)D(3) regulates 9, 13-di-cis-RA metabolism in vivo.     

1,25-Dihydroxyvitamin D3 inhibits synthesis and enhances degradation of proteoglycans in osteoblastic cells

The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, on the metabolism of proteoglycans by an osteoblastic cell line MC3T3-E1 were studied. Cells metabolically labeled with [35S]sulfate and/or [3H]glucosamine synthesized large and small dermatan sulfate proteoglycans and heparan sulfate proteoglycan. The incorporation of [35S]sulfate into proteoglycans for 1 h was reduced by 1,25-(OH)2D3 in a dose-dependent manner with a maximum reduction of 40% obtained at 10(-8)M 1,25-(OH)2D3. This effect was observed for all the proteoglycans with the decrease for the large dermatan sulfate proteoglycan most prominent. Treatment with 1,25-(OH)2D3 did not influence the degree of sulfation nor the molecular size of the glycosaminoglycan chains. Thus, the change in the incorporation of [35S] sulfate reflects net change in the synthesis of proteoglycans. When cells were treated with beta-D-xyloside, 1,25-(OH)2D3 also inhibited net synthesis of dermatan sulfate glycosaminoglycan chains on this exogenous substrate suggesting that it decreases the capacity of the cells for glycosaminoglycan synthesis. The incorporation of [3H]glucosamine into hyaluronic acid was also inhibited up to 70% by 10(-8) M 1,25-(OH)2D3. Treatment with 24,25-dihydroxyvitamin D3 did not cause significant changes in the proteoglycan synthesis. Degradation of proteoglycans associated with the cell layer was enhanced by treatment with 1,25-(OH)2D3 at 10(-8) M. Proteoglycans exogenously added to the culture were also degraded with a cell-mediated process which was stimulated by treatment with 10(-8) M 1,25-(OH)2D3. These results demonstrate that 1,25-(OH)2D3 reduces the synthesis and stimulates the degradation of proteoglycans in osteoblastic cells in culture.