Prostaglandins mediate the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes in a metabolite-specific and cell maturation-dependent manner.

Prior studies have shown that 1,25-(OH)2D3 stimulates alkaline phosphatase, phospholipase A2 (PLA2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E2 (PGE2) in growth zone chondrocytes. In contrast, 24,25-(OH)2D3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA2-specific activity and PGE2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH)2D3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH)2D3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH)2D3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH)2D3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA2 by either 1,25-(OH)2D3 or 24,25-(OH)2D3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE2 production. The rapid increase in 1,25-(OH)2D3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH)2D3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH)2D3-dependent increase in PGE2 production in growth zone cells and the 24,25-(OH)2D3-dependent decrease in PGE2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapid effects of 1,25-(OH)2D3 on growth zone cells, and contribute to the effects of 24,25-(OH)2D3 on resting zone cells; in both instances, the vitamin D metabolites exert their effects on PKC through changes in arachidonic acid via the action of PLA2. In addition, PKC by itself may mediate the production of PGE2.     

Effects of vitamin D metabolites on collagen production and cell proliferation of growth zone and resting zone cartilage cells in vitro

Previous studies have suggested that vitamin D metabolites directly influence the differentiation and maturation of chondrocytes in calcifying cartilage. Recently, this laboratory has shown that the response of chondrocyte plasma membrane and matrix vesicle enzymes to 1,25-(OH)2D3 and 24,25-(OH)2D3 is both cell and membrane specific. The current study demonstrates that cell replication and matrix protein synthesis are also modulated by vitamin D. Confluent, third-passage growth zone (GC) and resting zone (RC) costochondral chondrocytes were incubated in medium containing 10(-13)-10(-7) M 1,25-(OH)2D3 or 10(-12)-10(-6) M 24,25-(OH)2D3. The amount of collagenase-digestible protein (CDP) secreted into the media was inversely proportional to the concentration of fetal bovine serum (FBS). At 10% FBS, greater than 80% of the CDP was incorporated into the matrix. 1,25-(OH)2D3 stimulated CDP and percentage collagen synthesis by GC cells but had no effect on the synthesis of noncollagenous protein (NCP). 1,25-(OH)2D3 inhibited CDP and percentage collagen synthesis by RC cells but did not alter NCP synthesis. [3H]thymidine incorporation was inhibited in both cell types, whether confluent or subconfluent cultures were examined. At 10(-6) and 10(-7) M 24,25-(OH)2D3, there was a significant decrease in CDP production and percentage collagen synthesis by RC cells but no effect on NCP. However, at 10(-9) and 10(-10) M hormone there was an increase in NCP production but no effect on CDP, resulting in a decrease in percentage collagen synthesis. CDP and NCP production were unaffected by 24,25-(OH)2D3 in GC cells. High concentrations of hormone inhibited [3H]thymidine incorporation in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

1a,25-(OH)2D3 Regulates 25-Hydroxyvitamin D3 24R-Hydroxylase Activity in Growth Zone Costochondral Growth Plate Chondrocytes via Protein Kinase C

Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1alpha- and 24R-hydroxylase activities and metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1alpha,25-(OH)2D3 exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10(-9) - 10(-7) M 1alpha,25-(OH)2D3 or 24R,25-(OH)2D, for 24 hours, and 1alpha- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10(-10) - l0(-8) M 1alpha,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1alpha,25-(OH)2D3 (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)2D3-dependent effect on 24,25-(OH)2D3 production was determined by comparing the response to 1alpha,25-(OH)2D3 to the response to 1beta,25-(OH)2D3. 1alpha,25-(OH)2D3 increased 24R-hydroxylase specific activity in a dose-dependent manner, 24,25-(OH)2D3 production by intact cells was also increased. The effect of 1alpha,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1alpha,25-(OH)2D3 caused an increase; 1beta,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours. 1alpha-hydroxylase activity was unaffected by either metabolite at 24 hours. 1alpha,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1alpha,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1alpha,25(OH)2D3-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.     

The influence of vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (chondrocalcin)

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.     

Regulation of 25-hydroxyvitamin D3 metabolism in cultures of osteoblastic cells

This study was designed to investigate the mechanisms involved in the regulation of the conversion of 25-hydroxyvitamin D3 (25-OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] in primary cultures of osteoblastlike cells from neonatal mouse calvariae. These cells, when incubated with tritiated 25-OHD3 ([3H]25-OHD3), spontaneously synthesized [3H]24,25-(OH)2D3 20-50 times more efficiently than [3H]1,25-(OH)2D3 at a rate of conversion that was substrate dependent and linear from 1 to 36 h. Gas chromatography-mass spectrometry verified the identity of the dihydroxylated metabolites. The calcium ionophore A23187 (5 microM) consistently stimulated the synthesis of 1,25-(OH)2D3 while suppressing the production of 24,25-(OH)2D3. This effect was sustained for 36 h and was dose dependent for concentrations from 0.05 to 10 microM. Furthermore, A23187 stimulated cAMP production and indomethacin (50 ng/ml) blocked the A23187-induced production of cAMP and 1,25-(OH)2D3 but had no effect on the suppression of 24,25-(OH)2D3 by A23187. This led to other experiments to find out whether the stimulative effect of A23187 on 1,25-(OH)2D3 synthesis is mediated by prostaglandins or cAMP, or both. PGE2 (10(-8)-10(-6) M) increased the production of 1,25-(OH)2D3 and of 24,25-(OH)2D3. Forskolin (0.01-10 microM) and dibutyryl cAMP (0.1-10 mM) increased the production of both metabolites but to a lesser degree than PGE2. These data suggest that osteoblastlike cells are stimulated by A23187 to increase the synthesis of 1,25-(OH)2D3 through mechanisms involving prostaglandins and cAMP. The synthesis of 24,25-(OH)2D3 is suppressed by A23187 through different mechanisms.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Effect of 1,25-dihydroxyvitamin D3 on prostaglandin E2 production in cultured mouse parietal bones

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was tested for its effects on prostaglandin E2 (PGE2) production and bone resorption in cultured mouse parietal bones. We found that at 24 h 1,25-(OH)2D3 increased 45Ca release but did not affect PGE2 production. However, at 48 h 1,25-(OH)2D3 produced a dose-related increase in PGE2 production. PGE2 production was increased with 1,25-(OH)2D3 at 10(-10)-10(-8) M, and 45Ca release was increased with 1,25-(OH)2D3 at 10(-11)-10(-8) M. The effects of 1,25-(OH)2D3 on PGE2 production persisted in the presence of cortisol (10(-8) M), and the effects were greater in the presence of arachidonic acid (10(-5) M) or fetal bovine serum (10%). Human interleukin-1 alpha (IL-1, 1 ng/ml) and bovine parathyroid hormone-(1-34) (PTH, 10 ng/ml) increased PGE2 production earlier and to a greater extent than 1,25-(OH)2D3. The PGE2 response to IL-1 and PTH was not affected by 1,25-(OH)2D3 at 24 h, but at 48 h 1,25-(OH)2D3 (10(-8) M) increased the PGE2 response to both IL-1 and PTH. The stimulation of 45Ca release at 48 h by high concentrations of 1,25-(OH)2D3, PTH, or IL-1 was similar, and there was no evidence for an additive effect. To test for an effect of 1,25-(OH)2D3 on endogenous IL-1 production, experiments were performed in the presence of an IL-1 receptor antagonist (IL-1Ra, 1000 ng/ml), which has been found to block selectively IL-1 effects on bone resorption and PG production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D Metabolites Regulate Matrix Vesicle Metalloproteinase Content in a Cell Maturation-Dependent Manner

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcificationin vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-\. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1,25(OH)₂D₃ and 24,25 (OH)₂D₃. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10¹⁰-10^-7 M24,25(OH)₂D₃ or growth zone chondrocytes incubated with 10^-11-l0^-8 M 1,25(OH)₂D₃, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)₂D₃ regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A₂ specific activities as well as metalloprotemases which degrade proteoglycans.     

Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3.

We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1, 25(OH)2D3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3-mVDR). Fourth-passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone (RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1, 25(OH)2D3 with <0.1% affinity for the nVDR (2a, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-20-epi-22-oxa-25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in GC MVs. Like 1,25(OH)2D3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1, 25(OH)2D3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24, 25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24, 25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24, 25(OH)2D3 was also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose-dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1, 25(OH)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a-dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24, 25(OH)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation-dependent.     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

A direct effect of 24,25-(OH)2D3 and 1,25-(OH)2D3 on the modeling of fetal mice long bones in vitro

To examine the effects of 24,25-(OH)2D3 and 1,25-(OH)2D3 on fetal long bone modeling the radii and ulnae of 16 day fetal mice were grown in vitro for 2 days. Their growth, mineralization, and resorption were assessed by measuring diaphyseal length, calcium and phosphorus content, hydroxyproline-protein ratios, and the release of incorporated 45Ca. The results showed that 24,25-(OH)2D3 at concentrations of 10(-10)-10(-8) M stimulated the growth of the bones as indicated by their increased diaphyseal length, periosteal bone area, and hydroxyproline content. Calcium and phosphorus content was significantly increased; 45Ca release was unaltered. Bones incubated in media containing 10(-6) M 24,25-(OH)2D3 responded in a similar fashion to bones incubated in media containing 10(-10)-10(-8) M 1,25-(OH)2D3, with inhibition of bone growth as indicated by reduced diaphyseal length, periosteal bone area, hydroxyproline-protein ratios, and calcium and phosphorus content; 45Ca release was significantly increased. Neither metabolite affected total bone length. The results suggest a role for 24,25-(OH)2D3 in the growth of fetal mice bones in vitro and also confirm the findings from previous studies that 1,25-(OH)2D3 and high concentrations of 24,25-(OH)2D2 stimulate bone resorption.     

Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3.

In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 microm; SLA, 4.1 microm; and TPS, 4.9 microm. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-beta1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-beta1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-beta1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-beta1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-beta1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.     

Diversity in peritoneal macrophage response of CAPD patients to 1,25-dihydroxyvitamin D3

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. The effect of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] on the activity of peritoneal macrophages from CAPD patients and nonuremic controls was studied. 1,25(OH)2D3 had a biphasic effect on superoxide generation in the concentration range of 2.5 10(-9) M to 5 x 10(-6) M with a peak at 2 x 10(-8) M. The addition of 2 x 10(-8) M 1,25(OH)2D3 to nonuremic control macrophages for 24 hours caused a significant twofold increase in superoxide generation in response to phorbol myristate acetate (PMA), from 2.21 + 0.2 to 4.1 + 0.2 nmol/10(6) mac (P less than 0.001), and enhanced the bactericidal activity from 60 + 7% to 85 + 9% (P less than 0.005). CAPD patients were divided into two groups: Group A, patients with high peritonitis incidence (HPI); group B, patients with low peritonitis incidence (LPI). Macrophages from HPI patients show a lower bactericidal activity (37 +/- 5%) and were not affected by 1,25(OH)2D3 after 24 hours of treatment. The increase in macrophage activity was seen only after three days of incubation with the hormone. Macrophages from this group generated a high amount of prostaglandin E2 (PGE2) during the first 24 hours in culture (7.8 +/- 0.52 ng/ml as compared with 0.35 +/- 0.03 ng/ml in the controls).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures.

Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10(-6)-10(-9) M) and PGE1 (10(-6)-10(-7) M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption was increased (10(-7) M PGE2 and 10(-6) M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: regulation by 1,25-dihydroxycholecalciferol

To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD3, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on 3H-25OHD3 were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of 3H-25OHD3, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)2D3 (50 ng/day i.p. for 7 days) increased mean serum 3H-24,25-dihydroxycholecalciferol [3H-24,25(OH)2D3] from 2 +/- 2-210 +/- 80 fmol/ml (mean +/- 1 SD), increased mean serum 3H-25,26-dihydroxycholecalciferol [3H-25,26(OH)2D3] from 2 +/- 2-12 +/- 6 fmol/ml and lowered mean serum 3H-1,25(OH)2D3 from 210 +/- 40-4 +/- 4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)2D3 increased mean serum 3H-24,25-(OH)2D3 from 6 +/- 11-115 +/- 30 fmol/ml and increased mean serum 3H-25,26(OH)2D3 from 3 +/- 3-26 +/- 10 fmol/ml. Nephrectomy increased serum 3H-25(OH)D3 in untreated (from 1450 +/- 225-2675 +/- 225 fmol/ml serum) and 1,25(OH)2D3 treated rats (from 1600 +/- 175-3075 +/- 100 fmol/ml). 3H-1,25(OH)2D3 averaged 74 +/- 16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)2D3 production and for most 25,26(OH)2D3 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 1,25-dihydroxyvitamin D3 on mouse mammary tumor (GR) cells: evidence for receptors, cellular uptake, inhibition of growth and alteration in morphology at physiologic concentrations of hormone

Mammary glands are target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have examined a mouse mammary tumor cell line (GR) for receptors of 1,25(OH)2D3 and have examined alterations in the growth and morphology of these cells in response to 1,25(OH)2D3. GR cells contain a high affinity (Kd approximately 10(-11)), low-capacity receptor with a high specificity for 1,25(OH)2D3. The 1,25(OH)2D3 receptor in GR cells has a sedimentation coefficient of 3.5 and elutes from DEAE cellulose columns with approximately 0.15 M KCl. These properties of the receptor are similar to those reported for other 1,25(OH)2D3 receptors. 1,25(OH)2D3 is internalized by GR cells in situ and specifically bound 1,25(OH)2D3 is found predominantly, if not entirely, in the nucleus as determined by cell fractionation and autoradiographic techniques. The incubation of GR cells in culture for 7 days with 1,25(OH)2D3 markedly alters cell growth. Cell growth is retarded in a dose-dependent manner; physiologic concentrations (10(-10) M) of 1,25(OH)2D3 retard cell growth by approximately 50%. In addition, GR cells incubated with 10(-9) to 10(-8) M 1,25(OH)2D3 undergo marked morphological changes. The incubation of GR cells with other vitamin D metabolites such as 25-hydroxyvitamin D3 (25(OH)D3) at a concentration of 10(-9) M does not significantly alter cell growth or morphology. The presence of high affinity receptors for 1,25(OH)2D3, the specific internalization of 1,25(OH)2D3 predominantly into the nuclei, and the significant effects of physiological concentrations of 1,25(OH)2D3 on cell growth suggest a direct, specific, nuclear effect of 1,25(OH)2D3 on GR cells. The mouse mammary tumor model might be useful in examining the effect of 1,25(OH)2D3 on tumor formation.     

cAMP promotion of osteoclast-like cell development from mouse bone marrow cells requires a permissive action of 1,25-(OH)2D3.

Recruitment of osteoclasts from monocytic precursors is modulated by local signals. We previously showed that monoblastic differentiation in U937 cells is stimulated by 1,25-(OH)2D3 and cAMP in series. We investigate here the combined effects of these agents to stimulate differentiation of osteoclast-like cells from mouse marrow. Cells from mouse marrow were harvested and cultured in alpha-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase-containing multinuclear cells was measured after 8 days in culture by cytochemical staining. Continuous exposure of cultures to 10 nM 1,25-(OH)2D3 positively stimulated development of these cells after 8 days (101 +/- 3 cells per well, n = 74). No osteoclast-like cells were found when 1,25-(OH)2D3 was added for the first 4 days followed by 4 days more with no treatment. PGE2 (1 microM) as a single agent added during the last 4 days of culture was not able to recruit osteoclast-like cells. However, cultures exposed to 1,25-(OH)2D3 during the first 4 days and 1 microM PGE2 during the second 4 days developed osteoclast-like cells at 8 days [66 +/- 8% of the formation seen with 1,25-(OH)2D3 alone, p less than 0.05]. Dibutyryl cAMP (1 microM to 3 mM) was also not effective used as a single agent, but was able to stimulate formation of TRAP-positive multinuclear cells when 1,25-(OH)2D3 preceded its addition to culture medium. cAMP analogs therefore mimicked the effect of 1 microM PGE2, but these experiments do not allow us to assign the PGE2 action entirely to activation of cAMP second messenger.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that stimulation of 1,25(OH)2D3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis

When primary culture of C75BL6 mouse cortical kidney cells in serum-free medium were incubated with unlabeled 25(OH)D3, they produced a metabolite which co-migrated with authentic 1,25(OH)2D3 and which could be measured by competitive receptor assay. A metabolite co-migrating with authentic 10-oxo-19-nor-25-OH-D3 was also produced. However, when cultures were incubated with 25(OH)D3 for 1 hour or longer, 10-oxo-19-nor-25-OH-D accounted for less than 15% of the total 3H-1,25(OH)2D3 displacement activity. Production of 1,25(OH)2D3 increased with increasing content of the culture, with time of incubation, and with substrate concentration. The apparent Km was 1.4 +/- 0.6 microM and Vmax 2.6 +/- 0.4 pM/mg protein/hr. These cultures possessed a very high level of phosphodiesterase activity, as indicated by their high cyclic AMP (cAMP) response to IBMX. This high phosphodiesterase activity may have been responsible for the lack of stimulation of 1,25(OH)2D3 production by physiologic or near physiologic concentrations of parathyroid hormone (PTH) in the absence of IBMX. However, when IBMX 10(-6) M was present, bPTH 10(-9) M significantly increased production of both cAMP and 1,25(OH)2D3. There was a close correlation between 1,25(OH)2D3 production and cAMP content of the cultures (basal or stimulated). An incubation time of at least 4 hours was required for cAMP to increase 1,25(OH)2D3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH)2D3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect.(ABSTRACT TRUNCATED AT 250 WORDS)