Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

24,25-(OH)(2)D(3) regulates cartilage and bone via autocrine and endocrine mechanisms

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH)(2)D(3) in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH)(2)D(3) and to determine how 24(R),25(OH)(2)D(3) elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH)(2)D(3). 24(R),25(OH)(2)D(3) binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH)(2)D(3), but not 24S,25-(OH)(2)D(3), causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH)(2)D(3) does not cause translocation of PKC to the plasma membrane, but activates existing PKCalpha. There is a rapid decrease in Ca(2+) efflux, and influx is stimulated. 24(R),25(OH)(2)D(3) also reduces arachidonic acid release by decreasing phospholipase A(2) (PLA(2)) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE(2) that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE(2) decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCalpha and PKCzeta and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH)(2)D(3), and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCzeta is directly affected by 24(R),25(OH)(2)D(3) in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH)(2)D(3) causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1alpha,25(OH)(2)D(3) and lose responsiveness to 24(R),25(OH)(2)D(3), a characteristic of more mature growth zone (GC) chondrocytes. 1alpha,25(OH)(2)D(3) elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH)(2)D(3). These studies indicate that 24(R),25(OH)(2)D(3) plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.     

Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3

Vitamin D metabolites 1alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) regulate endochondral ossification in a cell maturation-dependent manner via membrane-mediated mechanisms. 24R,25(OH)(2)D(3) stimulates PKC activity in chondrocytes from the growth plate resting zone, whereas 1alpha,25(OH)(2)D(3) stimulates PKC in growth zone chondrocytes. We used the rat costochondral growth plate cartilage cell model to study how these responses are differentially regulated. 1alpha,25(OH)(2)D(3) acts on PKC, MAP kinase, and downstream physiological responses via phosphatidylinositol-specific PLC-beta; 24R,25(OH)(2)D(3) acts via PLD. In both cases, diacylglycerol (DAG) is increased, activating PKC. Both cell types possess membrane and nuclear receptors for 1alpha,25(OH)(2)D(3), but the mechanisms that render the 1alpha,25(OH)(2)D(3) pathway silent in resting zone cells or the 24R,25(OH)(2)D(3) pathway silent in growth zone cells are unclear. PLA(2) is pivotal in this process. 1alpha,25(OH)(2)D(3) stimulates PLA(2) activity in growth zone cells and 24R,25(OH)(2)D(3) inhibits PLA(2) activity in resting zone cells. Both processes result in PKC activation. To understand how negative regulation of PLA(2) results in increased PKC activity in resting zone cells, we used PLA(2) activating peptide to stimulate PLA(2) activity and examined cell response. PLAP is not expressed in resting zone cells in vivo, supporting the hypothesis that PLA(2) activation is inhibitory to 24R,25(OH)(2)D(3) action in these cells.     

24,25-Dihydroxyvitamin D(3) and bone metabolism

The 1alpha-hydroxylated metabolite of 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), is the biologically most active metabolite of vitamin D. The 24-hydroxylated metabolites were generally considered as degradation products of a catabolic pathway finally leading to excretion of calcitroic acid. Studies with analogues fluorinated at the C-24 position did not indicate a physiological function for 24R,25(OH)(2)D(3). Nevertheless throughout the years various studies showed biologic effects of other metabolites than 1alpha,25(OH)(2)D(3). In particular the metabolite 24R,25(OH)(2)D(3) has been functionally analyzed, e.g. with respect to a role in normal chicken egg hatchability and effects on chondrocytes in the resting zone of cartilage. Numerous studies have shown the presence of the vitamin D receptor in bone cells and effects of 1alpha,25(OH)(2)D(3) on bone and bone cells. Also for 24R,25(OH)(2)D(3) studies have been performed focusing on effects on bone and bone cells. The purpose of this review is to summarize the data regarding 24R,25(OH)(2)D(3) and bone and to evaluate its role in bone biology.     

Putative basal lateral membrane receptors for 24,25-dihydroxyvitamin D(3) in carp and Atlantic cod enterocytes: characterization of binding and effects on intracellular calcium regulation.

The vitamin D metabolite, 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)D(3)), was tested for its ability to specifically bind to basal lateral membranes isolated from intestinal epithelium of Atlantic cod (a seawater fish), carp (a freshwater fish), and chicken. Specific saturable binding was demonstrated in membranes from all three species. Membranes from Atlantic cod, carp, and chicken revealed K(d)'s of 7.3 +/- 0.9, 12.5 +/- 0.9 and 7.8 +/- 0.1 nM, and a B(max) for each species estimated to 57.9 +/- 2.9, 195.1 +/- 8.4 and 175 +/- 0.8 fmol/mg protein, respectively. Scatchard analyses indicated a convex curvature and Hill analyses revealed apparent Hill coefficients of 1.84 +/- 0.28, 1.80 +/- 0.29, and 1.78 +/- 0.27 for Atlantic cod, carp and chicken, suggesting a positive cooperative binding in all three species. Basal lateral membranes from Atlantic cod and carp were used to further characterize the binding moiety. In competition studies, basal lateral membranes from Atlantic cod or carp did not discriminate between 24R,25(OH)(2)D(3) and the 24S,25(OH)(2)D(3) isomer, whereas, 1,25(OH)(2)D(3) and 25(OH)D(3), were less effective in competing with [(3)H]24R,25(OH)(2)D(3) for binding to basal lateral membranes in Atlantic cod and carp. In both the Atlantic cod and carp enterocyte basal lateral membranes, the binding activity could be extracted equally well with high salt as with detergent, indicating a peripheral membrane protein rather than an integral membrane binding protein. Finally, isolated Atlantic cod and carp enterocytes were chosen for analyses of signal transduction events mediated by the putative receptor. In both species, 24R,25(OH)(2)D(3) but not 24S,25(OH)(2)D(3), suppressed Ca(2+)-uptake by enterocytes in a dose-dependent manner. Enterocytes from Atlantic cod and carp, acclimated to Ca(2+)-free media, responded by an intracellular Ca(2+)-release within seconds after addition of 24R,25(OH)(2)D(3) or 24S,25(OH)(2)D(3). The effects on intracellular Ca(2+)-release were dose-dependent for both metabolites. 24S,25(OH)(2)D(3) was effective at lower concentrations and triggered a higher response compared to 24R,25(OH)(2)D(3). These results suggest that the binding molecule(s) for 24R,25(OH)(2)D(3) and 24S,25(OH)(2)D(3) is/are capable of acting as a receptor, mediating rapid, non-genomic responses in intestinal cells.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

24R,25-(OH)(2)D(3) mediates its membrane receptor-dependent effects on protein kinase C and alkaline phosphatase via phospholipase A(2) and cyclooxygenase-1 but not cyclooxygenase-2 in growth plate chondrocytes

Recent studies have shown that 24R,25-(OH)(2)D(3) mediates its effects on growth plate chondrocytes via membrane receptors. This study examined the roles of phospholipase A(2) (PLA(2)) and cyclooxygenase (Cox) in the mechanism of action of 24R, 25-(OH)(2)D(3) in resting zone chondrocytes in order to determine whether the activity of one or both enzymes provides a regulatory checkpoint in the signaling pathway resulting in increased protein kinase C (PKC) activity. We also determined whether constitutive or inducible Cox is involved. Cultures were incubated with 24R, 25-(OH)(2)D(3) for 90 min to measure PKC or for 24 h to measure physiological responses ([(3)H]-thymidine incorporation, alkaline phosphatase-specific activity, [(35)S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, resting zone chondrocytes express mRNAs for both Cox-1 and Cox-2. Levels of mRNA for both proteins were unchanged from control levels after a 24-h incubation with 24R,25-(OH)(2)D(3). To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). Cox-1 inhibition resulted in effects on proliferation, differentiation, and matrix production typical of 24R, 25-(OH)(2)D(3). In contrast, inhibition of Cox-2 had no effect, indicating that 24R,25-(OH)(2)D(3) exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R,25-(OH)(2)D(3)-dependent increases in PKC. Activation of PLA(2) with melittin inhibited 24R, 25-(OH)(2)D(3)-dependent stimulation of PKC, and inhibition of PLA(2) with quinacrine stimulated PKC in response to 24R, 25-(OH)(2)D(3). Inclusion of resveratrol reduced the melittin-dependent inhibition of PLA(2) and caused an increase in quinacrine-stimulated PLA(2) activity. Metabolism of arachidonic acid to leukotrienes is not involved in the response to 24R, 25-(OH)(2)D(3) because inhibition of lipoxygenase had no effect. The effect of 24R,25-(OH)(2)D(3) was specific because 24S,25-(OH)(2)D(3), the biologically inactive stereoisomer, failed to elicit a response from the cells. These results support the hypothesis that 24R, 25-(OH)(2)D(3) exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2). PKC regulation may occur at multiple stages in the signal transduction cascade.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Membrane mediated signaling mechanisms are used differentially by metabolites of vitamin D(3) in musculoskeletal cells

1 alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) mediate their effects on chondrocytes and osteoblasts in part through increased activity of protein kinase C (PKC). For both cell types, 1 alpha,25(OH)(2)D(3) exerts its effects primarily on more mature cells within the lineage, whereas 24R,25(OH)(2)D(3) exerts its effects primarily on relatively immature cells. Studies using the rat costochondral cartilage growth plate as a model indicate that the two metabolites increase PKC activity by different mechanisms. In growth zone cells (prehypertrophic/upper hypertrophic cell zones), 1 alpha,25(OH)(2)D(3) causes a rapid increase in PKC that does not involve new gene expression. 1 alpha,25(OH)(2)D(3) binds its membrane receptor (1,25-mVDR), resulting in activation of phospholipase A(2) and the rapid release of arachidonic acid, as well as activation of phosphatidylinositol-specific phospholipase C, resulting in formation of diacylglycerol and inositol-1,4,5-tris phosphate (IP(3)). IP(3) leads to release of intracellular Ca(2+) from the rough endoplasmic reticulum, and together with diacylglycerol, the increased Ca(2+) activates PKC. PKC is then translocated to the plasma membrane, where it initiates a phosphorylation cascade, ultimately phosphorylating the extracellular signal-regulated kinase-1 and -2 (ERK1/2) family of MAP kinases (MAPK). PKC increases are maximal at 9 min, and MAPK increases are maximal at 90 min in these cells. By contrast, 24R,25(OH)(2)D(3) increases PKC through activation of phospholipase D in resting zone cells. Peak production of diacylglycerol via phospholipase D2 is at 90 min, as are peak increases in PKC. Some of the effect is direct on existing plasma membrane PKC, but most is due to new PKC expression; translocation is not involved. Arachidonic acid and its metabolites also play differential roles in the mechanisms, stimulating PKC in growth zone cells and inhibiting PKC in resting zone cells. 24R,25(OH)(2)D(3) decreases phospholipase A(2) activity and prostaglandin production, thereby overcoming this potential inhibitory component, which may account for the delay in the PKC response. Ultimately, ERK1/2 is phosphorylated. PKC-dependent MAPK activity transduces some, but not all, of the physiological responses of each cell type to its respective vitamin D metabolite, suggesting that the membrane receptor(s) and nuclear receptor(s) may function interdependently to regulate proliferation and differentiation of musculoskeletal cells, but different pathways are involved at different stages of phenotypic maturation.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

Physiological significance of C-28 hydroxylation in the metabolism of 1alpha,25-dihydroxyvitamin D(2).

In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1alpha, 25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] by identifying 1alpha,24(S),25,28-tetrahydroxyvitamin D(2) [1alpha,24(S),25, 28(OH)(4)D(2)] as a major renal metabolite of 1alpha,25(OH)(2)D(2) [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1alpha, 25(OH)(2)D(2). We perfused rat kidneys in vitro with 1alpha, 25(OH)(2)[26,27-(3)H]D(2) (5 x 10(-10)M) and demonstrated that 1alpha,24(R),25-trihydroxyvitamin D(2) [1alpha,24(R),25(OH)(3)D(2)] and 1alpha,24(S),25,28(OH)(4)D(2) are the only two major physiological metabolites of 1alpha,25(OH)(2)D(2). In the same perfusion experiments, we also noted that there is no conversion of 1alpha,25(OH)(2)D(2) into 1alpha,25,28-trihydroxyvitamin D(2 )[1alpha,25,28(OH)(3)D(2)]. Moreover, 1alpha,24(S),25,28(OH)(4)D(2) is not formed in the perfused rat kidney when synthetic 1alpha,25, 28(OH)(3)D(2) is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1alpha,25(OH)(2)D(2) occurs only after 1alpha,25(OH)(2)D(2) is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1alpha, 24(R),25(OH)(3)D(2) in rat kidney is not known. Recently, it was found that 1alpha,25(OH)(2)D(3)-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D(3) compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1alpha,24(R),25(OH)(3)D(2) to form 1alpha, 24(S),25,28(OH)(4)D(2). The biological activity of 1alpha,24(S),25, 28(OH)(4)D(2), determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1alpha,24(S),25,28(OH)(4)D(2) exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1alpha,25(OH)(2)D(2) in kidney under physiological conditions.     

Developmental changes in responsiveness to vitamin D metabolites

We have demonstrated that epiphyseal chondroblasts contain specific receptors for 24R,25-dihydroxy vitamin D3(24,25(OH)2D3) while diaphyseal osteoblasts contain specific receptors for 1 alpha 25-dihydroxy vitamin D3(1,25(OH)2D3). Both metabolites induce DNA synthesis and creatine kinase (CKBB) activity. We have also found that the responsiveness of rat kidney to these metabolites changes during development. In embryonic and early postnatal stages, the kidney responds to 24,25(OH)2D3, later to both 24,25(OH)2D3 and 1,25(OH)2D3, and the mature kidney only to 1,25(OH)2D3. These responses correlate with changes in the specific receptors present in the kidney. Furthermore, we have compared developmental changes in skeletal (epiphysis, diaphysis and mandibular condyle) and non-skeletal (kidney, cerebellum, cerebrum, liver and pituitary) tissue in both rat (a postnatal developer) and rabbit (a perinatal developer). Epiphyseal or diaphyseal chondroblasts at any stage of development were predominantly responsive to 24,25(OH)2D3, whereas osteoblasts were responsive to 1,25(OH)2D3. In contrast, condylar chondroblasts, kidney, cerebellum and pituitary responded to 24,25(OH)2D3 during early development and subsequently developed responsiveness to 1,25(OH)2D3. Using primary cell cultures from kidneys at different stages of maturation, we showed the same developmental pattern as in vivo. Chronic treatment of the cells with 24,25(OH)2D3, but not 1,25(OH)2D3, caused precocious development of responsiveness to 1,25(OH)2D3 in culture. We suggest that 24,25(OH)2D3 acts as a maturation factor, during early development in kidney, and probably in other tissues, possibly by induction of receptor to 1,25(OH)2D3, accompanied by down-regulation of its own receptor.     

Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25-dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding

We have previously described a significant decrease in the positive cooperativity level and affinity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding to its chick intestinal chromatin receptor induced in vitro by a physiological 10-fold molar excess of (24R)-25-dihydroxyvitamin D3 [24R,25(OH)2D3] [F. Wilhelm and A. W. Norman (1985) Biochem. Biophys. Res. Commun. 126, 496-501]. In this report, we have initiated a comparative study of the binding of 24R,25(OH)2[3H]D3 and 1,25(OH)2[3H]D3 to the the intestinal chromatin fraction obtained from vitamin D-replete birds. 24R,25(OH)2[3H]D3 specific binding to this chromatin fraction was characterized by a dissociation constant (Kd) of 34.0 +/- 6.4 nM, a positive cooperativity level (nH) of 1.40 +/- 0.13, and a capacity (Bmax) of 47 +/- 8 fmol/mg protein. The very low relative competitive index (RCI) of 24R,25(OH)2D3 (0.11 +/- 0.03%) for the 1,25(OH)2D3 binding site/receptor, as well as the inability of 1,25(OH)2D3 to displace 24R,25(OH)2D3 from its binding site at a physiological molar ratio of 1:10, strongly suggest the independence of 24R,25(OH)2D3 and 1,25(OH)2D3 binding sites. Stereospecificity of the 24R,25(OH)2D3 binding sites was attested by the displacement of only 45 +/- 6% of 24R,25(OH)2D3 specific binding by equimolar concentrations of 24S,25(OH)2D3. Collectively these results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25(OH)2D3 receptor.     

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.     

Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.     

Double bond in the side chain of 1alpha,25-dihydroxy-22-ene-vitamin D(3) is reduced during its metabolism: studies in chronic myeloid leukemia (RWLeu-4) cells and rat kidney

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1alpha,25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] like 1alpha,25(OH)(2)D(3), is also converted into the same final product, calcitroic acid. This finding indicated that 1alpha,25(OH)(2)D(2) also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1alpha,25(OH)(2)D(2) when compared to the side chain of 1alpha,25(OH)(2)D(3), has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1alpha,25(OH)(2)D(2). We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D(2) related compounds. We compared the metabolism of 1alpha,25-dihydroxy-22-ene-vitamin D(3) [1alpha,25(OH)(2)-22-ene-D(3)], a synthetic vitamin D analog whose side chain differs from that of 1alpha,25(OH)(2)D(3) only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1alpha,25(OH)(2)-22-ene-D(3) and 1alpha,25(OH)(2)D(3) are converted into common metabolites namely, 1alpha,24(R),25-trihydroxyvitamin D(3) [1alpha,24(R),25(OH)(3)D(3)], 1alpha,25-dihydroxy-24-oxovitamin D(3) [1alpha,25(OH)(2)-24-oxo-D(3)], 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D(3). This finding indicates that the double bond in the side chain of 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Along with the aforementioned metabolites, 1alpha,25(OH)(2)-22-ene-D(3) is also converted into two additional metabolites namely, 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Furthermore, we did not observe direct conversion of 1alpha,25(OH)(2)-22-ene-D(3) into 1alpha,25(OH)(2)D(3). These findings indicate that 1alpha,25(OH)(2)-22-ene-D(3) is first converted into 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3). Then the double bonds in the side chains of 1alpha,24,25(OH)(3)-22-ene-D(3) and 1alpha,25(OH)(2)-24-oxo-22-ene-D(3) undergo reduction to form 1alpha,24(R),25(OH)(3)D(3) and 1alpha,25(OH)(2)-24-oxo-D(3), respectively. Thus, our study indicates that the double bond in 1alpha,25(OH)(2)-22-ene-D(3) is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1alpha,25(OH)(2)-22-ene-D(3) metabolism indicating that prior C-24 hydroxylation of 1alpha,25(OH)(2)-22-ene-D(3) is required for the double bond reduction to occur.     

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.     

Acute stimulation of creatine kinase activity by vitamin D metabolites in the developing cerebellum

There is increasing evidence that vitamin D metabolites have a developmental function. We have investigated the influence of the vitamin D status on the activity of creatine kinase in the brain. Normally fed rats show an increase in the specific activity of cerebral and cerebellar creatine kinase during postnatal development. Vitamin-D-depleted rats failed to show this normal increase. Developing cerebellum, but not cerebrum, in both vitamin D-depleted rats and in normally fed animals, responded sequentially to a single injection of a vitamin D metabolite by displaying increased creatine kinase specific activity. In 5-25-day-old rats, 24R,25-dihydroxyvitamin D-3 significantly increased creatine kinase specific activity 24 h after injection. In contrast, 1,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity from 20 days after birth. A similar pattern of sequential responsiveness to vitamin D metabolites, but at an earlier age, was shown in the cerebellum of the rabbit, which is a 'perinatal brain developer' compared to the rat, a 'postnatal brain developer'. Because of the difficulty in obtaining vitamin D-depleted rabbits, studies were carried out in normally fed animals. In these rabbits, 24R,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity between 6 days before birth and 9 days after birth, while 1,25-dihydroxyvitamin D-3 caused an increase in cerebellar creatine kinase specific activity from 8 days after birth. These developmental differences found in creatine kinase basal activity and responsiveness are correlated with differences in cellular growth rates, both in the rabbit and in the rat, suggesting that vitamin D metabolites may be required for optimal cerebellar development.