24R,25-dihydroxyvitamin D3 promotes the osteoblastic differentiation of human mesenchymal stem cells.

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3.

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.

Regulation of parathyroid hypertensive factor secretion by vitamin D3 analogs in parathyroid cells derived from spontaneously hypertensive rats.

Parathyroid hypertensive factor (PHF) is a novel substance secreted by the parathyroid gland (PTG), which is elevated in 30-40% of all hypertensive patients; specifically, the low-renin subset. However, very little is known about the regulation of PHF secretion. Since the classical parathyroid regulator, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), may be elevated concurrent with or preceding the development of low-renin hypertension and elevated plasma PHF, we hypothesized that 1,25-(OH)2D3 would stimulate PHF release. To test this hypothesis, PTG organ and cell cultures, derived from spontaneously hypertensive rats (SHR) and the normotensive genetic control Wistar Kyoto (WKY) rats, were exposed to various vitamin D3 metabolites and PHF release measured by ELISA. 1,25-(OH)2D3 rapidly stimulated PHF release with enhanced sensitivity in SHR versus WKY cultures indicated by a leftward shift in the dose-response curve, whereas 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) had the converse effect. Vitamin D3 analog "BT," an agonist for the classical nuclear vitamin D receptor (1,25VDR(nuc)), was without effect suggesting a 1,25VDR(nuc)-independent mechanism and potential involvement of the plasma membrane-bound vitamin D receptor (1,25 D3-MARRS). Interestingly, protein expression of the 1,25 D3-MARRS was increased in SHR versus WKY parathyroid cells. In conclusion, these results support the idea that 1,25-(OH)2D3 may contribute to elevated plasma PHF in the SHR.

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.

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.

24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms.

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.

Calcium regulating activity of 26,27-dimethyl analog of 24R,25-dihydroxyvitamin D3.

To determine the possibility that methyl substitution in 26- and 27-positions of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] alters activities of the original compound, the effects of 24,25(OH)2D3 on calcium (Ca) regulating activity were compared with those of its methyl analog [24,25(OH)2(CH3)2D3] in addition to 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 24,25(OH)2D3 at 10(-6) M and 24,25(OH)2(CH3)2D3 at 10(-7) M and above significantly stimulated both bone resorption in neonatal mouse calvaria cultures and formation of osteoclast-like multinucleated cells (MNC) in mouse bone marrow cultures. A stimulative effect of 1,25(OH)2D3 on bone resorption and MNC formation was recognized in very low concentrations (10(-11) M and above). Although a potency of 24,25(OH)2(CH3)2D3 in stimulating bone calcium (Ca) mobilization and intestinal Ca transport was higher than that of 24,25(OH)2D3, the potencies of both compounds were similar to that of 1,25(OH)2D3 unlike in vitro experiments. As 1,24R,25-trihydroxy-26,27-dimethylvitamin D3 showed almost the same effect as 24,25(OH)2(CH3)2D3, the dihydroxy form is suggested to be hydroxylated at 1 alpha position and converted to trihydroxy form in vitamin D-deficient rats. From these results, methyl substitution in 26- and 27-position of 24,25(OH)2D3 was found to elevate Ca regulating activity of the original compound. In addition, it is suggested that the basis for a similarity in potency between 1,25(OH)2D3 and 24,25(OH)2D3 or its dimethyl analog in vitamin D-deficient rats is likely the result of 1 alpha-hydroxylation.

[Vitamin D].

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the active form of vitamin D3, stimulates intestinal calcium absorption and osteoclastic bone resorption, resulting in the elevation of plasma calcium. Recent studies have revealed that 1 alpha,25(OH)2D3 also promotes differentiation of various cells such as myeloid leukemia cells, lymphocytes, macrophages and skin keratinocytes. The target cells of 1 alpha,25(OH)2D3 possess 1 alpha,25(OH)2D3 receptors (VDR) which belong to the steroid thyroid retinoic acid receptor gene family. The complex of VDR and 1 alpha,25(OH)2D3 binds to vitamin D3-responsive elements (VDRE) present in the promoter region of target genes of 1 alpha,25(OH)2D3. In bone, osteoblasts possess VDR and synthesize several proteins, such as BGP, osteopontin and the third component of complement (C3), in response to 1 alpha, 25(OH)2D3. 1 alpha, 25(OH)2D3 is involved in the differentiation of osteoclast progenitors into mature osteoclasts directly and also by an indirect mechanism involving some proteins produced by osteoblasts. In this review article, we show the mode of action and the biological function of 1 alpha, 25(OH)2D3.

1,25-(OH)2D3 and 24,25-(OH)2D3 regulation of arachidonic acid turnover in chondrocyte cultures is cell maturation-specific and may involve direct effects on phospholipase A2.

Previous studies have shown that 1,25-(OH)2D3 stimulates phospholipase A2 (PA2) activity in growth zone chondrocytes (GC), but has no effect on the resting zone chondrocyte (RC) enzyme activity. 24,25-(OH)2D3 inhibits the RC enzyme but has no effect on the GC. This study examined whether the vitamin D metabolites affect arachidonic acid turnover in their contra-target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone. Acylation and reacylation were measured independently by incubating with p-chloromercuribenzoate. The results demonstrated that 1,25-(OH)2D3 has no effect on arachidonic acid turnover in RC, but stimulates turnover in GC. In contrast, 24,25-(OH)2D3 stimulates arachidonic acid turnover in RC, but inhibits both incorporation and release in GC. To determine whether direct interaction with PA2 is one mechanism by which 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate arachidonic acid release, snake venom (Niger niger) PA2 was incubated with the vitamin D metabolites. Enzyme specific activity was inhibited by 24,25-(OH)2D3 and stimulated by 1,25-(OH)2D3 in a time- and dose-dependent manner. These results suggest that at least part of the direct effect of vitamin D3 metabolites on cell membranes may be related to changes in PA2 activity. The regulation is related to the stage of differentiation of the target cell population. Changes in fatty acid acylation and reacylation may be one mode of vitamin D3 action in cartilage.

Effects of 1,25-dihydroxyvitamin D3 on in vitro lymphocyte reactions: arguments for a role at the maternofetal interface.

Calcitriol or 1,25-dihydroxyvitamin D3 is synthesized by successive hydroxylations of vitamin D in the liver and kidney and during pregnancy in the placenta and the decidua. The aim of the present study was to clarify the immunoregulatory role of calcitriol, if any, during pregnancy. Calcitriol concentrations of 10(-10) to 10(-8) M was shown to reduce the proliferation of allogeneically stimulated lymphocytes and cytotoxic cell generation in a dose-dependent manner. However, calcitriol did not inhibit IL-2-dependent proliferation of CTLL-2 cell line. Calcitriol reduced non-MHC restricted cytotoxicity. Calcitriol, therefore, might be involved in the successful engraftment and growth of the fetoplacental unit possibly synergized with other products of placental or decidual origin.