Original ArticlesProstaglandins 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
Introduction
Chondrocyte differentiation and maturation in the growth plate are modulated by metabolites of vitamin D.11 Experiments using costochondral growth plate chondrocyte cultures have shown that cellular response is cell maturation dependent and metabolite specific.6 1,25-(OH)2D3 affects primarily chondrocytes from the growth zone (prehypertrophic and upper hypertrophic cell zones), whereas 24,25-(OH)2D3 affects primarily cells from the resting zone. A variety of parameters are affected in a differential manner by the metabolites, including alkaline phosphatase,44 phospholipase A2 (PLA2),42 and protein kinase C (PKC)55-specific activities, arachidonic acid turnover,48, 51 and prostaglandin E2 (PGE2) production,47 calcium ion flux,34 and plasma membrane and matrix vesicle membrane fluidity.52
In addition, the effects of the metabolites are membrane specific. In cultures which have been exposed to 1,25-(OH)2D3 or 24,25-(OH)2D3 for 24 h, alkaline phosphatase activity is increased by 1,25-(OH)2D3 in growth zone chondrocytes, but not by 24,25-(OH)2D3.44 In resting zone chondrocytes, 1,25-(OH)2D3 has no effect, but enzyme activity is stimulated by 24,25-(OH)2D3. These effects are targeted to extracellular matrix vesicles, since the 1,25-(OH)2D3-stimulated increase in growth zone chondrocyte cultures was limited to this organelle and was not seen in isolated plasma membranes. Similarly, the 24,25-(OH)2D3-stimulated increase in resting zone cells was also limited to matrix vesicles.
It is likely that at 24 h, the increase in matrix vesicle enzyme activity is due, at least in part, to new production of alkaline phosphatase via traditional vitamin D receptor (VDR) mechanisms22 and its incorporation into new matrix vesicles. However, when isolated plasma membranes and matrix vesicles are treated directly with 1,25-(OH)2D3 or 24,25-(OH)2D3, a situation in which no new gene expression or protein synthesis is possible, cell maturation-dependent, metabolite-specific increases in resident alkaline phosphatase specific activity are seen,46 indicating that this enzyme is also modulated by nongenomic, membrane-mediated mechanisms which may not involve the VDR. Analogues of 1,25-(OH)h2D3 which exhibit <0.1% of the binding of 1,25-(OH)2D3 to the VDR also elicit these effects on alkaline phosphatase,24 supporting a VDR-independent mechanism.
Recent studies indicate that many of the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth plate cartilage involve rapid, membrane-mediated events.11 These responses may involve specific membrane receptors such as those identified in intestine and osteoblasts,2 which are distinct from the traditional VDR. Recent studies have demonstrated the presence of a membrane VDR for 1,25-(OH)2D3 in growth zone chondrocytes.38 In growth plate chondrocyte cultures, it is clear that the metabolites elicit specific signaling cascades that include activation of PKC. The time course of response is cell maturation specific.55 1,25-(OH)2D3 causes a rapid increase in PKC in growth zone chondrocyte cultures that can be seen by 3 min and is maximal at 9 min. This activation does not require new gene expression55; it is stimulated by arachidonic acid, PGE2, and diacylglycerol (DAG), indicating that PLA2, cyclooxygenase, and phospholipase C are involved.53 It is likely that this activation is membrane mediated because stimulation of isolated plasma membranes with 1,25-(OH)2D3 also results in increased PKC-α activity. Moreover, antibodies to the membrane VDR block the 1,25-(OH)2D3-dependent changes in PKC in matrix vesicles and plasma membranes.38
In contrast, 24,25-(OH)2D3 stimulates PKCα activity in resting zone chondrocyte cultures.55 This effect occurs after 9 min and is maximal at 90 min and requires both gene expression and protein synthesis. It is inhibited by arachidonic acid and PGE2,27 indicating that the mechanism differs from that of 1,25-(OH)2D3 in growth zone cells. Phospholipase C is not involved, although DAG levels increase,27 suggesting that phospholipase D action on phospholipids is responsible. 24,25-(OH)2D3 does elicit some nongenomic stimulation of PKC activity, since direct incubation of the vitamin D metabolite with isolated plasma membranes causes an increase in PKC-α.54
These studies suggest that differential modulation of phospholipid metabolism is one of the mechanisms by which vitamin D metabolites exert their differential effects on growth plate chondrocytes. For both types of cells, there is a rapid change in arachidonic acid turnover that is specific to the target cell. 1,25-(OH)2D3 causes an increase in arachidonic acid release within 5 min, followed by an increase in arachidonic acid incorporation in growth zone cells,48 but has no effect on either parameter in resting zone cells.51 24,25-(OH)2D3 causes an initial decrease in arachidonic acid release from resting zone cells.48 Release is not observed for the first 15 min, and as it increases, so does reincorporation. 24,25-(OH)2D3 has no effect on either parameter in growth zone cells.51
These data suggest that PLA2 is differentially modulated by the metabolites. Studies measuring PLA2 activity specifically confirm this.42 Moreover, these studies show that the differential effects of the vitamin D metabolites on PLA2 activity of the cells is also seen in isolated plasma membranes and matrix vesicles incubated directly with 1,25-(OH)2D3 or 24,25-(OH)2D3,46 again showing that modulation of this enzyme does not require a genomic response to either metabolite.
Release of arachidonic acid from phospholipids by PLA2 is presumed to be one of the rate-limiting steps in prostaglandin production.18 We have shown that 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate the production of PGE2 in a cell maturation-dependent manner similar to the metabolite-specific regulation of PLA2.47 This suggests that the effects of both vitamin D3 metabolites are mediated in part by changes in prostaglandin production and their subsequent effects on the cells.
There are considerable data showing that chondrocytes are responsive to prostaglandins and that chondrocyte differentiation is associated with alterations in prostaglandin synthesis and metabolism.16, 26, 32, 33, 35, 39, 49 It is becoming increasingly clear that cellular response to prostaglandins is a function of the maturation state of the cell. Production of cAMP in response to PGE2 varies with different stages of chondrocyte maturation in the growth plate.4, 15 This may explain some of the apparent conflicts in the literature. In one study, PGE2 caused an increase in [3H]thymidine incorporation as well as an increase in alkaline phosphatase activity of chick growth plate chondrocytes.31 However, in another study using chick growth plate cells, PGE2 increased [3H]thymidine incorporation and DNA synthesis, but decreased alkaline phosphatase activity and collagen production.40 Recently, we showed that PGE2 affects chondrocyte proliferation, differentiation, and matrix production by rat growth plate chondrocytes in a cell maturation-dependent manner, and that these effects are mediated by cAMP.43
We hypothesize that prostaglandins mediate the effects of vitamin D3 metabolites and act as autocrine and paracrine regulators of chondrocyte metabolism in the growth plate. The idea that changes in prostaglandins can act as second messengers of 1,25-(OH)2D3 and 24,25-(OH)2D3 in cartilage cells is supported by the fact that several factors: including interleukin-1, tumor necrosis factor, fibroblast growth factor, phorbol myristate acetate, and bradykinin stimulate the production of prostaglandins in chondrocytes.1, 3, 14, 23, 37 In addition, we hypothesize that there is an optimal concentration of prostaglandin, which mediates increased alkaline phosphatase and PKC activity. To achieve this optimal concentration, PLA2 is inhibited in resting zone cells which have higher basal PGE2 production, and it is stimulated in growth zone cells which have lower basal PGE2 production.46
In the present study, we evaluated whether prostaglandins mediate the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 on chondrocyte differentiation by measuring the specific activity of alkaline phosphatase in the presence of indomethacin, and determined whether this effect is metabolite, cell maturation, and membrane specific. Since 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate alkaline phosphatase in part through modulation of PKC,27, 55 we also determined whether the effects of the vitamin D3 metabolites on PKC are due to prostaglandin action on the cells. Finally, because the time course of PKC activation and PGE2 production in response to the vitamin D3 metabolites may differ, we hypothesize that the vitamin D metabolites modulate PLA2 activity; the resultant change in arachidonic acid regulates PKC and increased PKC modulates prostaglandin production. Therefore, we examined whether changes in PGE2 production in response to 1,25-(OH)2D3 and 24,25-(OH)2D3 are dependent on PKC.
Section snippets
Reagents
Vitamin D3 metabolites were generous gifts of Dr. Milan Uskokovic of Hoffman-LaRoche (Nutley, NJ). 1α,25-(OH)2D3 and 24R,25-(OH)2D3 stock solutions were dissolved in ethanol and diluted at least 1:5000 (v/v) with culture medium before addition to the cultures. PKC assay reagents were obtained from Gibco-BRL (Gaithersburg, MD). The protein content of each sample was determined using the bicinchoninic acid (BCA) protein assay reagent50 obtained from Pierce Chemical Company (Rockford, IL).
Growth zone chondrocytes
1,25-(OH)2D3 caused a dose-dependent increase in alkaline phosphatase-specific activity which was significant at concentrations of 10−8 to 10−9 mol/L (Figure 1, upper panel). Indomethacin significantly inhibited the basal-specific activity of alkaline phosphatase. Moreover, indomethacin also inhibited activity in cells treated with 1,25-(OH)2D3 to levels comparable to those seen in cells with no 1,25-(OH)2D3. 24,25-(OH)2D3 had no effect on alkaline phosphatase in the growth zone chondrocytes (
Discussion
Our results support the hypothesis that 1,25-(OH)2D3 mediates its effects on alkaline phosphatase-specific activity through increased prostaglandin production. Indomethacin consistently blocked the 1,25-(OH)2D3-dependent stimulation of enzyme activity. As noted previously,10 the effects of 1,25-(OH)2D3 were targeted to the matrix vesicles. We now show that this 1,25-(OH)2D3-dependent effect is mediated by prostaglandin.
24,25-(OH)2D3-dependent stimulation of alkaline phosphatase specific
Acknowledgements
The authors thank Sandra Messier for her assistance in the preparation of the manuscript. The research was supported by US PHS Grant DE-05937. This article is in partial fulfillment for the M.S. degree for Dr. Ridge Gilley, a fellow in the Air Force Institute of Technology. The opinions expressed are not necessarily those of the US Air Force.
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2013, Cellular SignallingCitation Excerpt :1α,25(OH)2D3 activates a number of rapid responses in its target cells. In chondrocytes and osteoblasts, 1α,25(OH)2D3 caused Pdia3-dependent activation of phospholipase A2 (PLA2) via phospholipase A2 activating protein (PLAA), resulting in release of arachidonic acid within seconds and subsequent production of prostaglandin E2 (PGE2) [18,19,33–35]. In addition, phosphatidylinositol-dependent phospholipase C, PKC and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) were rapidly increased downstream of PLA2 activation [10,33,36].
Phospholipase A<inf>2</inf> activating protein is required for 1α,25-dihydroxyvitamin D<inf>3</inf> dependent rapid activation of protein kinase C via Pdia3
2012, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :These actions of 1α,25(OH)2D3 can be blocked by antibodies to protein-disulfide isomerase family A, member 3 (Pdia3, also known as ERp60, ERp57, Grp58, and 1,25-MARRS), indicating that it is part of the receptor complex. Multiple studies have demonstrated the pivotal role PLA2 plays in 1α,25(OH)2D3-dependent membrane-mediated signaling [8,13–16]. Stimulation of PLA2 activity using agents such as bee venom melittin and snake venom mastoparan, mimics the effects of 1α,25(OH)2D3 on PKC in growth zone chondrocytes.
Mechanism of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling in musculoskeletal cells
2012, SteroidsCitation Excerpt :At the top of the growth plate is a zone of cartilage containing resting chondrocytes, surrounded by a hyaline cartilage-like matrix. These cells respond primarily to 24,25(OH)2D3 rather than 1,25(OH)2D3 [1,39,40]. Following a series of cell mitoses in the proliferating cell zone, the chondrocytes enter a prehypertrophic zone, where they undergo terminal differentiation.
Vitamin D metabolism, cartilage and bone fracture repair
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Protein-disulfide isomerase-associated 3 (Pdia3) mediates the membrane response to 1,25-dihydroxyvitamin D<inf>3</inf>in osteoblasts
2010, Journal of Biological Chemistry