Characterization of prostaglandin E2 receptors and their role in 24,25-(OH)2D3-mediated effects on resting zone chondrocytes

Abstract

Resting zone chondrocyte differentiation is modulated by the vitamin D metabolite, 24,25-(OH)(2)D(3), via activation of protein kinase C (PKC). In previous studies, inhibition of prostaglandin production with indomethacin caused an increase in PKC activity, suggesting that changes in prostaglandin levels may mediate the 24, 25-(OH)(2)D(3)-dependent response and act as autocrine or paracrine regulators of chondrocyte metabolism. Supporting this hypothesis is the fact that resting zone cells respond directly to prostaglandin E(2) (PGE(2)). The aim of the present study was to identify which PGE(2) receptor subtypes (EP) mediate the effects of PGE(2) on resting zone cells. Using primers specific for EP1-EP4, reverse transcription-polymerase chain reaction (RT-PCR) amplified EP1 and EP2 cDNA in a RT-dependent manner. A variant form of the EP1 cDNA, EPlv, was also amplified in an RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 24,25-(OH)(2)D(3)-mediated cell proliferation and differentiation. 17-phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, increased [(3)H]-thymidine incorporation in a dose-dependent manner and reversed the 24, 25-(OH)(2)D(2)-induced inhibition of [(3)H]-thymidine incorporation. SC-19220, an EP1 antagonist, caused a further dose-dependent decrease in 24,25-(OH)(2)D(3)-induced inhibition of [(3)H]-thymidine incorporation. PTPGE(2) also caused a biphasic increase in [(35)S]-sulfate incorporation and increased alkaline phosphatase enzyme activity at high concentrations (10(-8) M). 24, 25-(OH)(2)D(3)-induced alkaline phosphatase activity was synergistically stimulated in a dose-dependent manner by PTPGE(2). In contrast, 24,25-(OH)(2)D(3)-induced PKC activity was inhibited in a dose-dependent manner by PTPGE(2) and SC-19220, the EP1 antagonist, elevated PKC activity at high concentrations (10(-8) M). The EP2 agonist, misoprostol, only affected [(35)S]-sulfate incorporation, but in a dose-dependent manner. The EP3 and EP4 agonists had no effect on cell response. These results suggest that the EP1 receptor subtype mediates some of the PGE(2)-induced cellular responses in resting zone cells that lead to both increased proliferation and differentiation. Because 24,25-(OH)(2)D(3) inhibits PGE(2) synthesis in these cells, EP1-mediated induction of proliferation is blocked, encouraging cellular maturation and activation of PKC activity.

Prostaglandin E2 downregulates TNF‐α‐induced production of matrix metalloproteinase‐1 in HCS‐2/8 chondrocytes by inhibiting Raf‐1/MEK/ERK cascade through EP4 prostanoid receptor activation

“…However, we have not observed any involvement of EP2 receptor in PGE 2 ‐induced suppression of MMP‐1 release in HCS‐2/8 chondrocytes. Previous studies have shown the presence of both EP2 and EP4 receptors in chondrocytes [Del Toro et al, 2000; Miyamoto et al, 2003]. We have also demonstrated that in HCS‐2/8 chondrocytes EP2 receptor is implicated in TNF‐α‐stimulated release of m‐calpain [Fushimi et al, 2004].…”

“…PGE 2 was initially identified as a molecule, which stimulated bone resorption [Klein and Raisz, 1970]. In contrast, several studies have demonstrated that PGE 2 plays a key role in chondrocyte differentiation [Del Toro et al, 2000; Miyamoto et al, 2003]. Moreover, recent studies indicate that PGE 2 stimulates [Nishikawa et al, 2002] or downregulates [Case et al, 1990; Pillinger et al, 2003] the induction of MMPs.…”

“…On the other hand, EP2, EP3, and EP4 receptors can couple to adenylate cyclase to influence the intracellular cAMP levels. PGE 2 is involved in both bone formation and destruction [Suda et al, 1992; Del Toro et al, 2000]. PGE 2 is shown to enhance IL‐1β‐induced MMP‐3 production in human fibroblasts obtained from periodontically affected gingivae [Nishikawa et al, 2002].…”

Effects of 1?,25-(OH)2D3 on rat growth zone chondrocytes are mediated via cyclooxygenase-1 and phospholipase A2

“…Thus, Cox‐1 may contribute to the rapid increase in PKC that occurs in response to 1α,25‐(OH) 2 D 3 activation of the 1α,25‐(OH) 2 D 3 membrane vitamin D receptor by metabolizing arachidonic acid released by PLA 2 over the first 5 min [Schwartz and Boyan, 1988] and at the same time contributing to the downregulation of the rapid response. The PGE 2 produced as a consequence of the action of Cox‐1 can then act through its EP‐1 receptor [Del Toro et al, 2000] to amplify the rapid effects of 1α,25‐(OH) 2 D 3 on PKC. 1α,25‐(OH) 2 D 3 also induces new PKC expression and incorporation of PKCα into plasma membranes and PKCξ into matrix vesicles within 24 h of exposure to the hormone [Sylvia et al, 1996].…”

“…1α,25‐(OH) 2 D 3 causes a rise in the production of both prostaglandin E 1 (PGE 1 ) and prostaglandin E 2 (PGE 2 ) [Schwartz et al, 1992], and inhibition of prostaglandin production by indomethacin reduces the stimulatory effects of 1α,25‐(OH) 2 D 3 on PKC activity. Exogenous PGE 2 regulates PKC activity through the EP1 receptor present on growth zone cells [Del Toro et al, 2000].…”