Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

Abstract

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.

Osteoblast Response to Rest Periods During Bioreactor Culture of Collagen–Glycosaminoglycan Scaffolds

“…COX-2 levels were higher for all bioreactor groups compared with the static control, particularly after just 1 h of bioreactor culture. COX-2 is vital for the production of PGE 2 25 and the increase in COX-2 expression resulted in higher PGE 2 production in all flow groups over the static control at both 25 and 49 h. PGE 2 is involved in osteoblast differentiation 26 and may be linked to osteocalcin expression. 27,28 In 2D studies, it has been found that PGE 2 release is similar for continuous and short-term rest-inserted loading, a finding which is similar in 3D in this case.…”

Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3‐E1 osteoblast‐like cells

“…These results are confirmed in a report by Yoshida et al 49 AA is the substrate for PGE 2 synthesis 50 and has been implicated in osteoblastic differentiation. [49][50][51] It has been reported that PGE 2 , at high concentrations, inhibits ALP activity, 52,53 while blocking of endogenously produced PGE 2 by COX-blockers increases ALP activity. 49,53 It could therefore be speculated that the inhibitory effect of AA on ALP activity in our experimental conditions might be PGE 2 -mediated.…”

Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells

“…Recent studies using animals with deletion of either the EP2 or EP4 receptors or using selective agonists for these receptors have suggested that both EP2 and EP4 can mediate anabolic responses to PGE 2 (4)(5)(6)(7)(8)(9)(10), although the EP1 receptor may also be involved (11). Activation of EP2 and EP4 receptors increases cAMP and can also induce COX-2, which can result in auto-amplification of PGE 2 signaling (12).…”