| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 99 === Cyclooxygenase (COX) family, including COX-1, COX-2 and COX-3, is the central enzyme in catalyzing the conversion of arachidonic acid to prostaglandins (PGs). The enzyme activities can be blocked by anti-inflammatory drugs, such as glucocorticoid, non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 inhibitors, which are wildly used in orthopaedic patients to relieve pain and inflammation. However, reports indicated that the anti-inflammatory drugs suppress bone repair and remodeling in vivo. Osteoblast is an important cell involved in bone formation through proliferation, differentiation and mineralization. Our previous studies showed that anti-inflammatory drugs inhibit cell proliferation, arrest cell cycle, and up-regulate p27Kip1 in human osteoblasts (hOBs), suggesting that the up-regulation of p27Kip1 may contribute to anti-inflammatory drug-induced inhibition of proliferation in hOBs. However, the mechanism remains unclear. Akt/ Forkhead box O (FOXO) signaling is one of the key factors to suppress p27Kip1 promoter activity. Several studies also reported that anti-inflammatory drugs inhibit Akt signaling in various cancer cell lines. Based on the previous investigation, we hypothesized that Akt/FOXO/p27Kip1 signaling may play an important role in anti-inflammatory drug-induced suppression of proliferation in hOBs. In study I, we investigated the influences of three anti-inflammatory drugs, including indomethacin, celecoxib and dexamethasone, on the levels and/or activities of Akt, FOXO and p27Kip1 as well as the relationship between these factors and proliferation of hOBs. The results of study I showed that anti-inflammatory drugs suppressed the canonical level of phosphorylated Akt, accompanied by elevated FOXO3a level and increased promoter activity, mRNA expression and protein level of p27Kip1. Furthermore, FOXO silencing significantly attenuated the anti-inflammatory drug-induced up-regulation of p27Kip1 and suppression of proliferation in hOBs. The results of study I suggest that anti-inflammatory drugs suppress hOB proliferation, at least partly, through inactivating Akt, activating FOXO3a, and eventually up-regulating p27Kip1 expression.
Glucocorticoids were reported to suppress COX-2 synthesis. Both non-selective NSAIDs and COX-2 selective inhibitors are able to block COX-2 activity. Reports also indicated that functions of osteoblast might be regulated by COX-2. Accordingly, we suggest that COX-2 may be the key factor for anti-inflammatory drug-caused effects and COX-2, itself, may also contribute to the regulation of Akt/p27Kip1 signaling and proliferation in osteoblasts. Furthermore, these findings also indicated that COX-2 may be constitutively expressed in osteoblast to regulate proliferation under normal physiological condition. In study II, we investigated the mechanism of COX-2 on Akt signaling in hOBs and further identified the constitutively expressed COX-2 and its physiological role in osteoblastic proliferation. Results of the study II showed that osteoblasts adjacent to the trabecula, periosteum and endosteum in mouse femurs constitutively expressed COX-2, and co-expressed with p-Akt in osteoblasts. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. In conclusion, these studies represent the first prospect in human osteoblasts to demonstrate that Akt/FOXO3a/p27Kip1 signaling contributes to the suppressive effect of anti-inflammatory drugs on proliferation. Furthermore, our findings provide new insight into bone physiology; namely, that COX-2 is constitutively expressed in osteoblasts in the dynamic bone growth area, and facilitates osteoblast proliferation via PTEN/Akt/p27Kip1 signaling.
This study demonstrated that osteoblasts in the dynamic bone growth area constitutively express COX-2 under normal physiological condition. Furthermore, the constitutive COX-2 may contribute to regulate osteoblast proliferation via inhibit PTEN activity, promote Akt phosphorylation and suppress p27Kip1 protein level. Moreover, COX-2 may be the key factor of anti-inflammatory-influenced Akt/FOXO3a/p27Kip1 signaling.
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