消炎藥物及環氧合酶II影響人類成骨細胞增殖分子機制之探討

The Study of the Molecular Mechanisms of Anti-inflammatory Drugs and Cyclooxygenase-2 on Proliferation of Human Osteoblasts

李靜如(Ching-Ju Li)

指導教授 ： 何美泠

高雄醫學大學/醫學院/醫學研究所/博士(2010年)

https://doi.org/10.6832/KMU.2010.00140

摘要

Cyclooxygenase (COX)包含COX-1、COX-2與COX-3，為前列腺素生成的重要酵素。骨科臨床上常使用類固醇及非類固醇消炎藥物來抑制COX活性以抑制前列腺素的生成，進而達到消炎止痛的效果。然而，在許多的研究中發現，類固醇及非類固醇消炎藥物會抑制骨修復及骨生成作用。成骨細胞能經由增殖、骨分化及礦化的過程形成骨質，為骨生成過程中最重要的細胞之一。我們之前的研究發現，類固醇消炎藥物、非類固醇消炎藥物及COX-2選擇性抑制劑會抑制成骨細胞的增殖、並使其細胞週期停滯於G0/G1期並促進p27kip1 mRNA及protein的表現。因此我們推測，消炎藥物促進p27kip1的表現可能為其抑制人類成骨細胞增殖的重要機制之一。Akt/FOXO訊息傳遞路徑是已知調控p27kip1 promoter activity的主要機制之一。許多文獻亦指出，消炎藥物能抑制Akt傳遞路徑。因此，根據以上資料我們假設，消炎藥物可能透過影響人類成骨細胞Akt/FOXO 路徑的傳遞進而促進p27kip1的表現與抑制成骨細胞的增殖。本論文第一部分【Study I】探討了消炎藥物對Akt/FOXO/p27Kip1 signaling的影響。我們的結果顯示，消炎藥物抑制Akt phoshporylation、促進FOXO3a protein level並增加p27Kip1 promoter activity、mRNA與protein表現。此外，我們更進一步發現，FOXO silencing能顯著地抑制消炎藥物增加p27Kip1與抑制增殖的效果。根據本論文的第一部分研究，我們推測消炎藥物抑制成骨細胞增殖有一部份是透過抑制Akt並活化FOXO3a與p27Kip1所導致。
研究顯示類固醇消炎藥物能抑制COX-2之表現，而非類固醇消炎藥物則能抑制COX-2之活性。因此，我們進一步推測COX-2可能為此三類消炎藥物均促進p27Kip1與抑制成骨細胞增殖的主要機制。根據本論文第一部分的結果，我們假設COX-2 本身亦可能參與了Akt/p27Kip1與成骨細胞增殖的調控。因此，在一般生理狀況下成骨細胞可能能持續的表現COX-2，並且這些持續性表現的COX-2可能參與了成骨細胞增殖的調控。故我們探討成骨細胞是否有持續性的表現COX-2並參與了Akt signal進而調控人類成骨細胞的增殖。【Study II】的活體內研究顯示，trabecula、periosteum及endosteum的成骨細胞有持續性的表現COX-2。而且在活體內及活體外的研究均顯示，在成骨細胞上持續性表現的COX-2有與p-Akt co-expression的現象。我們進一步在活體外的研究中發現，COX-2 silencing顯著地促進人類成骨細胞PTEN activity並抑制p-Akt protein level與細胞增殖。重要的是，recombinant human COX-2 (rhCOX-2)的轉殖能回復COX-2 siRNA所抑制的PTEN phosphorylation。然而，無生物活性的rhCOX-2蛋白的轉殖則無法回復COX-2 siRNA所抑制的PTEN phosphorylation。相對於COX-2 siRNA的結果，我們發現COX-1 silencing對PTEN/Akt signaling及其下游之分子無顯著之影響。根據以上的結果，我們推測消炎藥物能透過抑制Akt活化FOXO3a進而促進p27Kip1表現。此外，我們進一步發現骨生成較活耀的區域中的成骨細胞有持續性的表現COX-2。我們推測PTEN/Akt signaling在調控成骨細胞的增殖上扮演了重要的角色。
統整以上，本研究顯示在骨生成較活耀區域中的成骨細胞會持續性的表現COX-2。此外，我們亦發現COX-2能透過抑制PTEN活性、促進Akt磷酸化與抑制p27Kip1蛋白的生成，進而促進成骨細胞的增殖。我們更進一步發現，消炎藥物抑制Akt/FOXO/p27Kip1訊息傳遞路徑進而抑制成骨細胞增殖可能是透過抑制COX-2所導致。

關鍵字

消炎藥物 ； 環氧合酶I ； 環氧合酶II ； PTEN ； Akt 訊息傳遞路徑 ； Forkhead box O (FOXO) ； p27Kip1 ； 增殖 ； 成骨細胞

並列摘要

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.

並列關鍵字

Anti-inflammatory drugs ； Cyclooxygenase-1 ； Cyclooxygenase-2 ； PTEN ； Akt signal transduction ； Forkhead box O (FOXO) ； p27Kip1 ； Proliferation ； osteoblast

參考文獻

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