趨化激素調控人類軟骨細胞表現間質分解酵素之分子機制探討

趨化激素調控人類軟骨細胞表現間質分解酵素之分子機制探討 Studies on the molecular mechanisms of matrix metalloproteinases regulation in chondrocytes by chemokines

中文摘要

趨化激素目前已經被證實在軟骨的降解中扮演著非常重要的腳色，但是其分子 機制至今仍然有許多未知及未被發現的地方。在此篇論文中將探討在正常人類軟 骨細胞以及人類軟骨癌細胞(SW1353)中趨化激素 Eotaxin-1 對於間質分解酵素 (MMP)表現的影響。

首先，在實驗中發現Eotaxin-1 對於 MMP-3 mRNA 的表現會隨著加入細胞的劑 量增加而增加。第二，p38 和 ERK 的抑制劑可以壓制經由 Eotaxin-1 刺激而增加 的MMP-3 mRNA。另一方面，cAMP 的拮抗劑 Rp-cAMP 和 PKA 的抑制劑 H-89 卻可以更增加經由Eotaxin-1 刺激而增加的 MMP-3 mRNA。經由以上的實驗數據 可知，主要調節MMP-3 表現的 Eotaxin-1 受器是抑制型的 G protein couple receptor。趨化激素 Eotaxin-1 在 EMSA 的試驗中發現，Eotaxin-1 也會活化轉錄因 子NF-κB 和 AP-1。

有趣的是在軟骨細胞萃取液中，我們探測不到經由Eotaxin-1 刺激所產生的

MMP-3 蛋白，經由偵測細胞培養液發現，Eotaxin-1 刺激的軟骨細胞，幾乎所有 的MMP-3 蛋白都分泌到了細胞外。從實驗的結果我們可以得知，調控 Eotaxin-1 分泌MMP-3 蛋白的訊息傳導路徑包括了 PLC-PKC、p38 和 JNK MAPK 路徑；且 從之前的論文文獻中得知，Eotaxin-1 會經由 Eotaxin-1 受器來活化下游的 Gβγ 次分子進而影響蛋白質的分泌。Eotaxin-1 不只可以調節軟骨細胞中 MMP-3 基因

的表現，更可以促使細胞中的MMP-3 蛋白分泌到細胞外，而這些可能是在骨關

節炎中軟骨降解的分子層面之關鍵。

英文摘要

It has been demonstrated that chemokines play an important role in cartilage

degradation, however, the underlying molecular mechanisms are not well studied. In this study, we investigated the effects of CC chemokines-eotaxin-1 on the MMP expression in both of human chondrocyte cell lines SW1353 and primary culture chondrocytes.

First, we found that eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Second, ERK inhibitor and p38 kinase inhibitor were able to repress MMP-3 mRNA expression induced by eotaxin-1. On the other hand, Rp- adenosine-3’,5’-cyclic monophosphorothioate (Rp-cAMPs), a competitive cAMP antagonist for cAMP-receptor proteins, and PKA inhibitor-H89 markedly enhanced the MMP-3 mRNA expression induced by eotaxin-1. There results suggest that MMP- 3 expression mainly mediated by eotaxin-1 receptor, which is a Gαi-coupled receptor.

Eotaxin-1 also activated the transcription factor AP-1 and NF-κB by EMSA experiment.

Interesting finding that no MMP-3 protein was detected in the cell lysate of eotaxin-1 treatment chondrocytes. However, most of MMP-3 protein was secreted into the culture medium in both of chondrocyte cell lines SW1353 and primary culture chondrocytes. We discovered that PLC-PKC cascade and p38 and JNK MAP kinase pathway regulated MMP-3 protein secretion induced by eotaxin-1. There results suggest that MMP-3 secretion mainly mediated by eotaxin-1 receptor, which activate Gβγ subunit. These results suggest that eotaxin-1 not only induced MMP-3 gene expression but also promoted MMP-3 protein secretion, and eotaxin-1 may be a key molecule in the cartilage degradation of arthritis.