Molecular mechanisms of anti-inflammatory actions of unfolded protein response in activated macrophages

目前已有研究指出，當巨噬細胞受感染或是在發炎的情形下會產生內質網壓力，然而對於內質網壓力在發炎中所扮演的角色仍不清楚。已知內質網壓力可誘導一些訊息傳遞路徑及活化基因轉錄因子，進而引起發炎反應。然而近年來也有研究結果顯示未折疊蛋白反應(unfolded protein response，UPR)具有抗發炎的作用，但詳細的分子機制尚需進一步的探討。巨噬細胞是宿主先天性免疫重要的第一道防線，目前對於內質網壓力和其他發炎刺激物質在巨噬細胞中互相作用後所產生的影響還不清楚。因此，在本篇研究中我們將利用tunicamycin (TM)，以及brefeldin A (BFA)為內質網壓力誘導劑，以老鼠巨噬細胞RAW264.7 及小神經膠質細胞BV-2 為模式來探討內質網壓力在內毒素lipopolysaccharide (LPS) 和interferons (IFNs) 所活化的發炎反應中所扮演的角色。由實驗結果得知，在老鼠巨噬細胞RAW264.7 中內質網壓力誘導劑TM 及BFA 能抑制LPS 誘導的iNOS 蛋白表現及NO 生成。此抑制作用是在iNOS 基因轉錄的層面，和細胞的生存率無關。此外，LPS 刺激3 小時後給予TM 及BFA 仍能抑制LPS 誘導的iNOS 及NO 表現，顯示出內質網壓力誘導劑的抑制作用可能是在LPS 活化的晚期訊息傳遞路徑或是在基因轉錄的層面上。由實驗結果證實，內質網壓力誘導劑對於LPS 所活化的早期訊息傳遞路徑，例如IKK 磷酸化，IkBa降解，p65 細胞核轉移以及MAPKs 活化等訊息傳遞上沒有影響。然而我們發現內質網壓力誘導劑會抑制LPS 活化的晚期NF-kB 家族轉錄因子和iNOS 啟動子區域的親和力，同時也抑制了RelB 細胞核轉移以及p300 這個共活化劑的表現。之前有報導指出p300 對於組蛋白H3 以及NF-kB 家族轉錄因子的乙醯化很重要，因此我們的實驗結果顯示出內質網壓力誘導劑能藉由影響p300 表現來抑制LPS 的發炎反應。此外，我們也發現內質網壓力誘導劑會抑制LPS 及IFNs 活化的STAT1 磷酸化。STAT1 磷酸化已經被報導對於LPS 活化iNOS 表現有協同的作用。當我們分別前處理細胞鈣離子敖合劑BAPTA/AM、攜鈣素抑制劑W7、以及不含鈣離子的細胞培養液，發現原本受內質網壓力誘導劑抑制的STAT1 磷酸化被回復了。結果亦顯示出內質網壓力誘導劑會造成細胞外鈣離子流入胞內，因此也證明了鈣離子確實會參與內質網壓力誘導劑抗發炎的作用。另外，我們亦利用廣效性的protein tyrosine phosphatases 抑制劑sodium orthovanadate 前處理細胞,發現內質網壓力誘導劑原本抑制STAT1 磷酸化的作用也被回復了。由此可知，內質網壓力誘導劑會活化細胞中某一種tyrosinephosphatase。由更進一步的實驗發現內質網壓力誘導劑會活化MKP-1 這個具有雙重protein phosphatase 功能的蛋白表現。MKP-1 已被報導能造成STAT1 去磷酸化，而此蛋白的表現會受到IFN-y負調控，但為一個能受calcineurin 正調控的蛋白。因此我們亦利用鈣離子敖合劑BAPTA/AM 及calcineurin 抑制劑前處理細胞,來釐清MKP-1 和鈣離子的關係。由實驗結果發現，內質網壓力誘導劑抑制STAT1 磷酸化的作用的確會受FK-506 回復，且內質網壓力誘導劑會增加MKP-1 的表現。綜合所有的實驗結果，我們發現在LPS 或IFNs 所活化的巨噬細胞中，未折疊蛋白反應的抗發炎作用主要是藉由影響RelB 細胞核轉移，p300 的表現以及活化鈣離子/攜鈣素/calcineurin 所調控的MKP-1 表現而產生的。本研究的結果不僅能深入了解未折疊蛋白反應的抗發炎作用機轉，更能提供新的標靶分子作為未來抗發炎藥物之研發的學理依據。Endoplasmic reticulum (ER) stress resulting from the accumulation of unfolded or misfolded proteins in the ER is known to be induced in infectious and inflammatory conditions. Once ER stress happens, unfolded protein responses (UPR) can be elicited trying to compensate for ER stress. Currently the role of ER stress in inflammatory response still remains elusive and debatable. ER stress-mediated activation of ER sensors can induce signal cascades and activate transcription factors for inflammatory gene responses. Some evidences nevertheless indicate anti-inflammatory outcome resulting from the UPR. Since macrophages are the first line of innate immunity, and real outcome upon ER stress interactive with other inflammatory stimuli has not been elucidated, in this study we used tunicamycin (TM) and brefeldin A (BFA) as ER stressors, and murine RAW264.7 macrophages and BV-2 microglia as cell models to clarify the role of UPR in lipopolysaccharide (LPS)- and interferons (IFNs)-mediated inflammatory responses. Our results revealed the ability of ER stressors (TM and BFA) to attenuate LPS-elicited iNOS gene expression and NO production, which was unrelated to cell viability. Moreover, post-treatment with TM or BFA at 3 h as compared to LPS still can inhibit LPS-induced iNOS/NO expression, suggesting that such inhibition occurs either at the late signals evoked by LPS or at the transcriptional machinery. Confirming our suggestion, we did not observe any effects of ER stressors on the early upstream signals evoked by LPS, including IKK phosphorylation, IkBa degradation, p65 nuclear translocation and three MAPKs activation. However, we found that ER stressors can block NF-kB binding to specific DNA elements in the iNOS promoter in late phase signaling evoked by LPS, as well as attenuate RelB nuclear translocation. Results suggest that inhibition of p300 expression, which is critical for histone H3 and NF-kB family acetylation, is involved in the anti-inflammatory actions of ER stressors. Furthermore, we also found that ER stressors could block LPS- and IFNs (a,b, and y)-mediated STAT1 phosphorylation; the latter has synergistic effect on LPS-induced iNOS expression. When treating cells with Ca2+ chelator BAPTA/AM, calmodulin inhibitor W7 or Ca2+ free medium, the inhibitory effect of ER stressors on STAT1 phosphorylation was abrogated. Moreover, UPR elicited influx of extracellular Ca2+. These results suggest the involvement of downstream effector of calmodulin in the negative regulatory effect of ER stressors. Studies with sodium orthovanadate, a broad-spectrum inhibitor of protein tyrosine phosphatases, suggest activation of tyrosine phosphatase accounts for the inhibitory effect of ER stressors on IFNs signaling. Furthermore, we observed that ER stressors could induce MKP-1 expression, which is a newly identified protein phosphatase targeting on STAT1, and can be down-regulated by IFN-y. Cells pretreated with BAPTA/AM or calcineurin inhibitor FK-506 could block the effect of TM on STAT1 phosphorylation. Taken together, these results suggest that attenuation of p300 expression, RelB nuclear translocation and induction of Ca2+/CaM/calcineurin-dependent MKP-1 contribute to the anti-inflammatory actions of ER stress in LPS- and IFNs-activated macrophages. This study not only provides advanced understanding of the anti-inflammatory mechanisms resulting from ER stress, but also provides defined targets for the development of anti-inflammatory agents