人類單核球細胞產生腫瘤壞死因子及基質金屬蛋白酵素之調控機轉

The regulatory mechanisms of the production of tumor necrosis factor-α and matrix metalloproteinase in human monocytic cells

周泳臣(Yung-Chen Chou)

指導教授 ： 蕭哲志

臺北醫學大學/醫學院/醫學科學研究所/博士(2011年)

摘要

腫瘤壞死因子(TNF)-α是一種發炎前(proinflammatory)細胞激素，在急性骨髓性白血病以及敗血症中它都扮演非常重要的角色。其在體內主要是由巨嗜細胞以及單核球細胞來製造與釋放，尤其是受內毒素(endotoxin)刺激時更加明顯。TNF-α蛋白在細胞中合成出來後會鑲在細胞膜上，其可以被TNF-α converting enzyme (TACE)酵素切下而釋放到細胞外。在過去文獻中發現高張壓力於活化的單核球細胞中會加強TNF-α的表現，但真正的機轉目前仍未被探討。已知細胞表面的TNF-α以及TACE蛋白在細胞活化時皆會進行內吞作用(endocytosis)而減少表現，恰巧高張環境也會干擾endocytosis的進行，因此本研究便進行一連串的實驗來探討高張環境加強TNF-α釋放可能涵蓋的機制。
TNF-α被釋放後可以引發許多發炎反應，並刺激基質分解酵素的產生。在許多病理過程中經常可以發現細胞外基質不正常的重塑作用(remodeling)，而這些基質的破壞主要是由基質金屬水解酶(MMPs)完成。本實驗發現，在接受LPS或PMA刺激的THP-1細胞中，蔗糖或NaCl所形成的高張環境皆可以顯著地加強其TNF-α的釋放。本研究也發現同樣皆具有內吞抑制作用的各種成分(包含高張蔗糖溶液)皆能增加PMA刺激時細胞膜上TACE的表現，但它們對活化細胞中的TNF-α釋放作用卻不一致。這樣的TNF-α釋放加強作用無法被TACE或IκB kinase抑制劑所抑制，但可以被蛋白合成抑制劑(cycloheximide)及MAP/ERK kinase抑制劑所阻斷。有趣的是TNF-α的mRNA表現不論是用LPS或PMA刺激，在高張環境下反而會明顯降低。而在LPS刺激下，THP-1細胞內的ERK1/2蛋白會在高張環境中重新再次磷酸化，而這樣的ERK活化對於TNF-α的加強釋放作用是必要的。
TNF-α所引發的基質金屬水解酶也參與了發炎和癌細胞侵襲轉移的過程。實驗中發現N-hydroxy cinnamoyl phenalkyl amides的類似物N-2-(4-bromophenyl) ethyl caffeamide (EK5) 在THP-1細胞中可以有效抑制TNF-α所引發的MMP-9蛋白表現。而不僅是MMP-9蛋白，其mRNA也同樣會被EK5依濃度梯度所抑制。NF-κB已知是一種製造MMP-9所需要的轉錄因子，其可以被各種發炎前細胞激素所活化並促進發炎反應。藉由轉染實驗送入的NF-κB報導基因，本研究發現EK5可以明顯壓制NF-κB的訊息傳遞，不過EK5對於NF-κB訊息傳遞上游的IκBα的降解以及NF-κB本身的轉位(translocation)卻沒有影響。有趣的是透過染色體免疫沉澱法(chromatin immunoprecipitation)，得知細胞內的p65蛋白和MMP-9的啟動子基因序列之間的結合會被EK5完全阻斷，不過p65上的磷酸化並不受藥物所影響。
總合結論，在LPS或PMA所活化的THP-1細胞中，高張環境所加強的TNF-α釋放需要ERK的活化，而且可能不需經由TACE的切除作用。而本研究所發現的這種轉錄後(post-transcriptional)調控方式可以引發已活化的血癌細胞製造出“巨量”的TNF-α，對於臨床上惡病質現象多了一個可深入探討的治療途徑。本研究也證明了EK5具有一個嶄新的分子機轉活性，其在人類單核球細胞中會阻斷核內NF-κB的訊息而抑制TNF-α所引發的MMP-9蛋白產生，至於其抗發炎的詳細機制仍需要更多的研究及動物實驗加以證實。

關鍵字

腫瘤壞死因子 ； 單核球 ； 高張 ； 內吞作用 ； 轉錄後調控 ； 第九型基質金屬蛋白酵素

並列摘要

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine and involved in the pathogenesis of acute myeloid leukemia and sepsis. It is primarily produced and/or secreted from macrophages and monocytes, especially in response to endotoxin stimulation. TNF-α is also synthesized as a transmembrane precursor (proTNF-α) and can be cleaved and released by TNF-α converting enzyme (TACE). Hypertonic stress enhances tumor necrosis factor (TNF)-α expression in activated monocytes. However, the underlying mechanism is unknown. The produced TNF-α is primarily cleaved and released by TNF-α-converting enzyme (TACE), and the surface expression of TACE is down-regulated by endocytosis. As hypertonicity inhibits endocytosis, we evaluated the mechanism of hypertonicity-induced TNF-α release from activated human monocytic THP-1 cells.
The released TNF-α could induce inflammation and activate cells to secrete proteolytic enzymes. Aberrant remodeling of the extracellular matrix occurs in many pathological processes, and its breakdown is mainly accomplished by matrix metalloproteinases (MMPs). Our results show that hypertonic sucrose and NaCl significantly enhanced TNF-α release from THP-1 cells upon LPS or PMA stimulation. Hypertonic sucrose and other endocytosis inhibitors increased surface expression of TACE, but their effects on TNF-α release were inconsistent. This enhancement by hypertonicity was not attenuated by inhibition of TACE or IκB kinase, but it was blocked by cycloheximide and a MAP/ERK kinase inhibitor. The LPS- or PMA-induced TNF-α mRNA expression was not increased; rather, it was inhibited by hypertonicity. ERK1/2 was re-activated after sucrose treatment in LPS-stimulated THP-1 cells.
TNF-α-induced MMPs would participate in the course of inflammation and tumor invasion. In another study, we investigated the intracellular mechanism for the inhibitory effects of an analogue of N-hydroxycinnamoylphenalkylamides, N-2-(4-bromophenyl) ethyl caffeamide (EK5), on TNF-α-stimulated expression of MMP-9 in THP-1 cells. Our results show that TNF-α-induced expression of MMP-9 at both mRNA and protein levels was completely blocked by EK5 in a concentration-dependent manner. Nuclear factor-κB (NF-κB), a key transcription factor for the production of MMP-9, can be activated by various proinflammatory cytokines and promotes inflammation. We found that EK5 markedly suppressed NF-κB signaling as detected by the NF-κB reporter gene assay but had no effects on the degradation of IκBα or translocation of NF-κB. Interestingly, chromatin immunoprecipitation results revealed that the association between p65 and MMP-9 promoter gene was completely abrogated by EK5, but the p65 phosphorylation was not affected.
In conclusion, hypertonicity-enhanced TNF-α protein synthesis from LPS- or PMA-activated THP-1 cells requires ERK activation and may proceed without TACE. A vast amount of TNF-α production was regulated by a crucial post-transcriptional manner in activated human monocytic leukemia cells, and it may possibly be contributed to the cachexia condition. Our findings also suggest that EK5 inhibits TNF-α-induced MMP-9 production through the nuclear-targeted down-regulation of NF-κB signaling in human monocytic cells and this may provide a novel molecular basis of EK5 activity. Further studies are needed to verify its anti-inflammatory effects.

並列關鍵字

TNF ； monocyte ； hypertonicity ； endocytosis ； post-transcriptional control ； MMP-9 ； NF-κB

參考文獻

Aggarwal, B. B. 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 3: 745-56.

Aggarwal, B. B., Vijayalekshmi, R. V. and Sung, B. 2009. Targeting inflammatory pathways for prevention and therapy of cancer: short-term friend, long-term foe. Clin Cancer Res 15: 425-30.

Aksoy, E., Vanden Berghe, W., Detienne, S., Amraoui, Z., Fitzgerald, K. A., Haegeman, G., Goldman, M. and Willems, F. 2005. Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4. Eur J Immunol 35: 2200-9.

Alfieri, R. R. and Petronini, P. G. 2007. Hyperosmotic stress response: comparison with other cellular stresses. Pflugers Arch 454: 173-85.

Angle, N., Hoyt, D. B., Cabello-Passini, R., Herdon-Remelius, C., Loomis, W. and Junger, W. G. 1998. Hypertonic saline resuscitation reduces neutrophil margination by suppressing neutrophil L selectin expression. J Trauma 45: 7-12; discussion 12-3.