- 學位論文
Poly(ADP-ribose) Polymerase-1調控巨噬細胞NLRP3發炎小體活化及紫外光B誘發角質細胞發炎反應之角色
The Roles of Poly(ADP-ribose) Polymerase-1 in Regulation of NLRP3 Inflammasome Activation in Macrophages and UVB-induced Inflammation in Keratinocytes
摘要
Poly(ADP-ribose) polymerase-1 (PARP-1)是廣為熟知的修復去氧核醣核酸(DNA)起始酵素，因為它可以催化二磷酸腺苷核糖的聚合反應，修飾並調控DNA修復相關的酵素。更多研究逐漸發現，PARP-1在許多疾病模式中會調控發炎反應，因此我們致力於研究PARP-1如何調控NLRP3發炎小體(inflammasome)活化以及紫外光B誘發之發炎反應。位於胞內的核苷酸結合寡聚化結構域樣受體NLRP3，由於它可以偵測到廣泛的病原體入侵以及源自於細胞損傷釋出的危險訊號因子，進而啟動下游的免疫反應，是很重要的先天免疫反應受體。我們的研究發現，在小鼠骨髓分化巨噬細胞中，NLRP3刺激劑誘發的caspase-1活化及白介素-1貝他(IL-1)釋放，都受到PARP-1基因缺損或是PARP-1抑制劑所抑制。然而，活化NLRP3發炎小體的第一步訊息並未受到PARP-1基因缺失影響。除此之外，三磷酸腺苷(ATP)誘發之胞內氧化自由基(ROS)，在PARP-1缺損之小鼠骨髓誘發巨噬細胞產生的較少，因而導致發炎小體複合體聚集較少。PARP-1會在ATP的刺激下從細胞核內轉移到細胞質，去催化NLRP3的聚(ADP-核糖基)化(PARylation)修飾，進而促進NLRP3發炎小體複合體的組成。PARP-1同時也做為NLRP3和TXNIP蛋白的橋樑，並參與在NLRP3/TXNIP複合體的生成，因此促進發炎小體的活化。我們的研究找到PARP-1調控NLRP3發炎小體活化的嶄新機制，因此PARP-1有潛力成為發展治療IL-1相關疾病的藥物標的。 紫外光會損傷皮膚，造成曬傷、發炎以及相關的皮膚疾病，在第二部分的研究中，我們想了解PARP-1如何調控紫外光B造成的皮膚損傷以及和表皮生長因子受體(EGFR)間的交互作用和其相關的發炎反應。我們發現PARP-1的基因缺損會惡化小鼠對於紫外光B誘發之發炎反應、水分散失以及背部皮膚損傷。在人類原發性角質細胞中，紫外光B會活化PARP-1並在PARP-1基因默化時造成更嚴重的DNA損傷。而紫外光B誘發之環氧合酶-2(COX-2)和基質金屬蛋白酶-1(MMP-1)的表現，會受到PARP-1基因靜默或其抑制劑所抑制，然而，腫瘤壞死因子阿法(TNF-和白介素-8 (IL-8)的生成卻因此而增強。由於EGFR基因靜默或是用其抑制劑gefitinib會抑制紫外光B誘發之COX-2、TNF-以及IL-8的表現量，顯示EGFR經由旁分泌的活化，會進而影響紫外光B誘發之發炎反應。西方墨點法的結果顯示，抑制PARP-1會降低紫外光B引發之EGFR和p38 MAPK活化，而p38 MAPK抑制劑會明顯抑制紫外光B誘發之發炎反應。值得注意的是，PARP-1或是EGFR的基因靜默都會降低紫外光B誘發之ROS生成，而抗氧化劑NAC會降低紫外光B引發的EGFR-p38 MAPK訊息傳遞和PARP-1活化。以上結果顯示，在EGFR、PARP-1和ROS之間有相互調控的迴路。因此，紫外光B的刺激下，PARP-1不只是參與DNA修復的功能，同時也會經由EGFR活化及ROS生成間的交互作用，造成下游p38 MAPK的訊息傳遞，進而調控角質細胞的發炎反應。
並列摘要
Poly(ADP-ribose) polymerase-1 (PARP-1) has been well known as the initiator of DNA repair by catalyzing the polymerization of ADP-ribose unit to DNA repair machinery. Emerging studies have shown that PARP-1 can also regulate inflammatory responses in various disease models. Thus we were interested in elucidating whether PARP-1 can regulate NLRP3 inflammasome activation and UVB-induced inflammation. The intracellular Nod-like receptor NLRP3 is one of the most crucial innate immune receptors because of its broad specificity in mediating immune response to pathogens invasion and danger signals associated with cellular damage. In our study, we found that NLRP3 stimuli-induced caspase-1 maturation and IL-1β production were impaired by PARP-1 knockout or PARP-1 inhibition in bone marrow-derived macrophages (BMDM). The step 1 signal of NLRP3 inflammasome activation was not affected by PARP-1 deficiency. Moreover, ATP-induced cytosolic ROS production was lower in Parp-1-/- BMDM resulting in the decreased inflammasome complex assembly. PARP-1 can translocate to cytosol upon ATP stimulation and trigger the PARylation modification on NLRP3, leading to NLRP3 inflammasome assembly. PARP-1 was also a bridge between NLRP3 and thioredoxin interacting protein (TXNIP) and participated in NLRP3/TXNIP complex formation for inflammasome activation. Overall, PARP-1 positively regulates NLRP3 inflammasome activation via increasing ROS production and interaction with TXNIP and NLRP3, leading to PARylation of NLRP3. Our data demonstrate a novel regulation mechanism for NLRP3 inflammasome activation by PARP-1, which serves as a potential target in the treatment of IL-1β associated inflammatory diseases. UV irradiation can injure the epidermis, resulting in sunburn, inflammation and cutaneous tissue disorders. In the second part of our study, we set out to understand how PARP-1 regulates UVB irradiation-induced skin injury and interplays with EGFR to mediate inflammation response. We found that PARP-1 deficiency exacerbated the UVB-induced inflammation, water loss and back skin damage in mice. In human primary keratinocytes, UVB can activate PARP-1 and enhance DNA damage upon PARP-1 gene silencing. PARP-1 silencing and PARP inhibitor olaparib can suppress UVB-induced COX-2 and MMP-1 expression, but enhance TNF-α and IL-8 expression. In addition, EGFR silencing or EGFR inhibition by gefitinib can decrease UVB-induced COX-2, TNF-α and IL-8 expression, suggesting EGFR activation via paracrine action can mediate UVB-induced inflammation responses. Immunoblotting data revealed that PARP-1 inhibition decreases UVB-induced EGFR and p38 MAPK activation. Pharmacological inhibition of p38 MAPK also dramatically led to attenuation of UVB-induced inflammatory gene expression. Of note, genetic ablation of PARP-1 or EGFR can attenuate UVB-induced ROS production, and antioxidant NAC can attenuate UVB-induced EGFR-p38 MAPK signaling axis and PARP-1 activation. These data suggest the regulatory loops among EGFR, PARP-1 and ROS upon UVB stress. PARP-1 not only serves DNA repair function but also orchestrates interactions to EGFR transactivation and ROS production, leading to p38 MAPK signaling for inflammatory gene expression in keratinocytes.