DJ-1缺失在神經退化疾病與癌症生成之探討

Abstract

失去DJ-1（PARK7）功能的突變與早發性巴金森氏症相關，但其分子機轉尚未清楚。因此，本篇主要目的是要比較野生型及DJ-1剔除鼠在脂多醣(LPS)刺激下，黑質中多巴胺神經的敏感性和探討所存在的細胞及分子機轉。我們的結果發現，在自然的狀況下，DJ-1剔除鼠黑質和DJ-1減少的微神經膠細胞(microglia)中的IFN-(樞紐性細胞激素)和I-TAC (下游調控者)表現上升，並且在DJ-1缺失的狀況下，脂多醣的刺激伴隨著更多的細胞激素/趨化激素增加。除此之外，直接在黑質內給予脂多醣刺激，造成DJ-1剔除鼠比野生鼠有更多的黑質中多巴胺神經死亡及紋狀體中多巴胺含量的減少。再者，微神經膠細胞易感於脂多醣刺激而釋放IFN-和I-TAC是透過強化NF-B的訊號傳遞，而這訊息可被NF-B抑制劑阻斷。在神經及微神經膠共同培養的細胞中，脂多醣所誘導的神經死亡可被DJ-1減弱的微神經膠細胞增強，透過中和性IFN-和I-TAC抗體的運用可阻斷這個現象。這些發現指出，DJ-1 缺失的微神經膠細胞會分泌較多的IFN-和I-TAC。此等發炎物質會加速神經的死亡。因此，基因缺陷(DJ-1)與發炎因子(脂多醣)間的交互作用能促進巴金森氏症病程的進展。 在腫瘤生成方面，DJ-1是一個致癌蛋白，能透過抑制細胞凋亡促進癌細胞的存活。然而，DJ-1也扮演調控介白素-1表現的角色，是否因DJ-1缺失而建立起的發炎微環境會影響癌症的發展仍然不清楚。因此，本研究將針對黑色素細胞在野生型及DJ-1剔除鼠進行轉移能力的比較。首先，在小鼠的股靜脈中打入B16F10黑色素細胞瘤細胞 (6×104)，並比較野生型及DJ-1剔除鼠間肺部腫瘤的形成ぶ肺部介白素-1和血清中細胞激素的量及MDSC細胞聚集的情形。第二，給予帶有腫瘤的小鼠介白素-1中和抗體，觀察是否介白素-1與癌症轉移相關。最後，分別給予Raw 264.7巨噬細胞株及B16F10黑色素細胞瘤細胞株，DJ-1的短髮夾核糖核酸和介白素-1重組蛋白來探討可能的分子機轉。 我們的研究結果指出介白素-1可增強細胞培養中黑色素細胞的生存ぶ群落形成，並且在DJ-1剔除鼠和DJ-1減量的巨噬細胞都有介白素-1增加的現象。而在DJ-1剔除鼠肺部中具免疫抑制性的MDSC細胞堆積和黑色素細胞瘤形成與介白素-1的增加有關，若給予DJ-1剔除鼠介白素-1的中和抗體可減少這兩個現象。總結來說，這些結果指出在DJ-1剔除鼠免疫抑制性的組織微環境中可增強癌症的轉移，並且介白素-1在促進癌症轉移上扮演重要的角色。 此外，除了腦下垂體可以分泌生長激素，其在肺臟的生長和功能有著自泌素的角色。然而，因內生性的缺失，引起肺臟生長激素不正常的表現，是否與腫瘤的生長有關仍然不清楚。從微陣列分析結果發現，在DJ-1剔除鼠肺臟中生長激素會增加，而類胰島素生長因子並無增加。我們進一步檢視生長激素對於黑色素細胞瘤細胞株的生長效應。生長激素能增加B16F10細胞的增生、群落形成和細胞侵襲能力。再者，生長激素可增加B16F10細胞表現基質金屬蛋白酶2 、9和13。最後，我們發現在C57/B6鼠施打生長激素會增加肺部腫瘤形成。總結，上述結果指出肺部生長激素表現量增加會增強B16F10細胞在肺部的惡性效應，並且在黑色素細胞瘤肺部轉移腫瘤生成的初步階段，扮演重要角色。 在我們的研究中發現，DJ-1缺失引發在組織微環境中細胞激素上昇，造成神經退化及促進腫瘤生長現象。DJ-1因此成為兩疾病之間一新關係，未來，抗發炎物質抑制劑的應用也許可為此類基因缺陷病人帶來更好的治療效果。

Mutation of DJ-1 (PARK7) has been linked to the development of early-onset Parkinson’s disease (PD). However, the underlying molecular mechanism is still unclear. This study is aimed to compare sensitivities of nigrostriatal dopaminergic neurons to lipopolysaccharide (LPS) challenge between DJ-1 knockout (KO) and wild-type (WT) mice, and explore the underlying cellular and molecular mechanisms. Our results found that basal levels of IFN- (the hub cytokine) and I-TAC (a downstream mediator) were elevated in substantia nigra of DJ-1 KO mice and in microglia cells with DJ-1 knockdown, and the release of cytokine/chemokine was greatly enhanced following LPS administration in DJ-1 deficient conditions. In addition, direct intranigral LPS challenge caused a greater loss of nigrostriatal dopaminergic neurons and striatal dopamine content in DJ-1 KO mice than in WT mice. Furthermore, the sensitization of microglia cells to LPS challenge to release IFN- and I-TAC was via the enhancement of NF-B signaling, which was antagonized by NF-B inhibitors. LPS-induced increase of neuronal death in neuron-glia co-cultures can be enhanced by DJ-1 knockdown in microglia, which was antagonized by neutralizing antibodies against IFN- or I-TAC. These results indicate that DJ-1 deficiency sensitizes microglia cells to release IFN- and I-TAC and causes inflammatory damage to dopaminergic neurons. The interaction between genetic defect (i.e. DJ-1) and inflammatory factors (e.g. LPS) may contribute to the development of PD. In cancer growth, DJ-1 is an oncoprotein which promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×104) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1) neutralizing antibody to see whether IL-1 is involved in the cancer metastasis. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1 to explore underlying molecular mechanisms. Our results showed that IL-1 enhanced survival and colony formation of cultured melanoma cells, and that IL-1 levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma nodule in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1 neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung metastasis of cancer, and IL-1playsan important role in promoting the cancer metastasis. Although growth hormone (GH) is mainly secreted from pituitary gland, it also plays an autocrine role in lung growth and pulmonary function. However, it is not clear whether unusual expression of GH in lung derived from endogenous defects is related to tumor growth. From microarray analysis, the results showed that GH but not insulin-like growth factor-1 (IGF-1) level was increased in the lung of DJ-1 knockout mice. We then examined the effect of GH on the growth of melanoma cells in the cell cultures. GH increased cell proliferation, colony formation and invasion of B16F10. Furthermore, GH also increased the expression of MMP-2, MMP-9 and MMP-13 in B16F10 cells. Finally, we found that administration of GH enhanced the lung nodules formation in C57/B6 mice. Taken together, these results indicate that increase of GH expression in lung may enhance the malignant effects of B16F10 cells and lung nodule formation of melanoma in early stage of pulmonary metastasis. In conclusion, we found that DJ-1 deficiency enhanced cytokine up-regulation in tissue microenvironment, caused neurodegeneration and promoted cancer cells growth. Therefore, DJ-1 becomes a new relationship between the two diseases and application of anti-inflammation strategy may have benefit for such genetic-disorder patients.