Pathogenic roles of decoy receptor 3 (DcR3) in tumor angiogenesis, osteoclast differentiation and inflammatory modulation

近來的研究指出腫瘤壞死因子受體家族的一員decoy receptor 3 (DcR3)，可以與 FasL, LIGHT 及TL1A 結合並中和這些屬於腫瘤壞死因子家族細胞激素的反應。DcR3會大量表現於一些腫瘤組織及癌症病人的血清中，因此DcR3被認為可藉由其中和FasL及LIGHT的作用來幫助腫瘤細胞躲避免疫攻擊，因此DcR3是一種新的免疫抑制因子。為了解DcR3對腫瘤發生是否有其他弁遄A本研究進行對DcR3在血管新生，嗜骨細胞生成及發炎反應三方面探討其病理角色。 由血管內皮細胞分泌的TL1A在調節內皮細胞凋亡扮演很重要的角色。之前的研究指出TL1A可以和death receptor 3 (DR3) 及 DcR3 結合。本研究證實了DcR3可以在人類臍靜脈內皮細胞誘發血管新生現象。DcR3促進人類臍靜脈內皮細胞增生，移行，並提升第二號基質金屬蛋白酵素的基因表現和酵素活性。此外，DcR3在體外細胞模式也促使人類臍靜脈內皮細胞分化成束狀類似血管結構及在體內動物模式產生血管新生現象。而TL1A抗體及DR3抗體在人類臍靜脈內皮細胞均可產生與DcR3極為類似的作用。反過來看，人類胸主動脈內皮細胞 不會表現TL1A，在這類細胞DcR3就不能產生細胞增生，移行，及類血管狀的分化。由這些結果說明了DcR3 可能不只是可以幫助腫瘤細胞躲避宿主免疫系統的攻擊而存活，在內皮細胞也可以經由阻斷TL1A 的作用而誘發血管新生作用。DcR3 幫助腫瘤細胞成長的病理學上的角色擴展了未來在抗血管新生治療上將它作為治療標的的可能性。 近來的研究顯示DcR3也可以經由反向訊息傳遞來調控單核球的弁遄C本實驗我們證實了DcR3可以誘導人類單核細胞，鼠類巨噬細胞株RAW264.7及骨髓細胞分化形成噬骨細胞。DcR3誘導分化形成的細胞顯示了噬骨細胞的特徵，包括了多核且巨大的外觀，噬骨作用，表現TRAP、CD51/61及MMP-9等嗜骨細胞特有的分子。給予DcR3可以經由ERK及p38 MAPK的訊息傳導路徑，誘發一個強力的致使噬骨細胞生成的細胞激素TNF-a 的釋出﹔若給予TNFR-Fc則可以阻斷DcR3所誘發的噬骨作用。由以上結果，我們推論DcR3經由雙重路徑反向訊息傳遞活化ERK及p38 MAPK進而刺激TNF-a 生成，因此DcR3是噬骨細胞生成的的重要調控因子。這個結果也說明了DcR3可能在癌細胞骨轉移時的噬骨活性上扮演一個重要的角色。 為了解DcR3在發炎反應的角色，在此我們利用人類臍靜脈內皮細胞偵測與單核細胞的附著作用。結果顯示，人類臍靜脈內皮細胞給予DcR3可以促進單核細胞株THP-1及U937貼附在內皮細胞。由流式細胞儀及酵素連結免疫吸附分析結果顯示DcR3處理過的人類臍靜脈內皮細胞顯著增加了第一型胞內黏著因子 (ICAM-1) 及第一型血管細胞黏著因子 (VCAM-1) 的表現，而在單核細胞本身則無增加的現象。我們也證實了 DcR3可以增加人類臍靜脈內皮細胞釋出白介素-8 (IL-8)。將第一型胞內黏著因子，第一型血管細胞黏著因子及白介素-8三者的抗體與 DcR3共同處理則幾乎可以完全阻斷 DcR3所引起促進單核細胞貼附的作用。反轉錄酶聚合酶鏈反應及指標性基因分析結果也說明了DcR3經由基因轉錄來增加黏著因子與白介素-8的表現。而給予PDTC的實驗也證實了NF-kB參與了這些因子的基因轉錄表現。DcR3所引起的IKK活化，IkB減少，p65的核轉移及NF-kB與DNA的結合活性均證實了這個論點。在先前的研究結果證明了DcR3可以經由拮抗內生性的TL1A而誘發血管新生現象，但是 TL1A 抗體在人類臍靜脈內皮細胞並不能產生類似DcR3所誘發促進細胞貼附的現象。此外，DcR3在不會表現TL1A的人類胸主動脈內皮細胞也不能引起細胞貼附現象。整體而言，本實驗我們闡明了DcR3在內皮細胞經由NF-kB的活化，增加了黏著因子與白介素-8的基因轉錄，進而促進單核細胞貼附到內皮細胞。這個作用並非拮抗TL1A而來，而是經由某一尚未被證實的標的。這個研究結果也說明了DcR3在發炎與癌症發展的相關連間扮演重要角色。Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily can prevent cytokine responses of FasL, LIGHT and TL1A by binding and neutralization. In addition, it has been demonstrated that DcR3 is up-regulated in various types of cancers, therefore DcR3 is regarded as an immuno-suppressor to down-regulate immunosurveillance of FasL and LIGHT functions. To understand the other possible roles of DcR3 in promoting tumor progress, this study is divided into three sections to delineate the pathological roles of DcR3 in angiogenesis, osteoclast formation and inflammation. TL1A is a member of the TNF superfamily and plays an important role in regulating endothelial cell apoptosis. Previous study shows TL1A is able to interact with death receptor 3 (DR3) and DcR3. Here we demonstrate that DcR3 is able to induce angiogenesis in human umbilical vein endothelial cells (HUVEC). DcR3 promotes HUVEC proliferation, migration and up-regulates matrix metalloproteinase-2 (MMP-2) mRNA expression and enzyme activity. Furthermore, DcR3 enhances EC differentiation into cord vascular-like structures in vitro as well as neovascularization in vivo. The effects of DcR3 on HUVEC are also mimicked by anti-TL1A and anti-DR3 antibodies. In contrast, human aortic endothelial cells (HAEC), which do not express TL1A, are not responsive to DcR3 treatment, including cell proliferation, migration and angiogenic differentiation. These data demonstrate DcR3 might not only help tumor cells to escape immune surveillance, but also induce angiogenesis by blocking TL1A action in EC. The pathological role of DcR3 in promoting cancer progress raises the possibility to target DcR3 for anti-angiogenic therapy in the future. Recent evidence indicates that DcR3 can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61 and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-a release through ERK, p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-a synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteoclastic bone metastases. To elucidate the role of DcR3 in inflammation, we herein show that HUVECs pretreated with DcR3 enhances the adhesion of the monocytic cells, THP-1 and U937. Flow cytometry and ELISA show that DcR3-treated HUVECs exhibit significant increases in ICAM-1 and VCAM-1 expressions, while the expressions of adhesion molecules in monocytes were not changed. We also show the ability of DcR3 to increase the secretion of IL-8 from HUVECs. Co-incubation of antibodies of ICAM-1, VCAM-1, and IL-8 abolish the DcR3-induced adhesion effect. RT-PCR and reporter assays reveal that the expressions of adhesion molecules and IL-8 are through gene transcription. Experiment with PDTC indicates involvement of the NF-κB signaling pathway. Supporting this notion, DcR3 induces IKK activation, IκB degradation, p65 nuclear translocation, and NF-κB-DNA-binding activity. The enhanced cell adhesion by DcR3 in HUVECs is not mimicked by the TL1A antibody, which has been shown to elicit angiogenesis through neutralizing endogenous TL1A. Moreover, DcR3-induced cell adhesion could be detected in HAEC where TL1A expression is lacking. Taken together, we demonstrate that DcR3 increases monocyte adhesion to EC via activation of NF-κB signaling, thereby transcriptionally upregulating adhesion molecules and IL-8 in EC. This novel action appears not to be due to TL1A neutralization, but occurs through an as yet undefined target(s). This study implicates a role of DcR3 in the relationship between inflammation and cancer development.Index Abbreviations ........................................ 3 Abstract ........................................ ... 5 Abstract in Chinese .................................. 7 Introduction ........................................ 9 Materials and Methods .............................. 18 Section I. Soluble Decoy Receptor 3 Induces Angiogenesis by Neutralization of TL1A, a Cytokine Belonging to Tumor Necrosis Factor Superfamily and Exhibiting Angiostatic Action Results ........................................... 32 Discussion ........................................ 37 Figures .......................................... 40 Section II. Decoy Receptor 3 Induces Osteoclast Formation from Monocyte/Macrophage Lineage Precursor Cells Results ............................................ 51 Discussion .......................................... 57 Figures ........................................... 62 Section III. Decoy Receptor 3 Increases Monocyte Adhesion to Endothelial Cells via NF-kB-Dependent Upregulation of ICAM-1, VCAM-1 and IL-8 Expression Results ............................................. 71 Discussion ......................................... 75 Tables ............................................. 78 Figures ............................................ 80 References ........................................ 8