Wnt5a Induces Endothelial Inflammation via β-Catenin−Independent Signaling Jihun Kim, Jungtae Kim, Dong Wook Kim, Yunhi Ha, Min Hwan Ihm, Hyeri Kim, Kyuyoung Song and Inchul Lee This information is current as of September 29, 2021. J Immunol 2010; 185:1274-1282; Prepublished online 16 June 2010; doi: 10.4049/jimmunol.1000181 http://www.jimmunol.org/content/185/2/1274 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/06/16/jimmunol.100018 Material 1.DC1 References This article cites 43 articles, 18 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/185/2/1274.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Wnt5a Induces Endothelial Inflammation via b-Catenin–Independent Signaling Jihun Kim,* Jungtae Kim,† Dong Wook Kim,† Yunhi Ha,‡ Min Hwan Ihm,† Hyeri Kim,‡ Kyuyoung Song,‡ and Inchul Lee* Wnt signaling has been implicated in certain inflammatory diseases. However, the biological role in the inflammatory regulation remains to be characterized. We investigated the regulation by Wnt signaling in endothelial cells, which are active participants and regulators of inflammation. Wnt5a induces cyclooxygenase-2 expression and enhances inflammatory cytokines rapidly, whereas Wnt3a shows limited effects, suggesting a role for b-catenin–independent Wnt signaling in the inflammatory endothelial activation. Pulse-like treatment of Wnt5a induces cyclooxygenase-2 more efficiently than continuous treatment. Wnt5a and TNF-a regulate subsets of cytokines overlapping, only partially, with each other. Calcium ionophore enhances endothelial inflammation similarly, whereas calcium chelators and protein kinase C inhibitor block Wnt5a-induced activation, suggesting Downloaded from a role for the Wnt/Ca2+/protein kinase C pathway in endothelial inflammatory regulation. Wnt5a activates RelA nuclear trans- location and DNA binding. Activated blood vessels, histiocytes, and synoviocytes express Wnt5a in atherosclerosis and rheumatoid arthritis but not in normal tissue, supporting the role of Wnt5a as an inflammatory mediator in vivo. Our data suggest that endothelial inflammation is regulated by a dual system consisting of b-catenin–independent Wnt signaling and TNF-a–mediated signaling. The Journal of Immunology, 2010, 185: 1274–1282. http://www.jimmunol.org/ nt/b-catenin signaling has been implicated in di- liferation and migration (16–18), as well as endothelial differentia- verse developmental and biological regulations (1–3). tion of embryonic stem cells (19). W b-catenin–independent Wnt signaling is also involved Inflammation is a critical defense mechanism against various in various biological functions, such as vertebral development, cell harmful stimuli. However, aberrant regulation may lead to various motility and adhesion, and cancer invasiveness (4–7). Recently, inflammatory diseases. NF-kB is a key transcriptional regulator Wnt5a, a prototype Wnt for b-catenin–independent signaling, has playing a central role in the onset of inflammation (20). Typically, been implicated in certain inflammatory diseases (8, 9). In NF-kB is activated by prototype inflammatory mediators, such as synoviocytes of rheumatoid arthritis, the expression of Wnt5a and TNF-a and IL-1b, via activation of IkB kinases (IKKs), which frizzled 5 is enhanced significantly (10), and the blockade of carry out the phosphorylation-dependent degradation of IkB in- by guest on September 29, 2021 signaling inhibits synoviocyte activation (11). Wnt5a and frizzled hibitors upon inflammatory stimuli (20–22). Recently, additional 5 are also expressed in activated macrophages, APCs, and tu- regulators of NF-kB were reported, indicating a complexity in the berculous granulomas (12). Wnt5a is induced by LPS/IFN-g in regulation of inflammation (23). Given the divergent biological re- human macrophages and is detectable in the sera of patients with quirements for inflammation and various clinical presentations severe sepsis (13). Those reports suggested a pathobiological role of inflammatory diseases, a complex regulatory mechanism, rather for Wnt signaling in human inflammatory diseases. However, it than a simple on–off function, would be required for subtle qual- remains to be characterized whether and how Wnt signaling is itative and quantitative regulations of inflammation. involved in inflammatory regulation. Recently, we reported that thyroid cancer-1 (TC1) (C8orf4) indu- Wnt signaling is highly dependent on the cell context (14). En- ces endothelial inflammation (24). Because TC1 is a regulator of dothelial cells are active participants and regulators of inflammatory the Wnt/b-catenin pathway (25, 26), we investigated a potential role processes (15). Wnt5a has been implicated in endothelial pro- for Wnt signaling in endothelial inflammatory regulation. Intrigu- ingly, we observed that b-catenin–independent signaling, rather than classical Wnt/b-catenin signaling, plays a significant role in *Department of Pathology, ‡Department of Biochemistry and Molecular Biology, and †Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College the inflammatory regulation of human endothelial cells. Our data of Medicine, Seoul, Korea suggested a complex dual-regulatory system of endothelial inflam- Received for publication January 19, 2010. Accepted for publication May 11, 2010. mation, consisting of b-catenin–independent Wnt signaling and This work was supported by a Doyak Research Program grant through the National prototype inflammatory mediator-dependent signaling. Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20090079398). Address correspondence and reprint requests to Dr. Inchul Lee, Department of Pathol- Materials and Methods ogy, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap- Cells and reagents Dong, Songpa-Gu, Seoul 138-736, Korea. E-mail address: [email protected] The online version of this article contains supplemental material. Humanaortic endothelial cells (HAECs)and HUVECs(Lonza, Walkersville, MD) were cultured in 0.1% gelatin-coated dishes containing EGM-2 basal Abbreviations used in this paper: CE, cytoplasm extract; COX, cyclooxygenase; GSK, medium (Lonza) at 37˚C in humidified atmosphere with 5% CO . Experi- glycogen synthase kinase; HAEC, human aortic endothelial cell; HSF1, heat shock 2 transcription factor 1; IKK, IkB kinase; NE, nuclear extract; NQO1, NADPH dehydro- ments were done using cells from passages six through nine. Human rWnt5a genase, quinone 1; PKC, protein kinase C; ROS, reactive oxygen species; TC1, thyroid and Wnt3a proteins (Millipore, Billerica, MA) and TNF-a (Sigma-Aldrich, cancer-1; WE, whole-cell extract. St. Louis, MO) were purchased commercially. BMS-345541, A23187, lith- ium chloride, BAPTA-AM, and SP600125 were also purchased from Sigma- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Aldrich. Go6953 was purchased from Calbiochem (San Diego, CA). An www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000181 The Journal of Immunology 1275 IgG1 mouse mAb against TNF-a was purchased from Santa Cruz Bio- [14C]Sucrose permeability test technology (Santa Cruz, CA; sc-52746). IgG1 mouse mAbs against heat 3 4 shock transcription factor 1 (HSF1) and NADPH dehydrogenase, quinone A total of 4 10 HUVECs/well were seeded on a Transwell filter (Corning, 1 (NQO1) (sc-52746; Santa Cruz Biotechnology) were used as controls. Lowell, MA) and incubated until a complete monolayer was formed, changing the media every 2 d, as described previously (24). Then, 100 mg/ml Wnt5a Plasmids and transfection and/or inhibitors were administered as described, and 50 ml(0.8mCi [0.0296 MBq]/ml) [14C]sucrose (Amersham Pharmacia Biotech) was added to the Mammalian expression vectors pCDNA3-HA-ICAT, pCDNA3-HA-GSK3b, upper compartment. After incubation for 30 min, the amount of radioactivity and pCDNA3-myc-Cby were cloned using pcDNA3 vector (Invitrogen, that diffused into the lower compartment was measured using a liquid scin- Carlsbad, CA). All clones were confirmed by DNA sequencing. HAEC tillation counter (Tri-Carb 3100TR; Packard Instrument, Meriden, CT). Experi- and/or HUVEC transfection was done using Effectene transfection reagent ments were repeated in triplicate. (Qiagen, Hilden, Germany), following the manufacturer’s instructions. For the transfection, 400 ng DNA was applied to 1.2 3 105 cells in a six-well Matrigel-invasion assay chamber. Controls were mock and/or vector transfected. The transfection Matrigel-invasion assay was performed using Matrigel Invasion Chambers efficiency was monitored using transfected b-galactosidase expression. (BD Biosciences,