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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 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 (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, San Jose, CA), according to the manufacturer’s proto- cols, as described previously (26). Briefly, 4 3 104 HAECs were placed in Real-time and semiquantitative RT-PCR the top chamber in the media containing 0.1% FCS; 100 ng/ml Wnt5a was added to the bottom chamber as a chemoattractant. After incubation for Real-time PCR was done as described previously (24). Total RNA was 12 h at 37˚C, cells on the top surface of the filter were wiped off with extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized a cotton-tipped swab, and the filter was fixed in methanol and stained using using Superscript II reverse transcriptase (Invitrogen). Quantitative PCR DiffQuik stain. The invasion rate was determined by counting cells at the was performed using a continuous fluorescence-detecting thermal cycler bottom of the filter. Experiments were repeated in triplicate.

ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Fos- Downloaded from ter City, CA) and SYBR Green real-time PCR master mix (Toyobo, Osaka, Measurement of intracellular reactive oxygen species Japan). Measurements were done in triplicate using b-actin as endogenous control. Semiquantitative RT-PCR was described previously (25, 26). PCR Reactive oxygen species (ROS) were detected using the cell-permeable indi- primers are summarized in the supplemental tables. cator, 5-(and-6)-carboxy-29,79-dichlorodihydrofluorescein diacetate. HAECs were treated with Wnt5a and loaded with 20 mM 5-(and-6)-carboxy-29,79- dichlorodihydrofluorescein diacetate for 30 min. Cells were collected, Western blotting and cytokine array washed with cold PBS, and analyzed using a FACScan flow cytometer. http://www.jimmunol.org/ Total or fractionated cell-protein samples were analyzed. Nuclear and cy- Statistical methods toplasmic fractions were separated using a fractionation kit from BioVision (Mountain View, CA). Samples were solubilized in lysis buffer and loaded All measurements are presented as the mean 6 SD. Significance was deter- (20 mg/lane) on 12% SDS-PAGE. Proteins were blotted onto nitrocellulose mined using ANOVA. membranes and probed using rabbit anti-cyclooxygenase (COX)-2, anti- RelA, anti–NF-kB p52, and anti–b-actin antisera (Santa Cruz Biotechnol- ogy). Anti–phospho-JNK antiserum was from Cell Signaling Technology Results (Danvers, MA). After anti-rabbit secondary Ab (Amersham Biosciences, Wnt5a upregulates inflammatory rapidly in endothelial Piscataway, NJ) was applied, blots were visualized using the ECL method (Amersham Biosciences). b-actin was used as loading controls. For the cells profiling of cytokine expression, 50 mg proteins were applied to a human Wnt5a expression was not detected by real-time PCR in HAECs or by guest on September 29, 2021 cytokine array (Raybiotech, Norcross, GA), according to the manufac- HUVECs under the culture condition (data not shown). HAECs turer’s instructions. were then treated using 100 ng/ml purified rWnt5a for up to 6 h, and the expression of inflammatory genes and cytokines was Immunofluorescence microscopy and immunohistochemical measured using real-time PCR. COX-2 was induced robustly in 1 h staining (Fig. 1A). Wnt5a did not upregulate COX-2 in other cell types, For immunofluorescence microscopy, cells grown on cover slips were such as SH-SY5Y, HeLa, HEK293T, and RAW264.7, suggesting immunostained using anti-RelA antiserum. After washing with PBS, FITC- that Wnt5a-induced inflammatory expression was specific labeled anti-rabbit Ig secondary Ab (Jackson ImmunoResearch Laborato- for endothelial cells (Supplemental Fig. 1A). IL-8, IL-6, IL-1a, ries, West Grove, PA) was applied. DNA staining was done using DAPI (Sigma-Aldrich). Cells were viewed using an Olympus BX51 fluorescence LLC2, TLR4, and TLR3 were also upregulated significantly in 1 h microscope. For controls, primary Abs were replaced with normal rabbit and then declined gradually for 6 h (Fig. 1A). In comparison, E- sera. selectin, ICAM1, and CX3CL1 were upregulated steadily for 6 h. Immunohistochemical staining of three atherosclerosis, three rheumatoid TC1 was upregulated similarly. The expression of endothelial arthritis, and control tissue samples was done using goat anti-Wnt5a serum (R&D Systems, Minneapolis, MN) and a Benchmark autostainer (Ventana NO synthase did not change significantly (Fig. 1A). Inducible Medical Systems, Tucson, AZ), as described previously (27). Ag retrieval NO synthase was not detected (data not shown). pretreatment was omitted, and slides were not heated .65˚C. This study The downstream regulation was also analyzed at the protein was approved by the Institutional Review Board of Asan Medical Center. level. Upon Western blotting, COX-2 was upregulated ∼45-fold Because it was a retrospective study without any influence on the diagnosis compared with control at 6 h, as measured by densitometry (Fig. and/or treatment, no written consent was required. The data were handled anonymously following guidelines of the Institutional Review Board. 1B). The expression of inflammatory cytokines was also analyzed using a cytokine array. Wnt5a enhanced the expression of cytokines, including G-CSF, GM-CSF, IL-1a, IL-3, IL-5, IL-6, IL-7, CCL2, ELISA-based NF-kB DNA-binding analysis and CCL8 in 24 h compared with control HAECs (Fig. 1C). We The DNA-binding capacity of p65-containing NF-kB dimers was assessed then compared the downstream regulation pattern of TNF-a,a using ELISA plates containing fixed NF-kB binding-site consensus prototype inflammatory mediator, with the Wnt5a-mediated regu- sequences, following the manufacturer’s instructions (Panomics, Fremont, CA), as described previously (24). Briefly, HAECs were treated using lation pattern. After 10 ng/ml TNF-a treatment for 24 h, GM-CSF, 100 ng/ml Wnt5a for 1 and 6 h. Ten micrograms of nuclear extract was CXCL1, CXCL2, IL-8, IL-10, and CCL2 were significantly up- diluted into binding buffer and incubated for 1 h at room temperature. regulated (Fig. 1D). Our data suggested that downstream regulation Following three washes, primary Ab specific for p65 was added to each profiles by Wnt5a and TNF-a overlapped only partially. To further well, incubated at room temperature for 1 h, followed by HRP-conjugated secondary Ab incubation and chromogen reaction. Experiments were done investigate a potential role for TNF-a in the Wnt5a-mediated in- in triplicate, and optical densities were measured using a SpectraMax flammatory regulation, we treated HAECs using Wnt5a and an microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). IgG1 mouse mAb against TNF-a, 100 ng/ml media. For controls, 1276 Wnt5a INDUCES ENDOTHELIAL INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Wnt5a regulates inflammation genes in HAECs. A, Real-time PCR of inflammation genes at 1, 3, and 6 h after treatment with 100 ng/ml Wnt5a. Experiments were done in triplicate. Measurements were done using SYBR Green real-time PCR using b-actin as endogenous control. Fold differences are presented as mean 6 SD compared with controls. B, Western blotting of COX-2 and densitometric analysis; 20 mg total proteins were loaded per lane, and b-actin was used as loading controls. C, Human cytokine array of HAECs treated with Wnt5a (100 ng/ml) for 24 h and control. 1, G- CSF; 2, GM-CSF; 3, IL-1a; 4, IL-3; 5, IL-5; 6, IL-6; 7, IL-7; 8, CCL2; and 9, CCL8. Fifty micrograms total proteins was loaded for each experiment. Positive and negative controls are indicated in the blot. D, Human cytokine array of HAECs treated with 10 ng/ml TNF-a for 24 h and control. 1, GM-CSF; 2, CXCL1; 3, CXCL2; 4, IL-8; 5, IL-10; and 6, CCL2. E, Real-time PCR of COX-2 expression upon Wnt5a treatment with or without mouse mAb against TNF-a, 100 ng/ml media. Isotype-controlled Abs against NQO1 and HSF1 were used as controls. F, HAEC monolayer permeability test. Complete monolayers of HAEC on a Transwell filter were treated using 100 ng/ml Wnt5a and Wnt3s and/or 10 ng/ml IL-1b for 6 h; the permeability using [14C] sucrose was done as described in Materials and Methods. Experiments were repeated in quadruplicate. G, Matrigel invasion assay was performed using Matrigel Invasion Chambers, as described in Materials and Methods. Experiments were repeated in triplicate. the same amounts of IgG1 mouse mAbs against NQO1 or HSF1, permeability of the HAEC monolayer by ∼50% compared with unrelated intracellular proteins, were applied similarly. The Wnt5a- the control (p , 0.01; Fig. 1F), whereas the same amount of Wnt3a mediated COX-2 upregulation was not inhibited by anti–TNF-a Ab did not enhance it significantly. The invasiveness of HAECs to compared with control Abs (Fig. 1E), suggesting that the Wnt5a- Matrigel was also enhanced significantly when Wnt5a was used mediated regulation was independent of TNF-a. as a chemoattractant (p , 0.05; Fig. 1G), suggesting the possi- In the culture media of HAECs treated using 100 ng/ml Wnt5a bility of a chemotactic activity of Wnt5a for endothelial cells. En- for 24 h, IL-1a, IL-3, and CCL2 were markedly increased com- dothelial cell migration via extracellular matrix has been impli- pared with the control, indicating enhanced secretion of inflamma- cated in angiogenesis and tissue repair (28). tory cytokines by endothelial cells (Supplemental Fig. 2). Wnt5a enhances endothelial monolayer permeability and Wnt5a and TNF-a regulate downstream genes differentially Matrigel invasiveness Our data suggested that Wnt5a and TNF-a regulated downstream We then investigated the effect of Wnt5a on endothelial perme- genes differentially, and the Wnt5a-mediated regulation was inde- ability, a hallmark of early-stage inflammation. The HAEC mono- pendent of TNF-a. To investigate downstream regulatory patterns layer was treated using 100 ng/ml Wnt5a, and permeability was further, we compared COX-2 and IL-8 regulation by various con- measured using [14C]sucrose. Wnt5a treatment for 6 h enhanced centrations of Wnt5a, Wnt3a, and TNF-a using real-time PCR. The Journal of Immunology 1277

FIGURE 2. Wnt5a and TNF-a regulate downstream genes differentially. A and B, Real-time PCR of COX-2 and IL-8 expression in HAECs after treatment using Wnt5a, Wnt3a, and TNF-a of various concentrations for 1 h. C and D, Pulse-like treatment of Wnt5a shows ef- ficient downstream regulation. Wnt5a (100 ng/ ml) was applied in three ways: continuous treat- ment for up to 3 h, alternating 30-min treatment and 30-min rest in Wnt5a-negative media (“pulse–rest”), and alternating 30-min rest fol- lowed by 30-min treatment (“rest–pulse”). The “pulse” media was saved for the next pulse treat- ment without adding Wnt5a. COX-2 and IL-8 expression was measured using real-time PCR. Downloaded from

Wnt5a began to enhance COX-2 expression at a concentration as minimal, even at high concentrations (Fig. 2A), suggesting that low as 1 ng/ml, and induced it exponentially at 100 ng/ml in 1 h. Wnt5a is a major regulator of COX-2 in endothelial cells. In

The induction rate varied, depending on cell passage. TNF-a en- contrast, IL-8 was upregulated by TNF-a from a low concen- http://www.jimmunol.org/ hanced COX-2 slightly at 100 ng/ml, and the effect of Wnt3a was tration much more efficiently than by Wnt5a or Wnt3a (Fig. 2B). by guest on September 29, 2021

FIGURE 3. Wnt5a induces endothelial inflammationviaWnt/Ca2+/PKC signaling. A, Real-time PCR of inflammation genes inHAECsat1,3,and6hafter0.1mM A23187 treatment. Measurements were done in triplicate. B, Wnt5a (100 ng/ml) was applied to HAECs pretreated with 1 mM BAPTA-AM for 2 h. Downstream gene expressions were measured at 0, 1, 3, and 6 h after Wnt5a treatment. For mock treatment, HAECs were not pretreated us- ing BAPTA-AM. C, Comparison of COX-2 and IL-8 expression after Wnt5a treatment for 1 h with or without BAPTA-AM pre- treatment. D, Real-time PCR measure- ments of COX-2 in HAECs treated with 100 ng/ml Wnt5a with or without 1 mM BAPTA-AM, 100 mM EGTA, and 10 mM Go6983 pretreatment for 2 h. Experiments were done in triplicate. E, HAEC mono- layer permeability test. HAECs were trea- ted with 100 ng/ml Wnt5a for 6 h with or without 1 mM BAPTA-AM pretreatment for 2 h. BAPTA-AM treatment alone for 2 h was analyzed for comparison. Experi- ments were done in quadruplicate. 1278 Wnt5a INDUCES ENDOTHELIAL INFLAMMATION

Our data suggested that Wnt5a and TNF-a preferentially regulated slightly compared with control (p , 0.01). BAPTA-AM pretreat- subsets of downstream genes. ment significantly blocked Wnt5a-mediated enhancement of the permeability (p , 0.001). Pulse-like Wnt5a treatment regulates downstream genes 2+ efficiently Wnt/Ca /protein kinase C signaling in endothelial inflammatory regulation To obtain a better understanding of the downstream regulatory 2+ mechanism by Wnt5a, we treated HAECs with Wnt5a in three ways: Wnt/Ca signaling is transmitted via protein kinase C (PKC) or continuous treatment for 3 h, 30-min treatments alternating with 30- calcium-dependent calcium/calmodulin-dependent kinase II (4, min rest periods in Wnt5a-negative media (“pulse–rest”), and al- 29). Go6983, an inhibitor of PKC isotypes a, b, g, d, and z, in- ternating 30-min periods of rest and treatment (“rest–pulse”). The hibited the expression of COX-2 in a dose-dependent manner expression of downstream genes was measured every hour using (Supplemental Fig. 3). Go6983 pretreatment (10 mM) for 2 h re- real-time PCR. The pulse treatment was applied repetitively using duced Wnt5a-mediated COX-2 expression significantly (Fig. 3D), the Wnt5a-containing media that was saved from previous pulses, suggesting that Wnt5a-induced endothelial inflammation is depen- 2+ without adding additional Wnt5a. In continuous and rest–pulse dent on Wnt/Ca /PKC signaling. treatments, COX-2 expression was upregulated initially, but it Wnt/planar cell polarity signaling includes small GTP- began to decline after 1 or 2 h (Fig. 2C). However, the pulse–rest binding proteins, JNK, and Rho-associated kinase (4, 30, 32). A treatment upregulated COX-2 continuously for 3 h to much greater JNK-specific inhibitor, SP600125, significantly blocked Wnt5a- levels than did continuous or rest–pulse treatments (Fig. 2C). mediated downstream regulation (Supplemental Fig. 4A). How-

IL-8 regulation tended to follow the COX-2 regulation pattern, ever, phospho-JNK was not increased in HAECs upon Wnt5a Downloaded from but it varied depending on treatment time (Fig. 2D). Our data treatment for 6 h (Supplemental Fig. 4B). The apparently incon- showed that Wnt5a signaling was transmitted in a pulse-like man- sistent data suggested that a possible off-target effect of SP600- ner, with a cumulative effect upon repeated pulses and the require- 125 should be excluded. Wnt5a treatment did not increase ROS ment for considerable recovery time from a pulse. Together, our in HAECs (Supplemental Fig. 5). ROS have been implicated in data strongly suggested a Ca2+-dependent signaling for inflamma- prolonged JNK activation (33).

tory gene regulation. Calcium oscillations were shown to increase http://www.jimmunol.org/ the efficiency and specificity of gene expression (29). Wnt5a-mediated inflammatory regulation depends on Ca2+ signaling b-catenin–independent Wnt signaling is transmitted via at least two routes: the Wnt/Ca2+ and Wnt/planar cell polarity pathways (4, 30). In the Wnt/Ca2+ pathway, the intracytoplasmic free calcium regulates calcium-dependent downstream signaling as a secondary messenger. Ca2+ signaling may be initiated rapidly with Ca2+ from by guest on September 29, 2021 endoplasmic reticulum stores and extracellular sources through calcium channels (31). To investigate the possibility of Ca2+-dependent regulation, we first analyzed the effect of 0.1 mM A23187, a calcium ionophore, on downstream regulation in HAECs. A23187 enhanced the ex- pression of inflammation genes that were similarly upregulated by Wnt5a (Figs. 1A,3A). Wnt5a and A23187 strongly in- duced COX-2 and IL-8, supporting a role for cytoplasmic Ca2+ in Wnt5a signaling. A23187 upregulated downstream genes steadily for 6 h, in contrast to the rapid increase-and-decrease pattern of the Wnt5a-mediated downstream regulation (Figs. 1A,3A), suggesting that Wnt5a signaling was dependent on a pulse-like upregulation of cytoplasmic Ca2+. Endothelial NO synthase was downregulated. We then investigated the effect of BAPTA-AM, a Ca2+ chelator that may cross the plasma membrane into the cell, on Wnt5a- mediated downstream regulation. The pretreatment using 1 mM BAPTA-AM for 2 h inhibited the Wnt5a-mediated transcription of downstream inflammatory genes (Fig. 3B), indicating that the FIGURE 4. Limited role of Wnt3a in endothelial inflammation. A, Im- 2+ Wnt5a-mediated inflammatory downstream regulation was Ca de- munofluorescence microscopy for b-catenin in HAECs treated using pendent. Notably, BAPTA-AM abolished COX-2 and IL-8 induction 100 ng/ml Wnt5a for 6 h showing b-catenin localization along the intercel- (Fig. 3C). EGTA, an extracellular calcium chelator, similarly in- lular border but not in nuclei. DAPI nuclear staining is shown in merged hibited Wnt5a-induced COX-2 expression (Fig. 3D). In HAECs image (original magnification 31000). B, Real-time PCR of inflammation pretreated using 100 mM EGTA, COX-2 expression was mildly genes in HAECs at 1, 3, and 6 h after 100 ng/ml Wnt3a treatment. Measure- enhanced after 1 h of Wnt5a treatment and downregulated again ments were done in triplicate. C, Semiquantitative RT-PCR measurement of inflammation genes in HAECs transfected using Wnt/b-catenin pathway after 2 h, suggesting a rebound of intracytoplasmic free calcium inhibitors GSK-3b, CTNNBIP1, and CBY1 for 24 h. Controls were mock from the endoplasmic reticulum store followed by its depletion. b 2+ and vehicle transfected. -actin was used as loading control. D, Human To further investigate the role of Ca in Wnt5a-mediated in- cytokine array of HAECs transfected using GSK-3b for 24 h; 50 mg total flammatory regulation, we analyzed the permeability of the HAEC proteins was loaded for each experiment. The control was vector transfected. monolayer treated using Wnt5a, with or without BAPTA-AM pre- 1, CXCL1; 2, IL-6; 3, IL-8; 4, CCL5; 5, oncostatin M; 6, fibroblast growth treatment (Fig. 3E). BAPTA-AM alone inhibited the permeability factor 4; 7, neurotrophin 4; and 8, CCL18. The Journal of Immunology 1279

Limited role of Wnt3a in endothelial inflammatory regulation with the mock- and empty vector-transfected control HAECs, as WeinvestigatedapotentialroleforclassicalWnt/b-cateninsignaling measured by RT-PCR (Fig. 4C). Cytokine-array analysis also in inflammatory regulation. Using immunofluorescence micros- showed upregulation of CXCL1, IL-6, and IL-8 proteins in GSK- copy, 100 ng/ml Wnt5a treatment did not induce detectable nuclear 3b–transfected HAECs (Fig. 4D). CTNNBIP1 and CBY1 bind translocation of b-catenin in HAECs after 6 h, suggesting that the b-catenin directly to inhibit the transcriptional activity (35, 36). Wnt5a-mediated regulation was b-catenin independent (Fig. 4A). Upon transfection, CTNNBIP1 and CBY1 enhanced the expression Wnt3a (100 ng/ml) upregulated COX-2, IL-8, and IL-1a minimally, of inflammatory genes, similarly to GSK-3b (Fig. 4C). Thus, all but they were downregulated after 1 h (Fig. 4B). IL-6, CCL2, and three Wnt/b-catenin pathway inhibitors showed similar enhance- TC1 were downregulated from the beginning. Our data suggested ment of inflammatory genes, suggesting a potential inhibition, that b-catenin–independent signaling played a major role in endo- rather than enhancement, of endothelial inflammation by Wnt/b- thelial inflammatory regulation, whereas the role of classical Wnt/ catenin signaling. b-catenin signaling was limited. k We further investigated the effects of Wnt/b-catenin pathway Wnt5a activates NF- B in endothelial cells inhibitors on inflammatory regulation. Glycogen synthase kinase We then investigated whether Wnt5a could activate NF-kB, a key (GSK)-3b was reported to upregulate a subset of inflammatory transcriptional regulator playing a central role in the onset of downstream genes in activated monocytes (34). Upon transfection inflammation. The NF-kB activity was analyzed using ELISA- using GSK-3b, many downstream genes, including COX-2, IL-6, based DNA-binding analysis for RelA-binding consensus sequence IL-1a, CCL5, ICAM-1, and E-selectin, were upregulated compared oligonucleotides. Wnt5a (100 ng/ml) enhanced the DNA-binding Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Wnt5a enhances NF-kB activity. A, ELISA-based DNA-binding analysis for RelA. Nuclear proteins from control HAECs and HAECs treated with Wnt5a for 1 and 6 h were applied to ELISA plates carrying RelA-binding consensus sequence oligonucleotides. Experiments were done in triplicate. B, Immunofluorescence microscopy using anti-RelA Ab and merged images with DAPI nuclear staining. HAECs were treated with 100 ng/ml Wnt5a for 30 and 60 min (original magnification 31000). C, Western blotting of RelA in WE, CE, and NE of HAECs after 1 h of Wnt5a treatment. Densitometric measurements using b-actin as loading control are presented in lower panel. D, Real-time PCR measurement of downstream genes in HAECs treated with 100 ng/ml Wnt5a with 1 mM BMS-345541 pretreatment for 2 h. No treatment was given to mock HAECs. E, Comparison of COX-2 and IL8 expression after Wnt5a treatment for 1 h with or without BMS-345541 pretreatment. CE, cytoplasm extract; NE, nuclear extract; WE , whole-cell extract. 1280 Wnt5a INDUCES ENDOTHELIAL INFLAMMATION activity ∼1.8- and 1.6-fold more than control HAECs after 1 and 6 h, Interestingly, Wnt5a was also expressed in endothelial cells of respectively (p , 0.001; Fig. 5A). Intracellular distribution of atherosclerotic vessels (Fig. 6B). Proliferating smooth muscle cells NF-kB proteins was also analyzed using immunofluorescence in atheromatous plaques were also positive for Wnt5a (Fig. 6C). microscopy. Nuclear translocation of RelA was evident in most Small blood vessels in rheumatoid arthritis also expressed Wnt5a cells after 30 min of Wnt5a treatment compared with control (Fig. 6D), whereas blood vessels of normal synovial tissue and de- HAECs (Fig. 5B). After 60 min, the intensity of nuclear RelA generative osteoarthritis did not (Fig. 6E). As reported previously immunostaining tended to increase, with occasional cells showing (10), activated synoviocytes in rheumatoid arthritis strongly ex- strong nuclear immunostaining (Fig. 5B). pressed Wnt5a (Fig. 6F). The distribution of RelA was also analyzed using Western blotting of fractionated HAEC samples. Nuclear RelA increased ∼4 times after 1 h of Wnt5a treatment, as measured by densi- Discussion tometry, compared with control, whereas whole-cell and cytoplas- Endothelial activation is critical for the initiation and progression mic RelA did not change (Fig. 5C). b-actin was used as loading of inflammation. A two-stage model of endothelial activation has controls; it was present in the nuclear and cytoplasmic fractions, been proposed for acute inflammatory regulation: nontranscrip- as described previously (37). NF-kB p52 was not detected on tional type I activation for immediate response and type II activa- Western blots, using nuclear fractions, upon repeated examina- tion for the inflammatory gene expression (15). Type I activation tions (data not shown), suggesting that Wnt5a signaling depended is initiated by a – interaction that enhances intra- on canonical NF-kB signaling, which is activated by IKK- cytoplasmic Ca2+ levels to mediate the activation. The inflamma- 2+ dependent IkB degradation (20–22). The pretreatment of HAECs tory activation by Wnt/Ca signaling is compatible with the Downloaded from for 2 h with 1 mM BMS-345541, an IKK inhibitor (38), blocked model, suggesting Wnt5a as a ligand for type I endothelial acti- the Wnt5a-mediated transcription of inflammatory genes (Fig. vation (Fig. 7). 5D), supporting the fact that Wnt5a-mediated inflammatory gene Our data also indicated that b-catenin–independent Wnt signaling regulation was NF-kB dependent. COX-2 and IL-8 induction was mediates type II endothelial activation, upregulating COX-2 and inhibited almost completely by BMS-345541 (Fig. 5E). key cytokines promptly. Type II activation is known to be regulated

by prototype inflammatory mediators, such as TNF-a and IL-1b, http://www.jimmunol.org/ Wnt5a expression in inflamed human tissue activating NF-kB for downstream gene regulation. Wnt5a and Downstream genes of Wnt5a, notably CCL2, CX3CL1, and TLR4, TNF-a seem to regulate subsets of downstream genes that partially have been implicated in atherosclerosis (39), which is an in- overlap with each other. Anti–TNF-a Ab does not inhibit Wnt5a- flammatory disease (40, 41). We then investigated the expression of induced COX-2 expression, suggesting that Wnt5a-induced inflam- Wnt5a in such inflammatory diseases as atherosclerosis and rheu- mation was independent of TNF-a. b-catenin–independent Wnt matoid arthritis using immunohistochemistry. Infiltrating histio- signaling also activated NF-kB slightly. Together, our data sug- cytes in atheromatous plaques expressed Wnt5a diffusely (Fig. 6A), gested that Wnt5a signaling may include other transcriptional regu- suggesting a pathobiological role for Wnt5a in vivo. Contrary to lators or regulatory mechanisms in addition to NF-kB-dependent a previous report (42), foamy macrophages in the lipid core were regulation. Further investigations are required. by guest on September 29, 2021 not immunostained for Wnt5a upon repeated experiments. Our data Our data suggested that endothelial activation is regulated by suggested that activated histiocytes and macrophages might have a dual system consisting of b-catenin–independent Wnt signaling different capabilities to induce and/or promote local inflammation. and TNF-a/IL-1b–mediated signaling. Such a complexendothelial- activation system would be advantageous for diverse and flexible regulation of inflammation, depending on the biological situation and requirement. Together, the immediate type I response and rapid COX-2 induction by Wnt/Ca2+ signaling might suggest an impor- tant biological role, particularly at the initiation stage of inflamma- tion. The local composition of Wnt5a and prototype inflammatory

FIGURE 6. Wnt5a expression in inflamed human tissue. A, Immuno- histochemical staining of an atheromatous plaque using anti-Wnt5a antise- rum in atherosclerotic plaques. Elongated histiocytes in the fibrous cap expressed Wnt5a (arrows), whereas lipid-laden macrophages in the lipid core did not (arrowheads) (original magnification 3400). B, Endothelial cells in aorta with atheromatous change expressed Wnt5a (arrows) (original magnification 3400). C, Proliferating smooth muscle cells in atheromatous plaques also expressed Wnt5a (arrows) (original magnification 3400). D, Capillary endothelial cells in rheumatoid arthritis showing Wnt5a expres- sion (arrows) (original magnification 3200). E, In contrast, blood vessels did not show Wnt5a immunostaining in normal synovium (arrows) (original magnification 3200). F, Activated synoviocytes in rheumatoid arthritis FIGURE 7. A model for cooperative activation of endothelial and in- strongly expressed Wnt5a (arrows) (original magnification 3200). flammatory cells mediated by Wnt5a and TNF-a. The Journal of Immunology 1281 mediators might determine the profile of inflammatory gene expres- 8. Sen, M., and G. Ghosh. 2008. Transcriptional outcome of Wnt-Frizzled in inflammation: evolving concepts. J. Immunol. 181: 4441–4445. sion and the pathobiological nature of inflammation in vivo. 9. George, S. J. 2008. Wnt pathway: a new role in regulation of inflammation. It is noteworthy that Wnt5a is expressed in human inflammatory Arterioscler. Thromb. Vasc. Biol. 28: 400–402. diseases, including atherosclerotic plaques and rheumatoid arthritis, 10. Sen, M., K. Lauterbach, H. El-Gabalawy, G. S. Firestein, M. Corr, and D. A. Carson. 2000. Expression and function of wingless and frizzled homologs in rheumatoid but not in normal tissue. Activated histiocytes, smooth muscle cells, arthritis. Proc. Natl. Acad. Sci. 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