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
J Immunol July 15, 2010, 185 (2) 1274-1282; DOI: https://doi.org/10.4049/jimmunol.1000181

Abstract

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 β-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-α 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 a role for the Wnt/Ca2+/protein kinase C pathway in endothelial inflammatory regulation. Wnt5a activates RelA nuclear translocation 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 β-catenin–independent Wnt signaling and TNF-α–mediated signaling.

Wnt/β-catenin signaling has been implicated in diverse developmental and biological regulations (1–3). β-catenin–independent Wnt signaling is also involved in various biological functions, such as vertebral development, cell motility and adhesion, and cancer invasiveness (4–7). Recently, Wnt5a, a prototype Wnt for β-catenin–independent signaling, has been implicated in certain inflammatory diseases (8, 9). In synoviocytes of rheumatoid arthritis, the expression of Wnt5a and frizzled 5 is enhanced significantly (10), and the blockade of signaling inhibits synoviocyte activation (11). Wnt5a and frizzled 5 are also expressed in activated macrophages, APCs, and tuberculous granulomas (12). Wnt5a is induced by LPS/IFN-γ in human macrophages and is detectable in the sera of patients with severe sepsis (13). Those reports suggested a pathobiological role for Wnt signaling in human inflammatory diseases. However, it remains to be characterized whether and how Wnt signaling is involved in inflammatory regulation.

Wnt signaling is highly dependent on the cell context (14). Endothelial cells are active participants and regulators of inflammatory processes (15). Wnt5a has been implicated in endothelial proliferation and migration (16–18), as well as endothelial differentiation of embryonic stem cells (19).

Inflammation is a critical defense mechanism against various harmful stimuli. However, aberrant regulation may lead to various inflammatory diseases. NF-κB is a key transcriptional regulator playing a central role in the onset of inflammation (20). Typically, NF-κB is activated by prototype inflammatory mediators, such as TNF-α and IL-1β, via activation of IκB kinases (IKKs), which carry out the phosphorylation-dependent degradation of IκB inhibitors upon inflammatory stimuli (20–22). Recently, additional regulators of NF-κB were reported, indicating a complexity in the regulation of inflammation (23). Given the divergent biological requirements for inflammation and various clinical presentations of inflammatory diseases, a complex regulatory mechanism, rather than a simple on–off function, would be required for subtle qualitative and quantitative regulations of inflammation.

Recently, we reported that thyroid cancer-1 (TC1) (C8orf4) induces endothelial inflammation (24). Because TC1 is a regulator of the Wnt/β-catenin pathway (25, 26), we investigated a potential role for Wnt signaling in endothelial inflammatory regulation. Intriguingly, we observed that β-catenin–independent signaling, rather than classical Wnt/β-catenin signaling, plays a significant role in the inflammatory regulation of human endothelial cells. Our data suggested a complex dual-regulatory system of endothelial inflammation, consisting of β-catenin–independent Wnt signaling and prototype inflammatory mediator-dependent signaling.

Materials and Methods

Cells and reagents

Human aortic endothelial cells (HAECs) and HUVECs (Lonza, Walkersville, MD) were cultured in 0.1% gelatin-coated dishes containing EGM-2 basal medium (Lonza) at 37°C in humidified atmosphere with 5% CO2. Experiments were done using cells from passages six through nine. Human rWnt5a and Wnt3a proteins (Millipore, Billerica, MA) and TNF-α (Sigma-Aldrich, St. Louis, MO) were purchased commercially. BMS-345541, A23187, lithium chloride, BAPTA-AM, and SP600125 were also purchased from Sigma-Aldrich. Go6953 was purchased from Calbiochem (San Diego, CA). An IgG1 mouse mAb against TNF-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; sc-52746). IgG1 mouse mAbs against heat shock transcription factor 1 (HSF1) and NADPH dehydrogenase, quinone 1 (NQO1) (sc-52746; Santa Cruz Biotechnology) were used as controls.

Plasmids and transfection

Mammalian expression vectors pCDNA3-HA-ICAT, pCDNA3-HA-GSK3β, and pCDNA3-myc-Cby were cloned using pcDNA3 vector (Invitrogen, Carlsbad, CA). All clones were confirmed by DNA sequencing. HAEC and/or HUVEC transfection was done using Effectene transfection reagent (Qiagen, Hilden, Germany), following the manufacturer’s instructions. For the transfection, 400 ng DNA was applied to 1.2 × 105 cells in a six-well chamber. Controls were mock and/or vector transfected. The transfection efficiency was monitored using transfected β-galactosidase expression.

Real-time and semiquantitative RT-PCR

Real-time PCR was done as described previously (24). Total RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). Quantitative PCR was performed using a continuous fluorescence-detecting thermal cycler ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Green real-time PCR master mix (Toyobo, Osaka, Japan). Measurements were done in triplicate using β-actin as endogenous control. Semiquantitative RT-PCR was described previously (25, 26). PCR primers are summarized in the supplemental tables.

Western blotting and cytokine array

Total or fractionated cell-protein samples were analyzed. Nuclear and cytoplasmic fractions were separated using a fractionation kit from BioVision (Mountain View, CA). Samples were solubilized in lysis buffer and loaded (20 μg/lane) on 12% SDS-PAGE. Proteins were blotted onto nitrocellulose membranes and probed using rabbit anti-cyclooxygenase (COX)-2, anti-RelA, anti–NF-κB p52, and anti–β-actin antisera (Santa Cruz Biotechnology). Anti–phospho-JNK antiserum was from Cell Signaling Technology (Danvers, MA). After anti-rabbit secondary Ab (Amersham Biosciences, Piscataway, NJ) was applied, blots were visualized using the ECL method (Amersham Biosciences). β-actin was used as loading controls. For the profiling of cytokine expression, 50 μg proteins were applied to a human cytokine array (Raybiotech, Norcross, GA), according to the manufacturer’s instructions.

Immunofluorescence microscopy and immunohistochemical staining

For immunofluorescence microscopy, cells grown on cover slips were immunostained using anti-RelA antiserum. After washing with PBS, FITC-labeled anti-rabbit Ig secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) was applied. DNA staining was done using DAPI (Sigma-Aldrich). Cells were viewed using an Olympus BX51 fluorescence microscope. For controls, primary Abs were replaced with normal rabbit sera.

Immunohistochemical staining of three atherosclerosis, three rheumatoid arthritis, and control tissue samples was done using goat anti-Wnt5a serum (R&D Systems, Minneapolis, MN) and a Benchmark autostainer (Ventana Medical Systems, Tucson, AZ), as described previously (27). Ag retrieval pretreatment was omitted, and slides were not heated >65°C. This study was approved by the Institutional Review Board of Asan Medical Center. Because it was a retrospective study without any influence on the diagnosis and/or treatment, no written consent was required. The data were handled anonymously following guidelines of the Institutional Review Board.

ELISA-based NF-κB DNA-binding analysis

The DNA-binding capacity of p65-containing NF-κB dimers was assessed using ELISA plates containing fixed NF-κB binding-site consensus sequences, following the manufacturer’s instructions (Panomics, Fremont, CA), as described previously (24). Briefly, HAECs were treated using 100 ng/ml Wnt5a for 1 and 6 h. Ten micrograms of nuclear extract was diluted into binding buffer and incubated for 1 h at room temperature. Following three washes, primary Ab specific for p65 was added to each well, incubated at room temperature for 1 h, followed by HRP-conjugated secondary Ab incubation and chromogen reaction. Experiments were done in triplicate, and optical densities were measured using a SpectraMax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

[14C]Sucrose permeability test

A total of 4 × 104 HUVECs/well were seeded on a Transwell filter (Corning, Lowell, MA) and incubated until a complete monolayer was formed, changing the media every 2 d, as described previously (24). Then, 100 μg/ml Wnt5a and/or inhibitors were administered as described, and 50 μl (0.8 μCi [0.0296 MBq]/ml) [14C]sucrose (Amersham Pharmacia Biotech) was added to the upper compartment. After incubation for 30 min, the amount of radioactivity that diffused into the lower compartment was measured using a liquid scintillation counter (Tri-Carb 3100TR; Packard Instrument, Meriden, CT). Experiments were repeated in triplicate.

Matrigel-invasion assay

Matrigel-invasion assay was performed using Matrigel Invasion Chambers (BD Biosciences, San Jose, CA), according to the manufacturer’s protocols, as described previously (26). Briefly, 4 × 104 HAECs were placed in 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 12 h at 37°C, cells on the top surface of the filter were wiped off with a cotton-tipped swab, and the filter was fixed in methanol and stained using DiffQuik stain. The invasion rate was determined by counting cells at the bottom of the filter. Experiments were repeated in triplicate.

Measurement of intracellular reactive oxygen species

Reactive oxygen species (ROS) were detected using the cell-permeable indicator, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. HAECs were treated with Wnt5a and loaded with 20 μM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate for 30 min. Cells were collected, washed with cold PBS, and analyzed using a FACScan flow cytometer.

Statistical methods

All measurements are presented as the mean ± SD. Significance was determined using ANOVA.

Results

Wnt5a upregulates inflammatory genes rapidly in endothelial cells

Wnt5a expression was not detected by real-time PCR in HAECs or HUVECs under the culture condition (data not shown). HAECs were then treated using 100 ng/ml purified rWnt5a for up to 6 h, and the expression of inflammatory genes and cytokines was measured using real-time PCR. COX-2 was induced robustly in 1 h (Fig. 1A). Wnt5a did not upregulate COX-2 in other cell types, such as SH-SY5Y, HeLa, HEK293T, and RAW264.7, suggesting that Wnt5a-induced inflammatory gene expression was specific for endothelial cells (Supplemental Fig. 1A). IL-8, IL-6, IL-1α, LLC2, TLR4, and TLR3 were also upregulated significantly in 1 h and then declined gradually for 6 h (Fig. 1A). In comparison, E-selectin, ICAM1, and CX3CL1 were upregulated steadily for 6 h. TC1 was upregulated similarly. The expression of endothelial NO synthase did not change significantly (Fig. 1A). Inducible NO synthase was not detected (data not shown).

FIGURE 1.
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 β-actin as endogenous control. Fold differences are presented as mean ± SD compared with controls. B, Western blotting of COX-2 and densitometric analysis; 20 μg total proteins were loaded per lane, and β-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-1α; 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-α 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-α, 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-1β 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 downstream regulation was also analyzed at the protein level. Upon Western blotting, COX-2 was upregulated ∼45-fold compared with control at 6 h, as measured by densitometry (Fig. 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-1α, IL-3, IL-5, IL-6, IL-7, CCL2, and CCL8 in 24 h compared with control HAECs (Fig. 1C). We then compared the downstream regulation pattern of TNF-α, a prototype inflammatory mediator, with the Wnt5a-mediated regulation pattern. After 10 ng/ml TNF-α treatment for 24 h, GM-CSF, CXCL1, CXCL2, IL-8, IL-10, and CCL2 were significantly upregulated (Fig. 1D). Our data suggested that downstream regulation profiles by Wnt5a and TNF-α overlapped only partially. To further investigate a potential role for TNF-α in the Wnt5a-mediated inflammatory regulation, we treated HAECs using Wnt5a and an IgG1 mouse mAb against TNF-α, 100 ng/ml media. For controls, the same amounts of IgG1 mouse mAbs against NQO1 or HSF1, unrelated intracellular proteins, were applied similarly. The Wnt5a-mediated COX-2 upregulation was not inhibited by anti–TNF-α Ab compared with control Abs (Fig. 1E), suggesting that the Wnt5a-mediated regulation was independent of TNF-α.

In the culture media of HAECs treated using 100 ng/ml Wnt5a for 24 h, IL-1α, IL-3, and CCL2 were markedly increased compared with the control, indicating enhanced secretion of inflammatory cytokines by endothelial cells (Supplemental Fig. 2).

Wnt5a enhances endothelial monolayer permeability and Matrigel invasiveness

We then investigated the effect of Wnt5a on endothelial permeability, a hallmark of early-stage inflammation. The HAEC monolayer was treated using 100 ng/ml Wnt5a, and permeability was measured using [14C]sucrose. Wnt5a treatment for 6 h enhanced permeability of the HAEC monolayer by ∼50% compared with the control (p < 0.01; Fig. 1F), whereas the same amount of Wnt3a did not enhance it significantly. The invasiveness of HAECs to Matrigel was also enhanced significantly when Wnt5a was used as a chemoattractant (p < 0.05; Fig. 1G), suggesting the possibility of a chemotactic activity of Wnt5a for endothelial cells. Endothelial cell migration via extracellular matrix has been implicated in angiogenesis and tissue repair (28).

Wnt5a and TNF-α regulate downstream genes differentially

Our data suggested that Wnt5a and TNF-α regulated downstream genes differentially, and the Wnt5a-mediated regulation was independent of TNF-α. To investigate downstream regulatory patterns further, we compared COX-2 and IL-8 regulation by various concentrations of Wnt5a, Wnt3a, and TNF-α using real-time PCR. Wnt5a began to enhance COX-2 expression at a concentration as low as 1 ng/ml, and induced it exponentially at 100 ng/ml in 1 h. The induction rate varied, depending on cell passage. TNF-α enhanced COX-2 slightly at 100 ng/ml, and the effect of Wnt3a was minimal, even at high concentrations (Fig. 2A), suggesting that Wnt5a is a major regulator of COX-2 in endothelial cells. In contrast, IL-8 was upregulated by TNF-α from a low concentration much more efficiently than by Wnt5a or Wnt3a (Fig. 2B). Our data suggested that Wnt5a and TNF-α preferentially regulated subsets of downstream genes.

FIGURE 2.
FIGURE 2.

Wnt5a and TNF-α 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-α of various concentrations for 1 h. C and D, Pulse-like treatment of Wnt5a shows efficient downstream regulation. Wnt5a (100 ng/ml) was applied in three ways: continuous treatment for up to 3 h, alternating 30-min treatment and 30-min rest in Wnt5a-negative media (“pulse–rest”), and alternating 30-min rest followed by 30-min treatment (“rest–pulse”). The “pulse” media was saved for the next pulse treatment without adding Wnt5a. COX-2 and IL-8 expression was measured using real-time PCR.

Pulse-like Wnt5a treatment regulates downstream genes efficiently

To obtain a better understanding of the downstream regulatory mechanism by Wnt5a, we treated HAECs with Wnt5a in three ways: continuous treatment for 3 h, 30-min treatments alternating with 30-min rest periods in Wnt5a-negative media (“pulse–rest”), and alternating 30-min periods of rest and treatment (“rest–pulse”). The expression of downstream genes was measured every hour using real-time PCR. The pulse treatment was applied repetitively using the Wnt5a-containing media that was saved from previous pulses, without adding additional Wnt5a. In continuous and rest–pulse treatments, COX-2 expression was upregulated initially, but it began to decline after 1 or 2 h (Fig. 2C). However, the pulse–rest treatment upregulated COX-2 continuously for 3 h to much greater levels than did continuous or rest–pulse treatments (Fig. 2C). IL-8 regulation tended to follow the COX-2 regulation pattern, but it varied depending on treatment time (Fig. 2D). Our data showed that Wnt5a signaling was transmitted in a pulse-like manner, with a cumulative effect upon repeated pulses and the requirement for considerable recovery time from a pulse. Together, our data strongly suggested a Ca2+-dependent signaling for inflammatory gene regulation. Calcium oscillations were shown to increase the efficiency and specificity of gene expression (29).

Wnt5a-mediated inflammatory regulation depends on Ca2+ signaling

β-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 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 μM A23187, a calcium ionophore, on downstream regulation in HAECs. A23187 enhanced the expression of inflammation genes that were similarly upregulated by Wnt5a (Figs. 1A, 3A). Wnt5a and A23187 strongly induced 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.

FIGURE 3.
FIGURE 3.

Wnt5a induces endothelial inflammation via Wnt/Ca2+/PKC signaling. A, Real-time PCR of inflammation genes in HAECs at 1, 3, and 6 h after 0.1 μM A23187 treatment. Measurements were done in triplicate. B, Wnt5a (100 ng/ml) was applied to HAECs pretreated with 1 μM 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 using BAPTA-AM. C, Comparison of COX-2 and IL-8 expression after Wnt5a treatment for 1 h with or without BAPTA-AM pretreatment. D, Real-time PCR measurements of COX-2 in HAECs treated with 100 ng/ml Wnt5a with or without 1 μM BAPTA-AM, 100 μM EGTA, and 10 μM Go6983 pretreatment for 2 h. Experiments were done in triplicate. E, HAEC monolayer permeability test. HAECs were treated with 100 ng/ml Wnt5a for 6 h with or without 1 μM BAPTA-AM pretreatment for 2 h. BAPTA-AM treatment alone for 2 h was analyzed for comparison. Experiments were done in quadruplicate.

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 μM BAPTA-AM for 2 h inhibited the Wnt5a-mediated transcription of downstream inflammatory genes (Fig. 3B), indicating that the Wnt5a-mediated inflammatory downstream regulation was Ca2+ dependent. Notably, BAPTA-AM abolished COX-2 and IL-8 induction (Fig. 3C). EGTA, an extracellular calcium chelator, similarly inhibited Wnt5a-induced COX-2 expression (Fig. 3D). In HAECs pretreated using 100 μM EGTA, COX-2 expression was mildly enhanced after 1 h of Wnt5a treatment and downregulated again after 2 h, suggesting a rebound of intracytoplasmic free calcium from the endoplasmic reticulum store followed by its depletion.

To further investigate the role of Ca2+ in Wnt5a-mediated inflammatory regulation, we analyzed the permeability of the HAEC monolayer treated using Wnt5a, with or without BAPTA-AM pretreatment (Fig. 3E). BAPTA-AM alone inhibited the permeability slightly compared with control (p < 0.01). BAPTA-AM pretreatment significantly blocked Wnt5a-mediated enhancement of the permeability (p < 0.001).

Wnt/Ca2+/protein kinase C signaling in endothelial inflammatory regulation

Wnt/Ca2+ signaling is transmitted via protein kinase C (PKC) or calcium-dependent calcium/calmodulin-dependent kinase II (4, 29). Go6983, an inhibitor of PKC isotypes α, β, γ, δ, and ζ, inhibited the expression of COX-2 in a dose-dependent manner (Supplemental Fig. 3). Go6983 pretreatment (10 μM) for 2 h reduced Wnt5a-mediated COX-2 expression significantly (Fig. 3D), suggesting that Wnt5a-induced endothelial inflammation is dependent on Wnt/Ca2+/PKC signaling.

Wnt/planar cell polarity signaling includes small GTP-binding proteins, JNK, and Rho-associated kinase (4, 30, 32). A JNK-specific inhibitor, SP600125, significantly blocked Wnt5a-mediated downstream regulation (Supplemental Fig. 4A). However, phospho-JNK was not increased in HAECs upon Wnt5a treatment for 6 h (Supplemental Fig. 4B). The apparently inconsistent data suggested that a possible off-target effect of SP600125 should be excluded. Wnt5a treatment did not increase ROS in HAECs (Supplemental Fig. 5). ROS have been implicated in prolonged JNK activation (33).

Limited role of Wnt3a in endothelial inflammatory regulation

We investigated a potential role for classical Wnt/β-catenin signaling in inflammatory regulation. Using immunofluorescence microscopy, 100 ng/ml Wnt5a treatment did not induce detectable nuclear translocation of β-catenin in HAECs after 6 h, suggesting that the Wnt5a-mediated regulation was β-catenin independent (Fig. 4A). Wnt3a (100 ng/ml) upregulated COX-2, IL-8, and IL-1α minimally, but they were downregulated after 1 h (Fig. 4B). IL-6, CCL2, and TC1 were downregulated from the beginning. Our data suggested that β-catenin–independent signaling played a major role in endothelial inflammatory regulation, whereas the role of classical Wnt/β-catenin signaling was limited.

FIGURE 4.
FIGURE 4.

Limited role of Wnt3a in endothelial inflammation. A, Immunofluorescence microscopy for β-catenin in HAECs treated using 100 ng/ml Wnt5a for 6 h showing β-catenin localization along the intercellular border but not in nuclei. DAPI nuclear staining is shown in merged image (original magnification ×1000). B, Real-time PCR of inflammation genes in HAECs at 1, 3, and 6 h after 100 ng/ml Wnt3a treatment. Measurements were done in triplicate. C, Semiquantitative RT-PCR measurement of inflammation genes in HAECs transfected using Wnt/β-catenin pathway inhibitors GSK-3β, CTNNBIP1, and CBY1 for 24 h. Controls were mock and vehicle transfected. β-actin was used as loading control. D, Human cytokine array of HAECs transfected using GSK-3β for 24 h; 50 μg total proteins was loaded for each experiment. The control was vector transfected. 1, CXCL1; 2, IL-6; 3, IL-8; 4, CCL5; 5, oncostatin M; 6, fibroblast growth factor 4; 7, neurotrophin 4; and 8, CCL18.

We further investigated the effects of Wnt/β-catenin pathway inhibitors on inflammatory regulation. Glycogen synthase kinase (GSK)-3β was reported to upregulate a subset of inflammatory downstream genes in activated monocytes (34). Upon transfection using GSK-3β, many downstream genes, including COX-2, IL-6, IL-1α, CCL5, ICAM-1, and E-selectin, were upregulated compared with the mock- and empty vector-transfected control HAECs, as measured by RT-PCR (Fig. 4C). Cytokine-array analysis also showed upregulation of CXCL1, IL-6, and IL-8 proteins in GSK-3β–transfected HAECs (Fig. 4D). CTNNBIP1 and CBY1 bind β-catenin directly to inhibit the transcriptional activity (35, 36). Upon transfection, CTNNBIP1 and CBY1 enhanced the expression of inflammatory genes, similarly to GSK-3β (Fig. 4C). Thus, all three Wnt/β-catenin pathway inhibitors showed similar enhancement of inflammatory genes, suggesting a potential inhibition, rather than enhancement, of endothelial inflammation by Wnt/β-catenin signaling.

Wnt5a activates NF-κB in endothelial cells

We then investigated whether Wnt5a could activate NF-κB, a key transcriptional regulator playing a central role in the onset of inflammation. The NF-κB activity was analyzed using ELISA-based DNA-binding analysis for RelA-binding consensus sequence oligonucleotides. Wnt5a (100 ng/ml) enhanced the DNA-binding activity ∼1.8- and 1.6-fold more than control HAECs after 1 and 6 h, respectively (p < 0.001; Fig. 5A). Intracellular distribution of NF-κB proteins was also analyzed using immunofluorescence microscopy. Nuclear translocation of RelA was evident in most cells after 30 min of Wnt5a treatment compared with control HAECs (Fig. 5B). After 60 min, the intensity of nuclear RelA immunostaining tended to increase, with occasional cells showing strong nuclear immunostaining (Fig. 5B).

FIGURE 5.
FIGURE 5.

Wnt5a enhances NF-κB 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 ×1000). C, Western blotting of RelA in WE, CE, and NE of HAECs after 1 h of Wnt5a treatment. Densitometric measurements using β-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 μM 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.

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 densitometry, compared with control, whereas whole-cell and cytoplasmic RelA did not change (Fig. 5C). β-actin was used as loading controls; it was present in the nuclear and cytoplasmic fractions, as described previously (37). NF-κB p52 was not detected on Western blots, using nuclear fractions, upon repeated examinations (data not shown), suggesting that Wnt5a signaling depended on canonical NF-κB signaling, which is activated by IKK-dependent IκB degradation (20–22). The pretreatment of HAECs for 2 h with 1 μM BMS-345541, an IKK inhibitor (38), blocked the Wnt5a-mediated transcription of inflammatory genes (Fig. 5D), supporting the fact that Wnt5a-mediated inflammatory gene regulation was NF-κB dependent. COX-2 and IL-8 induction was inhibited almost completely by BMS-345541 (Fig. 5E).

Wnt5a expression in inflamed human tissue

Downstream genes of Wnt5a, notably CCL2, CX3CL1, and TLR4, have been implicated in atherosclerosis (39), which is an inflammatory disease (40, 41). We then investigated the expression of Wnt5a in such inflammatory diseases as atherosclerosis and rheumatoid arthritis using immunohistochemistry. Infiltrating histiocytes in atheromatous plaques expressed Wnt5a diffusely (Fig. 6A), suggesting a pathobiological role for Wnt5a in vivo. Contrary to a previous report (42), foamy macrophages in the lipid core were not immunostained for Wnt5a upon repeated experiments. Our data suggested that activated histiocytes and macrophages might have different capabilities to induce and/or promote local inflammation. Interestingly, Wnt5a was also expressed in endothelial cells of atherosclerotic vessels (Fig. 6B). Proliferating smooth muscle cells in atheromatous plaques were also positive for Wnt5a (Fig. 6C).

FIGURE 6.
FIGURE 6.

Wnt5a expression in inflamed human tissue. A, Immunohistochemical staining of an atheromatous plaque using anti-Wnt5a antiserum 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 ×400). B, Endothelial cells in aorta with atheromatous change expressed Wnt5a (arrows) (original magnification ×400). C, Proliferating smooth muscle cells in atheromatous plaques also expressed Wnt5a (arrows) (original magnification ×400). D, Capillary endothelial cells in rheumatoid arthritis showing Wnt5a expression (arrows) (original magnification ×200). E, In contrast, blood vessels did not show Wnt5a immunostaining in normal synovium (arrows) (original magnification ×200). F, Activated synoviocytes in rheumatoid arthritis strongly expressed Wnt5a (arrows) (original magnification ×200).

Small blood vessels in rheumatoid arthritis also expressed Wnt5a (Fig. 6D), whereas blood vessels of normal synovial tissue and degenerative osteoarthritis did not (Fig. 6E). As reported previously (10), activated synoviocytes in rheumatoid arthritis strongly expressed Wnt5a (Fig. 6F).

Discussion

Endothelial activation is critical for the initiation and progression of inflammation. A two-stage model of endothelial activation has been proposed for acute inflammatory regulation: nontranscriptional type I activation for immediate response and type II activation for the inflammatory gene expression (15). Type I activation is initiated by a ligand–receptor interaction that enhances intracytoplasmic Ca2+ levels to mediate the activation. The inflammatory activation by Wnt/Ca2+ signaling is compatible with the model, suggesting Wnt5a as a ligand for type I endothelial activation (Fig. 7).

FIGURE 7.
FIGURE 7.

A model for cooperative activation of endothelial and inflammatory cells mediated by Wnt5a and TNF-α.

Our data also indicated that β-catenin–independent Wnt signaling mediates type II endothelial activation, upregulating COX-2 and key cytokines promptly. Type II activation is known to be regulated by prototype inflammatory mediators, such as TNF-α and IL-1β, activating NF-κB for downstream gene regulation. Wnt5a and TNF-α seem to regulate subsets of downstream genes that partially overlap with each other. Anti–TNF-α Ab does not inhibit Wnt5a-induced COX-2 expression, suggesting that Wnt5a-induced inflammation was independent of TNF-α. β-catenin–independent Wnt signaling also activated NF-κB slightly. Together, our data suggested that Wnt5a signaling may include other transcriptional regulators or regulatory mechanisms in addition to NF-κB-dependent regulation. Further investigations are required.

Our data suggested that endothelial activation is regulated by a dual system consisting of β-catenin–independent Wnt signaling and TNF-α/IL-1β–mediated signaling. Such a complex endothelial-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 important biological role, particularly at the initiation stage of inflammation. The local composition of Wnt5a and prototype inflammatory mediators might determine the profile of inflammatory gene expression and the pathobiological nature of inflammation in vivo.

It is noteworthy that Wnt5a is expressed in human inflammatory diseases, including atherosclerotic plaques and rheumatoid arthritis, but not in normal tissue. Activated histiocytes, smooth muscle cells, and synoviocytes express Wnt5a, supporting a pathobiological role for Wnt5a in inflammatory regulation in vivo. Infiltrating histiocytes are probably the major source of Wnt5a and prototype inflammatory mediators. Together, our data suggested a cooperative inflammatory activation between blood vessels and inflammatory cells. Intriguingly, activated vascular endothelial cells also express Wnt5a, suggesting an autocrine activation of endothelial cells (Fig. 7).

Classical Wnt/β-catenin signaling seems to have a limited or even negative role in inflammatory regulation. β-catenin inhibitors, including GSK-3β, CTNNBIP1, and CBY1, upregulate downstream inflammatory genes similarly, suggesting a potential inhibitory role for Wnt/β-catenin signaling in endothelial inflammatory regulation. GSK-3β was reported to upregulate a subset of inflammatory downstream genes in activated monocytes (34). Recently, it was reported that Wnt/β-catenin signaling was activated in endothelial cells of an experimentally rejected kidney model (43). However, in the model, it is not clear whether the endothelial β-catenin activation reflects a tissue repair or inflammatory activation. We previously reported that TC1 is a novel endothelial inflammatory regulator (24). Our data suggested that the inflammatory activation by TC1 is independent of its function as an enhancer of Wnt/β-catenin signaling (25, 26).

It is well established that Wnt signaling is cell-context dependent. Our data suggested that the inflammatory activation by β-catenin–independent Wnt signaling is specific for endothelial cells. Further investigations are required for the Wnt5a-mediated regulation of inflammatory cells. Wnt5a may have diverse biological roles in the regulation of endothelial cells. The Wnt5a-mediated upregulation of TLRs suggests a role for enhanced innate immunity against infection. The Matrigel invasiveness of endothelial cells may suggest a role for Wnt5a in angiogenesis and tissue repair. Given the association of Wnt5a with cancer invasiveness (6, 7), the regulation of tumor vascular endothelial cells by Wnt5a also needs to be investigated.

Acknowledgments

We thank Dr. Ki Hoon Han for ROS measurement in endothelial cells. Graphical artwork by Migyong Wu is appreciated.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by a Doyak Research Program grant through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20090079398).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this paper:

    CE
    cytoplasm extract
    COX
    cyclooxygenase
    GSK
    glycogen synthase kinase
    HAEC
    human aortic endothelial cell
    HSF1
    heat shock transcription factor 1
    IKK
    IκB kinase
    NE
    nuclear extract
    NQO1
    NADPH dehydrogenase, quinone 1
    PKC
    protein kinase C
    ROS
    reactive oxygen species
    TC1
    thyroid cancer-1
    WE
    whole-cell extract.

  • Received January 19, 2010.
  • Accepted May 11, 2010.

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