Wnt5a Regulates Odontoblast Inflammation by Promoting 1 CCL2 Expression

study aimed to investigate the role of Wnt5a in odontoblast inflammation. We measured and compared Wnt5a- or TNF-α-induced cytokine/chemokine expression in mouse odontoblast-like (17IIA11) cells by real-time PCR and Western blotting. Transwell assays were used to examine the effect of Wnt5a on RAW264.7 macrophage migration. We examined whether the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase 11 (MAPK) pathways were involved in the molecular mechanism of TNF-α or Wnt5a in 17IIA11 12 cells by Western blotting.

The Wnt signaling pathway is an evolutionarily conserved signaling pathway that regulates 29 embryonic development, tissue regeneration, and various types of inflammation [1][2][3][4][5]. The β-30 catenin-dependent Wnt signaling pathway is the canonical Wnt signaling pathway, while the β-31 catenin-independent pathway is the noncanonical Wnt signaling pathway [6]. 32 Wnt5a is a member of the Wnt signaling family and is involved in the activation of 33 noncanonical Wnt signaling; Wnt5a is also involved in regulating various type of inflammation, 34 including rheumatoid arthritis, periodontitis, and pulpitis [7][8][9][10][11]. In addition, Wnt5a induces the 35 expression of multiple cytokines/chemokines, including IL-6, IL-8, CXCL1,and CCL2,36 and indirectly activates the migration of immune cells in human dental pulp cell (hDPC) 37 populations [9,10]. 38 Pulp inflammation, also called pulpitis, is a major oral health problem caused by the invasion 39 of microorganisms and their components via dentinal tubules in the pulp [12] that frequently 40 leads to persistent and referred pain. Pulpitis often results in root canal therapy, whose 41 disadvantages include the loss of living pulp and increased tooth brittleness. Therefore, we hope 42 to provide a theoretical basis for the development of new treatments for pulpitis by 43 3/12 understanding pulpitis pathogenesis. 1 Odontoblasts are the first layer cells in dental pulp, and the main function of odontoblasts is 2 dentin formation. During pulpitis, odontoblasts are the first cells encountered by invading 3 bacteria, and the products of these bacteria are located at the pulp-dentin interface [13,14]. 4 Numerous studies have shown that odontoblasts are involved in pulp immune and inflammatory 5 responses to bacteria and their components [15,16]. In addition, odontoblasts produce Tumor 6 necrosis factors-alpha (TNF-α), IL-6, IL-8, CCL2, CXCL10 and IL-10 in response to 7 inflammatory stimuli [17,18]. However, it is unclear whether Wnt5a is involved in regulating 8 the inflammatory response of odontoblasts. 9 The mitogen-activated protein kinase (MAPK) pathway, including its three main members p38 10 MAP kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated 11 protein kinase 1/2 (ERK 1/2), is an important signaling pathway related to inflammation. A number 12 of studies have shown that MAPK is involved in the inflammatory response, and inflammatory 13 stimuli such as proinflammatory chemokines and cytokines can activate the MAPK pathway [19-14 21]. The nuclear factor-kappa B (NF-κB) pathway is another important signaling pathway 15 involved in regulating inflammation [22]. Furthermore, P65 phosphorylation is required for the 16 optimal activation of NF-κB-dependent gene transcription [23][24][25]. 17 TNF-α is a well-known inflammatory cytokine that can cause dental pulp inflammation [26]. 18 Both NF-κB and MAPK are critical for inducing the expression of genes involved in inflammation 19 [13,27] and are linked to Wnt5a-stimulated signaling [10,28]. In previous experiments, we 20 demonstrated that TNF-α upregulates the expression of Wnt5a in hDPC through the MAPK and 21 NF-κB pathways [9, 10]. However, whether Wnt5a regulates odontoblast inflammation and the 22 crosstalk between NF-κB and MAPK is poorly understood. 23 In this study, we investigated the role of Wnt5a in odontoblast inflammation and the 24 potential signaling pathways by which TNF-α upregulates Wnt5a expression in odontoblasts. 25

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Cell Lines and Culture 27 The 17IIA11 cell line was grown and maintained in Alpha Modification of Minimum 28 Essential Medium Eagle (α-MEM) supplemented with 10% fetal bovine serum (FBS) in a 29 humidified atmosphere containing 5% CO2 at 37 °C. 17IIA11 cells were treated with 30 recombinant human TNF-α (rhTNF-α, 10 ng/ml, Invivogen, San Diego, CA) or recombinant 31 human Wnt5a (rhWnt5a, 500 ng/ml, R&D Systems) for the indicated times. To examine the 32 signaling pathways, cells were pretreated with one of the following specific pathway inhibitors:  The RAW 264.7 macrophage cell line was purchased from the American Type Culture 37 Collection (ATCC). These cells were cultured in RPMI 1640 with 10% FBS, 2 mM L-38 glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere 39 containing 5% CO2 at 37 °C. 40 41

PCR. 30 31
Transwell Migration Assay 32 The migratory capacity of macrophages was examined using Transwell chambers (3-μm pore, 33 Corning) in triplicate. 17IIA11 cells were cultured with or without rhWnt5a treatment for 48 h 34 before supernatants were collected. Then, 200 μl of macrophages (2×10 6 /ml) was seeded in the 35 upper chamber in serum-free RPMI 1640, and 600 μl of culture supernatant from untreated or 36 Wnt5a-treated 17IIA11 cells was added to the lower well. rhWnt5a was also added to the 37 bottom well. After 4 h of incubation, the membranes were removed, and the underside of the 38 polycarbonate membrane was stained with 1% crystal violet for 30 min. Then, the number of 39 migrating cells was counted in five randomly selected fields under light microscopy. 40 41

Statistical Analysis 42
Each experiment was repeated at least three times, and the results are expressed as the 43 means ± standard deviation (SD). The statistical significance of differences was determined 44 using ANOVA. A value of p < 0.05 was considered statistically significant. 1 2

Wnt5a induced CCL2 expression and enhanced RAW264.7 cell migration 12
We investigated how Wnt5a responds to inflammatory stimuli and whether Wnt5a activates 13 the MAPK and NF-κB pathways in odontoblasts. First, we measured the cytokines and chemokines 14 that were induced by TNF-α and found that TNF-α upregulated the expression of IL-8, CXCL1, and 15 CCL2 (Fig. 3 a-c). Then, we treated 17IIA11 cells with Wnt5a and found that CCL2 was obviously 16 upregulated by Wnt5a (Fig. 3 d-e). Because Wnt5a induces CCL2 expression, we examined whether 17 macrophages were attracted to Wnt5a. Therefore, RAW264.7 cells were subjected to a Transwell 18 assay. We found that the supernatant of 17IIA11 cells treated with Wnt5a enhanced the migration 19 of RAW264.7 cells (Fig. 3 f-g). Moreover, Wnt5a activated the NF-κB and MAPK pathways in 20 odontoblasts (Fig. 3 ) 1 Figure 3. Wnt5a induces CCL2 expression and enhances macrophage migration. In addition, 2 Wnt5a activates the MAPK and NF-κB signaling pathways. a-e: The mRNA expression of cytokines 3 and chemokines. f, g: Transwell detected the migration of RAW 264.7 cells. h: On the left side is 4 the result that Western blot detected the protein expression of NF-κB and MAPK pathways (these 5 results are from different parts of the same gel); on the right is the ratio of phosphorylated protein 6 to non-phosphorylated protein of MAPK and NF-κB pathway related proteins. **, p<0.01 versus 7 the control. GAPDH was used as a control. 8 9

Summary of results 10
In this study, we first verified whether TNF-α induces the upregulation of Wnt5a and its 11 pathway in odontoblasts and then explored how Wnt5a responds to inflammatory stimuli in 12 odontoblasts. We found that TNF-α-induced Wnt5a upregulation occurs through MAPK-13 dependent NF-κB activation and that Wnt5a upregulates CCL2 expression. In addition, Wnt5a 14 enhanced macrophage migration (Fig. 4). 15 Studies have confirmed that Wnt5a can regulate a variety of inflammatory responses including 7 the response to pulpitis [9,10,31,32]. In hDPC, Wnt5a can not only promote the upregulation of 8 the expression of cytokines and chemokines induced by TNF-α but can also itself increase the levels 9 of cytokines and chemokines in DPCs [10]. Therefore, Wnt5a itself is an inflammatory factor. 10 Odontoblasts are the outermost cells of the dental pulp tissue and are the first line of defense for this 11 tissue against various external stimuli. However, whether Wnt5a is involved in odontoblast 12 inflammation is unclear. We found that TNF-α upregulated the expression of Wnt5a in odontoblasts, 13 and Wnt5a in turn upregulated the expression of CCL2 in odontoblasts. In addition, the supernatant 14 of 17IIA11 cells treated with Wnt5a enhanced the migration of RAW264.7 cells. Therefore, the 15 main role of Wnt5a in odontoblasts is to chemoattract immune cells to migrate to inflammation sites 16 by promoting the expression of chemokines. 17 Both NF-κB and MAPK are important signaling pathways in inflammation [13,27]. In hDPC, 18 TNF-α upregulates the expression of Wnt5a through the MAPK and NF-κB pathways [10]. However, 19 the crosstalk between the NF-κB and MAPK pathways and whether Wnt5a activates the MAPK and 20 NF-κB pathways in odontoblasts is unclear. In this study, we showed that TNF-α upregulates the 21 expression of Wnt5a through the MAPK-dependent NF-κB pathway. When Wnt5a is involved in 22 the inflammatory process, it also activates the MAPK pathway (including ERK1/2, P38, JNK) and 23 the NF-κB pathway [10,33]. Both the MAPK and NF-κB pathways are also involved in the 24 upregulation of cytokine and chemokine expression [9,34]. In this study, we demonstrated that 25 9/12 Wnt5a not only activates the MAPK and NF-κB pathways but also upregulates the expression of 1 CCL2 in odontoblasts. Therefore, Wnt5a may upregulate the expression of CCL2 via the MAPK 2 and NF-κB pathways . 3 In addition, Wnt5a is involved in the development of teeth and the differentiation of 4 odontoblasts [35,36]. Under inflammatory conditions, the expression of mineralization-related 5 genes is also upregulated through Wnt5a in hDPC [37,38]. In this study, we did not explore the 6 effects of inflammatory stimuli on the mineralization ability of odontoblasts. We intend to 7 investigate whether Wnt5a involved in the mineralization process of odontoblasts induced by 8 inflammation in subsequent experiments. 9 In summary, we have demonstrated that Wnt5a is induced by TNF-α via MAPK-dependent NF-10 κB activation in odontoblasts and that Wnt5a regulates the expression of CCL2. In addition, Wnt5a 11 indirectly promotes macrophage migration.