Egr-1 mediates low dose Arecoline induced human oral mucosa broblasts proliferation by transaction the expression of Wnt5a

High dose Arecoline (> 32 µg/ml) could inhibit but low dose Arecoline (< 16 µg/ml) could promote the proliferation of human oral broblasts. Wnt5a was both sucient and necessary for the promotion of brobasts proliferation. Egr-1, but not NF-κB, FOXO1, Smad2 or Smad3, mediated the expression of Wnt5a in brobasts. The specic siRNAs of Egr-1, Egr inhibitors or Wnt5a antibodies treatment blocked Arecoline induced Wnt5a upregulation and broblasts proliferation. transfected into cells Lipofectamine LTX. the dual-luciferase reporter assays, cells transfected 1 µg of a luciferase reporter plasmid 200 ng the pRL-CMV Renilla luciferase reporter plasmid (Promega). After transfection, cells in conditioned media or 24 h and then transferred to treatment media for 12 h. Firey luciferase activity was normalized to Renilla luciferase activity according to the protocol.

the control of brolasts proliferation. However, the relationship between Arecoline and Wnt, and the transcription factors that control Wnt expression in oral broblasts are still unclear.
In this study, we con rmed that low dose Arecoline promoted human oral broblasts proliferation, and found that Egr-1 up-regulated Wnt5a expression to mediate the proliferative effect of Arecoline. Collectively, these ndings establish Egr-1 and Wnt5a as new potential therapeutic targets for oral submucous brosis caused by chewing areca nut.

Low dose Arecoline induces the proliferation of broblasts
Previous studies have showed that Arecoline was cytotoxic to oral broblasts from the concentration of 50 µg/ml up [8]. However, it is true that the areca nut chewing increases the risk of oral cancer and oral submucous brosis. In fact, the salivary Arecoline level during chewing were about 0.1 µg/ml, and increased to about 0.3 µg/ml after chewing [20], which is much lower than the concentration used in most experiments.
In the present study, we intend to test the effect of different dose Arecoline (0.1, 0.5, 1, 4, 8, 16, 32 or 50 µg/ml) on the proliferation of human oral broblasts, half of the medium was changed every 24 hours. We con rmed that high dose Arecoline (32 µg/ml and 50 µg/ml) could inhibit the proliferation of broblasts (Fig. 1a). Our results also showed that Arecoline promoted the proliferation of broblasts at the concentrations from 0.1 µg/ml to 16 µg/ml ( Fig. 1b-1f), and the maximum proliferate effect was 8 µg/ml (Fig. 1e). These results showed that low dose Arecoline could promote the proliferation of human oral broblasts.

Arecoline Promotes Fibroblasts Proliferation By Inducing Wnt5a Expression
Earlier reports have shown that activation of Wnt/β-catenin signaling may promote the proliferation of broblasts by regulating the expression level of Wnt1, Wnt2, Wnt3a or Wnt5a [19]. To further study whether other Wnt isoforms take part in accommodation of human oral broblasts proliferation, the mRNA expression levels of all 19 Wnt gene family members in human oral broblasts exposed to 8 µg/ml Arecoline for 24 h were analyzed by RT-PCR. As illustrated in Table 1, Arecoline altered the transcription of ten Wnts (Wnt1, 2, 3a, 5a, 5b, 8b, 10a, 10b, 11 and 16). Of those, Wnt3a, Wnt5b, Wnt8b, Wnt10a, Wnt10b, Wnt11 and Wnt16 were expressed only at very low levels in both control and treatment group, whereas Wnt1, Wnt2 and Wnt5a expressed at higher levels. We next detected the protein expression level of Wnt1, Wnt2 and Wnt5a in 8 µg/ml Arecoline time course experiment. As expected, Arecoline signi cantly promoted the expression of Wnt1, Wnt2 and Wnt5a on the protein level (Fig. 2a). The values represent the mean ± S.E. of three independent experiments. * P < 0.05 To determine if Wnt1, Wnt2 or Wnt5a was required for Arecoline to promote the proliferation of broblasts, recombinant Wnt1, Wnt2 or Wnt5a protein and Wnt1, Wnt2 or Wnt5a antibody were used to treat human oral broblasts. Results showed that the proliferation of broblasts was not affected by Wnt1, Wnt2 protein or their antibody ( Fig. 2b and 2c); recombinant Wnt5a protein could increase the proliferation of broblasts (Fig. 2d); and the Arecoline-induced cell proliferation was inhibited by Wnt5a antibody (Fig. 2d). Furthermore, we found the speci c siRANs Wnt5a inhibited the Arecoline-induced cell proliferation, but the proliferation of broblasts was not affected by Wnt1 or Wnt2 siRNAs (Fig. 2e). Together, these results demonstrated that Wnt5a mediated the Arecoline-induced broblasts proliferation.
To con rm if Egr-1 is involved in Wnt5a regulation, broblasts were transfected with Egr-1 siRNAs. Arecoline induced Wnt5a expression was effectively blocked by siEgr-1 ( Fig. 3a and 3c), demonstrating that Egr-1 is involved in Wnt5a regulation. In support of this result, Egr-1 siRNAs signi cantly suppressed Wnt5a protein expression (Fig. 3c). Therefore, we concluded that Egr-1 is essential for transcriptional induction of Wnt5a in human oral broblasts.

Inhibition Of Egr Activity Prevents Arecoline Induced Fibroblast Proliferation
We next assessed the roles of Egr-1 in broblasts proliferation. Our results showed that Egr-1 knockdown inhibited the Arecoline-induced proliferation of broblasts (Fig. 4a). Furthermore, Mithramycin A (MMA) and Chromomycin A3 (CHA) were used to treat cells (MMA and CHA repress transcription by selectively displacing GC-rich DNA binding transcription factors, such as Egr-1 [23,24]). The results showed that MMA or CHA treatment blocked Arecoline induced Wnt5a upregulation and broblasts proliferation (Fig. 4b, 4c and 4d). These results indicated that the expression and activity of Egr-1 were required for Arecoline induced broblasts proliferation.

Discussion
Oral submucosal brosis (OSF) is one of the precancerous lesions of oral squamous cell carcinoma, which is related to the frequent chewing of Areca nut. OSF used to be reported mainly in Southeast Asia, but it is now found in the Asian immigrant populations of Britain and America and has become a global health problem [25]. The main pathological manifestation of OSF is abnormal accumulation of collagen in the lamina propria under the oral mucosa [26]. With the development of OSF, 3%-19% of patients may have cancer, and this probability increases year by year [27]. The habit of chewing areca nut is considered to be the most likely factor for the occurrence and malignant progress of OSF [28]. In 2003, the World Health Organization listed areca nut as a primary carcinogen.
Areca nut contains many substances, such as alkaloids, polyphenols and nitrosamines. Among them, the alkaloids are Arecoline, Tetrahydronicotinic acid, etc. [29]. Arecoline is the main carcinogens in Areca nut that induce OSF to develop into cancer by their toxic effects directly. However, many studies showed that Arecoline inhibited cell growth, proliferation and collagen synthesis in human oral broblasts in a dose dependent manner [7,8,30]. Chang et al. found that Arecoline was cytotoxic to human oral broblasts at a concentration higher than 50 µg/ml by depleting intracellular thiols and inhibiting mitochondrial activity [8,30]; Jeng et al. found Arecoline inhibited the migration, attachment, spreading, growth and collagen synthesis of human oral broblasts at concentrations of 0.4 mM (62 µg/ml) and 1 mM (155 µg/ml) [7]. In fact, Venkatesh et al. found that the salivary Arecoline level were about 0.1 µg/ml to about 0.3 µg/ml after chewing commercially available areca nut [20], which is much lower than the concentration used in most experiments. In this study, we found that low dose Arecoline could promote the proliferation of human oral broblasts. In according with our ndings, Xia et al. found a relative low dose (20 µg/ml) Arecoline treatment could increase the collagen production of oral broblasts [31]. Combined with these situations, we suggest that more studies should focus on the effect of low dose Areoline on oral diseases.
Recently, the role of the Wnt/β-catenin pathway is proving to be central to mechanisms of pulmonary, hepatic, renal and cardiac brosis [16,[32][33][34]. Wnt family has a large number of members, among which Wnt5a is closely related to brosis. Vuga et al. found Wnt5a played a role in broblast expansion and survival characteristics of idiopathic pulmonary brosis and other brotic interstitial lung diseases that exhibit usual interstitial pneumonia histological patterns [19]; Villar et al. suggested that the Wnt/βcatenin signaling pathway is activated very early in sepsis-induced acute respiratory distress syndrome and could play an important role in lung repair and brosis [35]; Abraityte et al. found Wnt5a is elevated in serum and myocardium of heart failure (HF) patients and promoted myocardial in ammation and brosis [36]; Martin-Medina et al. found Wnt5a was secreted on extracellular vesicles in lung brosis and induced by TGF-β in primary human lung broblasts [37]. We screened the expression of Wnt family members in broblasts after Arecoline stimulation and further determined their basic functions. Our results con rmed that Wnt5a also played a proliferation role in low dose Arecoline stimulated human oral broblasts. This result suggests that the treatment of oral submucous brosis may also refer to the treatment of other organ brosis.
Although many transcription factors have been reported to regulate Wnt5a expression [21,22], we nally con rmed that Egr-1 regulated Wnt5a expression. Interestingly, Egr-1 is a typical immediate early gene (IEG) [38]. IEGs are genes which are activated transiently and rapidly in response to a wide variety of cellular stimuli. These characteristics of Egr-1 are consistent with the wavy change of Arecoline concentration in areca nut chewer's saliva [20]. And in this study, we also produced an Arecoline concentration uctuation by replacing half of the cell culture medium every 24 hours. These phenomena are consistent with our results, which further con rms our results. Meaningfully, our results showed that MMA or CHA treatment blocked Arecoline induced broblasts proliferation. MMA is a U.S. Food and Drug Administration-approved drug that may against brosis, cancer and neurodegenerative diseases [39][40][41].
Therefore, additional exploration of its mechanisms in disease models may identify MMA as a promising drug candidate for the treatment of OSF and oral cancer.

Conclusions
The study demonstrated that high dose Arecoline could inhibit but low dose Arecoline could promote the proliferation of human oral broblasts. Egr-1 mediates low dose Arecoline induced human oral mucosa broblasts proliferation by transaction the expression of Wnt5a, and Egr inhibitors or Wnt5a antibodies are potential therapeutic drugs of OSF and oral cancer. Lipofectamine LTX (#15338100, Invitrogen) and Lipofectamine RNAiMAX (#13778150, Invitrogen) were used for transient gene or siRNA transfection of cells. The following primary antibodies were used: Wnt1 (#ab15251), Wnt2 (#ab109222), Wnt5a (#ab179824) and GAPDH (#ab181602) were from Abcam plc..

DNA Growth Assay
Following treatment of cells, the media was discarded, cells were solubilized for 30 min at 37 °C in 0.1% SDS and the amount of DNA was estimated using a Hoechst 33258 microassay.

Western Blotting
Protein from cells (30 µg) were separated by SDS-PAGE and transferred onto PVDF membranes. Then the members were blotted with primary antibodies at 4 °C overnight. Blots were incubated with HRPconjugated secondary antibody for 1 h. The proteins were visualized using the ECL Plus WB detection system (Pierce, Rockford, IL).

Dual-luciferase Reporter Assays
Constructs were transfected into cells using Lipofectamine LTX. For the dual-luciferase reporter assays, cells were transfected with 1 µg of a luciferase reporter plasmid and 200 ng of the pRL-CMV Renilla luciferase reporter plasmid (Promega). After transfection, cells were kept in conditioned media for 12 or 24 h and then transferred to treatment media for 12 h. Fire y luciferase activity was normalized to Renilla luciferase activity according to the protocol.

Statistical analysis
Data are presented as mean ± SEM. Statistical analyses were performed with GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) using ANOVA followed by post hoc tests as appropriate. Statistical signi cance was declared when p < 0.05. The experimenters were not blind to group assignment and no data were omitted. The datasets analyzed during the current study are available from the corresponding author on request.

Competing interests
The authors declare no con ict of interest.  Low dose Arecoline induces the proliferation of broblasts. Human oral broblasts were treated with different dose Arecoline for indicated times, then cell proliferation rate was quanti ed. * denotes p < 0.05.

Figure 2
Arecoline promotes broblasts proliferation by inducing Wnt5a expression. a. Human oral broblasts were treated with or without 8 μg/ml Arecoline for indicated times, then cell lysates were analyzed by Western blotting using indicated antibodies. b-d. Human oral broblasts were treated with 8 μg/ml Arecoline, recombinant Wnt1, Wnt2 or Wnt5a protein and Wnt1, Wnt2 or Wnt5a antibody for 48 h, then cell proliferation rate was quanti ed. e. Human oral broblasts were treated with 8 μg/ml Arecoline, Wnt1, Wnt2 or Wnt5a sepeci c siRNAs for 48 h, then cell proliferation rate was quanti ed. * denotes p < 0.05.

Figure 3
Egr-1 is necessary for the expression of Wnt5a. a. Human oral broblasts were transfected with Wnt5a promoter luciferase reporter plasmids, treated with 8 μg/ml Arecoline and p65, Smad2, Smad3 or Egr-1 siRNAs for 48 h, then luciferase reporter assays were performed to detect the activity of Wnt5a promoter.
b. Human oral broblasts were treated with 8 μg/ml Arecoline and p65, Smad2, Smad3 or Egr-1 siRNAs for 48 h, then RT-PCR assays were performed to detect the mRNA levels of Wnt5a. c. Human oral broblasts were treated with 8 μg/ml Arecoline and Egr-1 siRNAs for 24 h, then cell lysates were analyzed by Western blotting using indicated antibodies. * denotes p < 0.05. Inhibition of Egr activity prevents Arecoline induced broblast proliferation. a. Human oral broblasts were treated with 8 μg/ml Arecoline, Wnt1, Wnt2 or Wnt5a sepeci c siRNAs for indicated times, then cell proliferation rate was quanti ed. b. Human oral broblasts were transfected with Wnt5a promoter luciferase reporter plasmids, treated with 8 μg/ml Arecoline, 2 μM Mithramycin A (MMA) or 1 μM Chromomycin A3 (CHA) for indicated times, then luciferase reporter assays were performed to detect the activity of Wnt5a promoter. c-d. Human oral broblasts were treated with 8 μg/ml Arecoline, 2 μM MMA or