The molecular mechanism of GADD153 in apoptosis of keloid fibroblasts exposed to botulinum toxin type A

Abstract Apoptosis plays a key role in keloids. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor‐alpha (TNF‐α) antibodies reversed the BTXA‐induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti‐TNF‐α antibody treatment abolished the BTXA‐induced transcriptional activity of GADD153. The addition of TNF‐α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti‐TNF‐α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.

the pathophysiology of neurodegenerative Parkison's disease, 11 cardiac hypertrophy, 12 diabetic cardiomyopathy 13 and cancer. 14 C/EBP homologous protein, also known as growth arrest and DNA damage-inducing protein 153 (GADD153), is the main component of the ER stress-mediated apoptosis pathway. 15 As the primary element of the UPR, GADD153 plays two roles in regulating cell survival or cell death. 16 Under normal circumstances, the expression level of GADD153 in cells is very low, but if this protein is stimulated by genotoxic agents, calcium ionophores, lipopolysaccharides or nutrient deprivation, its expression level increases significantly. 17 c-Jun N-terminal kinase (JNK) and activator protein 1 (AP-1) play decisive roles in the regulation of GADD153 gene transcription. 18,19 Apoptosis plays an essential role in keloids. 20,21 Clinical studies have indicated that botulinum toxin A (BTXA) can induce the apoptosis of keloid fibroblasts and can mitigate hypertrophic scar formation. 22 Compared with normal adult fibroblasts, keloid fibroblasts have a higher rate of apoptosis in response to hypoxia and interferonγ. 23 It remains unknown whether BTXA can stimulate the expression of GADD153 to induce apoptosis of keloid fibroblasts.
In this study, we aimed to investigate 1) whether BTXA can induce 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ under 5% CO2/95% air in a humidified incubator. When confluent, keloid fibroblast monolayers were passaged every 9-10 days after trypsinization and used for experiments from the third to seventh passages.
The cells from the third to seventh passages were then cultured in DMEM containing 0.5% FBS, and the keloid fibroblasts were incubated for one additional day to render them quiescent before each experiment was initiated.

| Western blot analysis
Western blot was performed per the method of a previous study. 24 In brief, keloid fibroblasts were homogenized in modified RIPA buffer to purify the protein. An equal amount of protein (50 μg) was loaded, and anti-GADD153 antibodies (1:200 dilution; Santa Cruz Biotechnology) were used. Signals were visualized by chemiluminescent detection.
The protein loadings of the samples were further verified to be equal through the staining of monoclonal antibodies against α-tubulin. All Western blots were quantified using densitometry.

| Reverse transcription (RT) polymerase chain reaction (PCR)
In brief, total RNA was isolated from keloid fibroblasts using the single-step acid guanidinium thiocyanate/phenol/chloroform extraction method per a previously described method. 24

| Real-time quantitative PCR
Real-time quantitative PCR was performed per a previously described method. 24 The primers used for real-time quantitative

| Immunohistochemistry
Immunohistochemistry was used to detect the image of GADD153.

| Promoter activity assay
We used T-Pro NTR II transfection reagent (T-Pro Biotechnology, Taipei, Taiwan) to transform the GADD153 promoter construct containing the AP-1 binding site into keloid fibroblasts. The GADD153 promoter-containing plasmids were designed and transfected into keloid fibroblasts using liposomes as vectors. In addition, the GADD153 promoter construct containing the 5' AP-1 mutant binding site was also designed and transfected into keloid fibroblasts. Following the treatment of BTXA, keloid fibroblast extracts were prepared using a dual-luciferase reporter assay system (Promega) and measured for dual-luciferase activity using a luminometer (Turner Designs).

| Detection of TNFα concentration by enzyme-linked immunosorbent assay
In brief, TNFα recombinant protein (Calbiochem, San Diego, CA, USA) at different concentrations was added to the cultured medium. The level of IFNγ was measured using a quantitative sandwich enzyme immunoassay (Merck Millipore) per a previously described method. 24

| MTT assay
An MTT assay was used to determine keloid fibroblast viability after BTXA treatment. Keloid fibroblasts were adjusted to 3 × 10 4 cells/ml in DMEM medium. After treatment with BTXA, the medium was aspirated, 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt etrazolium bromide) solution was added, and incubation continued for 4 h. At the end of the incubation period, the MTT solution was removed from the attached cells and the converted dye crystals were dissolved with DMSO. The absorbance of the converted dye was measured at a wavelength of 570 nm.

| Cytotoxicity
A cytotoxicity study was performed per a previously described method. 25 To detect cell injury induced by BTXA, the cell death rate (%) after the addition of BTXA was monitored by trypan blue staining.

| Statistical analysis
All results are expressed as the mean ±standard error of the mean.
Statistical significance was evaluated in terms of the variance (GraphPad Software, San Diego, CA, USA). Dunnett's test was used to compare multiple groups to a single control group. The Turkey-Kramer test was used for pairwise comparisons between multiple groups after the ANOVA. A value of p < 0.05 was considered statistically significant.

| BTXA increased GADD153 protein and mRNA expression in keloid fibroblasts
As shown in Figure 1A   These findings suggest that the JNK pathway mediates the induction of GADD153 protein expression by BTXA in keloid fibroblasts.

| BTXA enhanced GADD153 promoter activity in keloid fibroblasts
To investigate whether GADD153 expression induced by BTXA was regulated at the transcriptional level, we used a luciferase reporter assay to investigate the genetic transcription activity of GADD153 in keloid fibroblasts after BTXA treatment. As shown in Figure 3A, the GADD153 promoter construct contained AP-1 (binding site: TGACTCA), specificity protein 1, nuclear factor 1, nuclear factor interleukin-6 (NF-IL6) and GC box binding sites. We also constructed a GADD153 promoter that contained an AP-1 mutation (binding site: As shown in Figure 3B, GADD153 promoter activity increased significantly after BTXA treatment (1 U/ml) for 16 h, but the same effect was not observed for the GADD153 mutant. Treatment with SP600125 and anti-TNFα antibodies before the addition of BTXA reversed the BTXA-induced promoter activity. These findings suggest that BTXA-induced GADD153 expression in keloid fibroblasts is mediated by the AP-1 pathway.

| BTXA-enhanced GADD153 protein expression in keloid fibroblasts through TNFα
As shown in Figure 4A, TNFα secreted from keloid fibroblasts after BTXA treatment markedly increased at 4 h and remained elevated for 24 h. This finding suggests that BTXA increased the secretion of TNFα from keloid fibroblasts.
To explore the effect of TNFα on GADD153 expression in keloid fibroblasts, we added different concentrations of TNFα to the cultured medium and incubated the solutions for 48 h. As shown in Figure 4B    We demonstrated that BTXA activates the expression of GADD153 protein via the JNK MAPK pathway in keloid fibroblasts. SP600125, a specific inhibitor of JNK, abolished the GADD153 protein expression induced by BTXA. However, specific inhibitors of ERK and p38 did not affect BTXA-induced GADD153 protein expression. Yun et al. reported that the host's response to BTXA is dependent on toll-like receptor 2 (TLR2), which is regulated by the JNK pathway. 26 In addition, another study demonstrated that 1) BTXA inhibited the formation of scar-related factors and the extracellular matrix in human scar fibroblasts and 2) the JNK pathway had a regulatory effect in this process. 27 These results suggest that BTXA is closely related to the regulation of JNK MAPK.
GADD153 promoter activity requires AP-1, the downstream target of JNK. 28 Through reporter gene testing, we demonstrated that the BTXA-induced transcriptional activity of the GADD153 promoter is AP-1 dependent. Our results indicate that the JNK pathway is the main pathway through which BTXA induces GADD153 expression. Previous studies have demonstrated that genistein exerts antiproliferative and proapoptotic effects on the expression of AP-1 subunits in keloid fibroblasts. 29 Wei et al. 30 suggested that a single complex, which contains AP-1 and SMAD binding complex components, is responsible for responding to serum transactivation. These aforementioned studies have indicated that the regulation of AP-1 plays a key role in keloid fibroblasts. However, previous research has demonstrated that the keloid regression induced by a flash lamp pulsed dye laser is mediated by the blockade of AP-1 transcription. 31 This discrepancy may be explained by the different experimental models used between studies. However, these conflicting results demonstrate that the regulatory role of AP-1 in keloid fibroblasts is still controversial.
Our results indicate that BTXA enhances the expression of TNFα in keloid fibroblasts. Previous research has also reported an increased gene expression of TNFα in keloid tissues. 32 In addition, our findings indicate that TNFα is involved in the expression of GADD153 In this study, we demonstrated that the BTXA-induced cell death and apoptosis of keloid fibroblasts is mediated by GADD153. This is finding is consistent with that of Zhanying et al. 37 that BTXA inhibits the proliferation of human skin keloid fibroblasts and promotes apoptosis by regulating ZEB2 targeting miR-1587/miR-2392. In addition, Gil et al. suggested that BTXA reduced the proliferation of human scar fibroblasts. 27 In this study, we demonstrated that the BTXA-induced apoptosis of keloid fibroblasts is regulated by JNK. Previous research has indicated that the inhibitory effect of BTXA in hypertrophic scar fibroblasts is closely related to the regulation of JNK. 27 We further demonstrated that BTXA-induced caspase 3 and 9 activity and that the addition of GADD153 siRNA reversed such BTXAinduced caspase 3 and 9 activity. Apoptosis can be divided into intrinsic and extrinsic pathways. Caspase 9 is related to the intrinsic pathway, which in turn induces the effect of caspase3, and ultimately leads to apoptosis. ER stress and GADD153 induces apoptosis through endogenous pathways. 38 Our results indicated BTXA-induced keloid fibroblasts apoptosis is through caspase 9 and caspase 3. On the other hand, CACNA1G-AS1 was previously reported to inhibit the expression of miR-205, promoting the inhibition of caspase 3 activity in human keloid fibroblasts. 21 Furthermore, Hao et al. 39 demonstrated that the inhibition of microRNA-21 increased caspase 3 and 9 activity in keloid fibroblasts. These studies have indicated that caspase 3 and 9 are closely related to the apoptosis of keloid fibroblasts. The present study suggests that GADD153 may be involved in the apoptosis of keloid fibroblasts treated with BTXA.
In summary, this study is the first to present evidence that BTXA induces GADD153 expression in cultured human keloid fibroblasts.

ACK N OWLED G EM ENT
This study was supported by the Shenzhen Key Medical Discipline Construction Fund (No. szxk026), Shenzhen, China.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest. Funding acquisition (equal); Resources (equal); Supervision (equal).