Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts

Abstract

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

Keywords: CCL20; TNF-α; cardiac fibroblasts; cardiac inflammation.

Figure 1

TNF-α induces CCL20 protein secretion and mRNA expression in HCFs. (A) Cells were incubated with TNF-α for the indicated time intervals (4, 6, 12, and 24 h). The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B,C) The mRNA levels of CCL20 were determined by real-time PCR. (B) Cells were incubated with TNF-α (5 ng/mL) for the indicated time intervals (0.5, 1, 2, 3, 4, and 6 h. (C) Cells were incubated with 0.3, 1, 5, and 15 ng/mL TNF-α for 2 h. (D) Cells were incubated with TNF-α (5 ng/mL) for the indicated time intervals (1, 2, 3, 4, and 6 h). The promoter activity of CCL20 was determined by a promoter assay kit. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone.

Figure 2

TNF-α-induced CCL20 expression via TNFR1 in HCFs. (A) Cells were pretreated with 1 and 0.1 μg/mL TNF receptor 1 antibody (TNFR1 Ab) or TNFR2 Ab for 1 h, and then incubated with 5 ng/mL TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. (B) Cells were pretreated with 0.1 and 0.01 μg/mL TNFR1 Ab for 1 h, and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or TNFR1 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of TNFR1 were determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 3

Involvement of EGFR in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 10 μM AG1478 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 10 μM AG1478 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or EGFR siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of EGFR were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 1 μg/mL TNFR1 Ab or 10 μM AG1478 for 1 h and then treated with TNF-α for the indicated time (0, 5, 10, 15, 30, and 60 min). The phosphorylation of EGFR was determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 4

Involvement of p38 MAPK in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 μM p38 inhibitor VIII (p38i VIII) for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 μM p38i VIII for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or p38α siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of p38α were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 10 μM AG1478 or 3 μM p38i VIII for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of p38 and EGFR was determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 5

Involvement of JNK1/2 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 μM SP600125 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 μM SP60012 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or JNK2 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of JNK2 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 10 μM AG1478 or 3 μM SP600125 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of JNK1/2 and EGFR was determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 6

Involvement of FoxO1 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 1 μM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 1 μM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or FoxO1 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of FoxO1 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 1 μM AS1842856, 3 μM p38i VIII, or 3 μM SP600125 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of FoxO1, p38, or JNK1/2 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with 3 μM p38i VIII, 3 μM SP600125, or 1 μM AS1842856 for 1 h and then stimulated with TNF-α for the indicated time intervals or 30 min. The binding of FoxO1 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 6

Involvement of FoxO1 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 1 μM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 1 μM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or FoxO1 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of FoxO1 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 1 μM AS1842856, 3 μM p38i VIII, or 3 μM SP600125 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of FoxO1, p38, or JNK1/2 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with 3 μM p38i VIII, 3 μM SP600125, or 1 μM AS1842856 for 1 h and then stimulated with TNF-α for the indicated time intervals or 30 min. The binding of FoxO1 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 7

Involvement of NF-κB p65 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 μM helenalin for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 μM helenalin for 1 h and then incubated with 5 ng/mL TNF-α for indicated time intervals. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of p65 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 3 μM helenalin, 1 μg/mL TNFR1 Ab, or 10 μM AG1478 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of p65 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with helenalin (3 μM) for 1 h or transfected with scrambled or p65 siRNA, and then incubated with TNF-α for 15 min. Cells were fixed and then labeled with an anti-phospho-p65 antibody and then a FITC-conjugated secondary antibody. The localization and expression of phospho-p65 were determined by immunofluorescent staining (green), and nuclei were stained with DAPI (blue). Scale bar: 50 µm. (F) Cells were pretreated without or with 3 μM helenalin for 1 h and then stimulated with TNF-α for the indicated time intervals or 15 min. The binding of NF-κB p65 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 7

Involvement of NF-κB p65 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 μM helenalin for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 μM helenalin for 1 h and then incubated with 5 ng/mL TNF-α for indicated time intervals. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of p65 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 3 μM helenalin, 1 μg/mL TNFR1 Ab, or 10 μM AG1478 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of p65 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with helenalin (3 μM) for 1 h or transfected with scrambled or p65 siRNA, and then incubated with TNF-α for 15 min. Cells were fixed and then labeled with an anti-phospho-p65 antibody and then a FITC-conjugated secondary antibody. The localization and expression of phospho-p65 were determined by immunofluorescent staining (green), and nuclei were stained with DAPI (blue). Scale bar: 50 µm. (F) Cells were pretreated without or with 3 μM helenalin for 1 h and then stimulated with TNF-α for the indicated time intervals or 15 min. The binding of NF-κB p65 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). ^# p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Figure 8

Schematic diagram illustrating the proposed signaling pathway involved in TNF-α-induced CCL20 expression and secretion in HCFs. TNF-α-induced CCL20 expression was, at least partially, mediated through binding to TNFR1 leading to transactivation of EGFR. Activated EGFR promoted the phosphorylation of p38 MAPK- or JNK1/2-dependent FoxO1 activation, which further bound with the FoxO1 response element (FRE) on the CCL20 promoter. In addition, TNF-α also turned on NF-κB transcription factors. Either FoxO1 or NF-κB activation could enhance the expression of CCL20 induced by TNF-α, which may be engaged in the inflammatory responses in HCFs. A better understanding of mechanisms underlying the regulation of the CCL20 gene by TNF-α will support more opportunities to develop anti-inflammatory therapeutic strategies for treating cardiac inflammation.