Epidermal growth factor upregulates the expression of A20 in hepatic cells via the MEK1/MSK1/p-p65 (Ser276) signaling pathway

Abstract

Tumor necrosis factor α-induced protein 3 (A20) suppresses inflammation by inhibiting the activation of nuclear factor kappa B (NF-κB). The aberrant expression of A20 is reportedly correlated with tumor development in human malignancies, including hepatocellular carcinoma (HCC). Proinflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-1, and lipopolysaccharide, may induce A20 expression. The present study revealed that epidermal growth factor (EGF) significantly increased A20 mRNA and protein levels in normal hepatic and hepatoma cells via the mitogen-activated protein kinase kinase-1 (MEK1)/mitogen- and stress-activated protein kinase-1 (MSK1)/phosphorylated (p)-p65 (Ser276) signaling pathway. A significant positive correlation was observed between the expression of EGF receptor and A20 in HCC and normal healthy liver tissues. The EGF-induced A20 upregulation was NF-κB-dependent and abolished by either the overexpression of the nuclear factor of a κ light polypeptide gene enhancer in a B-cell inhibitor α or treatment with the NF-κB inhibitor BAY11-7082. However, unlike TNF-α, EGF expression did not result in the upregulation of inflammatory molecules, including intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and monocyte chemoattractant protein-1. These results indicate that EGF preferentially upregulated the protective mediator A20 over proinflammatory factors. To our knowledge, the present study is the first to demonstrate that EGF induced A20 expression by activating the MEK1/MSK1/p-p65 (Ser276) signaling pathway without causing an apparent inflammatory response. These results may further extend our understanding of liver inflammation and tumor development.

Keywords: Tumor necrosis factor α-induced protein 3 (A20); epidermal growth factor (EGF); inflammation; liver; nuclear factor kappa B (NF-κB).

Figure 1

EGF upregulates A20 expression in QSG7701, HepG2, and HCCLM3 cell lines. A. Western blot of A20 in QSG7701, HCCLM3, and HepG2 cells stimulated with TNF-α (10 ng/ml), IL-6 (100 ng/ml), LPS (100 ng/ml), EGF (50 ng/ml) or HGF (50 ng/ml) for 6 hours. B. Western blot of A20 in QSG7701, HCCLM3, and HepG2 cells treated with EGF for different periods. C. The reverse transcription-quantitative PCR of A20 mRNA in QSG7701, HCCLM3, and HepG2 cells treated with EGF. Key: EGF, epidermal growth factor; A20, tumor necrosis factor α-induced protein 3; TNF-α, tumor necrosis factor-α; IL-6, interleukin 6; LPS, lipopolysaccharide; HGF, hepatocyte growth factor.

Figure 2

EGF-induced A20 upregulation is dependent on NF-κB activation. A. Western blot of A20 protein in QSG7701 cells with pCDNA3.1-IkBα or control plasmids treated with EGF for the indicated time. B. Western blot of A20 protein in QSG7701 cells pretreated with the NF-κB inhibitor BAY11-7082 at the indicated concentrations and followed by EGF treatment. C. Reverse transcription-quantitative PCR of A20 mRNA in QSG7701 cells pretreated with BAY 11-7082 or DMSO and after treatment with EGF. Key: EGF, epidermal growth factor; A20, tumor necrosis factor α-induced protein 3; IkBα, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α; DMSO, dimethyl sulfoxide. All data are expressed as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

EGF induced the expression of A20 through the MEK1/MSK1/p-p65 (Ser276) signaling pathway. Western blot of A20 protein in QSG7701 cells pretreated with (A) the PI3K inhibitor LY294002 or (B) the MEK1 inhibitor PD98059 followed by treatment with EGF for different periods. Key: EGF, epidermal growth factor; A20, tumor necrosis factor α-induced protein 3; MEK1, mitogen-activated protein kinase; MSK1, ribosomal protein S6 kinase A5; p, phosphorylated. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

EGF induced A20 expression via the MEK1/MSK1/p-p65 (Ser276) signaling pathway. A. EGF activated the MEK1/ERK signaling pathway, resulting in the activation of MSK1 and the subsequent phosphorylation of p65 at Ser276, increased p65 activity, and increased expression of A20. The inhibition of MEK1 by PD98059 resulted in reduced MSK1 phosphorylation, whereas the inhibition of MSK1 by H89 repressed the phosphorylation of p65 and the induction of A20. B. Western blot of A20 protein in QSG7701 cells pretreated with the MSK1 inhibitor H89 to inhibit the phosphorylation of p65 at Ser276 and then treated with EGF (50 ng/ml) for the indicated time. Key: EGF, epidermal growth factor; A20, tumor necrosis factor α-induced protein 3; MEK1; mitogen-activated protein kinase kinase; MSK1, ribosomal protein S6 kinase A5; p, phosphorylated. All data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Relationship between the expression of A20 and EGFR in HCC and liver tissues. mRNA was extracted from 22 pairs of (A) HCC and (B) adjacent normal tissues. The levels of A20 and EGFR were measured by reverse transcription-quantitative PCR. The correlation of A20 and EGFR expression in tumor (T) or peri-tumor (N) tissues was analyzed by Pearson’s correlation test. Key: A20, tumor necrosis factor α-induced protein 3; EGFR, epidermal growth factor receptor; HCC, hepatocellular carcinoma.

Figure 6

A20 induced by EGF contributed to the anti-inflammatory effect of this growth factor in hepatic cells. A. RT-qPCR analysis of ICAM-1, VCAM-1, and MCP-1 mRNA in QSG7701 cells treated with EGF or TNF-α at the indicated concentrations. B. RT-qPCR analysis of ICAM-1, VCAM-1, and MCP-1 mRNA in QSG7701-shcon and QSG7701-shA20 cells pretreated with EGF and then stimulated with TNF-α. Key: A20, tumor necrosis factor α-induced protein 3; EGFR, epidermal growth factor receptor; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; MCP-1, monocyte chemoattractant protein-1; RT-qPCR, reverse transcription-quantitative PCR.