Hepatitis B virus X protein up-regulates tumor necrosis factor-α expression in cultured mesangial cells via ERKs and NF-κB pathways

Abstract

Objective: To investigate the effects of hepatitis B virus (HBV) X protein (HBx) on the expression of tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (GMCs) and the underlying intracellular signal pathways.

Methods: The plasmid pCI-neo-X that carries the X gene of hepatitis B virus was transfected into cultured GMCs. HBx expression in the transfected GMCs was assessed by Western-blot. TNF-α protein and mRNA were assessed by ELISA and semi-quantitative RT-PCR, respectively. Three kinase inhibitors-U0126, an inhibitor of extracellular signal-regulated kinases (ERKs); lactacystin, an inhibitor of nuclear factor-κB (NF-κB); and SB203580, a selective inhibitor of p38 MAP kinase (p38 MAPK) were used to determine which intracellular signal pathways may underlie the action of HBx on TNF-α expression in transfected GMCs.

Results: A significant increase in HBx expression in pCI-neo-X transfected GMCs was detected at 36 h and 48 h, which was not affected by any of those kinase inhibitors mentioned above. A similar increase in the expression of both TNF-α protein and mRNA was also observed at 36 h and 48 h, which was significantly decreased in the presence of U0126 or lactacytin, but not SB203580.

Conclusions: HBx upregulates TNF-α expression in cultured GMCs, possibly through ERKs and NF-κB pathway, but not p38 MAPK pathway.

Keywords: Extracellular signal-regulated kinase; Glomerulonephritis; Heptitis B virus; Nuclear factor-κB; Tumor necrosis factor-α; X protein.

Figure 1.. Representative Western blot data, showing the time courses of the changes in the expression of HBx and TNF-α in GMCs after pCI-neo-X plasmid transfection.

Figure 2.. Effects of SB203580, U0126 and lactacystin on HBx expression in the HBV X gene-transfected GMCs.

-: without inhibitor; +: with inhibitor.

Figure 3.. Averaged levels of the HBx expression in the HBV X gene-transfected MGCs (HBx levels are normalized over β-actin, (n=4).

Figure 4.. Representative RT-PCR data showing the effects of different kinase inhibitors on TNF-α mRNA level in HBV X gene-transferred GMCs.

-: Without inhibitor; +: with inhibitor. M: Marker. Lane 1 and lane 6 show the GMC+pCI-neo control; the other lanes show TNF-α mRNA levels after successful GMC+pCI-neo-X transfection.

Figure 5.. Averaged data of TNF-α mRNA level, indicating that U0126, lactacystin, but not SB203580, significantly attenuated TNF-α mRNA levels in HBV X gene-transfected cells.

-: Without inhibitor; +: With inhibitor. Group 1 and group 6 refer to GMC+pCI-neo control. *P<0.05, compared with the data shown in lane 2 or lane 7. TNF-α mRNA levels are normalized over β-actin (n=4).

Figure 6.. Effects of the different kinase inhibitors on TNF-α expression as assessed by ELISA in HBV X gene-transfected cells (n=4). *P<0.05, compared with the GMC+pCI-neo-X group.