Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway

Abstract

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

Deletion and Functional Analysis of Hepatitis B Virus X Protein: Evidence for an Effect on Cell Cycle Regulators

“…HBx is a promiscuous trans-activator that modulates both viral and cellular promoters through a direct interaction with nuclear transcription factors or via cytoplasmic signal transduction [5]. The protein is shown to affect several cellular processes including reduction of adhesion of podocytes [6], modulation of apoptosis in kidney cell [7] and downregulation of microRNA-145 in HCC [8]. HBx affects cell cycle progression by inducing different signaling pathways, deregulating cell cycle checkpoints, and by protein degradation.…”

“…Some studies have reported suppression of apoptosis by HBx through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), cAMP response element-binding protein (CREB) and phosphatidylinositol 3-kinase (PI3K) pathways, and survivin protein [13][14][15]. Other studies have shown that HBx induces apoptosis by activation of caspases and c-Jun amino-terminal kinase (JNK) pathway [11,16].…”

Hepatitis B Virus Core Antigen Stimulates IL-6 Expression via p38, ERK and NF-κB Pathways in Hepatocytes

“…HBx, a regulatory protein, can stimulate multiple host pathways including p38, ERK, JNK and NF-κB [20, 42, 43]. Large surface protein activated p38 and NF-κB [44, 45], while C-terminally truncated middle surface protein activated ERK2 [46], NF-κB and other pathways [47].…”

Interference of Apoptosis by Hepatitis B Virus

“…Another death receptor, Fas, and its ligand FasL are upregulated in rat renal tubular epithelial cells (NRK-52E) transfected with HBX, and this increase is due to the activation of the MLK3-MKK7-JNK pathway [ 156 ]. The Fas sensitivity is reconstituted in HBX transgenic mice through the decrease of BCL-2, despite no direct interaction between HBX and BCL-2 family members [ 70 ].…”