Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway.
OBJECTIVE
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.
Department of Nephrology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, P.R. China.
Ping
P
He
Beiru
B
Zhang
Dajun
D
Liu
Xiaohui
X
Bian
Detian
D
Li
Yanqiu
Y
Wang
Guangping
G
Sun
Guangyu
G
Zhou
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053222:Fas Ligand Protein / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006515:Hepatitis B virus / Q000737:chemistry*; D026121:Indole Alkaloids / Q000494:pharmacology; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D048688:MAP Kinase Kinase 7 / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D048057:Mitogen-Activated Protein Kinase 10 / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000235:genetics*; D015534:Trans-Activators / Q000302:isolation & purification / Q000494:pharmacology*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins; D019014:fas Receptor / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D002227:Carbazoles; D053181:Caspase 8; D053222:Fas Ligand Protein; D026121:Indole Alkaloids; D048688:MAP Kinase Kinase 7; D020930:MAP Kinase Kinase Kinases; D048057:Mitogen-Activated Protein Kinase 10; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; D019014:fas Receptor; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C491898:Fas protein, rat; C505662:Faslg protein, rat; C057628:hepatitis B virus X protein; C049985:staurosporine aglycone; C505528:Casp8 protein, rat