Up-regulation of the lysyl hydroxylase 2 gene by acetaminophen and isoniazid is modulated by transcription factor c-Myb.
OBJECTIVE
Lysyl hydroxylase 2 (LH2), an isoform of hydroxylase, catalyses the hydroxylation of lysine residues in the telopeptide of collagen to form stable and irreversible cross-linkages in collagen. Increased activity of this enzyme in activated stellate cells in human liver has been proposed to relate to the promotion of hepatic fibrosis. In the present study, we examined the regulation of LH2 expression in drug-induced liver injury in order to clarify the mechanisms behind the hepatic fibrosis caused by certain drugs.


METHODS
The mRNA and protein expression of the target gene were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) with specific primers and Western blotting with a specific antibody, respectively.


RESULTS
The expression of LH2 was increased in HepG2 cells incubated with acetaminophen and isoniazid. This increase was accompanied by an increase in the expression of c-myeloblastosis viral oncogene homolog (Myb) mRNA. Over-expression of c-Myb in cells transfected with a c-Myb expression plasmid, pMbm I, caused an increase in the expression of LH2 mRNA. Mutation of the Myb-binding site in the promoter region of the LH2 gene resulted in a loss of transcriptional activation in the reporter gene assay.


CONCLUSIONS
These results suggest that c-Myb modulates the expression of the LH2 gene in HepG2 cells incubated with drugs causing hepatic fibrosis.
Kubota
Masafumi
M
Department of Medical Pharmaceutics, Gifu Pharmaceutical University, Japan.
Shinoda
Aya
A
Iguchi
Kazuhiro
K
Takahashi
Yukari
Y
Usui
Shigeyuki
S
Kiho
Tadashi
T
Hirano
Kazuyuki
K
D000082:Acetaminophen / Q000009:adverse effects*; D001665:Binding Sites; D015153:Blotting, Western; D056486:Chemical and Drug Induced Liver Injury / Q000235:genetics* / Q000378:metabolism; D005355:Fibrosis / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D007538:Isoniazid / Q000009:adverse effects*; D008099:Liver / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D010957:Plasmids; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D015854:Up-Regulation
D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D000082:Acetaminophen; C479138:PLOD2 protein, human; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D007538:Isoniazid