IFN-gamma upregulates expression of the mouse complement C1rA gene in keratinocytes via IFN-regulatory factor-1.
We examined the expression of the mouse complement component C1rA (mC1rA) in IFN-gamma-stimulated mouse keratinocytes (Pam 212) and found that it was upregulated. To analyze the mechanism involved, we cloned the 2,150 bp 5'-flanking region of mC1rA by the vectorette-PCR technique, and identified the transcription start site of mC1rA by rapid amplification of complementary DNA ends. Analysis of the 5' sequence revealed putative binding sites for activator protein 1, CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 1 (STAT-1), IFN-regulatory factor-1 (IRF-1), and others. We detected transcriptional activation dependent on this upstream region in reporter gene assays and Northern blots. To identify the cis-acting regulatory elements involved, we analyzed serial deletion constructs of the promoter using luciferase reporters. The -80 to -19 bp region, which contains a putative IRF-1 binding site, was required for both basal promoter activity and responses to IFN-gamma. The use of site-directed point mutations, electrophoresis mobility shift assays, and supershift assays indicated that the putative IRF-1 binding site was essential for both IFN-gamma-dependent and -independent transcriptional activity of the mC1rA promoter. We conclude that IFN-gamma stimulates mC1rA gene expression via IRF-1 in mouse keratinocytes.
Byun
Sung June
SJ
National Livestock Research Institute, Division of Animal Biotechnology, Suwon, South Korea.
Jeon
Ik-Soo
IS
Lee
Hyangkyu
H
Kim
Tae-Yoon
TY
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D015923:Complement C1r / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000502:physiology*; D007371:Interferon-gamma / Q000502:physiology*; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D018808:Transcription Factor AP-1; D007371:Interferon-gamma; D015923:Complement C1r