Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS.
The 230-kDa bullous pemphigoid antigen (BPAG1) is an integral component of hemidesmosomes. We have previously reported that interferon-gamma (IFNgamma) inhibits the transcription of the BPAG1 gene (1). Here we investigated the target sequences of IFNgamma-signal transduction pathway in the BPAG1 promoter in epidermal keratinocytes. Transient transfections with 5'-deletion constructs of BPAG1 promoter-luciferase reporter gene plasmids in cultured normal human epidermal keratinocytes (NHEK) allowed us to narrow the DNA region containing IFNgamma inhibitory element (IGIE) to between -1 and -89, upstream from the transcription initiation site (+1). Homology search in this region identified a chimeric sequence, consisting of IFN-stimulated responsive element (ISRE) with a partial 7-bp sequence of IFNgamma activation site (GAS), as identified in the guanylate-binding protein (GBP) gene, inserted at its center. Functional analysis of IGIE, inserted in front of the heterologous thymidine kinase promoter, indicated that IGIE acts as a down-regulatory element of the promoter through IFNgamma-dependent signal pathway. Transient transfection studies with BPAG1 promoter-reporter gene constructs containing mutated IGIE (with TT to GG transversions in the region of 5'ISRE, GAS, and 3'ISRE) demonstrated that disruption of the ISRE sequences, but not GAS, markedly suppressed the BPAG1 basal promoter activity and resulted in attenuated IFNgamma response in keratinocytes. Our findings provide novel insight into the mechanism of IFNgamma regulation in keratinocyte differentiation and proliferation.
Kaneko
Takahide
T
Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan.
Tamai
Katsuto
K
Matsuzaki
Yasushi
Y
Yamazaki
Takehiko
T
Nakano
Hajime
H
Kon
Atsushi
A
Hashimoto
Isao
I
Hanada
Katsumi
K
Kaneda
Yasuhumi
Y
Uitto
Jouni
J
D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000235:genetics; D015536:Down-Regulation; D000071821:Dystonin; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D015603:Keratinocytes / Q000737:chemistry / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020218:Response Elements; D015398:Signal Transduction
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C486268:DST protein, human; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D007371:Interferon-gamma; D004247:DNA; D008156:Luciferases