Functional analysis of a polymorphism in the promoter region of the IL-12/23p40 gene.
BACKGROUND
Human IL-12B gene on chromosome 5q31 encodes the common p40 subunit of IL-12 and IL-23. IL-12 is known to play critical roles in the generation of T-helper type 1 (TH(1)) cells, whereas IL-23 is involved in maintenance and/or population expansion of TH(17) cells. Although several reports suggested an association between a polymorphism (-6415CTCTAA/GC) in IL-12B and asthma, the molecular mechanism how this polymorphism is involved in allergic inflammation is still unclear.


METHODS
The transcription activity was analysed by reporter assay. A transcription factor binding to -6415 polymorphic site was identified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The amount of cytokines produced from peripheral monocytes were determined by ELISA.


RESULTS
Reporter assay showed that the transcription activity of the GC allele was higher than that of the CTCTAA allele. A transcription factor Sp1 bound to the region including the GC allele with a higher affinity than that of the CTCTAA allele in EMSA. In vivo binding of Sp1 to IL-12B gene carrying -6415GC was confirmed by ChIP assay. Overexpression of Sp1 up-regulated transcription activity of promoter carrying GC allele sequence, whereas the CTCTAA promoter was not affected by Sp1. We examined the correlation between -6415CTCTA/GC polymorphism and production of cytokine IL-12/23p40, IL-12p70, and IL-23 on peripheral blood monocytes, and monocytes with the GC/GC allele exhibited significantly higher expression of IL-12p70 protein than those with the CTCTAA/CTCTAA allele (P=0.009).


CONCLUSIONS
The -6415 polymorphism is involved in cytokine production potential by affecting Sp1-mediated transcription activity.
Shimokawa
N
N
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan.
Nishiyama
C
C
Hirota
T
T
Tamari
M
M
Hara
M
M
Ikeda
S
S
Okumura
K
K
Ogawa
H
H
D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D018664:Interleukin-12 / Q000378:metabolism; D053762:Interleukin-12 Subunit p40 / Q000235:genetics* / Q000378:metabolism; D053759:Interleukin-23 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection; D020298:U937 Cells
D053762:Interleukin-12 Subunit p40; D053759:Interleukin-23; D008070:Lipopolysaccharides; D016329:Sp1 Transcription Factor; D018664:Interleukin-12; D007371:Interferon-gamma