Inhibition of nuclear factor kappa B activation and inducible nitric oxide synthase transcription by prolonged exposure to high glucose in the human keratinocyte cell line HaCaT.
BACKGROUND
In human skin, inducible nitric oxide synthase (iNOS) appears to be a key enzyme during wound healing and has roles in protection from infection. We speculated that diabetic skin complications such as delayed wound healing and skin infection were due to iNOS activity altered by high glucose in skin keratinocytes.


OBJECTIVE
The purpose of this study was to see how high levels of glucose affect iNOS activity in the human keratinocyte cell line (HaCaT).


METHODS
HaCaT cells were exposed to high glucose for 1 day or 10 days. We measured nitric oxide (NO) end product nitrite in the culture medium using the Griess reagent, and intracellular tetrahydrobiopterin (BH(4), a cofactor of NOS) content by using high-performance liquid chromatography, analysed the expression level of iNOS mRNA by the reverse transcriptase-polymerase chain reaction method and evaluated the DNA binding activity of nuclear factor kappa B (NF-kappaB) by enzyme-immunoassay.


RESULTS
Short-term exposure (1 day) to a high level of glucose increased BH(4) and iNOS activity at the post-translational level. However, long-term exposure (10 days) to high glucose downregulates NF-kappaB binding activity and inhibits iNOS transcription and its activity.


CONCLUSIONS
Pretreatment with high glucose for 10 days down-regulated NF-kappaB activity and inhibited iNOS transcription and NO production, implying the involvement of a deficiency in NO synthesis in both skin infection and impaired wound healing in diabetic patients.
Nakai
K
K
Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Kita, Kagawa 761-0793, Japan.
Kubota
Y
Y
Kosaka
H
H
D000975:Antioxidants / Q000032:analysis; D001708:Biopterins / Q000031:analogs & derivatives* / Q000032:analysis; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D013997:Time Factors; D014158:Transcription, Genetic
D000975:Antioxidants; D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin; D005947:Glucose