1alpha,25(OH)(2)D(3) regulates NF-kappaB DNA binding activity in cultured normal human keratinocytes through an increase in IkappaBalpha expression.
NF-kappaB is a dimeric transcription factor which regulates transcription of a number of different genes including IL-8 and p53. In resting cells NF-kappaB is usually retained in an inactive state in the cytoplasm through binding to a member of the inhibitory kappaB (IkappaB) protein family. The purpose of this study was to determine the effect of 1alpha,25(OH)(2)D(3) on NF-kappaB activation in both unstimulated and stimulated (IL-1alpha) cultured normal human keratinocytes. NF-kappaB DNA binding activity was determined by EMSA using two different oligonucleotides containing the kappaB sequence from either the IL-8 or the p53 promoter. IkappaBalpha and p53 expression was determined by Western blotting and IL-8 expression by ELISA. In unstimulated keratinocytes no NF-kappaB binding to the IL-8 kappaB binding sequence was detectable, whereas stimulation with IL-1alpha (10 ng/ml) led to a significant ( P<0.05) induction of NF-kappaB binding. In contrast NF-kappaB binding to the p53 kappaB binding sequence was detectable in unstimulated cells, although it was significantly increased after IL-1alpha (10 ng/ml) stimulation. Incubation with 1alpha,25(OH)(2)D(3) (10(-8)-10(-7) M) was shown to significantly ( P<0.05) stimulate the expression of IkappaBalpha and in parallel experiments with normal human keratinocytes stimulated with IL-1alpha (10 ng/ml) a significant ( P<0.05) time and dose-dependent decrease in NF-kappaB binding to the IL-8 kappaB binding sequence and in IL-8 expression were seen. A less-pronounced decrease in NF-kappaB binding to the p53 kappaB response element was seen after preincubation with 1alpha,25(OH)(2)D(3) and IL-1alpha stimulation, and it did not result in any change in p53 expression. These results demonstrate that 1alpha,25(OH)(2)D(3) inhibits NF-kappaB binding to the IL-8 kappaB binding sequence more potently than binding to the p53 kappaB binding sequence. We propose that this selectivity may be mediated through an increased expression of IkappaBalpha which leads to an inhibition of specific NF-kappaB subunits resulting in a selective regulation of NF-kappaB-induced gene transcription.
Riis
Jette L
JL
Department of Dermatology, Aarhus Sygehus, Aarhus University Hospital, P.P. Orumsgade 11, 8000 Aarhus C, Denmark.
Johansen
Claus
C
Gesser
Borbala
B
Møller
Kristine
K
Larsen
Christian G
CG
Kragballe
Knud
K
Iversen
Lars
L
D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology*
C116441:2-methyl-1,25-dihydroxyvitamin D3; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; D016159:Tumor Suppressor Protein p53; D014807:Vitamin D; D004247:DNA