1?,25(OH)2D3 regulates NF-?B DNA binding activity in cultured normal human keratinocytes through an increase in I?B? expression

Abstract

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.

Upregulation of MMP‐9 production by TNFα in keratinocytes and its attenuation by vitamin D

“…Treatment with calcitriol did not affect either the IkBa degradation or recovery. These results are at variance with another study showing that calcitriol upregulated IkBa protein and mRNA levels in keratinocytes (Riis et al, 2004). Examining the involvement of NFkB activation in the induction of MMP-9 by TNFa, we found that addition of a specific NFkB inhibitor 1 h after exposure to TNFa, after full recovery of IkBa levels, sufficed to abolish MMP-9 induction.…”

Vitamin D Receptor Inhibits Nuclear Factor κB Activation by Interacting with IκB Kinase β Protein

“…Because the VDR can interact directly with p65 (21), the most commonly held mechanism to explain vitamin D inhibition of NF-B is that the VDR-p65 interaction blocks the nuclear translocation of p65/p50 (10,26). This mechanism, however, cannot explain how 1,25(OH) 2 D 3 stabilizes IB␣, which was reported in many studies (10,12,14,17,18) and is well known as a critical step in the inhibition of NF-B. In fact, in many studies (14,26), including this one, VDR-p65 interaction was not detectable, suggesting that VDR-p65 interaction is weak, if there is any.…”

“…nuclear translocation, blocks NF-B DNA binding, increases IB␣ levels, or stabilizes IB␣ protein (10,12,14,15,17,18). It has also been shown that 1,25(OH) 2 D 3 suppresses RelB transcription (19) and reduces p105/p50 and c-rel protein levels (20).…”

Increased NF-κB activity in fibroblasts lacking the vitamin D receptor

“…1,25(OH) 2 D 3 has also been shown to decrease the DNA binding capacity of NF-B in human fibroblasts (13). In human keratinocytes, 1,25(OH) 2 D 3 is able to reduce NF-B DNA binding activity by increasing I B␣ protein levels, which contributes to the reduction in IL-8 production (37). In pancreatic islet cells, a vitamin D analog is reported to significantly downregulate proinflammatory chemokine production, which is associated with upregulation of I B␣ transcription and arrest of NF-B p65 nuclear translocation (11).…”