Search

Summary1α,25(OH)2D3 and its analogs are potent mediators of keratinocyte differentiation in vitro. The precise mechanism of this action is still unknown. The nuclear transcription factor activator protein 1 seems to play an important role in keratinocyte differentiation. The purpose of this study was to investigate the effect of 1α,25(OH)2D3 on activator protein 1 DNA binding activity in cultured human keratinocytes. In a time-course study of human keratinocytes incubated with 1α,25(OH)2D3 (10-7-10-11 M) a significant dose-dependent increase in activator protein 1 DNA binding activity as determined by electrophoretic mobility shift assay was seen after 36 h. This increase was followed by a significant dose-dependent decrease in activator protein 1 DNA binding activity after 72 h. When differentiation was induced by raising the calcium concentration in the culture medium from 0.09 to 0.3 mM a similar increase in activator protein 1 DNA binding activity was observed after incubation for 48 h. Pharmacologic down-modulation of the protein kinase C activity with GF 109203X reversed the calcium-induced increase in activator protein 1 DNA binding activity and abolished keratinocyte differentiation as determined by a transglutaminase assay. In contrast, activator protein 1 DNA binding activity and keratinocyte differentiation were not affected when protein kinase C activity was down-modulated in the experiments with 1α,25(OH)2D3. The activator protein 1 complex in human keratinocytes consists of dimers of Fra-1, Fra-2, c-Jun, JunD, and c-Fos. Our results demonstrate that 1α,25(OH)2D3- and calcium-induced keratinocyte differentiation are accompanied by changes in activator protein 1 DNA binding activity. Protein kinase C activation appears to be essential for the calcium-dependent induction of keratinocyte differentiation, whereas a protein-kinase-C-independent activation of activator protein 1 DNA binding and keratinocyte differentiation is responsible for the 1α,25(OH)2D3-induced effects. [ABSTRACT FROM AUTHOR]

In the present study, keratinocytes were coincubated with human neutrophils to determine whether or not an increase in leukotriene B4 formation can occur. Human keratinocytes used were cultured in serum-free, low-calcium medium, whereas neutrophils were purified from heparinized venous blood. After coincubations, formation of leukotriene B4 was determined by reversed-phase high-performance liquid chromatography, coupled with its characteristic UV scan. Confirmation and quantification was by radioimmunoassay. Our data revealed that incubations of keratinocytes (1.5 × 106) alone stimulated with calcium ionophore resulted in no detectable amounts of leukotriene B4. In contrast, incubations of neutrophils (5 × 106) alone resulted in the generation of 62.2 ± 8.5 ng of LTB4. Coincubations of the neutrophils with keratinocytes (ratio 3:1) resulted in a 56-163% increase in leukotriene B4 formation. To delineate the source of the newly formed leukotriene B4, incubations of keratinocytes with leukotriene A4 revealed that keratinocytes can transform leukotriene A4 into leukotriene B4. These latter findings indicate that although keratinocytes cannot directly metabolize arachidonic acid into leukotriene B4 via the 5-lipoxygenase enzyme, they can transform neutrophil-derived leukotriene A4 into leukotriene B4, thus indicating the possible existence of a putative keratinocyte-leukotriene A4 hydrolase. It is therefore reasonable to speculate that the keratinocytes possess the capacity to generate leukotriene B4 in the epidermis when provided leukotriene A4 and thereby can amplify the inflammatory processes occurring during crophil exocytosis. [ABSTRACT FROM AUTHOR]

Nuclear factor-κB (NF-κB) is an inducible nuclear transcription factor regulating a range of cellular processes. An imbalance of the DNA binding activity of NF-κB may, therefore, be part of the pathophysiological mechanisms in psoriasis. The purpose of this study was to determine the NF-κB DNA binding activity in psoriatic skin using three differentκB sites and to determine how DNA binding activity was modulated by the anti-psoriatic drug calcipotriol. By electrophoretic mobility shift assay, we demonstrated that the NF-κB DNA binding to the p53κB site was decreased, whereas the NF-κB DNA binding to the interleukin-8 (IL-8)κB site was increased in lesional psoriatic skin compared with non-lesional psoriatic skin. No regulation was seen on the NF-κB DNA binding to the major histocompatibility complex class IκB site. These changes were paralleled by a similar decrease in p53 expression and an increase in IL-8 expression in involved psoriatic skin compared with uninvolved skin as determined by quantitative RT-PCR. The alteration in NF-κB DNA binding activity was neither accompanied by any change in the expression of the inhibitorκB (IκB) kinases, IKKα, IKKβ, and IKKγ nor in the expression of the NF-κB inhibitor proteins, IκBα and IκBβ. Immunofluorescence analysis revealed that p65 was sequestered in the cytoplasm of keratinocytes, whereas p50 exhibited a cytoplasmic as well as a nuclear localization. Interestingly, this distribution of p50 and p65 was similar in lesional and non-lesional psoriatic skin. Topical application of calcipotriol to lesional psoriatic skin for 4 d resulted in increased NF-κB binding to the p53κB site and decreased NF-κB binding to the IL-8κB site. Taken together, our data demonstrate that the NF-κB DNA binding activity is regulated in a specific manner in psoriatic skin depending on theκB sites investigated, and that topical treatment of psoriatic skin normalizes the abnormal NF-κB binding activity seen in lesional psoriatic skin. [ABSTRACT FROM AUTHOR]

Abnormal epidermal proliferation and differentiation characterize the inflammatory skin disease psoriasis. Here we demonstrate that expression of PPARδ mRNA and protein is markedly upregulated in psoriatic lesions and that lipoxygenase products accumulating in psoriatic lesions are potent activators of PPARδ. The expression levels of NF-κB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1β-induced translocation of p65 nor IL-1β-induced NF-κB DNA binding. We show that PPARδ physically interacts with the N-terminal Rel homology domain of p65. Irrespective of the presence of agonists none of the PPAR subtypes decreased p65-mediated transactivation in keratinocytes. In contrast p65, but not p50, was a potent repressor of PPAR-mediated transactivation. The p65-dependent repression of PPARδ- but not PPARα- or PPARγ-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-κB activation in chronic psoriatic plaques permitting unabated PPARδ-mediated transactivation contributes to the pathologic phenotype of psoriasis. [ABSTRACT FROM AUTHOR]

Investigate the mechanism by which 1alpha,25-dihydroxyvitamin D[sub3] modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Characteristics of stratified epidermis; Findings of western blotting and transfection experiments regarding dihydroxyvitamin; Effect of anti-annexin II antibody on the 1alpha,25(OH)[sub2]D[sub3]-induced phosphorylation.

Propositions about an abnormal fatty acid metabolism in atopic dermatitis patients prompted us to compare the phospholipid fatty acid composition and LTB4 release of neutropliils from 13 atopic dermatitis patients, as well as the adipose tissue triglyceride fatty acid composition, to that of 15 healthy controls matched by age, gender, and smoking habits. We found no differences in the tissue fatty acid composition between the two groups. The release of leukotrienc B4 from Ca-ionophore-stimulated neutrophils in patients was on the average only 42% (p <0.001) of that measured in the control group, despite the very similar arachidonic acid con- tents of these cells. Our study does not support the assumption of an abnormal fatty acid desaturation in atopic dermatitis patients. Rather, the capacity to release and/or convert arachidonic acid into leukotrienes in neutrophils appears to be affected by this disease. [ABSTRACT FROM AUTHOR]

IL-20 is a novel member of the IL-10 cytokine family with pleiotropic effects. Current knowledge of what triggers and regulates IL-20 gene expression is sparse. The aim of this study was to investigate the regulation of IL-20 expression in cultured normal human keratinocytes. The expression of IL-20 was rapidly induced by proinflammatory stimuli, in particular IL-1beta, IL-6, and UVB irradiation. Using kinase inhibitors and small-interfering RNA, we discovered that the p38 mitogen-activated protein kinase (MAPK) as well as inhibitory kappaB kinase-NF-kappaB signaling pathways are crucial for IL-20 expression. By electrophoretic mobility shift assay two kappaB-binding sites were identified upstream from the start codon in the IL-20 gene. Supershift analysis revealed binding of the p50/p65 heterodimer. Furthermore, the p38 MAPK was shown to exert its effects on IL-20 expression through activation of the downstream kinase mitogen- and stress-activated kinase 1 (MSK1), indicating transactivation of NF-kappaB driven IL-20 messenger RNA transcription as an important mechanism of action. IL-20 is assumed to be a key cytokine in the pathogenesis of psoriasis and possibly cancer, and therefore the p38 MAPK, MSK1, and NF-kappaB may be important new molecular targets for the modulation of IL-20 expression in these diseases.

Abnormal epidermal proliferation and differentiation characterize the inflammatory skin disease psoriasis. Here we demonstrate that expression of PPARdelta mRNA and protein is markedly upregulated in psoriatic lesions and that lipoxygenase products accumulating in psoriatic lesions are potent activators of PPARdelta. The expression levels of NF-kappaB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1beta-induced translocation of p65 nor IL-1beta-induced NF-kappaB DNA binding. We show that PPARdelta physically interacts with the N-terminal Rel homology domain of p65. Irrespective of the presence of agonists none of the PPAR subtypes decreased p65-mediated transactivation in keratinocytes. In contrast p65, but not p50, was a potent repressor of PPAR-mediated transactivation. The p65-dependent repression of PPARdelta- but not PPARalpha- or PPARgamma-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-kappaB activation in chronic psoriatic plaques permitting unabated PPARdelta-mediated transactivation contributes to the pathologic phenotype of psoriasis.