Activation of Keratinocyte Protein Kinase Cζ in Psoriasis Plaques Yuming Zhao, Rita Fishelevich, John P. Petrali, Lida Zheng, Malinina Alla Anatolievna, April Deng, Richard L. Eckert, Anthony A. Gaspari  Journal of Investigative Dermatology  Volume 128, Issue 9, Pages 2190-2197 (September 2008) DOI: 10.1038/jid.2008.81 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 NKT cells increased in psoriasis. (a) Increase of Vα24+Vβ11+ double positive NKT cells in the epidermis of a psoriatic plaque. All Vα24-positive cells in psoriasis were also Vβ11-positive. The isotype controls were negative and Vα24 and Vβ11 single labeling showed no isotype cross-reactivity (not shown). (b) CD2+Vα24+ double positive NKT cells (merge), as well as Va24 (red) or CD2 (green) single positive cells, in the psoriatic epidermis and dermal papilla. Bar=10μm. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Infiltrating lymphocytes in psoriatic plaques express increased invariant Vα24-JαQ transcripts. Relative Vα24-JαQ gene expression was measured using real-time PCR to analyze gene expression from six psoriasis (lesional and paired uninvolved skin). There is significant increase in relative Vα24-JαQ gene expression in psoriasis lesions (P<0.05, Wilcoxon signed-rank test). Values are mean±SD, error bar=1 SD. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CD1d gene expression is increased in psoriatic plaques. Increased CD1d mRNA in psoriatic plaques was shown compared with uninvolved skin in the six patients by real-time PCR (P<0.05, Wilcoxon rank test). Values are mean±SD, error bar=1 SD. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increased membrane expression of PKCζ by psoriatic plaques. (a) Immunofluorescence of PKCζ in psoriasis and normal control skin showed increased cytoplasmic and membrane staining for PKCζ in psoriasis. Bar=10μm. (b) Double labeling with anti-HLA-ABC antibody showed membrane localization of PKCζ in psoriasis lesion. (c) When mRNA from six pairs of both lesional and uninvolved skin specimens was examined by real-time PCR, PKCζ gene expression was increased in all psoriasis samples compared with uninvolved skin (P<0.05, Wilcoxon rank test). Values are mean±SD, error bar=1 SD. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increased phosphorylated PKCζ in membrane fractions in psoriasis plaques. Whereas no consistent difference was demonstrated in phosphorylated PKCζ in the whole lysates or in the cytosolic fractions of psoriasis and their uninvolved skin (n=6), the membrane fractions of psoriasis lesions (L) showed significant increase in phospho-PKCζ compared with their uninvolved counterparts (N). P<0.01, Student's t-test. Values are mean±SD, error bar=1 SD. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Increased PKCζ and membrane translocation in KCs stimulated with TNF. (a) Confocal images: KCs cultured on coverslips stimulated with TNF-α showed prominent membrane staining for PKCζ (arrowheads) than the unstimulated cells (arrows at the dim cell boundaries). Bar=10μm. The picture is representative of three separate experiments. (b) The cells with membrane staining were increased (48.9%) significantly in TNF-α-stimulated cells compared with the medium alone (22.1%; P<0.01 by Fisher's exact test). One hundred cells were counted for each condition. (c) The fluorescence staining intensity of TNF-α stimulated cells increased significantly than those in medium alone. The intensities were measured by image analysis system of the fluorescence micrographs. P<0.05, Student's t-test. (d) Electron microscopic images of HaCaT on culture inserts stimulated with or without TNF-α, 100ngml−1, for 10minutes. Cell stimulated with TNF-α showed stronger staining (arrowheads) for PKCζ in the plasma membrane as well as in the nuclear and perinuclear areas apart from cytoplasmic staining, compared with unstimulated control cells. Note: N denotes nucleus. Bar=1μm. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Increased phosphorylated PKCζ in membrane fractions of KCs stimulated with TNF. (a) KCs were stimulated with 100ngml−1 of TNF-α for 0, 2, 4, 6, 8, and 10minutes. While no significant change in the quantity of phospho-PKCζ was observed, pERK and pJNK were increased 6minutes after TNF-α stimulation. (b) Increased phospho-PKCζ in the membrane and nuclear, but not in the cytosolic fractions, of primary KCs stimulated with TNF-α 100ngml−1 for 5 and 10minutes (Student's t-test). These blots are representations of three separate experiments. Values are mean±SD, error bar=1 SD. Journal of Investigative Dermatology 2008 128, 2190-2197DOI: (10.1038/jid.2008.81) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions