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Cyclosporin A induces the unfolded protein response in keratinocytes

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Abstract

Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes has not been well elucidated as its function on T cells is well known. Previous study indicated that the expression of the unfolded protein response (UPR) markers, GRP78/Bip and HRD1 were poorly expressed in psoriasis vulgaris. To investigate if the UPR in keratinocytes is involved in the pathogenesis of psoriasis vulgaris we assessed immunocytochemistry of normal human skin and psoriatic lesions, quantitative PCR of keratinocyte cell line (HaCaT) treated with TGFβ. Moreover, to elucidate how CyA effects on the UPR in keratinocytes, we set out quantitative PCR and western blotting, HaCaT and squamous cell carcinoma cell lines (HSC-1) treated with CyA and CyA analog, cyclosporin D. Furthermore, the siRNA-mediated knockdown effect of cyclophilin (Cyp) A, Cyp B and Cyp C on HaCaT cells were also examined. As a result, the UPR was downregulated in keratinocytes from psoriatic lesions, characterized by immunocytochemical staining of GRP78/Bip, CHOP/GADD153, HRD1 and C/EBPβ. TGFβ induced UPR markers in HaCaT cells. CyA treatment and siRNA-mediated knockdown of Cyp B induced the UPR in HaCaT cells or HSC-1 cells. Altogether, we demonstrate that in psoriasis vulgaris CyA or reduction in Cyp B by RNA interference might induce the UPR in keratinocytes.

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Abbreviations

ATF6:

Activating transcription factor 6

Bip:

Immunoglobulin heavy chain-binding protein

C/EBPβ:

CCAAT/enhancer-binding protein beta

CHOP:

C/EBP homologous protein

ER:

Endoplasmic reticulum

GADD153:

Growth arrest and DNA damage inducible gene 153

GRP78:

Glucose-regulated protein 78 kDa

HRD:

Hydroxymethylglutaryl-CoA reductase degradation

PDI:

Protein disulfide isomerase

PERK:

PKR-like endoplasmic reticulum kinase

TGF-β:

Transforming growth factor-β

UPR:

Unfolded protein response

XBP-1:

X box-binding protein 1

CyA:

Cyclosporine A

Cyp:

Cyclophilin

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Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research C 20591319 (K.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant from the Nakatomi Foundation for the year 2007 (K.S.), a Grant from the Japanese Dermatology Association for the year 2008 (K.S.), and a Novartis CPCP Research Grant 2008 (K.S.).

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Authors and Affiliations

  1. Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Michiko Hibino, Kazumitsu Sugiura, Yoshinao Muro & Yasushi Tomita

  2. Department of Laboratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Yoshie Shimoyama

Authors
  1. Michiko Hibino
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  2. Kazumitsu Sugiura
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  3. Yoshinao Muro
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  4. Yoshie Shimoyama
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  5. Yasushi Tomita
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Corresponding author

Correspondence to Kazumitsu Sugiura.

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403_2010_1099_MOESM1_ESM.tif

DAB staining in a psoriatic lesion and a normal skin. DAB staining of epidermis of a psoriatic lesion (A) and a normal skin (B). Scale bars,100 μm. Derma-epidermal junction is dotted in both figures (TIFF 7082 kb)

403_2010_1099_MOESM2_ESM.tif

Lower concentration of CyA activated the UPR in HaCaT and HSC-1 cells. HaCaT cells were incubated with concentration of 0.5x106 and 107 M CyA for 4 h (A). The mRNA expression of CHOP/GADD153 (upper) and GRP78/Bip (lower) wasmeasured by qPCR. HSC-1 cells were incubated with concentration of 0.5x106 and 107 M CyA for 4 h (B). The mRNA expression of CHOP/GADD153 (upper) andGRP78/Bip (lower) was measured by qPCR. Data shown are the mean ± SD of four independent experiments (*0.01<p<0.05, **p<0.01) (TIFF 3161 kb)

403_2010_1099_MOESM3_ESM.tif

LDH release assay in HaCaT cells incubated with CyALDH release after stimulation with 106, 105, 104 M CyA for 6 h (A) or 12 h (B). Data from four experiments are expressed as a mean ± SD percentage of the maximal LDH release stimulated by 1% Triton X-100 treatment. No difference from control was found at any concentration of CyA for 6 or 12 h (TIFF 2465 kb)

403_2010_1099_MOESM4_ESM.tif

CyA activated the UPR in HSC-1 cells. Western blot analysis (upper) and quantitative densitometry (lower) of the protein expression of GRP78/Bip and CHOP/GADD153 in HSC-1 cells incubated with 105 MCyA for 48 h. Western blot analysis and laser densitometry were performed using immunoreactive GRP78/Bip, CHOP/GADD153 and β-actin antibodies. Data shown are the mean ± SD of four independent experiments (*0.01<p<0.05, **p<0.01) (TIFF 1901 kb)

403_2010_1099_MOESM5_ESM.tif

CyD activated the UPR in HSC-1 cells. Western blot analysis (upper) and quantitative densitometry (lower) of the protein expression of GRP78/Bip and CHOP/GADD153 in HSC-1 cells incubated with 105 MCyD for 48 h. Western blot analysis and laser densitometry were performed using immunoreactive GRP78/Bip, CHOP/GADD153 and β-actin antibodies. Data shown are the mean ± SD of four independent experiments (*0.01<p<0.05, **p<0.01) (TIFF 1905 kb)

403_2010_1099_MOESM6_ESM.tif

Reduction of cyclophilin B induced the UPR in HSC-1 cells. Western blot analysis (upper) and quantitative densitometry (lower) of the protein expression of GRP78/Bip and CHOP/GADD153 in HSC-1 cells transfected with CypB-siRNA or CD4-siRNA as a control. HSC-1 cells were incubated for 96 h with CypB-siRNA or CD4-siRNA. Western blot analysis and laser densitometry were performed using immunoreactive GRP78/Bip, CHOP/GADD153, Cyp B and β-actin antibodies for each group. Data shown are the mean ± SD of four independent experiments(*0.01<p<0.05, **p<0.01) (TIFF 2305 kb)

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Hibino, M., Sugiura, K., Muro, Y. et al. Cyclosporin A induces the unfolded protein response in keratinocytes. Arch Dermatol Res 303, 481–489 (2011). https://doi.org/10.1007/s00403-010-1099-3

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  • DOI: https://doi.org/10.1007/s00403-010-1099-3

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