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The Pathogenetic Role of IL-1β in Severe Epidermolysis Bullosa Simplex

2013; Elsevier BV; Volume: 133; Issue: 7 Linguagem: Inglês

10.1038/jid.2013.31

1523-1747

Verena Wally, Thomas Lettner, Patricia Peking, Doris Peckl‐Schmid, Eva M. Murauer, Stefan Hainzl, Helmut Hintner, Johann Bauer,

Autoimmune Bullous Skin Diseases

Dominantly inherited epidermolysis bullosa simplex type Dowling-Meara (EBS-DM) is caused by mutations in either the keratin 5 (K5) or the keratin 14 (K14) gene, which encode the major components of the intermediate filament (IF) network in basal keratinocytes. In healthy keratinocytes, heterodimers of K5 and K14 polymerize to form a cytoplasmic network extending to the cellular periphery and imparting stability to the basal layer of the stratified epithelia. In EBS-DM, causative mutations lead to disintegration and collapse of the IF network, which manifests cytologically as electron-dense aggregates at the periphery of the keratinocyte cytoplasm (Morley et al., 2003Morley S.M. D'Alessandro M. Sexton C. et al.Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations.Br J Dermatol. 2003; 149: 46-58Crossref PubMed Scopus (69) Google Scholar). EBS-DM cells exhibit increased fragility to mechanical stress as well as to heat and osmotic shock, making Dowling-Meara the most severe subtype of EBS. As a consequence, EBS-DM patients suffer from blistering and wounding of the skin and mucous membranes after even minor trauma (Stephens et al., 1993Stephens K. Sybert V.P. Wijsman E.M. et al.A keratin-14 mutational hot-spot for epidermolysis-bullosa simplex, Dowling-Meara - implications for diagnosis.J Invest Dermatol. 1993; 101: 240-243Abstract Full Text PDF PubMed Google Scholar; D'Alessandro et al., 2002D'Alessandro M. Russell D. Morley S.M. et al.Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock.J Cell Sci. 2002; 115: 4341-4351Crossref PubMed Scopus (89) Google Scholar; Coulombe et al., 2009Coulombe P.A. Kerns M.L. Fuchs E. Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility.J Clin Invest. 2009; 119: 1784-1793Crossref PubMed Scopus (154) Google Scholar; Jerabkova et al., 2010Jerabkova B. Marek J. Buckova H. et al.Keratin mutations in patients with epidermolysis bullosa simplex: correlations between phenotype severity and disturbance of intermediate filament molecular structure.Br J Dermatol. 2010; 162: 1004-1013Crossref PubMed Scopus (24) Google Scholar). Interestingly, upregulation of K14 at both the transcriptional and protein levels was shown recently in EBS-DM keratinocytes (Wally et al., 2010Wally V. Brunner M. Lettner T. et al.K14 mRNA reprogramming for dominant epidermolysis bullosa simplex.Hum Mol Genet. 2010; 19: 4715-4725Crossref PubMed Scopus (51) Google Scholar; Beriault et al., 2012Beriault D.R. Haddad O. McCuaig J.V. et al.The mechanical behaviour of mutant K14-R125P bundles and networks in NEB-1 keratinocytes.PLoS One. 2012; 7: e31320Crossref PubMed Scopus (24) Google Scholar). The JNK/MAPK stress pathway is also known to be upregulated in EBS-DM keratinocytes, and the cells show elevated JNK phosphorylation compared to clinically milder, K14-based EBS subtypes (D'Alessandro et al., 2002D'Alessandro M. Russell D. Morley S.M. et al.Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock.J Cell Sci. 2002; 115: 4341-4351Crossref PubMed Scopus (89) Google Scholar). Particularly with regard to a small molecule-based therapeutics, we have focused on the pathomechanism underlying K14 overexpression and aggregation. We have investigated the potential involvement of the pro-inflammatory cytokine IL-1β in EBS-DM pathology. IL-1β is secreted by keratinocytes (and other cell types) and binds to specific receptors of basal keratinocytes, activating those cells in response to wounding. Activated keratinocytes exhibit higher migratory potential and hyperproliferative activity, and secrete extracellular-matrix components and signaling polypeptides to the tissue microenvironment (Freedberg et al., 2001Freedberg I.M. Tomic-Canic M. Komine M. et al.Keratins and the keratinocyte activation cycle.J Invest Dermatol. 2001; 116: 633-640Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Defects in these processes could also contribute to the EBS-DM phenotype. To examine whether the presence of EBS-DM underlying mutations in the K14 gene is directly linked to the IL-1β-mediated JNK stress pathway, we compared K14, IL-1β, and JNK messenger RNA (mRNA) levels in two immortalized EBS-DM cell lines (KEB-7, EBDM-1), primary EBS-DM keratinocytes (PEBDM-1), and wild-type immortalized NEB-1 keratinocytes by using semiquantitative real-time PCR (SQRT-PCR). We also engineered NEB-1 keratinocytes to express a full-length K14 cDNA harboring an EBS-DM mutation (R125P) in exon 1 and examined those cells by SQRT-PCR as well. As expected, K14 expression was elevated in all EBS-DM keratinocytes. In addition, significant upregulation of JNK and IL-1β mRNA expression was detected (Figure 1a), except for JNK expression in PEBDM-1, where results varied in SQRT-PCR, resulting in a P-value of 0.34. However, PEBDM-1 keratinocytes, as well as all EBS-DM cell lines, showed a clear increase of phopho-JNK levels in western blot analysis (data not shown). Furthermore, upregulation of K14 protein was shown in all cell lines and PEBDM-1 keratinocytes (Figure 1b). In patient immortalized cell lines, increased production of secreted IL-1β was shown in an ELISpot assay (Figure 1c). With a view toward small-molecule therapeutics, we investigated the ability of IL-1β neutralizing antibody (IL-1βAb) and diacerein to inhibit IL-1β signaling. Diacerein, a component of rhubarb root, is reported to block the release of active IL-1β by inhibiting plasma membrane-bound IL-1 converting enzyme (Moldovan et al., 2000Moldovan F. Pelletier J.P. Jolicoeur F.C. et al.Diacerhein and rhein reduce the ICE-induced IL-1ß and IL-18 activation in human osteoarthritic cartilage.Osteoarthritis Cartilage. 2000; 8: 186-196Abstract Full Text PDF PubMed Scopus (78) Google Scholar; Verbruggen, 2000Verbruggen G. Chondroprotective drugs in degenerative joint diseases.Rheumatology. 2000; 45: 129-138Crossref Scopus (122) Google Scholar). We tested both substances in an in vitro setting to investigate their ability to ameliorate EBS-DM pathology at the cellular level. Treatment of KEB-7 and EBDM-1 with 2μgml−1 IL-1βAb for 24h resulted in significant downregulation of K14, JNK, and IL-1β expression compared to non-treated keratinocytes, as measured by SQRT-PCR (Figure 1d). Similarly, when the same cell lines were treated with 10μgml−1 diacerein, K14 and JNK expression levels went down (Figure 1e). However, because diacerein exerts its effect on IL-1β at the post-translational level, we observed no effect on IL-1β at the transcriptional level at 24h. At the protein level, both K14 and phospho-JNK levels decreased upon treatment with IL-1βAb or diacerein (Figure 1f). In contrast, in NEB-1 cells treated with recombinant IL-1β the expression levels of K14, IL-1β, and JNK all increased (data not shown). Because many genes encoding matrix-degrading proteins (eg, MMPs, KLKs) are targets of the IL-1β signaling pathway and are upregulated in EBS-DM (data not shown), we performed an invasion assay to measure keratinocyte invasiveness upon IL-1β inhibition (Liacini et al., 2002Liacini A. Sylvester J. Li W.Q. et al.Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1(AP-1) and nuclear factor kappa B (NF-kappaB) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes.Matrix Biol. 2002; 21: 251-262Crossref PubMed Scopus (373) Google Scholar). Whereas untreated EBDM-1 and KEB-7 cells showed significantly increased invasive potential over NEB-1 wild-type keratinocytes, a significant decrease in invasiveness was detected upon treatment with IL-1βAb (Figure 2a), accompanied by downregulation of matrix-degrading proteins (data not shown). In addition, we examined K14 aggregation in the cytoplasm of heat-shocked wild-type and EBS-DM keratinocytes in the presence or absence of IL-1βAb or diacerein (Figure 2b). Characteristic aggregates were detected by immunofluorescence microscopy in about 10% of all KEB-7 keratinocytes, accompanied by upregulation of K14, IL-1β, and JNK expression (data not shown). In contrast, no aggregates at all formed in NEB-1 cells or in KEB-7 cells treated with IL-1βAb or diacerein. This leads us to hypothesize that aggregation of K14 is a key event in the induction of IL-1β and the JNK stress pathway. To summarize, we showed here that, in EBS-DM keratinocytes, IL-1β signaling is constitutively activated, resulting in activation of the JNK stress pathway and overexpression of K14 and IL-1β itself in a positive feedback loop. Through this mechanism, expression of the dominantly interfering mutated K14 allele would also increase, potentially aggravating the EBS-DM phenotype. Therefore, therapeutic approaches to interrupt this positive feedback loop by inhibiting IL-1β using a neutralizing antibody or diacerein would seem worthwhile. Indeed, treatment of patient keratinocytes with these agents not only reduced the expression levels of K14 and IL-1β and the phosphorylation levels of JNK, but also stabilized the IF network upon heat shock. Our data are further corroborated by our recent demonstration that overall K14 expression levels in EBS-DM keratinocytes are reduced upon mRNA correction via spliceosome-mediated RNA trans-splicing (SMaRT), which shifts the expression ratio of wild-type versus mutated K14 alleles toward wild-type (Wally et al., 2010Wally V. Brunner M. Lettner T. et al.K14 mRNA reprogramming for dominant epidermolysis bullosa simplex.Hum Mol Genet. 2010; 19: 4715-4725Crossref PubMed Scopus (51) Google Scholar). We therefore propose that inhibition of IL-1β could be beneficial to EBS-DM patients by ameliorating the EBS-DM phenotype. This work was supported by DEBRA Austria.