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Identifying a Hyperkeratosis Signature in Autosomal Recessive Congenital Ichthyosis: Mdm2 Inhibition Prevents Hyperkeratosis in a Rat ARCI Model

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Identifying a Hyperkeratosis Signature in Autosomal Recessive Congenital Ichthyosis: Mdm2 Inhibition Prevents Hyperkeratosis in a Rat ARCI Model GYoussefet al. Mdm2 Upregulation in Autosomal Recessive Congenital Ichthyosis Lebre MC, van der Aar AM, van Baarsen L et al. and increases UV sensitivity in a human skin immune surveillance T cells. J Exp Med (2007) Human keratinocytes express func- model. JInvestDermatol130:2286–94 199:1265–75 tional Toll-like receptor 3, 4, 5, and 9. J Invest Reiss Y, Proudfoot AE, Power CA et al. (2001) CC Schuster C, Vaculik C, Prior M et al. (2012) Pheno- Dermatol 127:331–41 chemokine receptor (CCR)4 and the CCR10 typic characterization of leukocytes in prenatal McCully ML, Ladell K, Hakobyan S et al. (2012) ligand cutaneous T cell-attracting chemokine human dermis. J Invest Dermatol 132:2581–92 Epidermis instructs skin homing receptor (CTACK) in lymphocyte trafficking to inflamed Sigmundsdottir H, Pan J, Debes GF et al. (2007) expression in human T cells. Blood 120:4591–8 skin. J Exp Med 194:1541–7 DCs metabolize sunlight-induced vitamin D3 Mildner M, Jin J, Eckhart L et al. (2010) Knockdown Schaerli P, Ebert L, Willimann K et al. (2004) A to ‘program’ T cell attraction to the epidermal of filaggrin impairs diffusion barrier function skin-selective homing mechanism for human chemokine CCL27. Nat Immunol 8:285–93 Identifying a Hyperkeratosis Signature in Autosomal Recessive Congenital Ichthyosis: Mdm2 Inhibition Prevents Hyperkeratosis in a Rat ARCI Model Journal of Investigative Dermatology (2014) 134, 858–861; doi:10.1038/jid.2013.374; published online 17 October 2013 TO THE EDITOR Therefore, there is a need to develop keratin 1 expression, but keratin 10 reduc- Autosomal recessive congenital ichthyo- new treatments for ARCI. tion was observed only in Tgm1-kd sis (ARCI) is a Mendelian disorder of Current research on alternate ARCI cultures (Figure 1a and Supplementary keratinization. LI patients are born as treatments is focused on gene therapy Figure S1c online), suggesting both com- erythrodermic ‘‘collodion’’ babies but (Choate et al., 1996), or enzyme replace- mon and distinct programs of altered gene soon become covered with scales. The ment therapy, adding recombinant Tgm1 expression in the two models. Prin- most commonly mutated genes in ARCI to the skin (Aufenvenne et al., 2012). A cipal component analysis confirmed that, are Transglutaminase 1, Ichthyin, and greater understanding of the common although phenotypically similar, overall the Arachidonate Lipoxygenases 12b molecular mechanisms that lead to hyper- gene expression differences were diver- and 3 (Russell et al., 1995; Jobard keratosis would facilitate the discovery of gent (Figure 1b and Supplementary Figure et al., 2002; Fischer, 2009). Hyper- more directed therapies to reduce scaling S2 online). Despite these differences, we keratosis (scaling) is a cardinal feature in ARCI. Therefore, we investigated com- identified 155 genes that were commonly of disorders of keratinization, which are mon gene expression changes in two upregulated and 78 genes that were com- characterized by abnormal barrier different rat epidermal keratinocyte ARCI monly downregulated in both Tgm1- and function. For example, Tgm1-null mice models, to identify the common net- Alox12b-kd keratinocyte cultures (Supple- die perinatally because of transepi- works of differentially expressed genes mentary Table S1 online). We identified dermal water loss after birth, but that are required for hyperkeratosis and, upregulated genes related to the positive when this skin is grafted onto nude within these molecular networks, to iden- regulation of apoptosis and downregulated mice, they form scales similar to ARCI tify new ‘‘drug-able’’ targets for the reduc- genes related to protein phosphorylation patients (Kuramoto et al., 2002). The tion of hyperkeratosis. (Supplementary Figure S3 online). epidermis is hyperproliferative, pro- Two separate Arachidonate 12-lipoxy- The only significant network of inter- duces increased non-polar lipid, and genase (Alox12b) short hairpin RNA related genes was related to the genes the cornified layer is thickened. This (shRNA)–knockdown (kd) rat epider- involved in the positive regulation of scaling is the principle phenotype in mal keratinocyte lines (Supplementary apoptosis, and within this network we ARCI patients, and the time taken to Figure S1a and S1b online) were com- identified the p53 ubiquitin ligase remove scale significantly affects their pared with an existing Tgm1 shRNA–kd Mdm2 as an attractive candidate gene quality of life. Oral retinoids, the line (O’Shaughnessy et al., 2010). Both to target in order to reduce hyperker- current treatment for ARCI, efficiently Tgm1-andAlox12b-kd organotypic atosis, as mice overexpressing Mdm2 reduces scale formation but are also cultures were hyperkeratotic (Figure 1a). display a hyperkeratotic epidermis in a rarely associated with side effects, Comparison of Tgm1-kd with Alox12b-kd p53-independent manner (Alkhalaf some serious (Rood et al., 2007). cultures revealed a common increase in et al., 1999). Mdm2 upregulation in both Tgm1- and Alox12b-kd cultures was confirmed by quantitative reverse Abbreviations: ARCI, autosomal recessive congenital ichthyosis; kd, knockdown; pAkt, phosphorylated Akt transcriptase in real-time PCR (Figure 1c Accepted article preview online 4 September 2013; published online 17 October 2013 and d). In organotypic culture, cytoplasmic 858 Journal of Investigative Dermatology (2014), Volume 134 G Youssef et al. Mdm2 Upregulation in Autosomal Recessive Congenital Ichthyosis Scram Tgm1 kd Alox12b kd CHFR KRT1 Krt1 PC 1 and 2 USP7 0.6 RB1 MDM2 Tgm1 TP53INP1 siRNA Mdm2 0.2 Alox12b MDM4 EI24 siRNA PC2 –0.2 TP53 PSMD9 PSMC3 CDKN2A Scram –0.6 RPL5 Keratin 1 RPL11 –0.5 0.0 0.5 PSMC6 AKT1 PC1 Akt3 Dusp1 Keratin 10 ARCI skin Normal skin * 10 Scram Tgm1 kd 9 Alox12b kd 8 K1/Mdm2 5 4 * 3 Fold change Fold 2 Mdm2 1 0 Dusp1 Mdm2 Krt1 Akt3 Keratin 1 Figure 1. Keratin 1 and Mdm2 upregulation in ARCI models and patient samples. (a) Immunofluorescence of keratins 1 and 10 in scrambled (Scram) control, Tgm1-kd organotypic cultures, and Alox12b-kd organotypic cultures. (b) Principal component analysis comparing gene expression patterns between scrambled controls, Tgm1-kd cells, and Alox12b-kd cells. Divergence between Alox12b- and Tgm1-kd cells indicates different gene expression profiles. (c)Networkof gene–gene interactions of the commonly overexpressed genes involved in apoptosis; red genes are overexpressed in ARCI models. (d) Quantitative reverse transcriptase in real-time PCR analysis of apoptosis genes in the three scrambled controls, two Tgm1-kd keratinocyte cultures, and two Alox12b-kd keratinocyte cultures; error bars SD, *Po0.05 one-way analysis of variance. (e) Immunofluorescence of Mdm2 and keratin 1 in ARCI skin (3 of n ¼ 7) and a normal control. Bar ¼ 50 mm(a, e). ARCI, autosomal recessive congenital ichthyosis; kd, knockdown; siRNA, small interfering RNA. Mdm2 expression was increased and was Consistent with this, serine-phosphory- tosis. As Mdm2 is commonly upregu- more widespread (Supplementary Figure lated Akt (pAkt) was upregulated in both lated in cancer (Berger et al., 2004), S4 online). This expression pattern was kd organotypic cultures (Supplementary a number of Mdm2 inhibitors are cur- mirrored in ARCI patients in whom Mdm2 Figure S4 online). Therefore, in ARCI, we rently in phase I clinical trials (Secchiero was strongly upregulated in the spinous hypothesized that Mdm2 functioned et al., 2011) and therefore could be layer in 7/7 samples, clearly colocalizing through a p53/p63-independent pathway rapidly adopted to treat ARCI. Nutlin-3 with upregulated keratin 1 (Figure 1e). p53 involving increased Akt phosphorylation is a well-characterized and easily expression was undetectable in these to cause hyperkeratosis. available Mdm2 inhibitor, which acts cultures (not shown), and p63, which Mdm2 overexpression alone in kera- by preventing the interaction of Mdm2 Mdm2 can also degrade, was not reduced tinocytes did not result in either keratin with p53, as well as other protein targets in either kd culture (Supplementary 1 or pAkt upregulation (Figure 2a). We (Nicholson et al., 2012). Figure S4 online). Akt upregulation causes therefore tested whether the inhibition Treatment of Tgm1- and Alox12b-kd hyperkeratosis (Janes et al., 2004), and of upregulated Mdm2 in the diseased cells with nutlin-3 reduced both keratin Mdm2 activates Akt (Singh et al., 2013). state was sufficient to reduce hyperkera- 1 and pAkt expression levels (Figure 2b). www.jidonline.org 859 GYoussefet al. Mdm2 Upregulation in Autosomal Recessive Congenital Ichthyosis Untreated Nutlin-3 Tgm1 siRNA Alox12b siRNA Mdm2 Mdm2 Vector Mdm2 Scram Tgm1 siRNAAlox12b Scram Tgm1 siRNAAlox12b Scram siRNA Scram siRNA siRNA siRNA Mdm2 Mdm2 Keratin 1 Keratin 1 pSerAkt pSerAkt Total Akt Total Akt Gapdh Gapdh Untreated Nutlin treated Scram Tgm1 siRNA Alox12b siRNA Tgm1 siRNA Alox12b siRNA + Nutlin + Nutlin Scram Tgm1 siRNA Keratin 1 PCNA Alox12b siRNA 24% ± 1% 58% ± 4% * 58% ± 2% * 23% ± 6% 29% ± 1% * Figure 2. The Mdm2 inhibitor nutlin-3 reduces hyperkeratosis in ARCI models. (a–c) Western blot of Mdm2, keratin 1, pSerAkt, and total Akt in (a) rat epidermal keratinocytes overexpressing Mdm2, (b) Tgm1- and Alox12b-kd cells treated with nutlin-3, and (c) Tgm1- and Alox12b-kd cells expressing Mdm2 short hairpin RNA. Gapdh is the loading control. (d) Mdm2 immunofluoresence in Tgm1- and Alox12-kd cells with and without nutlin-3 treatment. (e) Histology of scrambled (Scram) control and kd cultures with or without nutlin-3 treatment. In addition, keratin 1 and proliferating
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