Proteomics of Skin Proteins in Psoriasis: From Discovery and Verification in a Mouse Model to Confirmation in Humans

Abstract

Herein, we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n = 3) were compared with littermate controls (n = 3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average p = 0.0082; cystatin A, human ortholog); slc25a5 (average fold change of 46.2 and an average p = 0.0318); serpinb3b (average fold change of 35.6 and an average p = 0.0345; serpinB1, human ortholog); and kallikrein related peptidase 6 (average fold change of 4.7 and an average p = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), stefin A1 (7.3-fold; p < 0.001), and slc25A5 (1.5-fold; p = 0.05) using qRT-PCR on a second cohort of animals (n = 8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; p < 0.05), 29,000-fold (stefinA1; p < 0.01), 322-fold (KLK6; p < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes versus healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3-fold), KLK6 (5.8-fold), and serpinB1 (76-fold; all p < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover, slc25a5, cystatin A, KLK6, and serpinB1 protein were all increased in lesional psoriasis skin compared with normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments. Herein, we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n = 3) were compared with littermate controls (n = 3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average p = 0.0082; cystatin A, human ortholog); slc25a5 (average fold change of 46.2 and an average p = 0.0318); serpinb3b (average fold change of 35.6 and an average p = 0.0345; serpinB1, human ortholog); and kallikrein related peptidase 6 (average fold change of 4.7 and an average p = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), stefin A1 (7.3-fold; p < 0.001), and slc25A5 (1.5-fold; p = 0.05) using qRT-PCR on a second cohort of animals (n = 8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; p < 0.05), 29,000-fold (stefinA1; p < 0.01), 322-fold (KLK6; p < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes versus healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3-fold), KLK6 (5.8-fold), and serpinB1 (76-fold; all p < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover, slc25a5, cystatin A, KLK6, and serpinB1 protein were all increased in lesional psoriasis skin compared with normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments. One in three individuals in the United States is afflicted with a skin disease, with ∼2–3% of the American population suffering from psoriasis (1.Helmick C.G. Sacks J.J. Gelfand J.M. Bebo Jr., B. Lee-Han H. Baird T. Bartlett C. Psoriasis and psoriatic arthritis: a public health agenda.Am. J. Prev. Med. 2013; 44: 424-426Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 2.Gelfand J.M. Weinstein R. Porter S.B. Neimann A.L. Berlin J.A. Margolis D.J. Prevalence and treatment of psoriasis in the United Kingdom: a population-based study.Arch. Dermatol. 2005; 141: 1537-1541Crossref PubMed Scopus (506) Google Scholar, 3.Kurd S.K. Gelfand J.M. The prevalence of previously diagnosed and undiagnosed psoriasis in US adults: results from NHANES 2003–2004.J. Am. Acad. Dermatol. 2009; 60: 218-224Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar) a chronic, immune-mediated inflammatory skin disease characterized by well-demarcated areas of “involved” red, raised, and scaly skin adjacent to areas of “uninvolved” normal appearing skin. The underlying cause of psoriasis remains unknown and the specific signals that trigger disease onset have yet to be identified; however, several lines of evidence suggest the involvement of antigen-specific T cells, although the antigens involved remain elusive (4.Valdimarsson H. Thorleifsdottir R.H. Sigurdardottir S.L. Gudjonsson J.E. Johnston A. Psoriasis–as an autoimmune disease caused by molecular mimicry.Trend Immunol. 2009; 30: 494-501Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A combination of human and animal studies have led to the understanding that in patients with a genetically susceptible background, some initiating stimulus, often a stressful event, an injury to the skin, or an infection, leads to a coordinated series of signaling events involving cytokines, resident skin cells, and skin-infiltrating immune cells, that once started, initiates a vicious pro-inflammatory hyperproliferative cycle. Once initiated, this cycle perpetuates sustained inflammatory responses. Intervention at several points in this cycle results in clinical resolution, however, durable remission and/or permanent clearance has not yet been achieved. Current psoriasis therapies are directed toward symptomatic relief and none of them represent a cure for this chronic illness. Current treatments include topical therapies, phototherapy, and systemic administration of immune-suppressants, anti-metabolites, oral retinoids, and biologics targeting immune cells or inflammatory cytokines (5.Tran B. Feldman S.R. Insight into psoriasis management: commercial perspectives for the U.S. psoriasis market.J. Dermatol. Treat. 2011; 22: 18-26Crossref PubMed Scopus (8) Google Scholar, 6.Menter A. Korman N.J. Elmets C.A. Feldman S.R. Gelfand J.M. Gordon K.B. Gottlieb A. Koo J.Y. Lebwohl M. Lim H.W. Van Voorhees A.S. Beutner K.R. Bhushan R. Guidelines of care for the management of psoriasis and psoriatic arthritis: Section 5. Guidelines of care for the treatment of psoriasis with phototherapy and photochemotherapy.J. Am. Acad. Dermatol. 2010; 62: 114-135Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Many of the most effective therapeutics however, also have the greatest adverse reactions; moreover, psoriasis can become resistant to specific therapies over time. Therefore, an ongoing need for discovery of new biological pathways and targets for psoriasis is obvious. We studied a mouse model of psoriasis using an unbiased proteomics screening approach to identify dysregulated peptides of interest in psoriasiform-inflamed mouse skin and ultimately compared these findings to psoriasis patient skin. We used in-gel label-free protein expression analysis to observe quantitative changes in protein expression. The peptides that were determined to be top-scoring from a statistical perspective and of biological interest were subjected to further analysis and confirmation using a targeted mass spectrometry approach along with qRT-PCR to assess gene expression in a distinct set of animal samples. Further validation of the translational importance of these novel proteins in primary keratinocytes from psoriasis skin as as human skin from psoriasis patient lesional and The results of these the of a discovery in-gel label-free expression model to identify proteins that are in the to that are in between control and genetically mouse skin and demonstrate the usefulness of mouse models and proteomic approaches for identifying novel proteins that are differentially regulated in human and targets for development of translational approaches to disease KC-Tie2 and littermate controls (n = were and skin as previously for in the label-free expression discovery J.A. J. A. not specific of leads to the development of J. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). A second distinct of (n = used for validation at the and protein animal were by the and and to the American for of protein samples that been and as previously J.A. J. A. not specific of leads to the development of J. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar) were and protein were determined using a as by the were for at using with a of The samples were to and with for with a of the gel-based of were of a The using and into distinct on protein molecular were using followed by and the were and using of for at were and at The were with followed by three to These were combined with the and peptides were at for analysis and in of for LC/MS/MS analysis. from the of KC-Tie2 and control (n = and qRT-PCR to the and as previously A. Gudjonsson J.E. of skin Immunol. 2013; PubMed Scopus Google Scholar) using the A1 KLK6 and adjacent to that used for and analyses and used to a second expression KC-Tie2 and control mouse skin (n = into and to from the the The to a and in to Once in and subjected to an of to a and of on for were at followed by this cycle with samples on between and and using the A. M. for 2009; PubMed Scopus Google Scholar). protein determined using a and of protein as previously J. J.E. A quantitative proteomic approach for protein of human Immunol. PubMed Scopus Google Scholar). LC/MS/MS using a an and an The of all samples. The mass the analysis using the A in and in were and in A at for using a with B. discovery by a of at over The at for followed by a at A for using a and the mass at a of and were using and in normal were from of to and to on were by were for and as mass spectrometry for the samples were the same as the discovery samples with of the LC/MS/MS that to the discovery for animal were and analyzed as a LC/MS/MS for a of LC/MS/MS One LC/MS/MS for animal in the set for a of LC/MS/MS The discovery set The were by The used mouse were as mass for mass for including of and of The discovery for these to be for the discovery of peptides with as previously J.M. A. and in human human primary analysis by label-free mass 2009; PubMed Scopus Google Scholar, H. of protein expression by mass PubMed Scopus Google Scholar). The that used for in the discovery set is in LC/MS/MS and for of LC/MS/MS and were and were by and mass by of in multiple a of a were with the mass and were for and using were into and with analysis of the using a and peptides from the same protein were to provide fold changes for protein The using the same and as with a discovery of for the areas from peptides of targets that were identified the were determined by and using the in the The mass spectrometry proteomics have been to the J.A. R. A. J.A. T. M. A. R. H. coordinated proteomics and PubMed Scopus Google Scholar) the with the and individuals with chronic to psoriasis and healthy controls were with including the of or psoriatic not to the patients systemic anti-psoriatic treatments for and topical treatments for to skin between psoriasis patients on of of and control skin were from the from all by the of the of or Moreover, all of the were in with clinical and to the of from skin and qRT-PCR to the and as previously A. Gudjonsson J.E. of skin Immunol. 2013; PubMed Scopus Google Scholar) using the KLK6 A and all on control human skin and psoriasis and involved lesional skin using primary targeting human KLK6 human A mouse and and psoriasis primary human were from skin (n = and to as previously A. Gudjonsson J.E. of skin Immunol. 2013; PubMed Scopus Google Scholar, A. Voorhees J.J. gene expression in human Dermatol. Full Text PDF PubMed Scopus Google Scholar, A. Gudjonsson J.E. A. and signaling in a Dermatol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. C. Voorhees J.J. Gudjonsson J.E. and a novel signaling that is in psoriasis and Immunol. 2011; PubMed Scopus Google Scholar). and qRT-PCR as previously A. Gudjonsson J.E. of skin Immunol. 2013; PubMed Scopus Google Scholar) using the same human To identify differentially peptides in KC-Tie2 mouse skin (n = 3) compared with littermate controls (n = we used a gel-based label-free expression approach to by fractionation of the by molecular over The were selected to differentially multiple skin as and to proteins of have been to in a combined in all samples with of were using label-free expression analysis by the of the and The and a to a for of peptides by the same protein were and a to identify peptides expression between control and KC-Tie2 mice. 5482 peptides mapping to 1281 proteins were identified and 105 proteins peptides that exhibited changes at the of p < with protein fold-changes and the 105 differentially a in were increased The proteins most p and in of and A were the p of KLK6 not = of biological interest and the fold change as a protein in our were selected for statistical or biological or with C. M. A. B. A. J. B. therapeutic for PubMed Scopus Google Scholar, A. of and by of psoriasis on the PubMed Scopus Google Scholar, J.E. J. Johnston A. T. Voorhees J.J. of the psoriatic in a regulated genes and with in Dermatol. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. of the psoriasis studies by gene set analysis 2010; PubMed Scopus Google Scholar) and A1 (average fold change average p = 0.0082; cystatin A, human ortholog); (average fold change average p = (average fold change average p = 0.0345; serpinB1, human ortholog); protein (average fold change average p = and related peptidase 6 average p = 0.2474; KLK6). KC-Tie2 mouse skin, approaches expression changes using qRT-PCR and label-free targeted mass spectrometry analysis. The to protein expression changes for we identified peptides with in the discovery analysis and we were by for an approach These were followed by changes in protein and gene expression of the identified targets in human psoriasis samples. a distinct of (n = we qRT-PCR on the identified targets of and confirmed increases in of the identified including (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), A1 (7.3-fold; p < 0.001), (1.5-fold; p = 0.05), and change in expression of from a of these (n = protein subjected to LC/MS/MS analysis using the for as to the to the targets identified in the and confirmed as increased at the were in all samples for the analysis. the proteins selected for label-free analysis identified for peptides for three peptides for KLK6 and three peptides for the identified proteins the in the gel-based 5482 of were used in the and the used as the the of peptides for the proteins selected for were observed with a of identified in the and identified in the gel-based discovery We observed an in protein for KLK6 using the with three peptides identified in the and identified in the discovery we were to peptides specific to using the a in for proteins between the discovery and analyses with of the peptides identified in the the we in the to protein we observed increases in the average protein of KC-Tie2 animals and control to be for for and for an not identified from the gel-based label-free expression are verified at the protein in control and KC-Tie2 mouse skin. changes are for peptides identified in control and KC-Tie2 of of used in samples for the mass not identified from the gel-based label-free expression are verified at the protein in control and KC-Tie2 mouse skin. changes are for peptides identified in control and KC-Tie2 of of used in samples for the mass not To the utility of a preclinical mouse model and our label-free proteomic screening approach in the of novel targets to human we the expression of our in psoriasis patient skin and primary keratinocytes from patient skin. with our in mouse skin, we increases all < 0.05) in gene expression between healthy controls used psoriasis psoriasis protein related peptidase mass and involved lesional psoriasis skin for KLK6 (5.8-fold), A (3-fold), and change in These increases in gene expression were also observed in primary keratinocytes from involved lesional psoriasis skin, we observed a and in = KLK6 = and A = compared with control keratinocytes from skin from individuals skin change observed for or psoriatic keratinocytes levels of gene compared with healthy control keratinocytes. keratinocytes were and from skin of healthy controls = and involved lesional psoriasis skin = and gene expression and to increases < 0.05) were observed for and KLK6 and increases in A p = of keratinocytes as a of increases were observed for and normal skin psoriasis skin lesional psoriasis skin protein kallikrein related peptidase 6 quantitative mass spectrometry The confirmation of changes in these proteins in psoriasis patient skin using skin and in proteins were increased in lesional psoriasis skin compared with normal skin with a in normal skin by keratinocytes and cells and this expression not change between and psoriasis skin. skin, increased in all of the and to the KLK6 levels were in and however, increases in KLK6 in the of in in and this increased in the of skin, between the in KLK6 in the of keratinocytes in the A in skin the most keratinocytes in the to a of cells at the most observed in skin. skin, increased of keratinocytes for A in the of the and with and also in keratinocytes in the in in the of healthy skin by keratinocytes and this not change between and psoriasis skin. skin, increased in the and also in immune cells in the in keratinocytes and cells in healthy skin, and levels in psoriasis skin. in involved lesional skin, to keratinocytes and cells, however, protein levels to of the of keratinocytes and in lesional psoriasis skin related to by control at and development for primary We a gel-based label-free proteomic approach to and identify differentially proteins in the skin of an mouse model of the KC-Tie2 mouse J.A. 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A is to have is an and a skin gene and is an of keratinocytes. cystatin A is of the genes in the for psoriasis C. C. C. A. J. analysis of cystatin A and protein genes at the psoriasis Dermatol. Full Text Full Text PDF PubMed Scopus Google and has been that the cystatin A a increased for psoriasis R. and over increased of evidence of gene 2011; PubMed Scopus (13) Google Scholar). The of cystatin in lesional mouse skin using an unbiased approach an validation of the KC-Tie2 mouse model and to identified peptides to importance be in human psoriasis. KLK6 also increased in KC-Tie2 mouse skin and lesional psoriasis and to in the of psoriasis skin, changes in skin of psoriasis. are M. T. R. and of kallikrein in human and Dermatol. Full Text Full Text PDF PubMed Scopus Google with and the most between immune and 2010; PubMed Google Scholar, of human 2009; Full Text Full Text PDF PubMed Scopus Google for in skin as M. A. A. C. A. kallikrein and systemic of Med. PubMed Scopus Google Scholar) and and M. T. A. H. peptidase skin is with of peptidase 6 and Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). is KLK6, although in psoriasis at the C. M. A. B. A. J. B. therapeutic for PubMed Scopus Google Scholar, A. of and by of psoriasis on the PubMed Scopus Google Scholar, J.E. J. Johnston A. T. Voorhees J.J. of the psoriatic in a regulated genes and with in Dermatol. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. of the psoriasis studies by gene set analysis 2010; PubMed Scopus Google Scholar) and to therapeutics is also J. M. A. H. of the with leads to of gene expression and in psoriatic skin, including Dermatol. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). We increases in KLK6 protein expression in the of psoriasis further increases in the keratinocytes of the and the immune of lesional skin. A A. A. A. M. to Psoriasis PubMed Scopus Google Scholar) KLK6 increases in to of with a of and we a label-free expression for identifying proteins in the skin of a mouse model of psoriasis and in the lesional skin of human psoriasis. We identified and verified in the KC-Tie2 mouse model and psoriasis patient skin increases in the proteins serpinB1 KLK6, cystatin A and These results demonstrate the usefulness of mouse models and proteomic approaches for identifying novel peptides/proteins that are differentially regulated in human psoriasis. with