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• W2783125175 abstract "The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-β signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis. The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-β signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis. The cellular microenvironment encompasses structural components, the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; BH, Benjamini-Hochberg; BM, basement membrane; C7, collagen VII; CAF, cancer-associated fibroblasts; DEJZ, dermal-epidermal junction zone; DEB, dystrophic epidermolysis bullosa; NHK, normal human keratinocytes; PCA, principal component analysis; SCC, squamous cell carcinoma; SILAC, stable isotope labelling by amino acids in cell culture; SOTA, self-organizing tree algorithm. 1The abbreviations used are: ECM, extracellular matrix; BH, Benjamini-Hochberg; BM, basement membrane; C7, collagen VII; CAF, cancer-associated fibroblasts; DEJZ, dermal-epidermal junction zone; DEB, dystrophic epidermolysis bullosa; NHK, normal human keratinocytes; PCA, principal component analysis; SCC, squamous cell carcinoma; SILAC, stable isotope labelling by amino acids in cell culture; SOTA, self-organizing tree algorithm., and soluble factors, e.g. growth factors (1.Hynes R.O. The extracellular matrix: not just pretty fibrils.Science. 2009; 326: 1216-1219Crossref PubMed Scopus (2290) Google Scholar). The ECM was initially regarded as purely structural scaffold keeping cells in place and ensuring tissue/organ integrity. Through the discovery of ECM-cell contact sites and underlying signaling events, the crucial role of the ECM for cell survival, proliferation and differentiation emerged (1.Hynes R.O. The extracellular matrix: not just pretty fibrils.Science. 2009; 326: 1216-1219Crossref PubMed Scopus (2290) Google Scholar, 2.Pozzi A. Yurchenco P.D. Iozzo R.V. The nature and biology of basement membranes.Matrix Biol. 2017; 57–58: 1-11Crossref PubMed Scopus (288) Google Scholar). Importantly, it is now clear that a dysregulated ECM can actively promote disease progression in humans (3.Bonnans C. Chou J. Werb Z. Remodelling the extracellular matrix in development and disease.Nat. Rev. Mol. Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (2278) Google Scholar). The skin contains a complex ECM, the dermal-epidermal junction zone (DEJZ), which binds the epidermis and the dermis and is generated by cell types of distinct epithelial and mesenchymal linages: keratinocytes, the main cell type of the epidermis, and fibroblasts, the main mesenchymal-derived cell type of the dermis. The DEJZ is vital for skin integrity. As part of the DEJZ the ECM protein collagen VII (C7) forms anchoring fibrils, which entrap dermal fibrils and establish stable dermal-epidermal adhesion (4.Has C. Nystrom A. Epidermal basement membrane in health and disease.Curr. Top. Membr. 2015; 76: 117-170Crossref PubMed Scopus (34) Google Scholar). Biallelic loss-of-function mutations of the gene COL7A1 encoding C7 cause dystrophic epidermolysis bullosa (DEB), an inherited skin fragility disorder characterized by a broad spectrum of clinical manifestations: skin blistering, abnormal wound healing, excessive scarring often resulting in aggressive skin cancer (5.Fine J.D. Eady R.A. Bauer E.A. Bauer J.W. Bruckner-Tuderman L. Heagerty A. Hintner H. Hovnanian A. Jonkman M.F. Leigh I. et al.The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB.J. Am. Acad. Dermatol. 2008; 58: 931-950Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). However, the variety and multitude of the symptoms are not easily explained by the underlying COL7A1 mutations. Toward this end “omics” approaches have been used to delineate the cellular and microenvironmental consequences of C7 loss. However so far, mainly the consequences of C7-deficiency on already transformed squamous cell carcinoma cells (6.Dayal J.H. Cole C.L. Pourreyron C. Watt S.A. Lim Y.Z. Salas-Alanis J.C. Murrell D.F. McGrath J.A. Stieger B. Jahoda C. et al.Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes.J. Cell Sci. 2014; 127: 740-751Crossref PubMed Scopus (18) Google Scholar, 7.Watt S.A. Pourreyron C. Purdie K. Hogan C. Cole C.L. Foster N. Pratt N. Bourdon J.C. Appleyard V. Murray K. et al.Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma.Oncogene. 2011; 30: 4666-4677Crossref PubMed Scopus (55) Google Scholar), and on dermal fibroblasts were addressed (8.Kuttner V. Mack C. Rigbolt K.T. Kern J.S. Schilling O. Busch H. Bruckner-Tuderman L. Dengjel J. Global remodelling of cellular microenvironment due to loss of collagen VII.Mol. Syst. Biol. 2013; 9: 657Crossref PubMed Scopus (82) Google Scholar, 9.Ng Y.Z. Pourreyron C. Salas-Alanis J.C. Dayal J.H. Cepeda-Valdes R. Yan W. Wright S. Chen M. Fine J.D. Hogg F.J. et al.Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.Cancer Res. 2012; 72: 3522-3534Crossref PubMed Scopus (95) Google Scholar). Transcriptome analyses have suggested that the gene expression profile of DEB fibroblasts resembles that of cancer-associated fibroblasts (CAFs) and that loss of C7 may generate a permissive microenvironment for tumor development (9.Ng Y.Z. Pourreyron C. Salas-Alanis J.C. Dayal J.H. Cepeda-Valdes R. Yan W. Wright S. Chen M. Fine J.D. Hogg F.J. et al.Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.Cancer Res. 2012; 72: 3522-3534Crossref PubMed Scopus (95) Google Scholar). Quantitative mass spectrometry (MS)-based proteomics studies using DEB fibroblasts focused on the extracellular consequences of the absence of C7 and revealed reduction of basement membrane proteins and increase of dermal matrix proteins, highlighting the multifaceted roles of C7 (8.Kuttner V. Mack C. Rigbolt K.T. Kern J.S. Schilling O. Busch H. Bruckner-Tuderman L. Dengjel J. Global remodelling of cellular microenvironment due to loss of collagen VII.Mol. Syst. Biol. 2013; 9: 657Crossref PubMed Scopus (82) Google Scholar). Reduced crosslinking of dermal ECM proteins because of lower abundance of transglutaminase 2, an interaction partner of C7, indicated altered mechanical properties of the fibroblast microenvironment because of loss of C7 (10.Kuttner V. Mack C. Gretzmeier C. Bruckner-Tuderman L. Dengjel J. Loss of collagen VII is associated with reduced transglutaminase 2 abundance and activity.J. Invest. Dermatol. 2014; 134: 2381-2389Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It has become apparent that progressive TGF-β-mediated soft tissue fibrosis plays a major role in disease evolution of DEB (11.Nystrom A. Thriene K. Mittapalli V. Kern J.S. Kiritsi D. Dengjel J. Bruckner-Tuderman L. Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms.EMBO Mol. Med. 2015; 7: 1211-1228Crossref PubMed Scopus (117) Google Scholar, 12.Odorisio T. Di Salvio M. Orecchia A. Di Zenzo G. Piccinni E. Cianfarani F. Travaglione A. Uva P. Bellei B. Conti A. et al.Monozygotic twins discordant for recessive dystrophic epidermolysis bullosa phenotype highlight the role of TGF-beta signalling in modifying disease severity.Hum. Mol. Genet. 2014; 23: 3907-3922Crossref PubMed Scopus (68) Google Scholar). In other diseases epithelial dysfunction is known to be a major instigator of fibrosis (13.Sakai N. Tager A.M. Fibrosis of two: Epithelial cell-fibroblast interactions in pulmonary fibrosis.Biochim. Biophys. Acta. 2013; 1832: 911-921Crossref PubMed Scopus (180) Google Scholar). Known for DEB is that C7 as a master regulator of laminin-332 deposition may exert control over the laminin-332-integrin α6β4 signaling axis (38.Nystrom A. Velati D. Mittapalli V.R. Fritsch A. Kern J.S. Bruckner-Tuderman L. Collagen VII plays a dual role in wound healing.J. Clin. Invest. 2013; 123: 3498-3509Crossref PubMed Scopus (132) Google Scholar). However, the potential contribution of these and additional epithelial alterations to progression of DEB remain elusive. Therefore, we performed a global transcriptome and proteome profiling comparing primary DEB keratinocytes to normal human keratinocytes (NHK), delineating C7-dependent molecular pathways dysregulated in disease. We combined high-throughput sequencing using RNAseq with stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative MS followed by bioinformatics analyses to address alterations in primary human keratinocytes and in skin of affected individuals. Loss of C7 did not only affect the composition of the cellular microenvironment, but it led to global changes in cell homeostasis on mRNA and on protein level. Altered abundance of ECM proteins and integrin receptors indicated dysregulated TGF-β activity caused by disruption of laminin-332-integrin α6β4 signaling. Indeed, TGF-β-dependent inflammatory and proteolytic processes were perturbed both in keratinocytes in vitro and in human DEB skin in vivo. Thus, we provide a detailed image of dysregulated molecular pathways in genetic skin fragility and show that loss of C7 leads to far more complex changes as anticipated. Importantly, our study reveals intrinsic epidermal alterations directly caused by C7 deficiency and increased TGF-β signaling in epidermal keratinocytes as instigators of dermal fibrosis. More information about procedures can be found online in the supplemental Information. Skin specimens were obtained from ten DEB patients (DEB1–10) for diagnostic purposes and the remaining material was used for research purposes after informed consent of the patients and approval of the Ethics Committee of the University of Freiburg. The study was conducted according to the Declaration of Helsinki. Specimens are stored in an internal collection. Immunofluorescence mapping revealed lack of C7 in the skin of all patients. Mutation analysis of the C7 gene, COL7A1 was performed as described before and disclosed mutations that lead to premature stop codons, thus confirming the diagnosis of severe generalized DEB (Table I) (14.Kern J.S. Gruninger G. Imsak R. Muller M.L. Schumann H. Kiritsi D. Emmert S. Borozdin W. Kohlhase J. Bruckner-Tuderman L. et al.Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort.Br. J. Dermatol. 2009; 161: 1089-1097Crossref PubMed Scopus (63) Google Scholar, 15.Kern J.S. Kohlhase J. Bruckner-Tuderman L. Has C. Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2006; 126: 1006-1012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To elucidate disease mechanisms DEB keratinocytes were compared with primary human keratinocytes (Ctrl1–12) derived from age-matched healthy donors (Table II). Cells used for each experiment are indicated in the respective figure according to the designations introduced in Tables I and II.Table ICharacteristics of primary DEB cells used in this study. n.d.: not detectedIDAge at biopsy/genderCOL7A1 mutationC7 levels (IF analysis)References (report of patients)DEB 18 years/mc.1934delCn.d.(15.Kern J.S. Kohlhase J. Bruckner-Tuderman L. Has C. Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2006; 126: 1006-1012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar)c.1934delCDEB 21 month/fc.425A>Gn.d.(15.Kern J.S. Kohlhase J. Bruckner-Tuderman L. Has C. Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2006; 126: 1006-1012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar)c.682 + 1G>ADEB 31 month/mc.425A>Gn.d.(14.Kern J.S. Gruninger G. Imsak R. Muller M.L. Schumann H. Kiritsi D. Emmert S. Borozdin W. Kohlhase J. Bruckner-Tuderman L. et al.Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort.Br. J. Dermatol. 2009; 161: 1089-1097Crossref PubMed Scopus (63) Google Scholar)c.5261dupCDEB 46 years/fc.425A>Gn.d.Patient not reported beforec.682 + 1G>ADEB 51 month/fc.4119 + 1G>Cn.d.Patient not reported beforec.8523_8536delDEB 63 years/fc.682 + 1G>An.d.Patient not reported beforec.5261dupCDEB 71 month/fc.425A>Gn.d.(15.Kern J.S. Kohlhase J. Bruckner-Tuderman L. Has C. Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2006; 126: 1006-1012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar)c.425A>GDEB 830 years/mc.426 + 1G>An.d.(15.Kern J.S. Kohlhase J. Bruckner-Tuderman L. Has C. Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2006; 126: 1006-1012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar)c.1474del11DEB 953 years/mc.425A>Gn.d.Patient not reported beforec.425A>GDEB 1047 years/mc.425A>Gn.d.Patient not reported beforec.425A>G Open table in a new tab Table IICharacteristics of primary control cells used in this studyIDAge at biopsy/ genderCtrl 12 years/mCtrl 24 years/mCtrl 33 years/mCtrl 420 years/fCtrl 513 years/fCtrl 61 year/mCtrl 738 years/fCtrl 84 years/mCtrl 96 years/mCtrl 1034 years/fCtrl 1113 years/fCtrl 121 years/m Open table in a new tab Patient DEB9 had a severe generalized DEB with lack of C7 in the affected skin, as shown in a skin biopsy taken from scarred, not acutely blistered or inflamed skin next to the left knee (supplemental Fig. S6). A skin biopsy taken from a clinically unaffected skin patch on the left knee revealed C7 re-expression at around 70% of the biopsy's width. A subsequent analysis of the COL7A1 gene from keratinocytes of the unaffected skin patch confirmed the correction of the COL7A1 gene on one allele (data not shown). In total, we analyzed 22 primary cell populations as listed in Tables I and II. Omics analyses were performed from two primary DEB and normal human keratinocyte populations, each. As DEB is a rare disease (less than 10 of one million newborns are affected), not more primary cells could be used for ‘omics’ analyses. For SILAC-based proteomics analyses two biological replicates each were analyzed using swapped labels. All statistical tests were corrected for multiple testing as outlined in the respective paragraphs. A SILAC approach was used to compare DEB keratinocytes to normal controls by MS-based proteomics. 5 × 105 fully SILAC-labeled keratinocytes were seeded on 10 cm2 cell culture dishes. On the following day, the medium was exchanged and 50 μg/ml ascorbate was added to allow proper maturation and secretion of collagens (16.Myllyharju J. Kivirikko K.I. Collagens, modifying enzymes and their mutations in humans, flies and worms.Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar). Keratinocytes were kept in culture for 6 days and fresh culture medium containing ascorbate was added daily. After 6 days, keratinocytes were washed 3 times with DPBS. The cells were removed from the underlying ECM through incubation with 0.5% Triton X-100 in DPBS for 10 s, followed by a washing step with DPBS and an incubation for 10 s with 20 mm NH4OH in DPBS (17.Vlodavsky I. Preparation of extracellular matrices produced by cultured corneal endothelial and PF-HR9 endodermal cells.Curr. Protoc. Cell Biol. 2001; (Chapter 10, Unit 10 14)Google Scholar). Cell lysates were concentrated by ultrafiltration using vivaspin columns (10 kDa MWCO). The remaining ECM was carefully washed 3 times with DPBS to eliminate intracellular contaminants and then solubilized with 4% SDS in 0.1 m Tris-HCl, pH 7.6 (18.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5042) Google Scholar). Aliquots of the different ECM samples or cell lysates were used for WB analysis or mixed 1:1:1 and prepared for MS analysis to test accurate mixing ratios. Mixing was adjusted for actual MS analysis afterward. Samples were heated in SDS-PAGE loading buffer, reduced with 1 mm DTT for 5 min at 95 °C and alkylated using 5.5 mm iodoacetamide for 30 min at room temperature. Protein mixtures were separated on 4–12% gradient gels. The gel lanes were cut into 10 equal slices, the proteins were in-gel digested with trypsin and the resulting peptide mixtures were processed on STAGE tips and analyzed by LC-MS/MS (19.Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3531) Google Scholar, 20.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar). MS measurements were performed on an LTQ Orbitrap XL mass spectrometer coupled to an Agilent 1200 nanoflow-HPLC. HPLC-column tips (fused silica) with 75 μm inner diameter were self-packed with Reprosil-Pur 120 ODS-3 to a length of 20 cm. Samples were applied directly onto the column without a pre-column. A gradient of A (0.5% acetic acid in water) and B (0.5% acetic acid in 80% acetonitrile in water) with increasing organic proportion was used for peptide separation (loading of sample with 2% B; separation ramp: from 10–30% B within 80 min). The flow rate was 250 nL/min and for sample application 500 nL/min. The mass spectrometer was operated in the data-dependent mode and switched automatically between MS (max. of 1 × 106 ions) and MS/MS. Each MS scan was followed by a maximum of five MS/MS scans in the linear ion trap using normalized collision energy of 35% and a target value of 5000. Parent ions with a charge state from z = 1 and unassigned charge states were excluded for fragmentation. The mass range for MS was m/z = 370–2000. The resolution was set to 60,000. MS parameters were as follows: spray voltage 2.3 kV; no sheath and auxiliary gas flow; ion-transfer tube temperature 125 °C. The MS raw data files were uploaded into the MaxQuant software version 1.4.1.2 for peak detection, generation of peak lists of mass error corrected peptides, and for database searches (21.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar). A full-length UniProt human database additionally containing common contaminants such as keratins and enzymes used for in-gel digestion (based on UniProt human FASTA version September 2013, 87,000 entries) was used as reference. Carbamidomethylcysteine was set as fixed modification and protein amino-terminal acetylation and oxidation of methionine were set as variable modifications. Triple SILAC was chosen as quantitation mode. Three missed cleavages were allowed, enzyme specificity was trypsin/P, mass tolerance for first search was 20 ppm, and the MS/MS tolerance was set to 0.5 Da. The average mass precision of identified peptides was in general less than 1 ppm after recalibration. Peptide lists were further used by MaxQuant to identify and relatively quantify proteins using the following parameters: peptide and protein false discovery rates, based on a forward-reverse database, were set to 0.01, minimum peptide length was set to 6, minimum number of peptides for identification and quantitation of proteins was set to one which must be unique, minimum ratio count was set to two, and identified proteins were requantified. The “match-between-run” option (2 min) was used. Annotated spectra of single peptide-based protein IDs are provided as Supplemental Files S1 and S2. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD005873 (22.Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. et al.2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar). DEB keratinocytes are characterized by a loss of C7 (Fig. 1A) and skin of affected individuals exhibits mechanical fragility, inflammation, excessive scarring and fibrosis (Fig. 1B). Whereas disease contributions of mesenchyme-derived cells have been extensively studied, the influence of epithelial cells remains obscure, which led us to investigate the epidermal responses to loss of C7 (see Tables I-II and Experimental Procedures for designation of used primary cells). To study the effects of C7 loss on the transcriptome, DEB keratinocytes from two donors and two age-matched controls were cultured to the same number of passages and the extracted RNAs were subjected to next-generation RNA sequencing. In total, expression of 39,293 gene variants was detected (supplemental Table S1) of which 300 were differentially regulated in DEB and control keratinocytes (BH-corrected q-value 0.1) (supplemental Table S2). To illustrate altered gene expression in DEB keratinocytes, we performed a cluster analysis of the differentially expressed genes and observed a greater variability between the two DEB cells than between the control cells (Fig. 2A). GO term analysis of differentially expressed genes revealed enrichment of GO terms like cell migration and ECM organization which are processes known to be affected by the loss of C7 (8.Kuttner V. Mack C. Rigbolt K.T. Kern J.S. Schilling O. Busch H. Bruckner-Tuderman L. Dengjel J. Global remodelling of cellular microenvironment due to loss of collagen VII.Mol. Syst. Biol. 2013; 9: 657Crossref PubMed Scopus (82) Google Scholar, 10.Kuttner V. Mack C. Gretzmeier C. Bruckner-Tuderman L. Dengjel J. Loss of collagen VII is associated with reduced transglutaminase 2 abundance and activity.J. Invest. Dermatol. 2014; 134: 2381-2389Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) (Fig. 2B). Expression of genes encoding laminin-332 (LAMA3/LAMB3/LAMC2) and integrin α6β4 (ITGA6/ITGB4), direct and indirect binding partners of C7, respectively, were downregulated in DEB keratinocytes. In contrast, the transcript encoding latent-transforming growth factor beta-binding protein 1 (LTBP1) was more abundant, indicating increased TGF-β secretion (23.Saharinen J. Keski-Oja J. Specific sequence motif of 8-Cys repeats of TGF-beta binding proteins, LTBPs, creates a hydrophobic interaction surface for binding of small latent TGF-beta.Mol. Biol. Cell. 2000; 11: 2691-2704Crossref PubMed Scopus (218) Google Scholar) and activation of a wound response program in cells devoid of C7 (Fig. 2C). Taken together, next-generation RNA sequencing revealed distinct changes of mRNA levels highlighting a perturbed transcriptional regulation because of loss of C7 in primary keratinocytes. Expression of genes encoding direct and indirect binding partners of C7 were found to be downregulated, whereas expression of genes encoding for proteins involved in response to injury and TGF-β signaling were upregulated in DEB keratinocytes. To complement transcriptome analyses and directly assess the extracellular consequences of loss of C7, we performed quantitative proteome analyses of the keratinocyte ECM using MS. Cells were cultured in the presence of ascorbate to ensure correct post-translational modification and stable triple-helix formation of collagens. After 6 days, cells were removed carefully from culture dishes using Triton/PBS followed by NH4OH/PBS (Fig. 3A) (17.Vlodavsky I. Preparation of extracellular matrices produced by cultured corneal endothelial and PF-HR9 endodermal cells.Curr. Protoc. Cell Biol. 2001; (Chapter 10, Unit 10 14)Google Scholar). Remaining insoluble proteins on the culture plate were harvested with 4% SDS and were defined as ECM. Efficiency of the protocol and purity of the ECM isolation were tested by Western blot (Fig. 3B). Intracellular proteins corresponding to the cell compartments nucleus (TBP), Golgi apparatus (GOLGA1), endoplasmic reticulum (HSPA5) and cytosol (GAPDH) were only detected in cell lysates. ECM proteins such as the transmembrane protein collagen XVII (COL17A1) and C7, both components of the DEJZ, fibronectin (FN1) and tenascin C (TNC) were found in both, cell lysate and ECM. Thus, intracellular proteins were depleted and extracellular components were enriched in the ECM fraction. The same primary keratinocytes as used for gene expression analyses were SILAC labeled to assess the effect of C7 deficiency on the abundances of extracellular proteins. A Super-SILAC mix of light isotope-labeled keratinocyte ECM derived from all four donors was generated and spiked into differentially heavy isotope-labeled ECM of control and DEB keratinocytes (Fig. 3C) (24.Geiger T. Cox J. Ostasiewicz P. Wisniewski J.R. Mann M. Super-SILAC mix for quantitative proteomics of human tumor tissue.Nat. Methods. 2010; 7: 383-385Crossref PubMed Scopus (432) Google Scholar). Samples were fractionated by SDS-PAGE and proteins in-gel digested with trypsin (19.Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3531) Google Scholar). Generated peptides were identified and quantified by reverse-phase LC-MS/MS. 1386 proteins were identified in two biological replicates each, of which 1237 could be quantified in at least one sample (average correlation coefficient = 0.66; supplemental Fig. S1 and supplemental Table S3; see supplemental File S1 for annotated spectra of single peptide IDs). Because of the high sensitivity of modern mass spectrometers, even small amounts of contaminating intracellular proteins are detected in the ECM fraction. To distinguish them and to obtain a comprehensive list of potential true ECM proteins, the original protein list was filtered: proteins defined in the “matrisome” and those annotated by the GO terms “extracellular” and/or “cell adhesion” were defined as the “ECM proteome” (25.Naba A. Clauser K.R. Hoersch S. Liu H. Carr S.A. Hynes R.O. The matrisome: in si" @default.
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• W2783125175 title "Combinatorial Omics Analysis Reveals Perturbed Lysosomal Homeostasis in Collagen VII-deficient Keratinocytes" @default.
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• W2783125175 doi "https://doi.org/10.1074/mcp.ra117.000437" @default.
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