Systematic identification and characterization of novel human skin-associated genes encoding membrane and secreted proteins

Abstract

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics.

Figure 1. Annotation of the 687 skin-associated genes (SAGs).

A) Functional classification. EDC; epidermal differentiation complex genes (see text). Categories with multiple functional classes: Metabolism (Amino acid metabolism, Enzyme, Lipid metabolism, Membrane regeneration/repair, Metabolism), Gene expression (Translation, Transcription factors), Signaling (Receptor, Signaling), Protein metabolism/trafficking (Chaperone, Protease/protease inhibitor, Protein kinase/phosphatase, Protein modification, Protein trafficking, Protein turnover), Nucleic acid binding/metabolism (DNA binding/metabolism, RNA binding/metabolism), Transport/channel (Channel, Transporter, Vesicular trafficking), Cell cycle/fate (Angiogenesis, Apoptosis, Cell cycle, Differentiation, Growth factor/growth factor binding protein, Hormone/hormone binding), Structural (Cytoskeleton, Structural), Cell adhesion (Cell adhesion, Gap junction, Tight junction, Extracellular matrix), Defense (Chemokine/chemokine receptor, Coaggulation, Cytokine/cytokine receptor, Immune response, Innate immunity+Cidal). B) Classification based on level of characterization of top 300 SAGs. See Table S1 for functional annotation for all 687 SAGs.

Figure 2. Expression profiles of top human skin-associated genes in the body index of gene expression (BIGE).

Affymetrix GeneChip data for the top 10 SAGs are shown as mean normalized relative expression (RU) (+) Standard deviation (y axis), plotted against the sample IDs from 105 human tissue and cell types grouped by system (x axis), as listed in Panel A. CNS, central nervous system; PNS, peripheral nervous system. Asterisk marks skin sample in each profile.

Figure 3. Semi-quantitative PCR confirmation of the specificity of expression of poorly characterized skin-associated genes (SAGs) in human total RNAs.

Expression levels of three control genes: (A) ALDOB, aldolase B fructose-biphosphate (not expressed in skin); (B) DCD, dermcidin; and (C) KRT1, keratin 1; and eight selected SAGs: (D) MUCL1, mucin-like 1; (E) WFDC5, WAP four-disulfide core domain 5; (F) TMEM45A, transmembrane protein 45A; (G) GPR115, G protein-coupled receptor 115; (H) CDHR1, cadherin-related family member 1; (I) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (J) C5orf46, chromosome 5 open reading frame 46; (K) GPR87, G protein-coupled receptor 87, were measured in healthy skin (eleven independent samples) and pooled total RNAs from spleen, kidney, brain and liver measured relative to control gene levels (18S RNA) and plotted as individual ratios, including, for skin samples, mean ratio values shown by the horizontal bar.

Figure 4. Expression of poorly characterized skin-associated genes in human skin-derived cell lines.

Semi-quantitative PCR analysis of expression of three control genes: (A) ALDOB, aldolase B fructose-biphosphate (not expressed in skin); (B) DCD, dermcidin; and (C) KRT1, keratin 1; and eight selected SAGs: (D) MUCL1, mucin-like 1; (E) WFDC5, WAP four-disulfide core domain 5; (F) TMEM45A, transmembrane protein 45A; (G) GPR115, G protein-coupled receptor 115; (H) CDHR1, cadherin-related family member 1; (I) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (J) C5orf46, chromosome 5 open reading frame 46; (K) GPR87, G protein-coupled receptor 87, were measured in low passage number primary human cells relative to control gene levels (18S RNA) and plotted as individual and mean ratios (horizontal bar).

Figure 5. Detection of selected poorly characterized SAG encoded proteins in normal human skin and primary keratinocytes.

Panels A–F; formalin-fixed paraffin embedded normal human skin sections were stained for DNA with DAPI and also with either SAG protein-specific (A, C, E), or isotype control antibodies (B, D, F) and visualized by immunofluorescent microscopy, 40× magnification. WFDC5, TMEM45A and GPR115 protein expression compared to beta actin was also detected in primary cultured human keratinocyte lysates using Western blotting (panels G, H and I respectively).

Figure 6. Regulation of poorly characterized skin-associated gene expression in primary human keratinocytes.

Semi-quantitative PCR analysis of expression of seven of the eight selected SAGs in primary human keratinocytes incubated with either cytokines (as indicated) or medium controls for 24 hours relative (RU) to control gene levels (18S RNA). Data shown are the mean ± SD. *p<0.05, **p<0.01 (Student’s t-test). SAGs tested were: (A) MUCL1, mucin-like 1; (B) WFDC5, WAP four-disulfide core domain 5; (C) TMEM45A, transmembrane protein 45A; (D) GPR115, G protein-coupled receptor 115; (E) CDHR1, cadherin-related family member 1; (F) SERPINB7, serpin peptidase inhibitor, clade B (ovalbumin), member 7; (G) GPR87, G protein-coupled receptor 87. C5orf46 expression was not detected in keratinocytes.

Figure 7. Expression of poorly characterized skin-associated genes in human healthy skin vs. skin diseases.

PSO, Psoriasis; AD, Atopic dermatitis; PN, Prurigo nodularis; LE, Lupus erythematosus; LP, Lichen planus; BCC, Basal cell carcinoma; AK, Actinic keratosis; SCC, Squamous cell carcinoma. Semi-quantitative PCR analysis of expression of eight selected SAGs; A–H, in healthy or diseased human skin biopsies relative to control gene levels (18S RNA), plotted as individual and mean ratios (horizontal bar). *p<0.05; **p<0.01; ***p<0.001 (Mann-Whitney-U test).