In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase

Abstract

Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm(2)), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.

Figure 1. Experimental design.

(A) Simulated solar irradiation (SSR) of abdominal areas, at doses of 2 and 4 J/cm², five hours before plastic surgery. Five women with a median age of 42 years, with phototype of II or III according to Fitzpatrick classification, were included in a photobiological study. (B) Microarray workflow. Non-irradiated and irradiated samples were separately compared to a reference, containing pooled samples (non-irradiated and irradiated). Significance Analysis of Microarrays (SAM) and a false discovery rate (FDR) of zero were used to identify transcripts that were differentially expressed between irradiated and non-irradiated conditions. SAM comparing 2 J/cm² with non-irradiated samples identified 288 differentially regulated genes; comparison between 4 J/cm²-irradiated and non-irradiated samples identified a set of 473 differentially regulated genes; and 3-class SAM identified a set of 476 genes (Table S1).

Figure 2. Gene and sample classification analyses.

Hierarchical Clustering (HC) (A) and Principal Component Analysis (PCA) (B) (Mev 4.4) were used to classify the differentially expressed genes after SAM normalization. The normalized expression for each gene (rows) in each sample (columns) is presented with a color code (green for down-regulated and red for up-regulated). Each HC analysis (A) formed groups confirmed by PCA (B), showing clearly distinct clusters for UV-irradiated and control samples.

Figure 3. Gene distribution as a function of UV treatment and chromosome location.

The Venn diagram (A) shows up- and down-regulated genes following 2 J/cm² and 4 J/cm² SSR. The diagram shows the number of genes in each group (UP or Down). The histogram (B) shows differentially expressed genes, demonstrating variation in response as a function of dose. The number of down-regulated genes is greater for the higher dose. Differentially regulated genes in response to UV exposure (C) do not show clustering for chromosomal location (the number of UV regulated genes is reported in Table S2).

Figure 4. GO tree for genes showing differential regulation only with a dose of 4 J/cm² SSR and qPCR validation.

Genes showing differential regulation only in response to the higher dose of SSR are annotated with several GO terms. Up-regulated genes (A) were associated with terms that included negative regulation of cellular processes, morphogenesis of anatomical structures, JAK-STAT signaling, nucleosome assembly and the immune response (inflammatory response and cell differentiation were also found for 1 MED (minimal erythema dose: 2 J/cm²), as indicated in red); for down-regulated genes (B), GO terms were predominantly associated with transcription and regulation, although three GO terms (in red) relating to “regulation of transcription” were also observed for 1 MED (Table S3). (C) Microarray data were validated for 11 genes (nine up and two down-regulated): mRNA was assayed by qPCR and normalized to the values for 18S mRNA. These genes were selected from the list of differentially expressed genes, for their classification as inflammatory response genes or their involvement in the p53 pathway. Error bars represent standard deviation. Stars indicate significant differences (two-tailed Student's t-test) between control and irradiated samples: * P<0.05; ** P<0.01; *** P<0.001.

Figure 5. The p38 pathway network for the 4 J/cm² SSR response.

Identification of p38-dependent genes ex vivo. Ex vivo study of the p38 pathway (A) in response to SSR using Ingenuity Pathway Analysis. Edges of the diagram are labeled with a description of the relationship between the nodes. Lines between genes represent known interactions, with solid lines representing direct interactions and dashed lines representing indirect interactions. Nodes are displayed using various shapes that represent the functional class of the gene product (see legend). Genes showing up-regulation of expression levels in response to SSR are in red. Skin samples from patients participating in the in vivo study were used for ex vivo analysis, with the same conditions as used in vivo. Genes belonging to the p38 pathway were identified using the specific kinase inhibitor SB203580. Genes not regulated by p38 are shown in B and those regulated by p38 are shown in C. Error bars represent standard deviations. Stars indicate significant differences (two-tailed Student's t-test) between DMSO-treated and SB203580-treated samples. * P<0.05; ** P<0.01; *** P<0.001 (Table S4). Values for irradiated DMSO and SB203580 samples were significantly different to values for their non-irradiated controls (data not shown).