T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis

IL17 stimulates proliferation, upregulates cytokine expression, and inhibits differentiation of normal keratinocytes. Tritiated thymidine incorporation was measured in WT and MyD88^−/− (A) or WT and EGFR^−/− (B) keratinocyte cultures treated with IL17 or IL22 for 24 hours, bars represent the mean ± SEM value of four replicates; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. C, Immunoblotting of total cell extracts from primary keratinocytes treated with IL17 for the indicated period. HSP90, heatshock protein 90; p-, phosphorylated. D, Heatmap representation of gene expression analysis performed by real-time PCR quantification from keratinocytes treated for 1, 3, 6, and 12 hours with IL17. E, Total SDS cell extracts from PBS, IL17- or IL22-treated keratinocytes were immunoblotted with specific antibodies recognizing early (K1 and K10) and late (filaggrin) markers of differentiation from cultures maintained under differentiating conditions (0.12 mmol/L Ca⁺⁺) for 24 hours. F, Real-time PCR quantification of K1 and K10 mRNAs in keratinocytes infected with GFP (control) or degradation-resistant IκBα (IκBsr Ad) adenovirus to block NF-κB activity and treated with IL17 under differentiating conditions (**, P < 0.01; ***, P < 0.001).

IL17 enhances transcriptional activity and cytokine release but not proliferation of RAS keratinocytes. A, Tritiated thymidine incorporation was measured in control or RAS-transduced WT and Myd88^−/− keratinocyte cultures treated with IL17 for 24 hours. Bars represent the mean ± SEM value of four replicates. B, Real-time PCR quantification of mRNAs from control and RAS-transduced keratinocytes stimulated with IL17 for 6 or 24 hours. Bars represent the mean ± SEM value of four replicates. C, Real-time PCR analysis of mRNA expression in control or RAS-transduced keratinocytes treated with PBS or IL1R antagonist (IL1ra, anakinra). D, Cytokine and chemokine concentrations in culture supernatants from control and RAS-transduced keratinocytes cultures collected 24 hours after TPA or IL17 treatments and assayed by multiplex ELISA. For A–E, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

IL17 enhances transcription of downstream effectors in mouse keratinocytes with endogenous mutant Ras activation and human keratinocytes transformed by human mutant RAS. A, Real-time PCR quantification of mRNAs from GFP and Cre adenovirus-transduced LSL-Hras^G12D mouse keratinocytes stimulated with IL17. B, Real-time PCR quantification of mRNAs from GFP, mutant HRAS^G12V, and KRAS^G12V lentivirus-transduced human keratinocytes. C, Real-time PCR quantification of mRNAs from GFP and mutant HRAS^G12V lentivirus-transduced human keratinocytes treated with human IL17A. For A–C, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

TPA, IL17, and the RAS oncogene are dependent on IkBz signaling for induction of tumor-promoting transcripts and tumor formation in vivo. Tritiated thymidine incorporation was measured in WT or Nfkbiz^−/− normal keratinocytes treated with IL17 for 24 hours (A) or RAS-transduced keratinocytes (B). Bars represent the mean ± SEM value of four replicates. C, Total SDS cell extracts from Nfkbiz^+/− and Nfkbiz^−/− keratinocytes were immunoblotted with specific antibodies recognizing K1 and K10 from cultures under basal (0.05 mmol/L Ca⁺⁺) conditions or treated with IL17 under differentiating conditions (0.12 mmol/L Ca⁺⁺). Real-time PCR quantification of mRNAs from Nfkbiz^+/− and Nfkbiz^−/− keratinocytes treated for 1, 3, 6, and 12 hours with IL17 (D) or from WT or Nfkbiz^−/− normal keratinocyte treated with TPA (E) or from control and RAS-transduced WT and Nfkbiz^−/− keratinocytes (F). Symbols represent the mean ± SEM value of three replicates. G, Immunoblotting of nuclear extracts from control and RAS-transduced WT and Nfkbiz^−/− keratinocytes. Arrow head denotes specific band. H, Primary keratinocytes Nfkbiz^+/− or Nfkbiz^−/− were transduced with a retrovirus expressing oncogenic RAS and grafted together with WT dermal fibroblasts onto the backs of athymic mice. Each dot represents mean volume of individual tumors at day 20 postgrafting. Data shown represent two independent experiments and are reported as mean ± SEM. For all panels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

The RAS transcriptome is dependent on IκBz. A, Heatmap visualization of relative expression of the top 82 genes whose expression is altered by at least 2-fold when comparing RAS-keratinocytes Nfkbiz^+/+ with RAS keratinocytes Nfkbiz^−/−. Genes are ordered by fold change. B, GSEA enrichment plots are shown for top-enriched or top-depleted gene sets in RAS-transduced Nfkbiz^−/− keratinocytes. The black line is the running enrichment score calculated along the ranked gene list; the vertical light blue bars in the plot indicate the position of the genes from the respective gene set. Supplementary Table S4 lists all GSEA estimates for these gene sets, including “leading edge” genes. C, Network visualization of top-enriched functional gene sets from MSigDB collections (GSEA FDR < 5.0%) in RAS-transduced Nfkbiz^−/− keratinocytes. Network nodes represent subsets of the enriched gene sets including GSEA core enriched genes (“leading edge”) and are shown in blue and red for gene sets underexpressed and overexpressed in the RAS-transduced Nfkbiz^−/− keratinocytes, respectively. Edges in the network represent mutual overlap of genes between the gene sets and the thickness of an edge is proportional to the combined similarity coefficient, ranging between 0.375 to 1.0. Supplementary Table S4 contains the underlying network data.