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Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes

Abstract

Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia1,2,3. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K4. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

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Figure 1: Loss of Krt17 attenuates HPV16Tg-induced skin tumorigenesis.
Figure 2: Aire expression, function and regulation in skin tumor keratinocytes.
Figure 3: K17 regulates Aire subnuclear distribution and localizes to the nucleus to promote gene expression.
Figure 4: K17 and Aire associate with target gene promoter regions.

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Acknowledgements

We thank members of the Coulombe laboratory for support, P. Peterson (Aire constructs; Tartu University), R. Foisner (subcellular fractionation), M. Vladut-Talor (central tolerance study) and J. Folmer (electron microscopy) for advice and assistance. These studies were supported by research grants CA160255 and AR044232 (to P.A.C.) and GM111682 (to F.W.) and by training grant CA009110 (to R.P.H.), all from the US National Institutes of Health.

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Authors and Affiliations

Authors

Contributions

R.P.H. and D.J.D. jointly led the characterization of skin tumorigenesis in the HPV mouse model, with assistance from M.C.H. and A.S.B. R.P.H. led the studies performed in A431 cells and mouse keratinocytes in culture, with contributions from J.T.J., B.-M.C., B.G.P., Y.G. and J.H. S.O. and D.Č. conducted central tolerance study with assistance from R.P.H. and A.S.B. J.M.T. provided human basal cell carcinoma samples. W.Z. and F.W. performed the flow cytometry analyses. F.W. provided guidance to R.P.H. for EMSA analyses. R.P.H., D.J.D. and P.A.C. contributed to experimental design and data analysis. R.P.H. and P.A.C. jointly wrote the manuscript.

Corresponding author

Correspondence to Pierre A Coulombe.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of HPVTg/+; Krt17−/− mice.

(a) Macroscopic images of HPV16Tg/+ mouse ears at P40, P70 and P120. (b) Immunostaining of HPV16Tg/+ ear tissue sections for K17 (red) and Hoechst (blue) at P20 and P40. (c) H&E staining of a wild-type P70 ear tissue section (this micrograph complements the data presented in Fig. 1b). (d) Immunostaining and quantification of vasculature in P70 ear tissue sections from wild-type (green bar), HPV16Tg/+ (blue bar) and HPV16Tg/+; Krt17−/− (open bar) mice. The elements stained are PECAM (green), K14 (red) and Hoechst (blue). n = 9 biological replicates per genotype. (e) Flow cytometry analysis of total cells isolated from P70 ear tissue for four distinct genotypes. Cells double-positive for CD45 and CD4, CD207, CD11b or CD11c were analyzed. Number indicates the percentage of double-positive cells per genotype. Data represent one of three biological replicates. (f) Immunoprecipitation of HPV16 E7 from P2 mouse skin keratinocytes. (g) p53 expression levels in P70 ear tissue. Actin was used as a loading control for total protein, and K14 serves as a loading control for epithelial cells. The K17 immunoblot establishes proper genotyping. PIS, preimmune sera. The blots in f and g represent one of six biological replicates. (h) TUNEL staining of P70 ear tissue, denoting apoptotic cells restricted to the suprabasal layers of epidermis, for both genotypes. Images represent one of six biological replicates. (i) Electron micrographs and aspect ratio quantification (box-and-whisker plot) of basal epithelial cells from 8-month-old ear tissue (8,400× magnification). n = 100 basal cells counted across 3 biological replicates per genotype.

Supplementary Figure 2 TPA- and HPV16-induced gene expression in skin tumor keratinocytes.

(a) Normalized expression for target genes in intact A431 keratinocytes stimulated with TPA relative to DMSO (solid red bars) and in TPA-treated A431 cells expressing either shControl or shKrt17 plasmid (open bars). n = 5 biological replicates. (b) Normalized expression for gene transcripts restored upon Krt17 re-expression (n = 7 biological replicates; blue bars) relative to vector control and compared to Krt42 re-expression (n = 5 biological replicates; red bars). (c) Normalized expression for distinct regions of the Aire transcript (exon 2 or 10 or a region spanning exons 13 and 14) in TPA-treated ears (blue bars) or thymus (red bars) relative to liver, in two different genetic backgrounds (C57BL/6, left; FVB/N, right). n = 4 biological replicates. (d,e) Sense-strand RNA in situ hybridization controls in skin tissue sections (complements data reported in Fig. 2c,d). Scale bars, 200 µm. (f) Coimmunoprecipitation of K17 with RFP-Trap pulldown of mCherry-Aire compared to unfused mCherry. GAPDH serves as a loading control for total protein. n = 1 of 7 biological replicates.

Supplementary Figure 3 Loss of Krt17 does not affect thymic Aire expression or function.

(a) Normalized expression for Aire in P10 thymic tissue between wild-type (blue bar), HPV16Tg/+ (red bar) and HPV16Tg/+; Krt17−/− (open bar) mice. *NS, no statistical significance. (b) RNA in situ hybridization for Aire in thymic tissue sections. Arrows denote regions of medullary thymus positive for hybridization. Scale bars, 200 µm. (c) H&E staining of lacrimal glands from aged (>4 month) C57BL/6 mice. Scale bars, 100 µm. (d) Histology scoring for the presence of lymphocytic infiltrate in multiple tissues from aged (>4 month) Krt17+/+, Krt17−/−, Aire+/+ and Aire−/− mice. *P values are from non-parametric ANOVA with Dunn’s post-testing. n.s., no statistical significance. (e) Summary of histology scoring (each pie represents an individual mouse) with each tissue represented by a different color. A filled wedge indicates >10% of the tissue exhibiting inflammation.

Supplementary Figure 4 Characterization of Aire and nuclear K17 punctae in tumor epithelia.

(a) A431 keratinocytes transfected with mCherry-Aire and immunostained in green with markers for lysosomes (LAMP-1), stress granules (eIF3η) or PML bodies (PML). Scale bars, 5 µm. (b) A431 keratinocytes stably expressing shRNA targeting Krt17 (shKrt17) or shControl plasmids transiently transfected with mCherry-Aire and immunostained for HSP70 (green). Scale bars, 20 µm. (c) Single-plane confocal images of HeLa cells stably expressing GFP-K17 and either mock treated or treated with LMB. Scale bars, 5 µm. (d) Single-plane confocal images through the nucleus of HeLa cells either mock treated or treated with LMB and immunostained for K17 (green) and Hoechst (blue) using mouse (left panels) or rabbit (right panels) anti-K17 antibodies. The z plane is on top. Arrows denote intranuclear punctae immunopositive for K17. Scale bars, 20 µm. (e,f) Single-plane confocal images of immortalized keratinocytes from wild-type (e) or Krt17−/− (f) mouse epidermis immunostained for K17 and K5. Scale bars, 20 µm. (g) Immunoblots for subcellular fractions of DMSO- or TPA-treated A431 cells transfected with mCherry-Aire compared to unfused mCherry control. Sol, soluble (GAPDH positive); NE, nuclear envelope enriched (nesprin 3 positive); Chr, chromatin enriched (histone H3 positive). (h) Immunoblots of subcellular fractions of DMSO- or TPA-treated A431 keratinocytes probed for various type I keratins, as indicated on the left. Sol, soluble (GAPDH positive); NE, nuclear envelope enriched (nesprin 3 positive); Chr, chromatin enriched (histone H3 positive). All images in ag are representative of three biological replicates. Blots in h are representative of five biological replicates.

Supplementary Figure 5 ChIP and EMSA assays.

(a) Immunoblot for K17 after immunoprecipitation of K17, relative to preimmune sera (PIS), from the nuclear extract used for the ChIP analysis presented in Figure 4. Input represents the sample removed before immunoprecipitation. The image represents one of seven biological replicates. (b) DNA amplicons from the K17 ChIP assay (Fig. 4) observed after equal numbers of qPCR cycles by DNA electrophoresis. The images represent one of three biological replicates. (c,d) 5′ upstream sequences of the C XCL10 (c) and CCL19 (d) transcripts. The locations of the forward and reverse primer sites used for the ChIP assays are depicted with colored arrows. NF-κB consensus sequences are denoted with bold text. The ATG translation start site is italicized. (e) EMSA analysis of radiolabeled Oct1 consensus oligonucleotides using nuclear extracts from A431 keratinocytes treated with TPA, relative to DMSO control. Supershift analysis was conducted with antibodies against K17, relative to preimmune sera (PIS) control. Cold competition was conducted using 50-fold excess of non-labeled oligonucleotide. The image represents one of three biological replicates. (f) Reciprocal coimmunoprecipitation demonstrating p65 enrichment with K17 immunoprecipitation relative to preimmune sera (PIS) (left panel; n = 7 biological replicates) and K17 enrichment with p65 immunoprecipitation relative to IgG control (right panel; n = 3 biological replicates).

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Hobbs, R., DePianto, D., Jacob, J. et al. Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes. Nat Genet 47, 933–938 (2015). https://doi.org/10.1038/ng.3355

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