MCPIP1 Controls the Effects of Myeloid Cells on Skin Carcinogenesis and Hair Growth

Monocyte chemotactic protein-induced protein-1 (MCPIP1), or Regnase-1, is an RNase with anti-inflammatory properties (Matsushita et al., 2009Matsushita K. Takeuchi O. Standley D.M. Kumagai Y. Kawagoe T. Miyake T. et al.Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay.Nature. 2009; 458: 1185-1190Crossref PubMed Scopus (514) Google Scholar). Expression of MCPIP1, encoded by the gene ZC3H12A, is impaired in several types of cancer (Miekus et al., 2019Miekus K. Kotlinowski J. Lichawska-Cieslar A. Rys J. Jura J. Activity of MCPIP1 RNase in tumor associated processes.Journal of Experimental & Clinical Cancer Research. 2019; 38: 421Crossref Scopus (16) Google Scholar). As the immune system is a key element in the etiology of cancers, we hypothesized that MCPIP1 of myeloid cells is important in the crosstalk between the immune system and cells of developing tumors. To test this hypothesis, we used mice lacking Mcpip1 in cells of myeloid origin (Mcpip1MKO), including macrophages and granulocytes, in a chemical carcinogenesis model of epithelial skin cancer. It was previously shown that McpipMKO mice produce aberrantly high amounts of cytokines and chemokines and develop systemic inflammation (Li et al., 2017Li Y. Huang X. Huang S. He H. Lei T. Saaoud F. et al.Central role of myeloid MCPIP1 in protecting against LPS-induced inflammation and lung injury.Signal Transduct Target Ther. 2017; 217066Google Scholar), including massive Th2-dominant skin inflammation (Szukala et al., 2023Szukala W. Pilarczyk-Zurek M. Folkert J. Kotlinowski J. Koziel J. Jura J. Depletion of Mcpip1 in murine myeloid cells results in intestinal dysbiosis followed by allergic inflammation.Biochim Biophys Acta Mol Basis Dis. 2023; 1869166764Crossref Scopus (0) Google Scholar). We also demonstrated that Mcpip1MKO mice develop severe skin irritation in response to dinitrofluorobenzene (DNFB), but only a moderate response in the imiquimod-induced psoriasis-like inflammation model (Szukala et al., 2023Szukala W. Pilarczyk-Zurek M. Folkert J. Kotlinowski J. Koziel J. Jura J. Depletion of Mcpip1 in murine myeloid cells results in intestinal dysbiosis followed by allergic inflammation.Biochim Biophys Acta Mol Basis Dis. 2023; 1869166764Crossref Scopus (0) Google Scholar). Mcpip1MKO (Zc3h12aloxP/loxPLysMCre) and control (Zc3h12aloxP/loxP) mice were subjected to the two-stage skin carcinogenesis protocol, initiated by a single application of 30 μg 7,12-dimethylbenz[a]anthracene (DMBA) and followed by biweekly treatments with 15 μg tetradecanoyl phorbol acetate (TPA) for 18 weeks (Figure 1a). In the control mice, the DMBA/TPA regimen resulted in the growth of papillomas, which were entirely absent in the Mcpip1MKO mice. Two further unexpected phenotypic differences were observed. While hair regrew in all control mice after shaving and DMBA/TPA-treatments, hair growth was impaired on the body surface subjected to the treatment in Mcpip1MKO mice. Furthermore, Mcpip1MKO mice developed more and larger macroscopically visible pigment spots in their skin than control mice (Figures 1b-d and Supplementary Figures S1a-c). Histologically, the pigment spots corresponded to accumulations of pigment in the dermis of Mcpip1MKO mice (Figures 1e-f and Supplementary Figure S1d). Markers of melanocytes were transciptionally upregulated in hyperpigmented lesions (Figure 1h) and also in lymph nodes (Supplementary Figure S1e). In line with the macroscopically obvious hair loss, the skin of DMBA/TPA-treated Mcpip1MKO mice almost completely lacked well-structured hair follicles (Figure 1g). We observed that many hair follicle remnants of Mcpip1MKO skin were surrounded by melanin (Supplementary Figure S1f). Global transcriptome analysis revealed profound gene expression differences between the skin of DMBA/TPA-treated control and Mcpip1MKO mice. Both the comparison of skin areas without (Figure 2a-c) and with (Supplementary Figures S2a-c and S3a-c) visible lesions (papillomas in control mice and hyperpigmented spots in Mcpip1MKO mice) demonstrated myeloid MCPIP-dependent effects on large sets of genes. Functional profiling and qRT-PCR indicated that the transcript levels of a plethora of chemokines, cytokines and other factors assigned mostly to leukocyte migration, cytokine-mediated signaling pathway such as regulation of T cell activation and response to interferon-gamma (IFN-γ) were elevated in Mcpip1MKO skin (Figures 2c-d and Supplementary Figures 2d-e). The recruitment of cytotoxic T cells (Cd8a+), granulocytes (Gr1+) and macrophages (F4/80+) into the DMBA/TPA-treated skin of Mcpip1MKO mice was enhanced as compared to treated control mice (Figure 2e). On the contrary, keratinocyte proliferation was markedly reduced in the skin of Mcpip1MKO mice (Figure 2f). Processes related to epithelial cell proliferation and Wnt signaling were significantly downregulated in the mRNA profiles of Mcpip1MKO mice (Supplementary Figure S3a-c). In addition, a number of keratins specific to hair follicles including Krt15, a marker of bulge stem cells, were markedly downregulated in Mcpip1MKO skin (Figure 2d and Supplementary Figure S3d). Our study indicates that loss of Mcpip1 in the cells of myeloid origin abrogates the susceptibility of mouse skin to the chemically induced papillomagenesis. We and others previously reported that DMBA/TPA treatment of Mcpip1eKO (epidermal knockout) mice induced the formation of squamous-like papillomas with a more aggressive phenotype than those developing in control mice (Morisaka et al., 2023Morisaka H. Takaishi M. Akira S. Sano S. Keratinocyte Regnase-1, a Downregulator of Skin Inflammation, Contributes to Protection against Tumor Promotion by Limiting Cyclooxygenase-2 Expression.Journal of Investigative Dermatology. 2023; 143: 731-739Abstract Full Text Full Text PDF Scopus (0) Google Scholar; Szukala et al., 2021Szukala W. Lichawska-Cieslar A. Pietrzycka R. Kulecka M. Rumienczyk I. Mikula M. et al.Loss of epidermal MCPIP1 is associated with aggressive squamous cell carcinoma.Journal of Experimental & Clinical Cancer Research. 2021; 40: 391Crossref Scopus (3) Google Scholar). Strikingly, the phenotype obtained by DMBA/TPA treatment of Mcpip1MKO mice was exactly opposite to the phenotype of equally treated Mcpip1eKO mice. The proliferation of keratinocytes was suppressed in Mcpip1MKO mice, and we speculate that IFN-γ is a key factor affecting epidermal proliferation. Cytotoxic CD8-positive T cells, which are present in high abundance in Mcpip1MKO skin, are the most plausible source of IFN-γ. It was shown that cytotoxic T cells expressing IFN-γ are the major anticancer component in the tumor microenvironment (Reina-Campos et al., 2021Reina-Campos M. Scharping N.E. Goldrath A.W. CD8+ T cell metabolism in infection and cancer.Nat Rev Immunol. 2021; 21: 718-738Crossref Scopus (139) Google Scholar), and CD8+ T cells suppress papillomagenesis in the chemical carcinogenesis model (Yusuf et al., 2008Yusuf N. Nasti T.H. Katiyar S.K. Jacobs M.K. Seibert M.D. Ginsburg A.C. et al.Antagonistic Roles of CD4+ and CD8+ T-Cells in 7,12-Dimethylbenz(a)anthracene Cutaneous Carcinogenesis.Cancer Res. 2008; 68: 3924-3930Crossref PubMed Scopus (44) Google Scholar). In our model, treatment with DMBA/TPA promoted hair loss in 100% of Mcpip1MKO mice but not in control mice. This phenotype is associated with a decrease in Krt15-positive hair follicle stem cells and resembles alopecia areata, a disorder that is caused by the damage of the hair follicles by IFN-γ producing T cells (Xing et al. 2014). We put forward the hypothesis that proinflammatory signals from Mcpip1-deficient myeloid cells cause the loss of follicular stem cells, thereby impairing hair growth. It is conceivable that inflammation-induced defects of the hair follicle architecture affect hair-associated melanocytes and force them to redirect the transfer of melanin from hair shafts to dermal pigment spots in mutant mice. As hair follicle stem cells are involved in DMBA/TPA-induced tumorigenesis (Trempus et al., 2007Trempus C.S. Morris R.J. Ehinger M. Elmore A. Bortner C.D. Ito M. et al.CD34 Expression by Hair Follicle Stem Cells Is Required for Skin Tumor Development in Mice.Cancer Res. 2007; 67: 4173-4181Crossref Scopus (112) Google Scholar), the degeneration of the hair follicles may contribute to the suppression of papillomas in Mcpip1MKO mice. In conclusion, our study reveals a critical role of Mcpip1 in regulating the effects of myeloid cells on the course of skin carcinogenesis and on hair follicles in the classical DMBA/TPA model (Supplementary Figure S4). Further studies in other experimental models are necessary to determine the molecular mechanisms of MCPIP1-dependent signaling from myeloid cells and their potential relevance for clinical dermatology. To obtain myeloid-specific knockout of the Mcpip1 protein (Mcpip1MKO), Zc3h12aloxP/loxP mice (control mice) were crossed with a LysM-Cretg/+ strain (The Jackson Laboratory), as described previously (Li et al., 2017Li Y. Huang X. Huang S. He H. Lei T. Saaoud F. et al.Central role of myeloid MCPIP1 in protecting against LPS-induced inflammation and lung injury.Signal Transduct Target Ther. 2017; 217066Google Scholar). All mice were on a C57BL background. The chemical DMBA/TPA approach was used to induce skin tumorigenesis as we previously described (Szukala et al., 2021Szukala W. Lichawska-Cieslar A. Pietrzycka R. Kulecka M. Rumienczyk I. Mikula M. et al.Loss of epidermal MCPIP1 is associated with aggressive squamous cell carcinoma.Journal of Experimental & Clinical Cancer Research. 2021; 40: 391Crossref Scopus (3) Google Scholar). The study was approved by the 2nd Ethical Committee of Jagiellonian University in Krakow (no. 93/2018). All procedures involving animals were carried out following the Guide for the Care and Use of Laboratory Animals (Directive 2010/63/EU of European Parliament). Other experimental details are available in the Supplementary Material. Datasets related to this article can be found at NCBI's Gene Expression Omnibus at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242128. The authors declare no conflicts of interest. Xing et al., 2014 20Xing L, Dai Z, Jabbari A, Cerise JE, Higgins CA, Gong W, et al. Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition. Nature Medicine 2014 20:9. Nature Publishing Group; 2014;20(9):1043–1049Google Scholar. The research and open access were supported by the Priority Research Area BioS under “Excellence Initiative-Research University” program at Jagiellonian University. AUTHOR CONTRIBUTION Conceptualization:A.L.-C.,W.S.,L.E.,J.J.; Data curation:A.L.-C.,W.S.,M.M.; Formal analysis: A.L.-C.,W.S.,K.G.,I.R.,M.M.; Funding Acquisition:A.L.-C.; Investigation:W.S.(lead),A.L.-C.,K.G.,I.R.; Methodology:W.S.,A.L.-C.; Project Administration:A.L.-C.,J.J.; Resources:J.J.(lead),M.M.,J.K.; Software:M.M.(lead),K.G.,W.S.; Supervision:A.L.-C.,J.J.; Validation:A.L.-C.; Visualization:W.S.(lead),A.L.-C; Writing-original draft:A.L.-C.(lead),W.S.,J.J.; Writing-review&editing:M.M.,L.E.,J.K. MATERIALS AND METHODS Chemically induced tumorigenesis The chemical DMBA/TPA approach was used to induce skin tumorigenesis as we previously described (Abel et al. 2009; Szukala et al. 2021). Briefly, 6- to 8-week-old control and Mcpip1MKO mice were shaved and treated the following day with a single dose of 30 μg DMBA (Sigma‒Aldrich) reconstituted in 200 μl acetone. Two weeks after the DMBA application, 15 μg TPA (Sigma‒Aldrich) in 200 μl acetone was applied topically twice per week for the subsequent 16 weeks. The number and volume of papillomas and/or the diameter of hyperpigmented spots were measured weekly. At 18 weeks after the DMBA application, the mice were sacrificed, and specimens of skin lesions (papillomas and hyperpigmented spots) and adjacent skin were collected and stored at -80°C. Histological and immunofluorescence staining Collected mouse tissue specimens were fixed for 2h in a 4% formaldehyde solution (ChemCruz, Dallas, TX, USA) before overnight incubation at 4°C in 30% sucrose. Then, the samples were frozen in OCT medium (Sakura, Torrance, CA, USA) at -80°C. Subsequently, 8-μm cryosections were cut using a Cryotome (Leica Biosystems, Wetzlar, Germany), rinsed and stored at -80°C for further staining. Obtained sections were stained with hematoxylin and eosin solutions (Sigma‒Aldrich) for general histology or a Fontana-Masson stain kit (Sigma‒Aldrich) according to the manufacturer’s protocols. For melanin bleaching, slides were first oxidized by a 15-minute incubation with a 1% aqueous potassium permanganate solution, washed with running tap water and decolorized by a 1-minute incubation with 2% aqueous oxalic acid (Sigma‒Aldrich), followed by counterstaining with hematoxylin and eosin. For immunofluorescence staining, antigen retrieval was performed by a 30-min incubation at 95°C in 10 mM citrate buffer (pH 6.0). Subsequently, the sections were blocked for 1 h with M.O.M. blocking reagent (Dako, Glostrup, Denmark) for a mouse primary antibody or with 5% horse serum containing 1% BSA and 0.005% Tween (Bio-Shop, Burlington, Canada) in PBS for others. Then, the slides were incubated with primary antibodies overnight at 4°C, rinsed with PBS and incubated with secondary antibodies and Hoechst 33258 (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. All the antibodies were diluted in the blocking buffer. Subsequently, the samples were washed with water and mounted with fluorescent mounting medium (Dako). Staining was visualized under a Leica DMC5400 fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using Leica Applications Suite X (Las X) image acquisition software. All figures were prepared using ImageJ (NIH, Bethesda, MD). The antibodies used in this study are listed in Supplementary Table S1. RNA isolation and quantitative real-time PCR Collected mouse skin samples were snap-frozen in liquid nitrogen and stored at -80°C for downstream analysis. For RNA isolation, the samples were homogenized in Fenozol (A&A Biotechnology, Gdansk, Polska) using a tissue homogenizer (OMNI International, Kennesaw, GA, USA). The total concentration of RNA was determined by measuring the absorbance using a NanoDrop 2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 μg of RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA), a deoxyribonucleotide triphosphate (dNTP) mix (EURx, Gdansk, Polska) and oligo(dT) (Sigma‒Aldrich). The reaction was carried out for 5 min at 65°C, 1 h at 42°C and 15 min at 72°C. The cDNA was then diluted 5 times and amplified with SYBR Green Master Mix (A&A Biotechnology) and 200 nM primers (Sigma‒Aldrich) using a QuantStudio3 thermocycler (Thermo Fisher Scientific). Relative mRNA levels were calculated by the 2-ΔCT method, and elongation factor 2 (Ef2) was used as a reference gene. The primer sequences and annealing temperatures are listed in Supplementary Table S2. ELISA Frozen skin samples were homogenized in PBS containing 1% Triton X-100 and centrifuged for 20 min at 4°C and 10000 x g. Then, the total protein concentration was measured using a bicinchoninic acid assay (Sigma‒Aldrich). The level of the cytokine Ifn-γ was measured in triplicate using a mouse Ifn-γ DuoSet ELISA kit (R&D Systems, Minneapolis, Canada) according to the manufacturer’s protocol. RNA-seq RNA samples were treated with DNaseI and concentrated using RNA Clean&Concentrator kit (Zymo Research, Orange, CA, USA) according to the protocol. A sequencing library was prepared from each RNA sample using the Ion AmpliSeq™ Transcriptome Mouse Gene Expression Kit according to the manufacturer’s protocol (Thermo Fisher Scientific). The libraries were prepared from 100 ng of total RNA. RNA was reverse-transcribed, and cDNA was then subjected to multiplex PCR to amplify the fragments of the target transcripts. The amplicons were then partially digested at the primer sequences, followed by adapter ligation to the amplicons and purification using the magnetic bead method. The molar concentration and size selection (125-300 bp) of each cDNA library was determined using the DNA HS Kit on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Each library was diluted to ∼55 pM before template preparation, and eight barcoded libraries were mixed in equal volumes and used for automatic template preparation on an Ion Chef (Thermo Fisher Scientific) instrument using reagents from the Ion PI Hi-Q Chef Kit (Thermo Fisher Scientific), Ion PI Hi-Q Sequencing 200 Kit and Ion PI v3 Proton Chip. The samples were sequenced on the Ion Proton System (Thermo Fisher Scientific) according to the manufacturer’s instructions. Raw reads were processed with TorrentSuite version 5.14.0 and mapped to the AmpliSeq mouse transcriptome reference. Gene abundances were quantified with htseq-count (HTSeq framework version 0.6). Differential gene expression analysis was performed with the R package DESeq2 version 1.18.1. Sequencing data have been deposited in NCBI's Gene Expression Omnibus (Barrett et al. 2013) and are accessible through GEO Series accession number GSE242128. Functional analysis of transcriptome data Functional annotation of DEGs (fold change > 2.0 and p adj < 0.05) was performed using the R package ClusterProfiler version 4.4 (Wu et a. 2021). Gene lists were searched using the Entrez gene annotation (ENTREZ_GENE_ID), with the Mus musculus background dataset used for analyses. Volcano plots and dot plots were created using the ggplot2 libraries in R. Statistical analysis All graphs were created and statistical analyses were performed using GraphPad Prism 8 and CorelDraw 2021. Statistical significance is indicated by asterisks in the figures, with the following definitions: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Abel EL, Angel JM, Kiguchi K, DiGiovanni J. Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications. Nat Protoc. 2009;4(9):1350–62 Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, et al. NCBI GEO: archive for functional genomics data sets--update. Nucleic Acids Res. 2013;41(Database issue):D991-5 Szukala W, Lichawska-Cieslar A, Pietrzycka R, Kulecka M, Rumienczyk I, Mikula M, et al. Loss of epidermal MCPIP1 is associated with aggressive squamous cell carcinoma. Journal of Experimental & Clinical Cancer Research. 2021;40(1):391 Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting The Table S1. of antibodies used for immunofluorescence in a Table S2. of primers used for size in a Supplementary Figure S2. Functional and molecular analysis of upregulated DEGs in Mcpip1MKO and DMBA/TPA-treated RNA-seq analysis of DEGs in DMBA/TPA-treated skin lesions from Mcpip1MKO spots) compared to the control mice Volcano and Gene enrichment analysis of upregulated the expression levels of DEGs related to Gene of T cell analysis of and expression Ifn-γ protein levels were determined using an ELISA Data are shown as the test was used to Figure image Figure Functional and molecular analysis of downregulated DEGs in Mcpip1MKO and DMBA/TPA-treated enrichment analysis of downregulated in Mcpip1MKO DMBA/TPA-treated skin compared to the control mice skin and skin lesions from Mcpip1MKO spots) compared to the control mice the expression levels of DEGs related to Gene cell the expression levels of DEGs hair follicle Figure image Figure of control and Mcpip1MKO skin response to chemical Figure image

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