Linear ubiquitin chain assembly complex plays an important role in regulating TNF-α signaling activation by modifying target proteins with linear (M1-linked) ubiquitin chains. In this study, we report that the epidermis-specific knockout (KO) of RNF31, the catalytic subunit of linear ubiquitin chain assembly complex, results in an early postnatal lethality in mice due to severe skin inflammation. The inflammation was mainly triggered by TNF-α–induced apoptosis in RNF31 KO keratinocytes. Mechanistically, the deficiency of RNF31 not only impaired TNF-α–induced NF-κB activation, but also significantly increased apoptosis. Consistently, deleting TNF receptor 1 could rescue the lethality of RNF31 epidermis-specific KO mice and also the skin inflammation. Collectively, our study provides an in vivo insight that linear ubiquitination is critical for maintaining the homeostasis of keratinocytes, which will shed light on designing therapeutic compounds to treat skin inflammation.

This article is featured in In This Issue, p.3869

The skin forms the primary barrier between the host’s body and its environment to protect the host from infection by microbial pathogens (1). Epidermis is the outermost structure of the skin that is directly exposed to the environment. Previous studies indicate that epidermal keratinocytes play a critical role in regulation of immune homeostasis and inflammation in the skin. Disruption of skin homeostasis could result in acute and chronic inflammatory skin diseases, such as atopic dermatitis and psoriasis (2, 3), because danger-associated molecular patterns releasing from dead skin cells or infected microbial pathogens can trigger the host’s innate immune responses, leading to severe skin inflammation in the host. Therefore, there must be a tight regulation for maintaining and re-establishing healthy skin homeostasis after injury.

Infliximab, a type of TNF-α Ab, has a promising clinical efficacy for psoriasis patients, which highlights that the TNF-α pathway has an important function in the regulation of skin homeostasis (4). TNF-α can trigger two opposing cellular responses: NF-κB activation and cell death (58). Studies in genetic mouse models have shown that keratinocytes-specific deletion of IκB kinase 2 (IKK2), NF-κB essential modifier (NEMO), or transgenic expression of the mutated superrepressor IκBα, all of which inhibit NF-κB activation, could result in the development of TNF-α–dependent psoriasis-like inflammatory skin lesions (911). Another study reported that TNF-α in keratinocytes could trigger IL-24–dependent psoriasis-like skin inflammation in mice (12). Moreover, TNF-α signaling-regulated survival factors could protect epidermal keratinocytes from necroptosis or apoptosis in vivo to prevent skin inflammation (1315). Therefore, it is clear that TNF-α signaling plays a critical role in the regulation of skin cell homeostasis and skin inflammation. However, the exact molecular mechanism by which the TNF-α pathway controls skin inflammation is not fully defined.

Recent studies have revealed that M1-linked (also known as linear) ubiquitination is a novel type of posttranslational modification that regulates TNF-α–mediated NF-κB activation through targeting two key components, receptor-interacting serine/threonine kinase (RIP) 1 and NEMO (1619). M1-linked ubiquitination chains are formed between the carboxyl group of the C-terminal glycine of ubiquitin and the N-terminal methionine (Met1) of another ubiquitin. To date, the linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex for M1-linked ubiquitination, and it consists of catalytic subunit RNF31 (HOIP) and two associated molecules, HOIL-1 and Sharpin (2022). Genetic studies demonstrated that Sharpin-deficient mice could develop chronic proliferative dermatitis because of TNF-α–mediated apoptosis and necroptosis (19, 23, 24). Interestingly, the skin inflammation in Sharpin-deficient mice could be rescued by TNF receptor 1 (TNFR1) knockout (KO) (25, 26), and this phenomenon indicates that M1-linked ubiquitination is critical for preventing skin inflammation by regulating the TNF-α signaling pathway. Another study reported that Sharpin could negatively regulate TCR signaling to control regulatory T cell function, which contributed to inflammation in Sharpin-deficient mice (27). In contrast to Sharpin, HOIL-1 KO mice develop well and have no obvious autoimmune disease, including skin inflammation (16). However, RNF31-deficient mice are embryonically lethal (28), which prevents further investigation of the impact of LUBAC-mediated M1-linked ubiquitination on skin inflammation and homeostasis. Therefore, the exact role of M1-linked ubiquitination regulating apoptosis leading to skin inflammation remains to be determined.

In this study, we generated the RNF31-conditional KO mice and genetically deleted RNF31 in mouse epidermis to investigate the function of RNF31 in skin. We found that these mice developed fatal early-onset skin inflammation, which was triggered by TNF-α–induced apoptosis. Consistently, TNFR1 deficiency could rescue the lethality and skin inflammation in epidermis-specific RNF31 KO (E-KO) mice. Together, these findings provide new insights into the regulatory role of M1-linked ubiquitination in maintaining skin homeostasis, and targeting this process may be a novel therapeutic approach for skin inflammation originated from dysregulation of cell death.

RNF31-flox mice were generated by Crispr-Cas9 technique. Briefly, two sgRNAs, which are located in intron5–6 and intron11–12, were designed, and the targeting donor construct was designed to introduce two loxP sites. Then, the donor vector, the mRNA of the sgRNAs, and Cas9 were injected together into the fertilized eggs of C57BL/6 mice. The corrected floxed founder mice were genotyped by genomic PCR and Southern blot. Wild-type (WT) C57BL/6 mice and TNFR1 KO mice were purchased from Jackson Laboratory and bred in the facility. Keratin 14 (K14)-Cre mice were kindly provided by Prof. G. Ma from Shanghai Jiao Tong University. All mice were housed in the specific pathogen-free animal facilities in Tsinghua University. All mouse experiments were performed in compliance with institutional guidelines and according to the protocol approved by the Institutional Animal Care and Use Committee of Tsinghua University.

The mouse TNF-α recombinant protein was purchased from R&D (410-MT-010). The de novo protein synthesis inhibitor cycloheximide (CHX) (C7698-1G) was purchased from Sigma. Abs against RNF31 (ab85294) and Sharpin (ab125188) were purchased from Abcam. Abs specific for PARP (9542), caspase-3 (9665), caspase-8 (9746), IκB-α (4814), phosphorylated IκB-α (9246), phosphorylated JNK (9255), JNK (4672), and cFLIPS/L (56343) were obtained from Cell Signaling Technology. Abs against β-tubulin (BE0025-100) were obtained from Easybio. An Ab specific for RIP1 (610459) was obtained from BD Biosciences.

Cells were lysed in lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, and a protease inhibitor mixture (Roche). Cell lysates were then subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was sequentially probed with primary Abs and HRP-conjugated secondary Abs. ECL substrates (Pierce) were used to visualize the specific bands on the membrane.

Total cells or skin tissues RNA was isolated using TRIzol (Invitrogen) and reverse transcribed using SuperScript III (Invitrogen). Quantitative real-time PCR (RT-PCR) was performed using Power SYBR Green PCR Master Mix (Genestar). The amounts of transcript were normalized to those for Gapdh. Melting curves were run to ensure amplification of a single product. The primers used are listed in Supplemental Table I.

RNA sequencing (RNA-seq) was performed by Novogene. Quality control was performed by running the samples on an Agilent Bioanalyzer 2100 using an RNA 6000 Nano chip. An RNA-seq library was generated from poly(A)–enriched RNA using an Illumina stranded RNA sample prep kit according to the manufacturer’s instructions and was subsequently sequenced for 50 nt from paired end on an Illumina HiSeq 2000. After removing the adaptor and low-quality reads by using cutadapt (1.9.1) with default option, the clean reads were aligned to the mouse Grcm38 with geocode GRCm38.87 by hisat2 (2.0.4). After alignment, the BAM files for each individual alignment were used to analyze the differential expression of genes with Cufflinks (version 2.2.1). Heat maps were generated with the gplots package in R (version 3.2.3). The RNA-seq data have been deposited in the Gene Expression Omnibus under accession number GSE111297, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111297.

Primary keratinocytes were isolated from the skin of newborn mice according to the previous reported protocol (29). The total number of live keratinocytes was counted after trypan blue staining. The primary keratinocytes were cultured in Defined Keratinocyte-SFM medium (10744019; Life Technologies) and then stimulated with TNF-α for indicated times.

For histopathology analyses, skin and other organs were fixed in 4% paraformaldehyde solution, processed according to standard procedures, embedded in paraffin, and sectioned. Five micrometers-thick sections were stained with H&E, immunohistochemistry, and immunofluorescence. We used the following Abs: Ki67 (GB13030-2), F4/80 (GB11027), Ly6G (GB11229), CD11c (GB11059), K10 (ab76318; Abcam), K14 (ab7800; Abcam), Loricirin (ab176322; Abcam), cleaved caspase-3 (9664; CST), and cleaved caspase-8 (9496; CST).

All values in this article were given as mean ± SEM, unless stated otherwise. All experiments were reproduced at least three independent times. Statistical significance was calculated by two-tailed unpaired t test using GraphPad Prism software. Statistical significance was set based on p values. NS = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

RNF31 KO mice are embryonically lethal at midgestation because of endothelial cell death mediated by TNFR1 (28). Thus, to better study the function of RNF31 in vivo, we first generated RNF31-flox mice by using CRISPR-Cas9 technology (Fig. 1A). Then, we crossed RNF31-flox mice with K14-Cre mice (designed as RNF31E-KO for simplicity), which express Cre recombinase under the control of human K14 promoter. Genomic PCR and immunoblotting analysis showed efficient deletion of the RNF31 allele and specific KO of the full-length RNF31 protein in the epidermis of these mice (Fig. 1B, 1C). RNF31E-KO mice were born at the expected ratio and were also macroscopically indistinguishable from littermate control mice. But after postnatal day (P) 2, RNF31E-KO mice displayed a slower growth rate than WT littermates, and the black hair of these deficient mice could not develop normally (Fig. 1D). These mice also developed severe spontaneous damage in skin after P2 (Fig. 1D, black arrow). Finally, RNF31E-KO mice died around 4–6 d after birth (Fig. 1E). These results suggested a critical role of RNF31 in maintaining skin homeostasis.

FIGURE 1.

Loss of RNF31 in mouse keratinocytes results in severe skin inflammation. (A) Schematic overview of strategy to generate RNF31-flox mice by CRISPR-Cas9 technology. Briefly, two designed sgRNAs are located in intron5–6 and intron11–12, and the targeting donor construct was designed to introduce loxP sites. (B) Genomic PCR analysis of 1-d-old littermate mice. Mice 1 and 2 are RNF31fl/+K14cre, Mice 4 and 5 are RNF31fl/flK14cre, Mouse 3 is RNF31fl/+K14+, and Mouse 6 is RNF31fl/flK14+. (C) Western blotting detection of the expression level of RNF31 in lysates of control and RNF31E-KO primary keratinocytes from 1-d-old littermate mice (mouse genotyping from B). (D) Representative macroscopic images of 4-d-old WT and RNF31E-KO littermate mice. Black arrows denote severe spontaneous damage in skin after P2. (E) Survival curve of WT and RNF31E-KO mice (n = 9 per group). (F) H&E staining, Loricrin, K10, K14, and Ki67 staining of skin sections from WT and RNF31E-KO littermate mice at P1, P3, and P5. P1, n = 3; P3, n = 3; P5, n = 3. (G) Microscopic quantification of the epidermal thickness from H&E results in (F) (n = 3). Values shown are mean ± SEM. Scale bars, 50 μm (H&E) and 100 μm (Immunofluorescence) in (F). In (G), statistical significance was determined using two-tailed unpaired t test. ns = p > 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

Loss of RNF31 in mouse keratinocytes results in severe skin inflammation. (A) Schematic overview of strategy to generate RNF31-flox mice by CRISPR-Cas9 technology. Briefly, two designed sgRNAs are located in intron5–6 and intron11–12, and the targeting donor construct was designed to introduce loxP sites. (B) Genomic PCR analysis of 1-d-old littermate mice. Mice 1 and 2 are RNF31fl/+K14cre, Mice 4 and 5 are RNF31fl/flK14cre, Mouse 3 is RNF31fl/+K14+, and Mouse 6 is RNF31fl/flK14+. (C) Western blotting detection of the expression level of RNF31 in lysates of control and RNF31E-KO primary keratinocytes from 1-d-old littermate mice (mouse genotyping from B). (D) Representative macroscopic images of 4-d-old WT and RNF31E-KO littermate mice. Black arrows denote severe spontaneous damage in skin after P2. (E) Survival curve of WT and RNF31E-KO mice (n = 9 per group). (F) H&E staining, Loricrin, K10, K14, and Ki67 staining of skin sections from WT and RNF31E-KO littermate mice at P1, P3, and P5. P1, n = 3; P3, n = 3; P5, n = 3. (G) Microscopic quantification of the epidermal thickness from H&E results in (F) (n = 3). Values shown are mean ± SEM. Scale bars, 50 μm (H&E) and 100 μm (Immunofluorescence) in (F). In (G), statistical significance was determined using two-tailed unpaired t test. ns = p > 0.05, ***p < 0.001, ****p < 0.0001.

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The immunohistological analysis of skin sections from RNF31E-KO and WT mice showed that the structure of skin was normal and had no significant difference between RNF31E-KO and WT mice at P1 (Fig. 1F, 1G). We stained the skin sections with K14 and keratin 10, which are expressed in basal and suprabasal keratinocytes, respectively, as well as loricrin, a marker of terminal keratinocyte differentiation. These epidermal differentiation markers in RNF31E-KO mice were normal at P1 (Fig. 1F). Also, the immunostaining results of proliferative marker Ki67 showed a comparable number of proliferated keratinocytes at P1 in RNF31E-KO mice compared with littermates control (Fig. 1F). These results suggested that RNF31-mediated M1-linked ubiquitination is not required for the normal differentiation and proliferation of the epidermis. But as the mice grow, RNF31 deficiency in the epidermis significantly changed the structure of the skin and increased the thickness of the epidermis at P3 and P5 (Fig. 1F, 1G). From immunofluorescence analysis, the hyperplastic epidermis in RNF31E-KO mice showed increased expression of K14 in suprabasal keratinocytes but decreased expression of K10 and loricrin. At P5, even the expression of K10 and loricrin were nearly completely lost (Fig. 1F). And consistent with the epidermal hyperplasia, the level of Ki67 was greatly increased in the hyperplastic skin patches of RNF31E-KO mice at P3 and P5 (Fig. 1F). Above all, these results suggested that the inflammatory response triggered by RNF31 deficiency in keratinocytes is responsible for the development of skin lesions in RNF31E-KO mice.

To gain insights into how loss of RNF31 resulted in severe skin inflammation, we performed RNA-seq using total skin tissues from 3-d-old WT and RNF31E-KO mice. Loss of RNF31 significantly affected the genes expression pattern in skin cells (Supplemental Fig. 1A). To get an overview of the transcriptome, we performed gene ontology analysis of genes with at least a 2-fold change using DAVID Bioinformatics Resources 6.7. Upregulated genes in the skin of KO mice were mainly enriched in immune and inflammatory responses, including the chemokine signaling pathway, TNF signaling pathway, cytokine/cytokine receptor pathway, and NF-κB signaling pathway (Fig. 2A, Supplemental Fig. 1B), which is likely due to the RNF31 deficiency in keratinocyte, triggering innate immune and inflammatory responses. To further explore the function of RNF31 in the regulation of immune response, we analyzed the putative upregulated cytokines and chemokines and confirmed the results by RT-PCR (Fig. 2B, Supplemental Fig. 1C). The RT-PCR results were consistent with the RNA-seq data. Because the major role of chemokines is to act as a chemoattractant to guide the migration of immune cells, we hypothesized that the increased chemokines in skin may recruit more immune cells migrating to the skin. In agreement with our hypothesis, infiltrating macrophages (F4/80 staining), dendritic cells (CD11c staining), and neutrophil cells (Ly6G staining) were significantly increased in skin epidermis and dermis of RNF31E-KO mice (Fig. 2C). A previous study reported that two calprotectin S100A8-S100A9 were upregulated in psoriatic epidermis of human patients and mouse models (30). In RNF31E-KO mice, S100A8 and S100A9 were also remarkably upregulated (Supplemental Fig. 1D). Taken together, these data reveal that specific deletion of RNF31 in skin epidermis could promote severe skin inflammation.

FIGURE 2.

RNF31 deficiency in keratinocytes triggers skin inflammation. (A) RNA-seq was performed using total skin tissues RNA isolated from 3-d-old WT and RNF31E-KO littermate mice. Shown are heat maps for selected gene panels, mainly including cytokines and chemokines. (B) RT-PCR confirmation of relative expression level of selected genes shown in (A). Values shown are mean ± SEM. (C) F4/80 (macrophages marker), CD11c (dendritic cells marker), and Ly6G (neutrophils marker) staining of skin sections from 3-d-old WT and RNF31E-KO littermate mice. Representative images of at least three replicates are shown. Scale bars, 20 μm. In (B), statistical significance was determined using a two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

RNF31 deficiency in keratinocytes triggers skin inflammation. (A) RNA-seq was performed using total skin tissues RNA isolated from 3-d-old WT and RNF31E-KO littermate mice. Shown are heat maps for selected gene panels, mainly including cytokines and chemokines. (B) RT-PCR confirmation of relative expression level of selected genes shown in (A). Values shown are mean ± SEM. (C) F4/80 (macrophages marker), CD11c (dendritic cells marker), and Ly6G (neutrophils marker) staining of skin sections from 3-d-old WT and RNF31E-KO littermate mice. Representative images of at least three replicates are shown. Scale bars, 20 μm. In (B), statistical significance was determined using a two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

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As previous studies have reported that the loss of the LUBAC component could sensitize cells to cell death (19, 28, 31), we hypothesized that loss of RNF31 in epidermis might promote cell death, leading to severe skin inflammation. To test this hypothesis, we detected the apoptosis markers, cleaved caspase-3 and cleaved caspase-8, in the epidermis from 3-d-old mice (Fig. 3A). The immunostaining results showed a significantly increased level of cleaved caspase-3 and cleaved caspase-8 in RNF31E-KO mice compared with littermates control. This indicates that loss of RNF31 increased cell apoptosis of keratinocytes in vivo. Next, we isolated primary keratinocytes from the skin of 1-d-old WT and RNF31E-KO mice (Fig. 3B). One day after birth, RNF31E-KO mice did not show any obvious skin inflammation, but the number of isolated survival keratinocytes from KO mice was obviously less than WT mice (Fig. 3C). This indicated that RNF31 deficiency triggers cell death before skin inflammation. Similar to previous findings (28), the cause of cell death may be the autocrine TNF-α from cells in skin. After TNF-α and CHX stimulation, more cleaved caspase-3, cleaved caspase-8, and cleaved PARP in KO keratinocytes could be detected than those in WT keratinocytes (Fig. 3D). We also found the expression level of Sharpin was significantly decreased in RNF31 KO keratinocytes (Fig. 3D), suggesting that RNF31 deficiency could affect the stability of the LUBAC complex. Consistent with our previous findings (32), RNF31 could be cleaved in apoptosis condition (Fig. 3D).

FIGURE 3.

RNF31 deficiency promotes cell apoptosis in vivo. (A) Cleaved caspase-3 and cleaved caspase-8 staining of skin sections from 3-d-old WT and RNF31E-KO littermate mice. Scale bar, 20 μm. (B) The brief flow diagram of isolating primary keratinocytes from 1-d-old mice. (C) Total cell number of survival primary keratinocytes that isolated from skin of 1-d-old WT and RNF31E-KO mice (n = 8 per group). Values shown are mean ± SEM. (D) Western blotting analysis of the indicated proteins in lysates of WT and RNF31 KO primary keratinocytes from 1-d-old littermate mice treated with TNF-α (20 ng/ml) and CHX (10 μg/ml). (E) Western blotting analysis of the indicated proteins in lysates of WT and RNF31 KO primary keratinocytes isolated from 1-d-old littermate mice treated with TNF-α (20 ng/ml). (F) RT-PCR results of WT and RNF31 KO primary keratinocytes isolated from 1-d-old littermate mice treated with TNF-α (20 ng/ml) for 6 h. In (C) and (F), statistical significance was determined using a two-tailed unpaired t test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

RNF31 deficiency promotes cell apoptosis in vivo. (A) Cleaved caspase-3 and cleaved caspase-8 staining of skin sections from 3-d-old WT and RNF31E-KO littermate mice. Scale bar, 20 μm. (B) The brief flow diagram of isolating primary keratinocytes from 1-d-old mice. (C) Total cell number of survival primary keratinocytes that isolated from skin of 1-d-old WT and RNF31E-KO mice (n = 8 per group). Values shown are mean ± SEM. (D) Western blotting analysis of the indicated proteins in lysates of WT and RNF31 KO primary keratinocytes from 1-d-old littermate mice treated with TNF-α (20 ng/ml) and CHX (10 μg/ml). (E) Western blotting analysis of the indicated proteins in lysates of WT and RNF31 KO primary keratinocytes isolated from 1-d-old littermate mice treated with TNF-α (20 ng/ml). (F) RT-PCR results of WT and RNF31 KO primary keratinocytes isolated from 1-d-old littermate mice treated with TNF-α (20 ng/ml) for 6 h. In (C) and (F), statistical significance was determined using a two-tailed unpaired t test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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Because LUBAC could target NEMO and RIP1 to regulate TNF-α–induced NF-κB activation, we detected TNF-α–induced NF-κB and MAPK signaling in primary keratinocytes. However, we found that IκBα degradation was partially delayed. Phosphorylation of p65, JNK, p38, and ERK was also partially reduced in KO keratinocytes (Fig. 3E). We also detected NF-κB–targeted cytokines and chemokines in primary keratinocytes after TNF-α stimulation. These cytokines and chemokines were all significantly decreased in KO keratinocytes (Fig. 3F). Therefore, loss of RNF31 in keratinocytes partially affects TNF-α–induced NF-κB and MAPK signaling.

It has been reported that ablation of TNFR1 could markedly reduce cell death and restore normal vascularization in RNF31-deficient mice (28). Previous studies also reported that TNF-α or TNFR1 KO can significantly rescue the skin inflammation in Sharpin-deficient mice (19, 25). To determine whether the severe skin inflammation in RNF31E-KO mice was resulted from the TNFR1-mediated apoptosis, we crossed the mice with TNFR1 KO mice to see whether TNFR1 deficiency could rescue the skin inflammation. Interestingly, TNFR1 KO could almost fully rescue the skin inflammation of RNF31E-KO mice at early stage. The rescued mice could survive well, and the body size was comparable with littermates control. We could not observe obvious skin inflammation in the rescued mice, except for some wrinkled hairs in the head region at 3 wk old (Fig. 4A). H&E staining results of skin and ear sections also showed that the rescued mice have normal skin structure and similar thickness of epidermis compared with the littermates control (Fig. 4B, 4C). The immunofluorescence of skin differentiation marker showed that K10, Loricrin, and K14 are comparable between rescued mice and littermates control (Fig. 4D). These results suggested that TNFR1 not only rescued the skin inflammation, but also rescued the defects of skin proliferation and differentiation. Previous studies reported that TNFR1-induced signaling in nonepidermal cells could induce extracutaneous organ inflammation in Sharpin-deficient mice (26). To elucidate the possibility of whether other organs still have inflammation in RNF31E-KOTNFR1−/− mice, we stained the sections of lung, spleen, liver, and kidney. And the H&E staining results revealed normal structure and no obvious inflammation in rescued mice compared with littermates control (Fig. 4E). Taken together, these data indicated that the severe skin inflammation of RNF31E-KO mice was predominantly regulated by TNFR1-mediated cell apoptosis (Fig. 4F).

FIGURE 4.

TNFR1 deficiency in keratinocytes prevents skin inflammation in RNF31 E-KO mice. (A) Representative macroscopic images of 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (B) H&E staining of skin and ear sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (C) Microscopic quantification of the epidermal thickness from H&E results in (B) (n = 3). Values shown are mean ± SEM. (D) Immunofluorescence staining of loricrin, K10, and K14 in skin and ear sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (E) H&E staining of lung, spleen, liver, and kidney sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (F) The proposed model of RNF31 in the process of regulating skin homeostasis. Scale bars, 50 μm in (B) and 100 μm in (D) and (E). In (C), statistical significance was determined using two-tailed unpaired t test (ns = p > 0.05).

FIGURE 4.

TNFR1 deficiency in keratinocytes prevents skin inflammation in RNF31 E-KO mice. (A) Representative macroscopic images of 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (B) H&E staining of skin and ear sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (C) Microscopic quantification of the epidermal thickness from H&E results in (B) (n = 3). Values shown are mean ± SEM. (D) Immunofluorescence staining of loricrin, K10, and K14 in skin and ear sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (E) H&E staining of lung, spleen, liver, and kidney sections from 3-wk-old control (RNF31fl/+K14cre -TNFR1−/−) and RNF31E-KO-TNFR1−/− littermate mice. (F) The proposed model of RNF31 in the process of regulating skin homeostasis. Scale bars, 50 μm in (B) and 100 μm in (D) and (E). In (C), statistical significance was determined using two-tailed unpaired t test (ns = p > 0.05).

Close modal

Host skin is the first line of defense to prevent infection by pathogenic microorganisms. Skin cell homeostasis is maintained by the normal survival and differentiation of keratinocytes. Dysregulation of these processes in keratinocytes may result in apoptosis and necrosis of skin cells. Previous studies indicate that TNF-α–mediated cell death in epidermal keratinocytes is a potent trigger of skin inflammation (33). Consistently, the genetic deletion of Fas-associated death domain protein (13) and caspase-8 (14), two key components of the TNF receptor signaling pathway in skin, induces severely inflammatory skin lesions and inflammation because of RIP3-mediated programmed necrosis in these mice. Epidermal deletion of cFLIP in mice (cFLIPE-KO) led to embryonic lethality (15), whereas the inducible deletion of cFLIP in adult mice epidermis could also result in severe inflammation in skin with apoptotic but not necroptotic cell death. These findings supported that TNF-α–mediated signaling is critical in the regulation of skin homeostasis.

Various studies indicated that LUBAC is involved in the regulation of cell survival signaling pathways. RNF31 deficiency leads to embryonic lethality, whereas depletion of TNF receptor partially rescues this lethality (28), indicating that RNF31 deficiency-associated lethality is mainly due to its role in TNFR1 signaling. Sharpin deficiency results in hypersensitization of epidermal keratinocytes to TNF-α–induced cell death, leading to skin inflammation (25, 26). But another study reported that Sharpin could regulate TCR signaling independent of M1-linked ubiquitination, which controls regulatory T cell function leading to skin inflammation in Sharpin-deficient mice (27). And HOIL-1 KO mice show no obvious skin inflammation (16). These studies indicated that the function of LUBAC-mediated M1-linked ubiquitination in skin inflammation is complicated. We generated RNF31-conditional KO mice, specifically deleting RNF31 in the epidermis. This strategy could help us to specifically investigate the function of LUBAC-mediated M1-linked ubiquitination in skin.

Although previous studies and our data suggest that LUBAC-dependent M1-linked ubiquitination contributes to the regulation of cell survival, the molecular mechanism by which LUBAC regulates cell survival has not been defined. RNF31E-KO mice have a phenotype similar to NEMOE-KO mice (11). This may indicate that NF-κB activation through LUBAC is critical for skin inflammation. But our data and other studies showed that RNF31 deficiency only partially impaired or delayed TNFR1-mediated NF-κB activation (28, 34). However, NEMO deficiency could almost completely inhibit TNFR1-mediated NF-κB activation. Another study in IKK2E-KO mice showed slightly milder skin inflammation compared with our RNF31E-KO mice (9), and IKK2 is also more critical in TNFR1-mediated NF-κB activation. Therefore, these results suggest that RNF31 may directly regulate cell survival, which is independent of NF-κB activation. In our recent findings (D. Joo, Y. Tang, and X. Lin, unpublished observations), we found that RNF31 could partially regulate cFLIP stability by conjugating M1-linked ubiquitin chains to cFLIP, which stabilized cFLIP upon stimulation with TNF-α. These findings are also consistent with the phenotype in cFLIPE-KO mice (15). Further study is in needed to confirm this new mechanism.

We found that RNF31 deficiency in keratinocyte had limited effect on skin development, but the inflammatory response triggered by RNF31 deficiency resulted in the development of skin lesions in RNF31E-KO mice. The isolated primary keratinocytes from RNF31E-KO newborn mice showed obvious apoptosis even without stimulation by exogenous TNF-α. Therefore, we proposed that keratinocytes and innate immune cells in the epidermis or dermis may encounter commensal pathogens, which initiate the basal level of innate immune responses leading to secretion of TNF-α, which induces apoptosis of keratinocytes in RNF31E-KO mice. Consistently, TNFR1 deficiency can rescue the skin inflammation of RNF31E-KO mice.

As described in this article, we applied the genetic approach in a conditional KO mouse model to specifically study the RNF31- and LUBAC-mediated M1 ubiquitination in maintaining skin homeostasis. Our results highlighted the critical role of RNF31 in regulating skin inflammation. Some clinical studies have shown that LUBAC component polymorphisms or deficiency could result in severe inflammation in human patients (34, 35). Our findings indicate that the LUBAC component may be a good diagnostic marker or therapy target for some clinical diseases. Therefore, it will be interesting to examine whether any skin inflammatory diseases contain RNF31 mutations in future studies.

We thank Prof. Gang Ma from Shanghai Jiao Tong University for kindly sharing K14-cre mice.

This work was supported by National Natural Science Foundation of China Grants 81570211 (to X.L.) and 31670904 (to X.Z.).

The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111297) under accession number GSE111297.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CHX

cycloheximide

E-KO

epidermis-specific KO

IKK2

IκB kinase 2

K14

keratin 14

KO

knockout

LUBAC

linear ubiquitin chain assembly complex

NEMO

NF-κB essential modifier

P

postnatal day

RIP

receptor-interacting serine/threonine kinase

RNA-seq

RNA sequencing

RT-PCR

real-time PCR

TNFR1

TNF receptor 1

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data