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. 2022 Sep 2:10:961675.
doi: 10.3389/fcell.2022.961675. eCollection 2022.

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1

Ke Wang  1 , Hu-Ning Xu  1 , Yi-Wen Wang  1 , Jian Mao  1 , Da Liu  1 , Xiao-Jing Zhu  1 , Yu-Sheng Cong  1   2 , Miao Wang  1
Affiliations

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1

Ke Wang et al. Front Cell Dev Biol. .

Abstract

Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

Keywords: Ufl1; Ufl1f/f KRT14Cre/+; Ufmylation modification; endothelin-1; pigmentation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Generation of Ufl1 f/f KRT14Cre/+ mice. (A) Targeting strategy to generate Ufl1 conditional knockout mice. Ufl1 f/f mice were crossed with KRT14Cre/+ transgenic mice to obtain conditional skin knockout mice. (B) PCR-based genotyping of wild-type and floxed alleles of Ufl1 and KRT14-Cre allele. (C) q-PCR analysis of Ufl1 gene expression in the skin of 3-month-old CTRL and CKO mice (n = 6). (D) Ufl1 expression was detected by Western blot in the skin of 3-month-old CTRL (Ufl1 f/+, Ufl1 f/f or Ufl1 f/+ KRT14Cre/+), CKO (Ufl1 f/f KRT14Cre/+) mice. (E) Immunohistochemical staining (IHC) of Ufl1 proteins in sections of ear skin from CTRL and CKO. ***p < 0.001, unpaired Student’s test. Error bars represent ± s.d. The scale bar represents 100 μm.
FIGURE 2
FIGURE 2
Loss of Ufl1 causes severe skin abnormalities in mice. (A) Images of mice with the indicated genotypes. (B) Body weight of mice in A. (C) Paws, tails and ears of the indicated mice as shown in A. (D,E) Tails and ears sections from CTRL and CKO mice were stained with hematoxylin and eosin (H&E) and epidermal thickness. (F,G) Masson-Fontana staining of the tail and ear sections of mice in C, and melanin quantification of the indicated tail and ear samples. Arrowheads mark the dermal-epidermal border in panels. SC, stratum corneum. ***p < 0.001, unpaired Student’s test. Error bars represent ± s.d. The scale bar represents 100 μm.
FIGURE 3
FIGURE 3
Global transcriptome analysis of ear. (A) Volcano plot of downregulated (blue) and upregulated (red) transcripts in CKO mice skin. Genes in melanin biosynthesis differentially expressed are marked by red lines. (B) q-PCR confirmation of mRNA expression of melanin biosynthesis-related genes. (C,D) Pearson’s correlation between Ufl1 and OCA2 or SLC45A2 in The Cancer Genome Atlas (TCGA) SKCM. Plots show the Spearman’s correlation and OCA2 or SLC45A2 with UFL1 mRNA level from RNA-seq data in TCGA melanoma calculated. R and p values are shown. *p < 0.05, ***p < 0.001, unpaired Student’s test. Error bars represent ± s.d.
FIGURE 4
FIGURE 4
Ufl1 deficiency promote the expression and secretion of Endothelin-1 (ET-1). (A) Immunofluorescence staining for mice ears sections. The images showed ET-1 staining (green), and DAPI staining (blue) as a nuclear counterstain (upper panel). (B) Western blot analysis of ET-1 expression in the ears of 3-month-old CRTL and CKO mice (n = 3). (C) Western blot analysis of ET-1 expression in the HaCaT cells with Ufl1 depletion. si-RNA, small interfering RNA; NC, negative control. (D,E) ET-1secretion in the plasma and ears was analyzed by ELISA (n = 6). *p < 0.05, ***p < 0.001, unpaired Student’s test. Error bars represent ± s.d. The scale bar represents 100 μm.
FIGURE 5
FIGURE 5
Ufl1 deletion affects Endothelin-1 synthesis. (A) Western blot analysis of the mutual interactions between Ufl1, DDRGK1 and ET-1 in HEK293T cells by co-immunoprecipitation. (B) In vitro binding assays between ET-1 and Ufl1 or DDRGK1. (C) Ufmylation of ET-1 was analyzed by western blot with anti-UFM1 antibody in HEK293T cells expressing the Ufmylation system components. (D) In vitro Ufmylation assay of ET-1. Us., UFMylation system components (consisting of UBA5, UFC1, UFL1, DDRGK1 and UFM1). (E) ET-1 stability was examined by western blot in UFL1-depletion HaCaT cells (sh-UFL1) compared to control HaCaT cells (sh-NC). The cells were treated with 100 µg ml−1 cycloheximide (CHX) for the indicated times. The graph represents quantification of the ET-1 protein levels.
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