OSMRβ mutants enhance basal keratinocyte differentiation via inactivation of the STAT5/KLF7 axis in PLCA patients

Primary localized cutaneous amyloidosis (PLCA) is a skinlimited disorder characterized by deposition of amyloid material in the superficial dermis. According to clinical characteristics, PLCA is divided into lichen, macular, and nodular amyloidosis. PLCA is found worldwide but has a higher incidence in South America and Southeast Asia, such as in Brazil and China (Chang et al., 2014; Tey et al., 2016). The etiology of PLCA is complicated, involving environmental factors, the immune state, and genetic factors (Tanaka et al., 2009; Katayama et al., 2019). A genome-wide scan revealed that mutations in several genes are involved in the development of PLCA, including oncostatin M receptor (OSMR) (Arita et al., 2008), interleukin 31 receptor A (IL31RA) (Shiao et al., 2013), and glycoprotein Nmb (GPNMB) (Yang et al., 2018). Recently, we demonstrated that the c.1538G>A (p. G513D) and c.2081C>T (p.P694L) mutations of OSMR were the most frequent mutations in a Chinese PLCA population (Lu et al., 2019). It has been reported that OSM maintains hair follicle stem cell and muscle stem cell quiescence by binding to heterodimeric receptors comprising gp130 and OSMRβ (Sampath et al., 2018; Wang et al., 2019). Additionally, OSM signaling plays crucial roles in the regulation of cardiomyocyte differentiation and cellular plasticity (Kubin et al., 2011). Whether OSMRβ-mediated cell differentiation plays a role in PLCA remains unexplored. To answer this question, we compared the RNA expression profiles between PLCA patients and healthy controls. Interestingly, Gene Ontology (GO) analysis of the dysregulated genes revealed that most of them were associated with keratinocyte differentiation processes (Fig. 1A). Consistent with the above findings, in PLCA patients with OSMR mutations, epidermal keratinocyte differentiation was enhanced, with increased expression of FLG and LOR, compared to that in healthy controls (Fig. 1B). Furthermore, immunofluorescence analysis suggests that the expression of Ki67, an indicator of cell proliferation, was also enhanced in the epidermis of PLCA patients with OSMR mutations (Fig. 1C and 1D). To further determine the biological functions of OSMRβ protein in the skin, Osmr C57BL/6 mice were produced using the CRISPR/Cas9 system (Figs. 1E, 1F and S1A). Unfortunately, no PLCA-like phenotype was observed in these mice under physiological or pathological conditions (including UVA exposure and an itch challenge; data not shown). Hair follicle cycle changes were observed between WT and Osmr mice at post-natal day 30 (P30) using hematoxylin and eosin staining (Fig. S1B), which is consistent with previous reports (Wang et al., 2019). More importantly, the tail epidermal thickness was significantly

The cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 in air.
The cDNA fragments of human wildtype (WT) OSMR (hOSMR-WT) and human OSMR with the p.G513D or p.P694L mutation (hOSMR-pG513D and hOSMR-pP694L, respectively) were chemically synthesized (BGI, China). A myc tag coding region was fused to the 5' end, after the signal peptide, of the WT and mutated OSMR cDNA fragments. The cDNA fragment of human KLF7 was obtained by reverse transcription (RT)-PCR using HaCaT cell mRNA as the template. A 3×flag coding region was fused to the 5' end of the KLF7 cDNA fragment.

Construction of overexpression and knockout HaCaT cell lines
HaCaT cells with OSMR or KLF7 knocked out were generated using the CRISPR Reads were aligned to the Ensembl human GRCh38.p13 reference genome or the mouse GRCm38.p6 reference genome with Hisat2 (v2.0.1). SAM files were generated from alignment results using SAM tools. Read counts were obtained with HTSeq (v0.6.1) with the union option. Differential expression was determined using the R/Bioconducter package DESeq2.

EdU incorporation assay
For the in vivo proliferation analysis, 5-ethynyl-2'-deoxyuridine (EdU, Sigma, 900584-50MG, USA) was dissolved in sterile PBS to a concentration of 10 mg/ml. A single dose of 50 mg/kg was injected into the mice intraperitoneally 24 h prior to sacrifice.

Histopathology and immunofluorescence
For the immunofluorescence analyses, 3D skins, human skin biopsies or mice skin tissues were submerged in 4% paraformaldehyde for 1 h and then embedded and frozen in optimal cutting temperature (OCT) compound at -80°C. Samples were sectioned at 8-μm thickness, blocked with 2% BSA in PBS/0.3%Triton-X, and labeled with primary antibodies overnight at 4°C. The following day, the unbound primary antibodies were washed off and fluorescence-conjugated secondary antibodies (1:500) were added. Images were acquired on an A1+ confocal microscope (Nikon, USA). For histology, formalin-fixed paraffin-embedded sections of skin were rehydrated in increasingly dilute ethanol concentrations and stained with hematoxylin & eosin or Congo red.

Luciferase reporter assay
Luciferase reporter assays were performed as described previously (Arcidiacono et al., 2018). Briefly, HEK293T cells were pretreated with STAT3, STAT5, ERK1/2, or AKT inhibitors. After 12 h, the cells were stimulated with OSM, transfected with 500 ng of the luciferase reporter plasmid containing full-length or different truncated KLF7 promoters in 24-well plates. The assays were performed using a Luciferase Assay Kit (E1910; Promega, USA) according to the manufacturer's protocol.

Western blotting
Cells were harvested in RIPA lysis buffer containing a protease inhibitor cocktail. Western blotting was performed as described previously (Liu et al., 2018). The blots were probed with specific primary antibodies against STAT3

Quantitative real-time RT-PCR analysis
Total RNA samples were isolated from cells, 3D skins or mice tissue using

Flow cytometry analysis
To

Statistical Analysis
The quantitative data were tested for normality using the Shapiro-Wilk test.
One-way ANOVA with Bonferroni's multiple comparisons test was used where applicable. Supplementary

Supplemental Table 2. Primers, sgRNAs, and siRNAs
GCUCGGCAGUGGACAUCUUTT siKLF7#2 GCUCUUCUCUAGACAGCUATT       In physiological condition, OSM binds to OSMR on the keratinocyte cell membrane and activates STAT5 in the cytosol, and STAT5 translocates to nucleus and activates KLF7, which inhibits the expression of key genes of keratinocyte differentiation. In pathological condition of PLCA patients with loss-of-function OSMR mutants, OSM is unable to phosphorylate STAT5 and activate KLF7, and fails to maintain a low expression level of key genes of keratinocyte differentiation.