Screening for critical genes related to psoriasis pathogenesis
To screen for differentially-expressed genes between psoriasis lesion and non-lesion tissues that might exert a crucial effect on the pathogenesis of psoriasis, we downloaded and analyzed various online microarray expression profiles and RNA-seq data (Fig.1A). First, we collected 7 sets of mRNA expression microarray chip data reporting the differentially expressed genes between psoriasis lesion skins and normal control tissue (non-involved/non-lesion skin) (GSE13355, GSE14905, GSE30999, GSE34248, GSE41662, GSE50790, GSE6710) from the gene expression omnibus database of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/geo/) and 3 sets of RNA-seq sequencing data (E-GEOD-54456, GSE114286, GSE121212) from the European Bioinformatics Institute's Expression Atlas database (https://ebi.ac.uk/gxa/home). The details of the data involved are shown in Table S1. Then, we use the limma or DE2Seq package in the R program language to normalize chip data and perform differential expression analysis. The RANK value (ie, the FDR value or adjusted p-value) was calculated using R package, and 320 genes with FDR values less than 0.01 and average |log2FC|> 0.56 were obtained, including 261 genes that were up-regulated and 59 genes that were down-regulated. Heatmap based on seven sets of online microarray expression profiles (GSE13355, GSE14905, GSE30999, GSE34248, GSE41662, GSE50790, and GSE6710) and three sets of RNA-seq data (E-GEOD-54456, GSE114286, and GSE121212) showed the top twenty differentially-expressed genes (ten upregulated and ten downregulated) in psoriasis lesion skin tissues compared to that in non-lesion skin tissues (Fig.1B). To further identify critical genes related to psoriasis severity, we analyzed the correlation between the expression of top ten downregulated genes and the PASI scores in each sample included in GSE85034 (total sample size = 179) and the expression of four genes, namely BTC, BCAR3, CHP2, and CTNNBIP1, was significantly negatively correlated with PASI score in each sample (data not shown). As is well-known, CTNNBIP1 targets two different β-catenin armadillo regions via its N-terminal and C-terminal domains, disrupting the interaction between β-catenin and TCF [12, 15, 16], therefore exerting a negative regulatory effect on Wnt signaling. Based on the essential role of Wnt/β-catenin signaling pathway within psoriasis pathogenesis [24, 25], we selected CTNNBIP1 for further experiments. According to GSE85034, CTTNBIP1 expression was significantly negatively correlated the PASI score in each sample (Fig.1C).
According to NCBI online database, the sequencing results of 27 tissues from 95 persons demonstrated that CTNNBIP1 expression within skin tissue samples showed to be remarkably increased compared to that within other tissue samples (Fig.S1). Online data we selected, including GSE85034 (Fig.1D), GSE63741 (Fig.1E), GSE30768 (Fig.1F), GSE30999 (Fig.S2A), GSE6710 (Fig.S2B), GSE121212 (Fig.S2C), E-GEOD-54456 (Fig.S2D), GSE13355 (Fig.S2E), all reported that the expression of CTNNBIP1 showed to be obviously downregulated within psoriasis lesion tissue samples, in comparison with that in non-lesion tissue samples.
CTNNBIP1 expression and protein levels within psoriasis lesion and non-lesion tissue samples
Before investigating the specific effect of CTNNBIP1 on psoriasis, we first evaluated the expression of CTNNBIP1 within tissue samples. H&E staining showed that, compared to the non-lesion skin tissues, there were obvious erythema, plaque, infiltration, and scales in psoriasis lesions (Fig.2A). In the meantime, IHC staining showed that CTNNBIP1 protein was decreased in psoriasis lesion tissue samples than that within non-lesion tissue samples (Fig.2A). Consistently, Immunoblotting revealed that CTNNBIP1 protein levels were significantly reduced in psoriasis lesion than those within non-lesion tissues (Fig.2B), and real-time PCR revealed that the mRNA expression of CTNNBIP1 showed to be remarkably downregulated within psoriasis lesion tissue samples than that in non-lesion tissue samples (Fig.2C). Also similar as online data, Pearson’s correlation analysis demonstrated that the expression of CTNNBIP1 had a negative correlation with PASI scores in psoriasis lesion samples (Fig.2D). These data suggest that CTNNBIP1 expression and protein levels are decreased in psoriasis lesion tissues and might play a role in psoriasis pathogenesis.
Changes in CTNNBIP1 expression in response to therapies
To further confirm that CTNNBIP1 is involved in psoriasis pathogenesis, we monitored CTNNBIP1 expression within non-lesion skin and lesion skin tissue samples in response to different therapies. According to GSE85034, the expression of CTNNBIP1 showed to be markedly downregulated within lesion tissue samples than that in non-lesion tissue samples, while continually increased from the 2nd to the 16th week after adalimumab/methotrexate treatment (Fig.3A). Similarly, dynamic observation on five cases of psoriasis patients underwent methotrexate treatment found that CTNNBIP1 expression was downregulated in psoriasis lesion tissues while rescued 20 days after treatment (Fig.3B). According to GSE30768, CTNNBIP1 expression was significantly downregulated in four cases of lesion skin tissues compared to that in non-lesion tissues, while upregulated in non-relapse lesion area upon efalizumab treatment and failed to be rescued by efalizumab treatment in relapse area (Fig.3C). These data suggest that CTNNBIP1 upregulation might be related to psoriasis improvement.
Effects of knocking down CTNNBIP1 upon IMQ-induced psoriasis-like dermatitis in mice
Next, we investigated the specific effects of CTNNBIP1 on psoriasis pathogenesis by establishing IMQ-induced psoriasis-like dermatitis model in mice. Based on the Materials and methods section, we divided mice into 4 groups, and the appearance of mice back skin within four groups was shown in Fig.4A. Knocking down CTNNBIP1 in control mice could induce psoriatic changes on mice skin, while IMQ-induced damages on mice skin were further aggravated by CTNNBIP1 knockdown (Fig.4A). Histopathologic analyses on four groups by H&E staining showed that psoriatic phenotypes, including erythema, plaque, infiltration, and scales, began to appear in CTNNBIP1 knocked-down control mice and IMQ-treated mice while these phenotypes were aggravated in CTNNBIP1 knocked-down IMQ mice (Fig.4B). We performed real-time PCR (Fig.4C) and Immunoblotting (Fig.4D) to confirm the in vivo knockdown of CTNNBIP1.
Effects of knocking down CTNNBIP1 on HaCaT cell apoptosis and proliferation
First, we confirmed the effects of CTNNBIP1 on IMQ-induced psoriasis-like dermatitis in mice. Next, we investigated the in vitro effects of CTNNBIP1 upon keratinocytes. We transfected si-CTNNBIP1 to generate CTNNBIP1 silence in HaCaT cells, and performed Immunoblotting to verify the transfection efficiency (Fig.5A-B). Next, the cell viability, the cell apoptosis and the DNA synthesis capacity of CTNNBIP1-silenced HaCaT cells were determined using Flow cytometry, EdU, and MTT assays. CTNNBIP1 silence significantly inhibited HaCaT cell apoptosis (Fig.5C-D), promoted the DNA synthesis capacity (Fig.5E), and promoted the cell viability (Fig.5F). These data indicate that CTNNBIP1 silence could promote the over proliferation of keratinocytes.
CTNNBIP1 silence promotes β-catenin nucleus translocation-mediated TCF transcriptional activity
As for the underlying mechanism, CTNNBIP1 could disrupt β-catenin-TCF interaction and reduce TCF transcriptional activity [12, 15, 16]. Thus, next, we investigated whether CTNNBIP1 silence could affect the nucleus translocation of β-catenin and TCF4 transcription of downstream genes, such as c-Myc  and cyclin D1 [10, 11]. We also monitored changes in the late keratinocyte differentiation marker filaggrin [26, 27]. In CTNNBIP1-silenced HaCaT cells, the plasma protein levels of β-catenin showed to be obviously upregulated (Fig.6A-B) while the total cellular protein levels of β-catenin showed no obvious alterations (Fig.6C-D). Meanwhile, IF staining showed that β-catenin protein tended to distribute in the cell nucleus in CTNNBIP1-silenced HaCaT cells (Fig.6E). Following increased β-catenin nucleus translocation, the transcriptional activity of TCF4 was also significantly enhanced (Fig.6F). Consistently, β-catenin/TCF-complex downstream cyclin D1, c-Myc, and Ki-67 proteins were significantly increased (Fig.6G-H). These data indicate that CTNNBIP1 silence promotes β-catenin nucleus translocation and enhances TCF4 transcription of downstream cyclin D1, c-Myc, and Ki-67.
Expression and correlation of c-Myc and cyclin D1 with CTNNBIP1 in tissue samples
As a further confirmation of above-described in vitro findings, we examined the protein content and distribution of c-Myc and cyclin D1 within psoriasis lesion and non-lesion tissues. Both these two downstream genes showed to be upregulated within psoriasis lesion tissue samples than those within non-lesion tissues (Fig.7A-B). Consistently, MYC and CCND1 mRNA expression showed to be markedly upregulated within psoriasis lesion tissues than that in non-lesion tissues (Fig.7C and E). CTNNBIP1 mRNA expression had a negative correlation with MYC and CCND1 mRNA expression, respectively, within tissues as analyzed by Pearson’s correlation analysis (Fig.7D and F).