IL23R (Interleukin 23 Receptor) Variants Protective against Inflammatory Bowel Diseases (IBD) Display Loss of Function due to Impaired Protein Stability and Intracellular Trafficking

Abstract

Genome-wide association studies as well as murine models have shown that the interleukin 23 receptor (IL23R) pathway plays a pivotal role in chronic inflammatory diseases such as Crohn disease (CD), ulcerative colitis, psoriasis, and type 1 diabetes. Genome-wide association studies and targeted re-sequencing studies have revealed the presence of multiple potentially causal variants of the IL23R. Specifically the G149R, V362I, and R381Q IL23Rα chain variants are linked to protection against the development of Crohn disease and ulcerative colitis in humans. Moreover, the exact mechanism of action of these receptor variants has not been elucidated. We show that all three of these IL23Rα variants cause a reduction in IL23 receptor activation-mediated phosphorylation of the signal-transducing activator of transcription 3 (STAT3) and phosphorylation of signal transducing activator of transcription 4 (STAT4). The reduction in signaling is due to lower levels of cell surface receptor expression. For G149R, the receptor retention in the endoplasmic reticulum is due to an impairment of receptor maturation, whereas the R381Q and V362I variants have reduced protein stability. Finally, we demonstrate that the endogenous expression of IL23Rα protein from V362I and R381Q variants in human lymphoblastoid cell lines exhibited lower expression levels relative to susceptibility alleles. Our results suggest a convergent cause of IL23Rα variant protection against chronic inflammatory disease.

Keywords: genetic polymorphism; glycosylation; inflammatory bowel disease (IBD); interleukin 23 receptor; protein trafficking (Golgi); receptor.

FIGURE 1.

Characterization of IL23R activation mechanism model. A, schematic representation of allosteric IL23R activation. IL23R exists as a heterodimer before interaction with IL23 cytokine similar to the EpoR receptor (36, 62). The extracellular domain exists in a conformation that keeps the intracellular domains and associated JAK2 and TYK2 separated from each other by ∼70 Å. Upon binding to IL23, the extracellular dimer is reorganized and brings the intracellular cytoplasmic domains within ∼ 40 Å of each other, allowing for interaction between and activation of their associated kinases. For identification of IL23R activation mechanism, the extracellular and transmembrane domains of IL23R and IL12Rβ1 are fused to Renilla luciferase (Rluc) complementary N- and C-terminal fragments, Rluc F[1] and F[2], via flexible linkers consisting of 1, 2, or 4 five-amino acid repeats to generate the following: IL23R and IL12Rβ1 fused to 5-, 10-, and 20-amino acid linkers that are in turn fused to the Rluc fragments. HEK293 cells transfected with these receptor fusions and PCA were detected by measuring the luminescence. When the receptors are fused via a 5-aa linker, folding of the Rluc reporter protein from the fragments only occurs for the IL23 hormone-bound active form of the receptor dimer. If receptors fused to Rluc fragments via 10- or 20-aa linkers, Rluc reporter protein can fold from the fragments in either inactive or active dimer conformations. The structural model of Renilla luciferase was rendered using information from 2pSj.pdb (63) using PyMOL 1.3. B, Rluc enzyme assay detected the IL23R activation in HEK293 cells. Cells were co-transfected with IL23Rα and IL12Rβ1 fused to Rluc PCA fragments via various linker lengths. The next day the cells were incubated with and without 100 ng of IL23 cytokine followed by measurement of Rluc enzyme activity using benzyl coelenterazine as the substrate and detection using a luminometer. The signal is expressed as relative light units (RLU). C, Rluc PCA reveals the interaction between IL23Rα and their subunits and their associated kinases (JAK2 and TYK2). HEK293T cells were transfected with pCDNA3.1 harboring the cDNA of full-length IL23R receptor subunits and the kinases JAK2 and TYK2 fused to Rluc fragments to detect the interaction as measured by PCA (RLU) of Rluc. D, JAK2 and TYK2 association in the presence of IL23 binding to IL23R was detected by Rluc PCA. Rluc fragments were fused to JAK2 and TYK2 and were co-transfected in HEK293T cells stably expressing IL23R subunits followed by measuring Rluc luminescence. Statistical significance denoted by the asterisk (*) was calculated by Student's t test, where p < 0.05. E, IL23Rα variants show the reduction in activation by IL23. IL23Rα, its, variants and IL12Rβ1 were fused to Rluc fragments to detect activation by IL23 (10 ng/ml), and luminescence was measured as in B. The change in RLU between cells not treated and treated with IL23 was calculated, and activation was thus observed as a function of change in RLU. Statistical significance denoted by the asterisk (*) was calculated by ANOVA, where p < 0.01. F, G, and H, IL23Rα variants retain their ability to interact with IL12Rβ1, JAK2, and TYK2. IL23Rα and variants Rluc fragment 2 (F[2]) fusions were co-transfected with either IL12Rβ1 fused to Rluc fragment 1 F[1], IL12Rβ1 F[1] (F), JAK2 F[1] (G), and TYK2 F[1] (H), and reconstitution of Rluc enzyme activity was measured to assess their interaction. Rluc enzyme activity (RLU) is expressed as percentage relative to IL23Rα. Data and S.D. are representative of three independent experiments.

FIGURE 2.

IL23Rα protective variants R381Q, G149R, and V362I display reduction in IL23R signaling. A, HeLa cells were co-transfected with plasmids carrying IL23Rα or variants and IL12Rβ1. After washing with PBS to remove media and overnight growth in serum-free medium, transfected cells were incubated with or without IL23 (15 ng/ml) for 30 min. Cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to phospho-STAT3 (pSTAT3) and STAT3. Representative blots from three independent experiments are shown here. B, corresponding densitometry of the levels of pSTAT3 in IL23Rα and IL23Rα variants obtained from Western blots above. The signal of pSTAT3 detected on Western blot was measured as pixels using ImageJ 1.46r (National Institutes of Health) and were further normalized to levels of total STAT3. Data and S.D. are representative of three independent experiments. Statistical significance calculated by ANOVA is denoted by the asterisk (*), where p < 0.01 is compared with IL23Rα; however, R381Q was significantly different from V362I, with a p < 0.05. C, HeLa cells were co-transfected with plasmids carrying STAT4, IL23Rα or variants, and IL12Rβ1. As stated for STAT3 experiments (A), the cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to phospho-STAT4 (pSTAT4) and STAT4. D, corresponding densitometry of the levels of pSTAT4 in IL23Rα and IL23Rα variants obtained from Western blots of C. The signal of pSTAT4 detected on Western blot was measured as pixels using ImageJ 1.46r (National Institutes of Health) and were further normalized to levels of total STAT4. Data and S.D. are representative of three independent experiments. Statistical significance calculated by ANOVA is denoted by the asterisk (*), where p < 0.01 is compared with IL23Rα; in addition, R381Q was significantly different from G149R and V362I with a p < 0.01. EV denotes empty vector.

FIGURE 3.

IL23-induced STAT3 phosphorylation is dependent on the presence of both IL23Rα and IL12Rβ1 and IL23 cytokine. A, HeLa cells were co-transfected with plasmids carrying either IL12Rβ1 or IL12Rβ1 together with IL23Rα. After washing with PBS to remove medium and overnight growth in serum free medium, transfected cells were incubated with or without IL23 (15 ng/ml) for 30 min. Cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to phospho-STAT3 (pSTAT3) and tubulin. EV denotes empty vector. B, HeLa cells were co-transfected with plasmids carrying IL23Rα or variants and IL12Rβ1. After washing with PBS to remove medium and overnight growth in serum free medium, transfected cells were incubated with or without IL23 (15 ng/ml) for 30 min. Cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies specific to IL23Rα and IL12Rβ1.

FIGURE 4.

IL23Rα-protective variants display different receptor maturation. A, glycosylation states of IL23Rα stably expressed in HEK293 cells. Cell lysates were treated with Endo H or with peptide N-glycosidase and neuraminidase and resolved by SDS-PAGE and Western blot using antibodies against IL23Rα. Endo H-resistant receptors are mature in glycosylation, whereas Endo H-sensitive receptor corresponds to immature glycosylated receptor. B, Western blots of cell lysates from HEK293 transfected with IL23Rα common or protective variants. The amount of cDNA transfected is noted in parentheses. Mature (M IL23Rα) or immature IL23Rα (IM IL23Rα) receptors are indicated to the right. Molecular markers are displayed to the left of the blots in kDa. C, the ratio of mature IL23Rα (M) to immature IL23Rα (IM) was calculated. Data represented are the means ± S.D. calculated from three independent experiments. Statistical significance-calculated ANOVA is denoted by the asterisk (*), where p < 0.01.

FIGURE 5.

Localization of IL23Rα common and protective variants in HEK293 cells. A, confocal microscopy images of cells expressing N-terminal FLAG-tagged receptors in HEK293 cells. Cells were imaged for vYFP fluorescent protein to show IL23Rα expression levels; Hoeschst (Nuc-Blue, Molecular Probes), to identify the nucleus and antibody toward FLAG tag, was used to localize IL23Rα. Overlay images (Merge) from Hoechst and anti-FLAG staining is shown to demonstrate IL23Rα localization. Scale bar, 10 μm. B, mean fluorescent intensities from the corresponding confocal images were used to quantify IL23Rα, which were normalized to the total expression of IL23Rα and its variants using levels of vYFP fluorescent protein as described under “Experimental Procedures.” Different fields of images were quantified, and S.E. values are n = 3. Statistical significance calculated by ANOVA is denoted by asterisk (*), where p < 0.01 compared with IL23Rα.R381Q was significantly different from G149R and V362I, and G149R is significantly different from V362I with a p < 0.01. C, localization of IL23Rα variant-vYFP fusions; green, IL23Rα; red, ER. Hoeschst staining was used to identify the nucleus, and cell light ER-RFP was used to identify ER. Scale bar, 10 μm. D, ratio of total fluorescence from v-YFP-tagged IL23Rα and ER-localized v-YFP-tagged IL23Rα is calculated as described under “Experimental Procedures.” On average, 10 cells were quantified for each variant, and the S.E. was calculated. Statistical significance calculated by ANOVA is denoted by the asterisk (*) compared with IL23Rα, where p < 0.05; in addition, R381Q was significantly different from G149R, with a p < 0.05, and G149R was significantly different from V362I, with a p < 0.01.

FIGURE 6.

BFA treatment of HEK293 cells stably expressing IL23Rα and variants results in partial O-glycosylation. HEK293 cells expressing IL23Rα and its variants when exposed to BFA (1 μg/ml) for 5 h is shown. Cell lysates from two independent experiments were subjected to SDS-PAGE, and Western blotting with antibodies to IL23Rα show that the immature receptors in both wild type and variants are able to be partially O-glycosylated when the responsible enzymes found in Golgi are available to them. Arrows indicate the mature (M IL23Rα) and immature (IM IL23Rα) and the aberrant IL23Rα (abIL23Rα) that is partially O-glycosylated.

FIGURE 7.

Unfolded protein response is up-regulated in cells expressing the IL23Rα G149R variant. A, HEK293 cells stably expressing IL23Rα and its variants were harvested, and cell lysates were electrophoresed on SDS-PAGE followed by Western blot. Antibodies specific to ER stress and unfolded protein response markers indicate that BiP abundance is higher in IL23Rα G149R compared with the common variant, suggesting that IL23Rα G149R is retained in the ER. BiP abundance could also be induced in HEK293 cells expressing IL23Rα when these cells are treated with tunicamycin (1 μg/ml) for 5 h. B, the viability of cells expressing IL23Rα variants is not affected. HEK293 cells stably expressing IL23Rα and variants were plated in 96-well plate (20,000 cells) in triplicate. The next day the cells were assayed for levels of mitochondrial dehydrogenase activity by using MTT (64). Data and error bars represent the S.D. from three independent experiments.

FIGURE 8.

IL23Rα R381Q and V362I were not stable. A, HEK293 cells stably expressing IL23Rα and IL23Rα variants were treated with cycloheximide (500 μm) for various times. Representative Western blot analysis of the cell lysate is shown here. B, IL23Rα mature (M) and immature (IM) levels over time under cycloheximide exposure are represented in percentages, which were calculated by taking IL23Rα levels at time 0 as 100%. One-phase exponential decay fit of the data was performed using GraphPad Prism 6.0, and the R² value for the receptors are as follows: IL23Rα M (0.7212), IL23Rα IM (0.654), R381Q M (0.7460), R381Q IM (0.8905), G149R M (0.7655), G149R IM (0.876), V362I M (0.9559), V362I IM (0.9388). Error values were S.E. where n = 3. C. Half-life of IL23Rα and variants was calculated by using GraphPad Prism 6.0. Error values were S.D. where n = 3. Statistical significance calculated by ANOVA is denoted by the asterisk (*) compared with IL23Rα where p < 0.01; in addition, R381Q was significantly different from G149R and V362I with a p < 0.01, and G149R was significantly different from V362I with a p < 0.01.

FIGURE 9.

The rates of maturation of R381Q and G149R are reduced compared with that of the common variant IL23Rα. A, HEK293 cells stably expressing IL23Rα and IL23Rα variants were treated with cycloheximide (CLX, 500 μm) for 4 h followed by washing off the cycloheximide and incubated for the indicated time points. Representative Western blot analysis of the cell lysates is presented. B, synthesis of IL23Rα mature (M) and immature (IM) levels over time after cycloheximide exposure are represented in pixels. Linear regression analysis of the data were used to calculate the rate using GraphPad Prism 6.0, and the R² value for the receptors are as follows: IL23Rα M (0.8591), IL23Rα IM (0.7436), R381Q M (0.9256), R381Q IM (0.9054), G149R M (0.8357), G149R IM (0.9543), V362I M (0.8428), V362I IM (0.8117). The error values are S.E. where n = 3. C, ratio of the rate for IL23Rα and variants between their mature and immature receptor synthesis is represented here. Error values are S.D. where n = 3. Statistical significance calculated by ANOVA is denoted by the asterisk (*) compared with IL23Rα, where p < 0.01. In addition, R381Q was significantly different from V362I, with a p < 0.01, and G149R was significantly different from V362I, with a p < 0.01.

FIGURE 10.

Expression levels of IL23Rα-protective variants are lower in NIDDK cell lines. Western blotting of cell lysates from NIDDK cell lines shows the expression of IL23Rα and its variants R381Q and V362I at endogenous levels using antibodies specific to IL23Rα. IL12Rβ1 antibody was used for the loading control. Representative Western blot from three independent experiments is shown, and cells that are homozygous and heterozygous to the IL23Rα allele are described as homo and hetero, respectively.