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Alternative splicing directs two IL-20R2 isoforms and is responsible for the incomplete gene knockout via the exon I ablation

Abstract

Two heterodimeric receptors consisting of interleukin (IL)-20R2 are shared by three of the IL-20 family of cytokines, IL-19, IL-20 and IL-24. Along with IL-22, these cytokines are downstream effectors of IL-23 and have been implicated in keratinocyte functions and the pathogenesis of psoriasis. Surprisingly, whereas knocking out either the IL-23 or IL-22 gene abolished imiquimod-induced psoriatic phenotypes in mice, similar attempt for IL-20R2 had little effect. Here, we report that the apparent disparity may result from a new IL-20R2 isoform encoded by an alternatively spliced transcript which survived the previous attempt for IL-20R2 gene knockout via the exon I deletion.

Introduction

Psoriasis is a chronic skin disease characterized by abnormal inflammation of the epidermis.1 Following stimuli such as trauma or infection in genetically predisposed individuals, resident dendritic cells and macrophages become activated and produce interleukin (IL)-23 and TNF-α, which lead to the activation of TH17 cells and monocytes.1, 2, 3 These cells then produce IL-17 and IL-22,4, 5 as well as IL-20 family of cytokines,6 which in turn activate keratinocytes through their tissue-specific receptors. These cytokines, in particular IL-24, then lead to proinflammatory cytokine production such as MCP-1 by keratinocytes via both autocrine and paracrine manners, which further recruit more leukocytes to the dermal layer, thus creating a vicious cycle of inflammation within the skin lesion.7 This cascade of events culminates in epidermal remodeling with altered proliferation and differentiation of keratinocytes.

The IL-20 subfamily, which is comprised of IL-19, IL-20, IL-22, IL-24 and IL-26, signals through different heterodimeric receptors with shared subunits, and is part of the larger IL-10 family of cytokines.8, 9 IL-19, IL-20 and IL-24 require the IL-20 receptor β-subunit (IL-20R2) for signaling,10, 11, 12 whereas IL-22 and IL-26 signal through receptor complexes consisting of IL-10R2.13, 14 IL-20R2 can form a functional heterodimeric receptor with either the IL-20 receptor α-subunit (IL-20R1) to bind to IL-19, IL-20 and IL-24, or with the α-subunit of the IL-22 receptor (IL-22R1), which also permits IL-20 and IL-24 signaling.9, 15 Although abnormal expression of these cytokines has been linked to psoriasis, more direct evidence for probable causal effects have come from both transgenic mice and gene knockout studies. While both IL-17 and IL-23 transgenic exhibited systemic inflammation,16, 17 transgenic mice overexpressing IL-22, IL-20 and IL-24 all exhibited similar psoriatic skin phenotype and died upon birth.7, 10, 18 IL-23 functions upstream of both IL-17/IL-22 lineage via T cells and IL-19, 20 and IL-24 lineage via macrophages or potentially other cell types in inducing psoriasis.6, 19 As predicted, gene knockout of IL-23 and IL-22 both abolished imiquimod (IMQ)-induced psoriatic skin phenotype.5, 20

In this study, we attempted the same experiment with previously published IL-20R2 knockout mice,21 but failed to see inhibition of psoriatic skin phenotype caused by IMQ. A closer inspection of these mice revealed that the exon I deletion, which was intended to behead the receptor protein by removing the signal peptide, resulted in an incomplete knockout of the IL-20R2 gene owing to alternative splicing of the mRNA in mouse skin. Although these mice completely lost the known IL-20R2 transcript, we discovered that a new mRNA transcript encoding essentially the intact IL-20R2 except with a different signal peptide survived the gene targeting. Thus, a more accurate assessment of the roles for IL-19, IL-20 and IL-24 in the pathogenesis of psoriasis still awaits a complete IL-20R2 gene knockout in mice.

Results

IL-20R2−/− mice showed little phenotypic difference from WT in IMQ-induced psoriasis

To assess whether the lack of IL-20R2 could inhibit the biological functions of IL-19, IL-20 and IL-24 in vivo, we obtained the IL-20R2 knockout mice previously published in Journal of Immunology.21 Wild-type (WT) and IL-20R2−/− mice were first genotyped and verified by PCR with specific primer pairs annealing to both outside and within the target sequence as previously described (Figure 1a).21 To our surprise, following the established protocol,22 daily applications of IMQ cream failed to yield any phenotypic difference between WT and IL-20R2−/− mice in both the time of onset and degree of skin psoriasis (Figure 1b). Two or 3 days after the start of IMQ application, the skin of the WT and IL-20R2−/− mice started to display signs of erythema, scaling and thickening. From days 2 to 3 onward, inflammation became obvious, which continued to increase in severity up to the end of the experiment. Mice treated with control Vaseline cream did not show any sign of inflammation. IL-20R2−/− mice under both C57BL/6 and BALB/c genetic backgrounds gave the same phenotype indistinguishable from that of WT.

Figure 1
figure1

IL-20R2 Knockout mice exhibit little inhibitory effect in IMQ-induced psoriatic phenotype. (a) Genotype confirmation of the WT and IL-20R2−/− mice. PCR primer pairs UW27/28 and UW69/hl51 were specific to WT (top) and IL-20R2−/− (bottom) mice, respectively.21 (b) The WT and IL-20R2−/− mice (both BALB/c and C57BL/6) showed little difference in IMQ-induced psoriatic phenotype. Representative photographs were shown for the WT and IL-20R2−/− mice at day 7 following the treatment with either IMQ or Vaseline as a negative control.

IL-20R2−/− mice exhibited competence in ligand binding in vivo

The lack of phenotype differences above could be explained by two possibilities. First it could suggest that the three cytokines signaling through IL-20R2 are not involved in IMQ-induced psoriasis. But to reach such a conclusion, one must make sure that these IL-20R2−/− mice were indeed null for IL-20R expression, a fact that was not firmly established via the analysis of either ligand binding or receptor protein expression, other than RT-PCR of samples from an atypical target tissue, liver.10, 21 To determine whether the IL-20R2−/− mice have completely lost the receptor expression at the protein level in its most relevant target tissue, skin, we used AP-tagged mouse IL-24 (mIL-24-AP) as a probe to conduct in situ receptor-binding analysis using paraffin-embedded mouse skin samples.7 The correct expression of mIL-24-AP and AP alone control as secreted proteins was confirmed by western blot analysis before in situ staining experiments (Figure 2a). To our surprise, like WT mice, the IL-20R2−/− mice were stained positive for ligand (mIL-24-AP) binding in the epidermis, whereas AP alone showed little binding (Figure 2c). To ensure that the ligand binding was specific, we also used an Fc-tagged mouse IL-24 (mIL-24-Fc), which was purified by protein A affinity chromatography and confirmed by affinity binding to soluble IL-20R2-AP fusion protein (Figure 2b), to compete for mIL-24-AP binding. The result showed that mIL-24-AP ligand binding was indeed receptor-specific, because it could be completely competed by a 10-fold excess of mIL-20-Fc, but not by an irrelevant Fc fusion (TNFR2-Fc) at the same concentration (Figure 2c). These results suggest that either the IL-20R2 gene might have not been completely knocked out in these IL-20R2−/− mice or there could exist another yet-to-be identified new receptor in mouse epidermis.

Figure 2
figure2

IL-20R2 knockout mice remain proficient in IL-24 ligand binding in the epidermis. (a) Expression and characterization of mouse IL-24-AP (mIL-24-AP) fusion protein. Ten microliters of 1 unit per milliliter of either mIL-24-AP or AP control protein were detected by western blot analysis using antibody specific to AP. (b) Expression and characterization of mouse IL-24-Fc (mIL-24-Fc) fusion protein. Two micrograms of either IL-24-Fc or TNFR2-Fc as a negative control were separated on a SDS-PAGE and either stained by Coomasie Blue or detected by in situ affinity staining with human IL-20R2-AP fusion protein7 after transferred to a PVDF membrane. (c) Paraffin-embedded skin sections of WT (top) and IL-20R2−/− (bottom) mice were analyzed by either hematoxylin and eosin staining or in situ ligand-receptor binding to either mIL-24-AP or AP control to detect the expression of IL-24 receptors. Note the intense staining of the epidermis by mIL-24-AP of both WT and IL-20R2 knockout mice. The specificity of mIL-24-AP staining was confirmed by competition with 10-fold excess of mIL-24-Fc, which essentially completely blocked the mIL-24-AP binding to the epidermis. The same amount of TNFR2-Fc served as a negative control for the specificity for the competition experiment.

IL-20R2 mRNA and protein expression were detected from the skin of IL-20R2−/− mice

We first looked at whether the previous attempt to knockout the IL-20R2 gene in mice resulted incomplete ablation of the IL-20R2 mRNA transcript. On the basis of the gene targeting strategy with which the signal peptide-encoding the Exon 1 along with some downstream Intron 1 were deleted and replaced by TK-NeoR gene (Figure 3a), there should not have been any IL-20R2 mRNA transcription left, let alone functional receptor protein. Besides confirmation of the gene-targeting construct for the IL-20R2−/− mice at the genomic level (Figure 1a), we were also able to show that, unlike in WT mice, no IL-20R2 mRNA transcript containing the Exon 1 sequence could be detected from the skin of the IL-20R2−/− mice by RT-PCR using an Exon 1-specific upstream primer (Figure 3b). To determine whether any IL-20R2 mRNA transcript could have survived without the Exon 1, we then designed an RT-PCR primer (P2) to amplify the transcript downstream of the Exon 1 sequence. The result indicated that there was a detectable level of such IL-20R2 mRNA transcript from the skin samples of IL-20R2−/− mice under either C57BL/6 or BALB/c genetic background, albeit the expression level was lower than that in the WT mice (Figure 3b). The choice for the downstream RT-PCR primer (P3) being located in exon 5 was intended to avoid the amplification of the genomic DNA which consists of large introns (Figure 3a). Western blot analysis of the skin samples with IL-20R2-specific antibody as well as in situ ligand affinity staining using mIL-24-AP both revealed that IL-20R2 protein expression could be detected at around 160 Kda in IL-20R2−/− mice under non-reducing condition, and the receptor expression level was lower than that of the WT mice, consistent with the mRNA expression (Figure 3c). Under reducing condition, IL-20R2 protein expression was also detected from the WT mice with an apparent molecular weight slightly below 50 Kda, which is consistent with the size of the monomeric receptor subunit (Figure 3c). The expression level of IL-20R2 under reducing condition was much lower in the IL-20R2−/− mice, as seen under the non-reducing condition, whereas little mIL-24-AP in situ binding could be detected for the monomeric form of the receptor subunit (data not shown).

Figure 3
figure3

IL-20R2 mRNA and protein expression were detected from the skin of IL-20R2−/− mice. (a) Schematic diagrams of the IL-20R2 gene with intron/exon structures and primer locations for RT-PCR in the contest of the WT (left) and IL-20R2−/− (right) genetic loci. Seven exons (Exon 1–7) corresponding to the mRNA and protein-encoding domains of IL-20R2308 were indicated by gray rectangles. Known protein domains were marked by abbreviations: SS, signal peptide; FN, fibronectin-binding domain; P, primer binding site; UTR, untranslated region; TK-Neo, neomycin resistance gene under control of thymidine-kinase promoter. (b) Semi-quantitative RT-PCR analysis of IL-20R2 mRNA expression in the skin of WT and IL-20R2−/− BALB/c mice. DNA-free total RNA from the skin samples were reverse-transcribed followed by PCR using primer pairs as indicated. Although no IL-20R2 mRNA expression was seen in IL-20R2−/− mice with primers P1 and P3, which targeted the Exon I-containing transcript, IL-20R2 mRNA expression was detectable with primers P2 and P3, which targeted the Exon 2-containing transcripts. RPL-19 was used as internal control for equal RNA loading for the RT-PCR. (c) Fifty micrograms of total proteins from WT and IL-20R2−/− mouse skins were separated on non-reducing or reducing SDS-PAGE and analyzed by either western blot using mouse IL-20R2-specific antibody or in situ ligand affinity staining using mIL-24-AP. Antibody against GAPDH and AP alone were used as negative controls for equal sample loading and specificity in receptor binding, respectively. Non-reduced IL-20R2 protein was detectable in IL-20R2−/− mice, and the expression level appeared lower in IL-20R2−/− mice compared with WT mice.

Detection of a new IL-20R2 transcript via alternative splicing in mouse skin

To determine whether the remaining IL-20R2 mRNA transcript from the KO mice could still encode a functional IL-20R2 receptor, we conducted a 5′ RACE to obtain the mRNA sequence upstream of the Exon 2 from the knockout mice using RT primer located within the Exon 2. A 319 bp 5′ RACE cDNA fragment (race319) was recovered which showed 65 bp of overlapping sequence with the Exon 2, plus additional 254 bp upstream sequence matching that from the Intron 1 immediately upstream of the Exon 2 (Figure 4a). As a negative control, mock RT-PCR reaction (-reverse transcriptase) in the 5′ RACE did not result in any cDNA amplification (data not shown). This finding suggests that the Intron 1/Exon 2 splice site was intact for the transcript detected. Combining the cDNA sequence from the 5′ RACE with that of the Exon 2 and downstream cDNA of the known IL-20R2 transcript, we found that this transcript could encode an open reading frame of 302 amino acid residues with a new signal peptide sequence encoded by the Intron 1 in-frame fused to the open reading frame from the Exon 2 (here designated as IL-20R2302 isoform). To distinguish from this newly found IL-20R2 transcript, we named the previously known IL-20R2 transcript as IL-20R2308 isoform. As the cDNAs amplified by RT-PCR primers downstream of the Exon 2 could not differentiate which isoform-encoding transcripts were amplified (Figures 3b and 4b), we next designed a series of RT-PCR primers to determine whether the newly discovered IL-20R2302 transcript was specific to the knockout mice or it was also expressed by the WT animals. First, we showed that an RT-PCR primer (P4) specific to the Intron 1 sequence uncovered by the 5′ RACE in combination with a downstream primer annealing to the 3′ UTR region (P5) were able to amplify a full-length cDNA encoding IL-20R2302 from both WT and the KO mice (Figure 4b), albeit the level of expression was lower in the KO mice. Primers used in Figure 3 were also used as controls for the specific detection of either IL-20R2308 or both IL-20R2302/IL-20R2308 transcripts. The 1017 bp full-length IL-20R2302-encoding cDNA was cloned and completely sequence-verified to be as predicted (GenBank Accession No.: KT878835).

Figure 4
figure4

Identification of a new IL-20R2 transcript (IL-20R2302) via alternative splicing in mouse skin. (a) A schematic diagram of the mIL-20R2 gene locus and a predicted new mRNA transcript structure via alternative splicing (Exon 1 skipping). Primer locations for PCR and the location of the mRNA sequence identified by the 5′ RACE were as indicated. Known introns/exons from the IL-20R2302 transcript and their encoded protein domains were as described in Figure 3, except the skipping of the Exon 1 led to a new Exon 2, which contained a different signal peptide (SS*) in-frame linked to the coding region from the original Exon 2. (b) Semi-quantitative RT-PCR analysis of the full-length IL-20R2302 mRNA transcript in the skins of WT and IL-20R2−/− BALB/c mice. DNA-free total RNA samples were reverse-transcribed followed by PCR using primer pairs as indicated. An upstream primer (P4) located within the Intron 1 region immediate upstream the Exon 2 and a downstream primer (P5) located in the 3′ UTR region were used in RT-PCR to confirm the existence of the predicted IL-20R2302 transcript. As a control, IL-20R2308-specific transcript was detected only in the WT mice by an Exon 1-specific upstream primer (P1) as shown in Figure 3 in combination with P5 primer. Both IL-20R2302 and IL-20R2308 transcripts were detected by RT-PCR using an upstream primer (P2) located in Exon 2 in combination with the same downstream P5 primer. Primers specific to RPL-19 transcript were used as control for equal RNA loading. (c) Mapping of the 5′ splice site junction of the IL-20R2302 mRNA transcript by RT-PCR analysis. A series of upstream primers were designed from the Intron 1 sequence and used for RT-PCR in combination with a downstream primer located within the Exon 2 (P6). Two upstream primers (P7 and P8) were found to flank the new splice site within the Intron 1. P7 primer was able to detect the IL-20R2302 transcription, whereas primer P8 could not. PCR amplification of the corresponding genomic DNA loci from the WT BALB/c mice served as a control for the integrity of the same primer pairs used for both RT-PCR and genomic DNA amplification. (d) Quantitative PCR analysis of IL-20R2 mRNA transcription in skin of WT and IL-20R2−/− BALB/c mice. Primers P1 and P9 were specific to IL-20R2308, and P10 and P9 were specific to IL-20R2302. Samples were normalized to the housekeeping gene RPL-19. No mRNA was detectable with primers P1 and P9 in IL-20R2−/− mice, and IL-20R2302 was downregulated by over threefold in IL-20R2−/− mice in comparison with WT mice using primers P10 and P9. Statistical significance of differences in IL-20R2 mRNA fold was analyzed using the T test. Values of P<0.01 (*) were considered statistically significant.

To find out whether the IL-20R2302 transcript came from alternative splicing, we tried to map the 5′ boundary of the mRNA and compared it against the IL-20R2 genomic DNA locus. To this end, a series of 5′ primers located at different regions within the Intron 1 but further upstream of the P4 primer binding site were used in combinations with a downstream primer located in the Exon 2 (P6). The 5′ end of the new IL-20R2302 transcript was mapped by RT-PCR to a genomic region between the P7 and P8 primers, when used in combination with P6 primer (Figure 4c). Genomic PCR with the same primer pairs were shown as a control, indicating that the splice site is likely located in between the P7 and P8 primer binding sites. We then conducted qPCR analysis for the relative mRNA expression of IL-20R2308 and IL-20R2302 using allele-specific primer pairs. The upstream primer P1 was located within the Exon 1, thus specific for IL-20R2308; primer P10 was designed to be located within the Intron 1 sequence of IL-20R2308, thus specific to IL-20R2302 transcript; the downstream primer P9 was located within the Exon 2 region, which is common to both alleles of the IL-20R2 transcripts. It was found that indeed no IL-20R2308 mRNA expression could be detected from the IL-20R2−/− mice. In contrast, the mRNA expression of IL-20R2302 was downregulated in IL-20R2−/− mice by over threefold in comparison with that of the WT, which is consistent with the result from in situ ligand binding, suggesting incomplete knockout in IL-20R2 gene (Figure 4d).

Our result suggests that the IL-20R2302 transcript seemed to arise from an alternative splicing, resulting in the skipping of the splice site between the Intron 1 and Exon 2. The resulting new transcript without the Exon 1 encodes an IL-20R2 isoform consisting of 302 amino acid residues. Protein sequence alignment with the known IL-20R2 sequence revealed that the original Exon 1 which encodes for the signal peptide comprising of 26 amino acids was replaced by a new Intron 1-encoded signal peptide sequence consisting of 18 amino acid residues (Figure 5a). So other than the difference in signal peptide, the two IL-20R2 transcripts have the identical downstream amino acid sequence. Hydropathy plot analysis showed that the new signal peptide exhibited a higher hydrophobicity than that of the known isoform and may guide a better membrane targeting for the receptor (Figure 5b).

Figure 5
figure5

Amino acid sequence alignment of the coding regions of IL-20R2308 and IL-20R2302. (a) Amino acid sequence alignment of IL-20R2308 and IL-20R2302. (b) Comparison of hydropathy plots for IL-20R2308 and IL-20R2302. Note that the two transcripts differ only in the signal peptide region and IL-20R2302 has a shorter signal peptide sequence with a higher hydrophobicity score than that of IL-20R2308.

To find out whether the IL-20R2302 receptor splice allele indeed encodes a functional receptor, we tried to analyze its ability to bind to AP-tagged ligands. Because mouse IL-24-AP (mIL-24-AP) gave high background cell surface staining on COS-E5 cells (data not shown), which would compromise the in situ receptor binding assay, we tested whether IL-20 could be used as a ligand instead. After confirming that hIL-20-AP, like mIL-24-AP, could bind to mouse IL-20R2308 in vitro, we transfected COS-E5 cells with both IL-20 receptor heterodimers (IL-20R1/IL-20R2308 and IL-20R1/IL-20R2302). Compared with cells transfected with vector control, cells transfected with either IL-20R1/IL-20R2308 or IL-20R1/IL-20R2302 gave numerous IL-20-AP-positive cells with cell surface staining, confirming that the IL-20R2302 receptor is functional in ligand binding (Figure 6).

Figure 6
figure6

Ligand–receptor binding analysis for IL-20R2302. (a) Both IL-20-AP and IL-24-AP fusion proteins could bind to mouse IL-20R2308 in vitro. 0.12 unit of either mIL-20R2308-AP or AP as a negative control were separated on a non-reducing SDS-PAGE and detected by either western blot analysis with an AP antibody or in situ affinity staining with IL-20-AP and mIL-24-AP fusion proteins after transferring to a PVDF membrane. (b) Cell surface staining assay for IL-20R2302 receptor binding. COS-E5 cells transfected with either mouse IL-20R1/IL-20R2308 or IL-20R1/IL-20R2302 heterodimeric receptors were stained with IL-20-AP and viewed under a light microscope (× 10 and × 40). COS-E5 cells transfected with pCDEF vector alone served as a negative control. Cells transfected with both IL-20 receptor heterodimers (IL-20R1/IL-20R2308 and IL-20R1/IL-20R2302) gave numerous positive cells with cell surface staining, confirming that the IL-20R2302 receptor is functional in ligand binding. The results shown were representative of at least three independent experiments.

Discussion

In this study, we have demonstrated that a previous attempt to knockout the IL-20R2 gene in mice via the Exon 1 deletion resulted in incomplete ablation of the receptor expression in mouse skin owing to alternative splicing of the mRNA. Although the Exon 1 deletion indeed wiped out IL-20R2308 expression, the skipping of the Exon 1 resulted in a new IL-20R2 transcript (IL-20R2302) that is not an artifact generated by the gene knockout process, but rather, like the IL-20R2308 transcript, is normally expressed in the mouse skin. This new IL-20R2 transcript (IL-20R2302) contains an Intron 1-encoded signal peptide in-frame fused to the receptor-coding region from Exon 2, which results in essentially the same functional receptor competent in ligand binding. However, the fact that the new IL-20R2302 transcript visualized by the semi-quantitative RT-PCR was expressed at a lower level in the KO mice in comparison with that from the WT seemed to implicate that the gene targeting process had, to some extent, affected its transcription or splicing. Although accurate quantification of the expression levels for IL-20R2308 and IL-20R2302 transcripts could not be assessed with different primer pairs targeting respective targets, relative expression levels compared with the same internal control transcript RPL-19 indicated that the expression of IL-20R2308 transcript seemed to be much higher than that of IL-20R2302 in the WT mice (Figure 4d). However, we could not completely rule out the existence of another yet-to-be identified functional receptor, because the KO mice showed similar signal intensity in the skin as that of WT mice in in situ receptor binding with AP-tagged ligands (Figure 2b). Also of interest is our finding that both western blot analysis and ligand-AP affinity staining detected the IL-20R2 receptor complex under non-reducing condition with an estimated molecular weight of 160 Kda, which is much bigger than the predicted molecular weight of 38 Kda for IL-20R2 subunit. This suggested that the signal detected might come from disulfide bond-linked heterodimeric receptor complexes consisting of IL-20R2 and IL-20R1 or IL-20R2 and IL-22R1, or both, all of which would have predicted molecular weights over 100 Kda prior to glycosylation. Indeed, under reducing condition, the IL-20R2 antibody detected a protein species with an apparent molecular weight slightly below 50 Kda, which is similar to the size of monomeric IL-20R2 subunit and consistent with our previous finding that IL-20R2 is directly involved in ligand binding (Figure 3c).11

Although IL-22 KO abolished IMQ-induced psoriatic phenotype in mice, a therapeutic antibody against human IL-22 failed to demonstrate any efficacy in a human clinical trial for the treatment of psoriasis.23 This is in contrast to IL-23, which has been proven to be an excellent therapeutic target for psoriasis, as anti-IL-23 antibody therapy is now approved for clinical use.23 It should be noted that the failure in clinical trials of anti-IL-20 antibody in the treatment of psoriasis24 could be due to the redundancy in ligand signaling from IL-20R2-containing receptor complexes. Thus, a conclusive functional analysis of IL-20 family of cytokines, in particular IL-19, IL-20 and IL-24, all of which depend on IL-20R2 for signaling, still awaits a complete knockout of the receptor gene through better targeting strategies. Such analysis may not only resolve whether there exist additional receptor(s) for IL-20 family of cytokines, but also shed light on whether blocking these cytokines as a whole could be beneficial for treating autoimmune diseases such as psoriasis and rheumatoid arthritis.

Materials and methods

Mice and IMQ treatment

Breeding pairs of IL-20R2−/− mice with the exon I deletion under both C57BL/6 and BALB/c genetic background were obtained from the laboratory of Dr Ursula M Wegenka at Ulm University, Germany.21 WT control parental C57BL/6 and BALB/c mice were purchased from Beijing HFK Bioscience CO., LTD (Beijing, China). All animals were kept under standard pathogen-free conditions in the animal care center at Sichuan University and received humane care. Animal experiments were approved by a state-appointed board on animal ethics and were performed according to international guidelines for animal experimentation. For IMQ-induced psoriasis in mice, IL-20R2−/− C57BL/6 (or BALB/c) mice and WT controls at 8–11 weeks of age each received a daily topical application of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals, E. Fougera and Co., Melville, NY, USA) on their shaved back for six consecutive days per published protocol.22 As negative controls, mice were treated with Vaseline Lanette cream (Fagron, St Paul, MN, USA).

Genotying for IL-20R2−/− mice

Total DNA of WT and IL-20R2−/− mice were purified from tails using QIAamp DNA Mini Kit (QIAGEN, Shanghai, China). The mIL-20R2 genotyping in WT and KO mice was carried out by PCR using primers according to the published procedure.21 WT mice were identified by PCR primers UW27 and UW28; while IL-20R2−/− mice were identified by primers UW69 and hl51 (Table 1).

Table 1 Sequence of PCR primers

AP-Tag and Fc-Tag fusion proteins

cDNA encoding mouse IL-24 cDNA was gene synthesized by GeneScript (Piscataway, NJ, USA), and cloned into the HindIII-BglII sites of pAP-Tag2 and pGH-Fc expression vectors (GenHunter, Nashville, TN, USA) to obtain mouse IL-24-AP (mIL-24-AP) and IL-24-Fc (mIL-24-Fc) fusion constructs, respectively. Similarly, human or mouse IL-20R2 cDNA were used as templates to amplify the ecto-domain of the receptors,25 which were then cloned into pAP-Tag2 to obtain hIL-20R2-AP and mIL-20R2-AP fusions. The fusion proteins were then transfected into GH-CHO (dhfr−/−) cell lines (GenHunter) and cultured according to the manufacturer’s instruction. Human soluble TNF-RII-Fc fusion protein (TNFR2-Fc) was obtained from GenHunter.

Histological analysis and in situ ligand-receptor staining

Skin samples from the back of the mice were fixed in 10% formalin, paraffin-embedded, sectioned at 5 μm and stained with hematoxylin and eosin. For in situ ligand-receptor staining, the sections were rehydrated and stained with 1 U ml−1 of AP alone or corresponding AP fusion proteins essentially as previously described.7 For competition experiments, 10 μg ml−1 of the corresponding Fc fusion proteins were included during AP fusion protein binding.

Western blot analysis and ligand-receptor affinity-staining

Total proteins from WT and IL-20R2−/− mouse skins were extracted by solution C (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% NP40, pH 7.0). After separation of 50 μg of each sample on a SDS-PAGE followed by western blot transfer to a PVDF membrane (GenHunter), the blots were probed either with 1:500 dilution of a mouse IL-20R2 antibody (cat. no. RS-2620R, ABCAM, Cambridge, MA, USA) or 1 U ml−1 of the corresponding AP fusion proteins. As a loading control, a 1:5000 dilution of HRP-GAPDH antibody (cat. no. KC-5G5, KangChen Bio-tech, Shanghai, China) was used. For the ligand–receptor affinity blotting, 0.12 Unit of AP or mouse IL-20R2-AP (mIL-20R2-AP) fusion proteins were separated on a non-reducing SDS-PAGE and transferred to a PVDF membrane (GenHunter). The blots were probed with 1:2500 dilution of a AP antibody (GenHunter) or 1 U ml−1 of either the mIL-24-AP or human IL-20-AP (hIL-20-AP).7 IL-20R2 and AP antibodies were visualized with anti-rabbit IgG-HRP (Southern BioTech, Birmingham, AL, USA), while mIL-24-AP, hIL-20R2-AP and hIL-20-AP fusion proteins were visualized with AP Assay Reagent S (GenHunter) as previously described.26

Reverse transcription-PCR

Total RNA was prepared from mouse skin using RNApure Reagent (GenHunter). Genomic DNA was removed by DNase I digestion using the MessageClean kit (GenHunter). Two micrograms of each DNA-free total RNA were reverse-transcribed using RevertAid H Minus Reverse Transcriptase, according to the manufacturer’s instructions (Thermo-Fisher, Pittsburgh, PA, USA). RT-PCR was performed in a final reaction volume of 20 μl with 2 μl of cDNA, 2 μl 10 × PCR buffer (TaKaRa, Kusatsu, Japan), 2U rTaq- DNA-Polymerase (TaKaRa), 20 pM of each primer and 200 μM dNTPs (GenHunter). RT-PCR primers used are listed in Table 1 or as published previously.21 The housekeeping gene RPL-19 (ribosomal protein L19) was used as a control for sample normalization. RT-PCR was carried out on a T-100 thermcycler (Bio-Rad, Hercules, CA, USA) as follows: 94 °C for 3 min, followed by 94 °C, 15 s; 58 °C, 30 s; 72 °C, 1 min; for 40 cycles followed by final extension at 72 °C, 5 min. The full-length IL-20R2302 cDNA was amplified by RT-PCR from the DNA-free total RNA of WT mouse skin using primer pairs P4 and P5. The cDNA was cloned into pKillin PCR cloning vector27 and completely sequenced using primers pQE-F and pQE30-R.

5′ RACE of IL-20R2 mRNA

Total RNA from mouse skin was purified and reverse-transcribed as in RT-PCR using primer GSP1 for targeting the 5′ region of the IL-20R2 mRNA. Following the first-strand cDNA synthesis, removal of dNTP and remaining primers by ExoSAP-IT (Affymetrix, Santa Clara, CA, USA), poly-C tailing of the 3′ end of the cDNA was carried out using terminal deoxynucleotidyl transferase (Affymetrix), according to the manufacturer’s instructions. Primers for fluorescent PCR amplification used were: GSP2 and CGHG9H (an equal mixture with the following three anchored primers CGHG9A, CGHG9C and CGHG9T) labeled with Cy-3, which emits red fluorescence. After separating the PCR products using a 6% denaturing polyacrylamide gel, the resulting amplified cDNA detected by the Typhoon FLA 9500 laser fluorescence scanner (GE, Pittsburgh, PA, USA) was recovered and reamplified following the protocol of fluorescent differential display.28 The cDNAs were then verified by DNA sequencing.

Quantitative PCR

Quantitative real-time PCR was performed using CFX Connect (Bio-Rad). RNA extraction and reverse transcription were performed as described in SRT-PCR. To compare the transcription level of IL-20R2 gene in WT and IL-20R2−/− mouse skins, we used qPCR primers (listed in Table 1) specific to either IL-20R2308 (P1 and P9) or IL-20R2302 (P10 and P9). The cDNA obtained from 1 μg RNA was subjected to real-time PCR using the Bio-Rad SsoFast EvaGreen supermix and standard Q-PCR protocol according to instruction of the supplier. QPCR data were analyzed by the CFX software supplied by Bio-Rad. All samples were checked for transcription efficiencies near 100% by serial dilutions. Ct values of all samples were normalized to the Ct values of the housekeeping gene RPL-19 (ΔCt) and values of WT and IL-20R2−/− were plotted as fold expression of the baseline 2−ΔCt. Statistical significance of differences in IL-20R2 mRNA fold was analyzed using the T test.

In situ cell staining for ligand/receptor binding

COS-E5 cells were seeded at the density of 1 × 105/well in six-well plates and grown to 50% confluence before transfection. Forty-eight hours after transfections with pCDEF vector alone or pCDEF-IL-20R1 (ref. 11) in combinations with either IL-20R2308 or IL-20R2302 to for heterodimeric receptors, in situ receptor binding assays were carried out in duplicate with 1 U ml−1 of hIL-20-AP, followed by visualization with AP Assay Reagent S (GenHunter) as previously described.11

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Acknowledgements

We would like to thank Dr Ursula Maria Wegenka for making the IL-20R2 knockout mice available and her kind advice for this study. This work was supported in part by a 863 grant (2012AA02A305) and grants (2012ZX09103301; 2011ZX09401005) from the Chinese Ministry of Science and Technology (PL), a 973 grant (2012CB910700) from the Chinese Ministry of Education (PL), and a grant (81171955) from the Chinese Natural Science Foundation (PL). We thank Jamie Walden and Jonathan Meade from GenHunter Corporation for proofreading the manuscript.

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Zhou, H., Liu, X., Yu, R. et al. Alternative splicing directs two IL-20R2 isoforms and is responsible for the incomplete gene knockout via the exon I ablation. Genes Immun 17, 220–227 (2016). https://doi.org/10.1038/gene.2016.13

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