Interleukin-20 receptor expression in the trabecular meshwork and its implication in glaucoma

Abstract

Purpose: To determine whether interleukin-20 receptors (IL-20R) are expressed in trabecular meshwork cells and the effect of a T104M mutation in IL-20R2 on downstream cellular functions.

Methods: Evaluation of signal transducer and activator of transcription (STAT)3 phosphorylation and generic matrix metalloproteinase (MMP) activity in primary open angle glaucoma (POAG) dermal fibroblasts (pHDF) with the T104M IL-20R2 mutation were compared with normal human dermal fibroblasts (HDF). Expression of IL-20R1 and IL-20R2 in human trabecular meshwork (HTM) cells was determined by immunohistochemistry and western immunoblotting.

Results: A T104M mutation in IL20-R2 was identified in a large POAG family in which the GLC1C locus was originally mapped. pHDFs harboring this mutation had significantly increased phosphorylated STAT3 (pSTAT3) activity compared with normal HDFs. However, stimulation with either IL-19 or IL-20 for 15 min resulted in significantly decreased levels of pSTAT3 in pHDFs compared with controls. Generic MMP activity was significantly decreased in pHDFs compared with controls after stimulation with IL-20 for 24 h. Both IL-20R1 and IL-20R2 receptors were expressed in HTM cells by western immunoblot and immunofluorescence, and they appeared to be up-regulated in response to cytokine treatment.

Conclusions: A T104M mutation in IL-20R2 significantly impacts the function of this receptor as shown by decreased pSTAT3 levels and generic MMP activity. Reduced MMP activity may affect the ability of glaucoma patients to alter outflow resistance in response to elevated intraocular pressure.

FIG. 1.

Interleukin-20 receptor (IL-20R)2 co-receptor family. Ligands are shown above their respective receptors. The T104M mutation in IL-20R2 is indicated by a star with its impact on binding to IL-20, IL-19, and IL-24. Signaling through the class 2 receptor involves Janus kinase (JAK) and signal transducer and activator of transcription (STAT)3 activation through phosphorylation. Activation of STAT3 can lead to altered matrix metalloproteinase (MMP) levels and activity.

FIG. 2.

Pedigree of the GLC1C family. Below the symbol is the identification number of those family members for whom we have DNA and/or clinical information. Below the identification number is the IL-20R2 genotype (C/T indicates IL-20R2 mutation, C/C indicates normal IL-20R2 genotype) and age of last exam or age of diagnosis, if they have primary open angle glaucoma (POAG). No DNA was available for individual 2004; therefore, the IL-20R2 genotype is not listed. The left upper corner is black if they have been diagnosed with POAG. A black dot in the middle of the symbol indicates that they are a suspect. The genotype is not shown for those individuals for whom we have no DNA sample.

FIG. 3.

Quantitation of STAT3 activation in POAG (n=2) and normal human dermal fibroblasts (HDF; n=3) with and without cytokine stimulation (100 and 200 signify different doses of IL-20 in ng/mL). Phosphorylated STAT3(Y705) was quantitated by ELISA assay, and absorbance data were normalized to pan-STAT3. Results show the average±standard error of the mean (SEM). ANOVA determined significance: *P<0.05 between cytokine-treated and -untreated cells; **P<0.05 comparing POAG versus normal cells with the same cytokine treatment.

FIG. 4.

MMP activity in the conditioned media of normal (n=3) and glaucoma (n=2) HDFs treated with cytokines for 24 h (100 and 200 signify different doses of IL-20 in ng/mL). Results are shown as a percentage of the untreated control±SEM. *P<0.03 by ANOVA.

FIG. 5.

Western immunoblots and densitometry of human trabecular meshwork (HTM) cells treated with cytokines for 24 h (100 and 200 signify different doses of IL-20 in ng/mL). (A) A representative immunoblot of IL-20R2 protein levels in HTM cell lysates. (B) Densitometry of gel bands presented as a percentage of untreated control cells±SEM. *P<0.04 by ANOVA; n=4. (C) A representative immunoblot of IL-20R1 protein levels in HTM cell lysates. (D) Densitometry of gel bands presented as a percentage of untreated control cells±SEM. *P<0.05 by ANOVA; n=3.

FIG. 6.

Immunofluorescence and confocal microscopy of IL-20R2 and IL-20R1 in HTM cells. Representative images of IL-20R2 (A–D) and IL-20R1 (E–H) localization in HTM cells stimulated for 24 h with vehicle control (A, E), IL-20 at 100 ng/mL (B, F), IL-19 (C, G), and IL-24 (D, H). DAPI was used to stain the nuclei (blue). Scale bar=20 μm.