The P2X(7) receptor regulates proteoglycan expression in the corneal stroma

Abstract

Purpose: Previously, the authors demonstrated that the lack of the P2X(7) receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X(7) receptor on components of the corneal stroma extracellular matrix.

Methods: Unwounded corneas from P2X(7) knockout mice (P2X(7) (-/-)) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma.

Results: P2X(7) (-/-) mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X(7) (-/-) mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X(7) (-/-) compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X(7) (-/-) mice, indicating that there are more heparan sulfate proteoglycans in the latter.

Conclusions: Our results indicate that loss of P2X(7) alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma.

Figure 1

P2X₇^−/− stromas show altered expression of collagen and related proteins. Real Time PCR results±SEM of at least three independent experiments were calculated using the ΔΔCt method and normalized to 18S rRNA. All sample results are presented relative to the median WT sample, normalized to 1. Negative controls were performed without reverse transcriptase. * p<0.03, Student’s t-test.

Figure 2

Altered expression of proteoglycan core proteins in P2X₇^−/− stromas. Real Time PCR results±SEM of at least three independent experiments were calculated using the ΔΔCt method and normalized to 18S rRNA. All sample results are presented relative to the median WT sample, normalized to 1. Negative controls were performed without reverse transcriptase. * p<0.05, Student’s t-test.

Figure 3

KSPG localization is altered in P2X₇^−/− stromas. Frozen corneas were sectioned and stained with antibodies specific for keratocan and lumican, followed by FITC-conjugated IgG (green) and counterstained with To-Pro 3AM for nuclei (blue). Negative controls were incubated with secondary antibody only and counterstained with To-Pro 3AM. Images are representative of three independent experiments. Keratocan (inset): enlarged regions from central stroma with enhanced signal to show detail of localization. Scale bar: 10 μm.

Figure 4

Decorin localization is altered in P2X₇^−/− stromas. Frozen corneas were sectioned and stained with antibody against decorin, FITC-conjugated IgG, and To-Pro 3AM. Negative controls were incubated with non-immune IgG instead of primary antibody, and To-Pro 3AM. Images are representative of three independent experiments. E=Epithelium, S=Stroma. Scale bar: 50 μm.

Figure 5

Electron micrographs of electron dense filaments stained with Cuprolinic blue on stromal collagen. Samples of A: WT and B: P2X₇^−/− corneas were digested with either Keratanase I, Chondroitinase ABC, or both, before Cuprolinic blue staining and imaging with TEM. Undigested and stained tissue are included. The scale bar seen in the undigested column represents 500 nm, and applies to all images in the Undigested, Keratanase I, and Chondroitinase ABC columns. The scale bar seen in the Keratanase I and Chondroitinase ABC columns represent 200 nm and applies to images in that column only. Descemet’s membrane is marked with “D” and is visible in the WT posterior images.

Figure 6

Sulfation of glycosaminoglycans in WT and P2X₇^−/− corneal stromas. Collagen fibril length was measured, and the number of proteoglycan (PG) units along that length was counted for each non-overlapping region (Anterior, Middle, Posterior) and summed for the entire cornea (Overall). Results were normalized to number of PG units per 100 nm collagen for A: Undigested corneas, B: Corneas digested with Keratanase I, C: Corneas digested with Chondroitinase ABC, and D: Corneas digested with both Keratanase I and Chondroitinase ABC. A minimum of 75 measurements were performed for each region of each group and digestion condition, and results were averaged and presented as ±SEM ** p<0.0001 and * p<0.05, one-way ANOVA followed by Tukey’s post-hoc test.

Figure 7

Perlecan localization is altered in P2X₇^−/− stromas. Frozen corneas were sectioned and stained with antibody against perlecan, FITC-conjugated IgG, and To-Pro 3AM. Negative controls were incubated with non-immune IgG instead of primary antibody, and To-Pro 3AM. Images are representative of three independent experiments. A: Perlecan expression is increased throughout the stroma in P2X₇^−/− corneas. Inset: enlarged regions from the central stroma with enhanced signal to show detail of localization. Scale bar: 10 μm. B: Perlecan is localized to the basement membrane in WT corneas (arrow) but not in P2X₇^−/− corneas. Scale bar: 500 μm.

Figure 8

Schematic of WT and P2X₇^−/− corneal stromas. Rods represent collagen fibrils, small circles represent SLRPs, and rectangles represent perlecan.