Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death

Changes in Epithelial and Stromal Corneal Stiffness Occur with Age and Obesity

The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and compare them to 15-week pre-Type 2 diabetic obese mouse. Nanoindentation was performed on corneal epithelium in situ and then the epithelium was abraded, and the procedure repeated on the basement membrane and stroma. Confocal imaging was performed to examine the localization of proteins. Stiffness was found to be age and obesity dependent. Young’s modulus was greater in the epithelium from 15-week mice compared to 8-week mice. At 15 weeks, the epithelium of the control was significantly greater than that of the obese mice. There was a difference in the localization of Crb3 and PKCζ in the apical epithelium and a lack of lamellipodial extensions in the obese mouse. In the pre-Type 2 diabetic obese mouse there was a difference in the stiffness slope and after injury localization of fibronectin was negligible. These indicate that age and environmental changes incurred by diet alter the integrity of the tissue with age rendering it stiffer. The corneas from the pre-Type 2 diabetic obese mice were significantly softer and this may be a result of changes both in proteins on the apical surface indicating a lack of integrity and a decrease in fibronectin

HCLEs express a full length and truncated P2X₇ mRNA transcript and protein.

<p><b>A.</b> mRNA was purified from total RNA of HCLE and IMR90 cells cultured for 72 hours by twice passing over an oligo-dT column. Purity was confirmed by methylene blue staining of nytran filter following transfer (note lack of rRNA in mRNA lanes). <b>B</b>. Northern blot analysis of HCLE and IMR90 mRNA from 72 hour cultures. The probe used was generated by PCR amplification of a consensus region in all P2X₇ variants. The relative position of 18s rRNA is indicated and the smaller transcript expressed by epithelial cells is indicated (arrow). <b>C.</b> Expression of P2X₇ and P2X_7j mRNA transcript over a 72 hour incubation. Relative expression was determined using the ΔΔCT method. <b>D.</b> Expression of P2X₇ receptor by IMR90 cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. <b>E.</b> Expression of P2X₇ receptor by HCLE cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. Hetero- and homotrimers are identified on crosslinking gels. Epithelial cells were cultured for 72 hours, incubated in situ in the presence of formaldehyde, lysed and incubated at 65°C or 95°C to maintain or break crosslinks. Inset – equivalent experiment resolved by SDS-PAGE (8%) and immunoblotted with anti-P2X₇. Data is representative of a minimum of 3 experiments.</p

Confluent monolayer corneal epithelial cells do not form large pores when stimulated with BzATP.

<p>HCLE or IMR90 cells were cultured and stimulated with control media or media containing 100 µM BzATP in the presence of EtBr or ToPro-3 and imaged over 20 min while maintained at 37°C and 5% CO₂ in an environmental chamber on a Zeiss LSM 510 confocal microscope. Representative images are of cells after 20 min BzATP with EtBr (EtBr) or BzATP with ToPro-3 (ToPro)(Scale bar  = 50 µm). Control treated cells in the presence of EtBr are shown (Control). ToPro-3 is detected in IMR90 cells and only detected in HCLE cells being shed. The time to initial EtBr uptake is presented. Images are representative of 3 independent experiments.</p

P2X₇ activation induces phosphorylation of ERK and cell migration.

<p><b>A.</b> Cells were treated in the presence or absence of BzATP for 5 min, lysed and equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-p-ERK. Blots were stripped and reprobed with anti-ERK and pERK normalized to ERK. Significance was determined by Student's test (p<0.05). Blot is representative of 3 independent experiments. <b>B.</b> Transwell migration assays were performed for 8 hr in the presence of BzATP or binding buffer (control). Migrated cells were stained with propidium iodide, counted in 6 randomly chosen fields (1.48 mm²) and averaged. Data are representative of 3 independent experiments and are presented as mean +/− SEM. Significance was determined by Student's t-test: **p<0.001. <b>C.</b> Downregulation of P2X₇ attenuates wound healing in a representative experiment. HCLE cells transfected with control (non-targeting siRNA) or siRNA targeted to P2X₇ receptor were cultured to confluence. Unsupplemented media or media with 100 µM BzATP were added prior to injury. Slides were placed on a heated stage in an environmental chamber at 37°C and 5% CO₂. Scratch wounds were made and contiguous images were taken every 20 min over 20 hr. Data are representative of 3 independent experiments. <b>D</b>. Percent wound closure from directed migration experiments at endpoint are presented as mean +/− SEM. Significance was determined by ANOVA followed by Tukey posthoc test: **p<0.001. <b>E.</b> HCLE cells transfected with control (non-targeting siRNA) or siRNA targeted to P2X₇ receptor were cultured to confluence. Real time RT-PCR was performed and relative expression of the indicated receptors was determined using the ΔΔCT method. The average relative expression of 3 independent experiments +/− SEM is presented. Significance was determined by Student's test: **p<0.004. <b>F.</b> Parallel experiments to <b>E</b>. were conducted and cultures were lysed and equivalent amounts of protein resolved by SDS-PAGE and immunoblotted with antibodies directed to P2X₇ and P2X₄ receptors.</p

Diabetic corneal epithelium displays enhanced P2X₇ receptor expression compared to control non-diabetic corneal epithelium.

<p>Central corneal epithelium from 6 samples of control and diabetic were analyzed using real time PCR. Relative expression of the P2X₇ receptor (all variants), P2X_7j variant receptor, and Ki67 were determined using the ΔΔCT method. The average relative expression of 6 independent samples +/− SEM is presented. Significance was determined by Student's t-test: **p<0.002, *p<0.05 respectively.</p