Protein Tyrosine Phosphatase 1B (PTP1B) Expression in Human Corneal Endothelial Cells

Abstract

Purpose:: Human corneal endothelial cells (HCEC) proliferate in response to EGF; however, the response is relatively weak, particularly in cells from older donors (>50 years old). Protein tyrosine phosphatase (PTP) activity is an important mechanism that helps negatively regulate proliferation. PTP1B is known to regulate the activity of the EGF receptor (EGFR). These studies determined whether PTP1B is expressed in HCEC and compared the EGFR and PTP1B expression in HCEC cultured from young and older donors.

Methods:: Donor corneas were obtained from National Disease Research Interchange. Ex vivo corneas were used for PTP1B immunocytochemical (ICC) analysis (Donor age: 18, 59, 61 and 66 years old). Secondary antibody alone acted as a negative control. Results were analyzed using fluorescence confocal microscopy. HCEC were cultured from 4 young donors (2 newborns and donors 2 and 3 years old) and 4 older donors (60, 66, 74 and 78 years old). Protein was extracted from passage 2 HCEC 7 days after cells reached confluence. Western blots were prepared according to published protocols to analyze EGFR and PTP1B expression. Semi-quantitative analysis of protein bands was conducted using NIH ImageJ software. Beta-actin was used for normalization.

Results:: Whole mount ICC of human corneas showed clear, punctate PTP1B localization in the cytoplasm of HCEC from all corneas. In Western blot analysis of PTP1B, the primary antibody recognized both a phosphorylated and non-phosphorylated form. Total PTP1B expression (calculated from the density of the phosphorylated plus non-phosphorylated forms) was significantly higher in older donors than in young donors (p=0.024<0.05). No significant difference was observed in EGF receptor expression between the two age-groups (p=0.444>0.05).

Conclusions:: Although EGF receptors are similarly expressed in HCEC from both young and older donors, PTP1B, a negative regulator of EGF receptor activity, is more highly expressed in HCEC from older donors. These results suggest that the decreased proliferative response of HCEC previously observed in older donors could be due to increased activity of PTP1B. This hypothesis is currently being tested.

Keywords: cornea: endothelium • cornea: basic science