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D.L. Harris, N.C. Joyce; Effects of PTP1B Inhibition on Rat Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2323.
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Growth factor induced cell proliferation is mediated through receptor tyrosine kinases (RTKs), such as epidermal growth factor (EGF) receptor, resulting in tyrosine phosphorylation (Tyr–PO4). Tyr–PO4 of cadherin–based junctional proteins can disrupt cell–cell contacts. Both types of Tyr–PO4 events can be inhibited by protein tyrosine phosphatases (PTPs), potentially indicating a pathway to regulate contact–dependent inhibition of proliferation as is found in corneal endothelial cells (CECs). Past studies have shown that a non–specific inhibitor of PTPs such as sodium orthovanadate (SOV) releases cell contacts and promotes cell cycle entry. The current study employs a specific PTP1B inhibitor (CinnGel 2Me; BioMol International) to test its effect on rat corneal endothelial cells.
Rat corneas were obtained and either processed for whole mount immunocytochemistry (ICC) or cultured for RT–PCR, ICC or western blot. Expression of PTP1B in rat CECs was evaluated using all of the above methods with G3PDH used as a control for PCR and beta–actin or non–muscle myosin for western blot. In addition, beta–catenin and PTP1B staining was performed on whole mounts. After 24 hr serum starvation, addition of CinnGel 2Me for a total of 60 min prior to the start of the experiment, and then addition of EGF at varying times, important proteins involved in downstream signaling pathways were compared with controls. Those included western blots detecting levels of phospho–EGF receptor (Tyr 992) and activation of MAPK signaling with antibodies against ERK2 and phospho–ERK.
PTP1B is localized just subjacent to the plasma membrane in rat whole mounts and diffusely cytoplasmic with a perinuclear concentration in subconfluent CECs. Double staining for beta–catenin and PTP1B shows beta–catenin outlining the cell borders whereas PTP1B is subjacent to the membrane. PTP1B expression was equivalent by RT–PCR in subconfluent versus confluent CECs whereas it was 3 fold higher by western blot. With the addition of the inhibitor and in the presence of EGF, both phospho–EGF receptor (Tyr 992) and phospho–ERK activation are increased and prolonged over controls for up to 2 hrs.
Specific inhibition of the protein tyrosine phosphatase, PTP1B, results in increased downstream signaling of EGF receptor and ERK, suggest that treatment of corneal endothelium with this inhibitor may promote cell cycle entry.
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