Adenosine A1 Agonists Induce the Secretion of MMP–2 From Trabecular Meshwork Cells Through the Activation of PKC

Abstract

Abstract: : Purpose: Previous studies have shown that the activation of PKC is an early step in the signaling events associated with adenosine A1 agonist–induced secretion of MMP–2. The objective of this study was to evaluate the role of individual protein kinase C (PKC) isoforms in the signaling pathways that modulate adenosine A1 agonist–induced MMP secretion from human trabecular meshwork cells. Methods: Primary cultures of human trabecular meshwork cells were used in this study. PKC isoforms were detected by Western blotting using PKC–isoform specific antibodies. To evaluate MMP–2 secretion, cells were serum–starved for 16 hour and then treated with the adenosine A1 agonist cyclohexyladenosine (CHA; 1 µmol/L) for 2 hours. The media was then concentrated and analyzed for MMP–2 by Western blot. Transfection of the siRNA, targeting the endogenous gene for PKC isoform, was carried out in 70–80% confluent cell cultures using 1–100 nmol/L siRNA duplexes for each PKC isoform. Results: In human trabecular meshwork cells, seven PKC isoforms were identified: PKCα, Δ, ε, , µ, and . The addition of CHA for 5 minutes resulted in the rapid and complete translocation of PKCα from the cytosol to the membrane. The addition of CHA for 2 hours increased MMP–2 secretion by 104 ± 24%. This increase in MMP–2 secretion was blocked by the addition of the PKCα/ß inhibitor, Go–6976. The addition of specific siRNA for PKCα produced a selective 80 to 85% knock–down of PKCα and blocked the CHA–induced secretion of MMP–2. Conclusions: Human trabecular meshwork cells express multiple PKC isoforms. Addition of the adenosine A1 agonist, CHA, stimulates the secretion of MMP–2 from trabecular meshwork cells. This secretory response involves the activation of PKCα. These results confirm the existence of functional adenosine A1 receptors in the trabecular cells; and activation of these receptors in vivo may modulate outflow facility through the PKCα–dependent secretion of MMP–2 secretion.

Keywords: trabecular meshwork • signal transduction: pharmacology/physiology • adenosine