Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium

The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to control ion concentrations in the fiber mass and the Na,K-ATPase response may reflect the critical contribution of the epithelium to lens ion homeostasis.