Abstract
Purpose :
Sprouty (Spry) and Spred have been identified as regulators of RTK-mediated MAPK/ERK signaling in many developmental processes. They are expressed in lens epithelium, and are proposed to function as inhibitors of cellular proliferation. To better understand their specific role in lens biology, we overexpressed Spry and Spred in lens epithelial cells to compare their impact on FGF-induced lens cell proliferation.
Methods :
Rat lens epithelial explants were transduced with different adenoviruses (Ad5eGFP, Ad5ΔSpry2, Ad5Spry1, Ad5Spry2 and Ad5Spred2). Ad5eGFP, with no specific antagonist inserted, and Ad5ΔSpry2, coding for a non-functional form of Spry2, were used as negative and positive controls, respectively. Transduced cells were treated with a proliferating dose of FGF2 (5ng/ml) for 24 hours. EdU- and/or BrdU-incorporation were used to measure the percentage of proliferating cells. Immunofluorescence labeling and western blotting were used to examine changes to levels of phosphorylated ERK1/2 (pERK1/2) under these conditions.
Results :
Cells transduced with Ad5eGFP and Ad5ΔSpry2 proliferated similarly in response to FGF (student’s t-test, p>0.5; n=5). Overexpression of Spry1 did not significantly impact on FGF-induced lens cell proliferation; however, both Ad5Spry2- and Ad5Spred2-transduced cells significantly reduced cell proliferation relative to control cells. The different Sprys and Spred2 showed different inhibitory effects on FGF-induced pERK1/2. In Spry1-overexpressing cells, pERK1/2 was suppressed early with FGF treatment, but increased and was sustained from 4hr onwards. Spry2-overexpressing cells had peak levels of pERK1/2 by 10 mins with FGF; however, this dropped by 30 mins and remained low throughout the culture period, similar to Spred2-overexpressing cells where pERK1/2 levels increased over 2hr before decreasing by 4hr.
Conclusions :
Sprys and Spred2 differentially regulate FGF-induced ERK1/2 in lens cells, and this reflects their ability to block cell proliferation. The lesser effect of Spry1 in blocking cell proliferation was consistent with its failure to suppress pERK1/2. Spry2 and Spred2 suppressed pERK1/2 through most of the culture period, reflective of their similar antagonistic abilities on lens cell proliferation. This differential regulation of pERK1/2 and lens cell proliferation highlights the importance of pERK1/2 duration and intensity for regulating cell proliferation in lens.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.