Negative regulation of lens fiber cell differentiation by RTK antagonists Spry and Spred

Abstract

Sprouty (Spry) and Spred proteins have been identified as closely related negative regulators of the receptor tyrosine kinase (RTK)-mediated MAPK pathway, inhibiting cellular proliferation, migration and differentiation in many systems. As the different members of this antagonist family are strongly expressed in the lens epithelium in overlapping patterns, in this study we used lens epithelial explants to examine the impact of these different antagonists on the morphologic and molecular changes associated with fibroblast growth factor (FGF)-induced lens fiber differentiation. Cells in lens epithelial explants were transfected using different approaches to overexpress the different Spry (Spry1, Spry2) and Spred (Spred1, Spred2, Spred3) members, and we compared their ability to undergo FGF-induced fiber differentiation. In cells overexpressing any of the antagonists, the propensity for FGF-induced cell elongation was significantly reduced, indicative of a block to lens fiber differentiation. Of these antagonists, Spry1 and Spred2 appeared to be the most potent among their respective family members, demonstrating the greatest block in FGF-induced fiber differentiation based on the percentage of cells that failed to elongate. Consistent with the reported activity of Spry and Spred, we show that overexpression of Spry2 was able to suppress FGF-induced ERK1/2 phosphorylation in lens cells, as well as the ERK1/2-dependent fiber-specific marker Prox1, but not the accumulation of β-crystallins. Taken together, Spry and Spred proteins that are predominantly expressed in the lens epithelium in situ, appear to have overlapping effects on negatively regulating ERK1/2-signaling associated with FGF-induced lens epithelial cell elongation leading to fiber differentiation. This highlights the important regulatory role for these RTK antagonists in establishing and maintaining the distinct architecture and polarity of the lens.

Keywords: FGF; Lens fiber differentiation; RTK-Antagonists; Spred; Sprouty.

Fig. 1. Inhibition of FGF-induced fiber cell elongation by Spry

Representative micrographs of P10 rat lens epithelial explants transfected (Lipofectamine^®) with pLXSG (control, A, B and B′), pLXSG-Spry1 (C, D and D′), pLXSG-Spry2 (E, F and F′) or pLXSG-Y55A-Spry2 2 (control, G, H and H′), and cultured with or without FGF (100ng/ml) for 5 days. β-catenin immunolabeling (red) outlined the cell membranes. Arrows indicate cells with a fiber cell morphology. Higher magnification insets are shown. Scale bar: 50μm, insets, 25 μm.

Fig. 2. Inhibition of FGF-induced fiber cell elongation by Spred

Representative micrograph of P10 rat lens epithelial explants transfected with pLXSG-Spred1 (A, B and B′), pLXSG-Spred2 (C, D and D′) or pLXSG-Spred3 (E, F and F′) using Lipofectamine^®, and cultured with FGF (100ng/ml, B, B′, D, D′, F and F′) or without FGF (A, C and E) for 5 days. β-catenin immunolabeling (red) outlined the cell membranes. Arrows indicate cells with a fiber cell morphology. Higher magnification insets are shown. Scale bar: 50μm, insets, 25μm.

Fig. 3. Spry and Spred inhibit FGF-induced elongation of lens epithelial cells

EGFP-positive cells from the transfected lens explants (n=5 explants/group) were scored based on cell morphology. Data represents mean ± s.e.m with statistical tests performed using student’s t-test (*=p<0.001; ns=not significant).

Fig. 4. Spry negatively regulates FGF-induced ERK1/2 phosphorylation

Immunoblotting of phosphorylated ERK1/2 from non-transduced FGF-treated P10 rat lens epithelial explants (A) or transduced with Ad5Spry2 (B) or Ad5-Y55A-Spry2 (C). Representative western blots of phosphorylated (p)- or total (t)-ERK1/2 with GAPDH loading control. (D) Quantitative analysis of pERK1/2 levels in Spry2- and Y55A-Spry2-overexpressing cells. Both pERK1/2 and tERK1/2 were standardised to GAPDH, with the ratio of pERK1/2 against tERK1/2 in Y55A-Spry2-overexpressing cells at 0 min set to a value of 1, against which all other ratios were determined. Data represents mean ± s.e.m with statistical tests performed using student’s t-test (*=p<0.001; ns=not significant).

Fig. 5. Inhibition of FGF-induced fiber cell elongation by Spry or Spred

Representative micrographs of P10 rat lens epithelial explants transfected with Ad5eGFP (control; A, B, B′ and K), Ad5Spry1 (C, D, D′ and L), Ad5-Y55A-Spry2 (E, F, F′ and M), Ad5Spred2 (G, H, H′ and N) or Ad5-Y55A- Spry2 (control; I, J, J′ and O), and then treated with no FGF (A, C, E, G and I) or with 50 ng/mL FGF (B, B′, D, D′, F, F′, H, H′, J, J′, K-O) for 5 days. β-crystallin staining is shown in K-O. Arrows indicated elongate cells. Scale bar: 50 μm.

Fig. 6. Spry suppression of FGF-induced Prox1 expression

Representative western blots for Prox1 (upper panel) and GAPDH (loading control; lower panel) from lysates of P10 rat lens epithelial explants overexpressing Spry2 (A) or Y55A-Spry2 (B), and cultured with FGF for 0 to 10 hrs. (C) Quantitative analysis of western blots of Prox1 (normalised to GAPDH). Data represents mean ± s.e.m with statistical tests performed using student’s t-test (*=p<0.05; ns=not significant).