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FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling

Frank J. Lovicu* and John W. McAvoy

Save Sight Institute, and Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney, NSW, Australia



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  		Fig. 1. FGF2 induced phosphorylation of p42/p44 MAPK in a dose-dependent manner. Representative immunoblots of phospho-ERK (upper panel) and total ERK (lower panel) from lens epithelial explants exposed to no FGF2 (lane 1), 5 ng/ml FGF2 (lane 2) or 100 ng/ml FGF2 (lane 3) for 15 minutes at 37°C.

 


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  		Fig. 2. FGF2-induced phosphorylation of p42/p44 MAPK. (A) Representative immunoblot of phospho-ERK from lens epithelial explants exposed to different concentrations of FGF2 for 2 hours at 37°C. Lens explants exposed to either no FGF2 (B,E), 5 ng/ml FGF2 (C,F) or 100 ng/ml FGF-2 (D,G) for 2 hours at 37°C, immunolabelled for phospho-ERK (B-D) or counterstained with Hoechst dye (E-G). Scale bar: 40 µm.

 


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  		Fig. 3. UO126 blocks FGF-induced phosphorylation of p42/p44 MAPK. (A) Representative immunoblots of phospho-ERK (top) and total ERK (bottom) from lens epithelial explants exposed to no FGF2 (lanes 1 and 3) or 100 ng/ml FGF2 (lanes 2 and 4) for 15 minutes at 37°C, in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of UO126. (B) Lens explant exposed to 100 ng/ml of FGF2 in the presence of UO126, immunolabelled for phospho-ERK (right), counterstained with Hoechst dye (left). Control explants were treated with an equivalent volume of DMSO without UO126. Scale bar: 40 µm.

 


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  		Fig. 4. UO126 blocks FGF-induced phosphorylation of p42/p44 MAPK and BrdU-incorporation. Representative micrographs of cells in lens explants exposed to no FGF (A-C,G-I) or 5 ng/ml FGF-2 (D-F,J-L) in the presence of DMSO (A,B,D,E,G,H,J,K) or DMSO with UO126 (C,F,I,L), immunolabelled for phospho-ERK (B,C,E,F) or BrdU-incorporation (H,I,K,L), counterstained with Hoechst dye (A,D,G,J). After 2 days culture, a low dose of FGF could still induce an increase in ERK phosphorylation (E), together with a marked increase in BrdU-incorporation (K). In the presence of UO126, however, phosphorylation of ERK and the number of cells that incorporated BrdU was markedly reduced both in control explants (C,I), and in explants treated with a low dose of FGF2 (F,L). Scale bar: 40 µm.

 


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  		Fig. 5. FGF-induced fibre cell elongation and multilayering accompanied phosphorylation of ERK and could be blocked by UO126. Representative micrographs of cells in lens explants not exposed to FGF2 (A,B) or exposed to 100 ng/ml FGF2 (C-F) in the presence of DMSO (A-D) or DMSO with UO126 (E,F), immunolabelled for phospho-ERK (B,D,F) or counterstained with Hoechst dye (A,C,E). After 5 days culture with FGF, cells have elongated and multilayered as shown by the overlapping large ovoid nuclei (C). These cells also displayed a marked increase in phospho-ERK labelling (D) compared with epithelial cells of control explants (B). This FGF-induced activation of ERK was blocked in the presence of UO126 (F), which not only resulted in inhibition of cell elongation (compare D with F) but also multilayering of the cells (compare C with E). Scale bar: 50 µm.

 


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  		Fig. 6. UO126 blocks FGF-induced cell multilayering in lens explants. Representative sections of explants cultured for either 3 (C,D) or 5 days (A,B,E,F) with no FGF2 (A,B) or 100 ng/ml of FGF2 (C-F) in the presence of DMSO (A,C,E) or DMSO with UO126 (B,D,F). FGF2 induced a progressive thickening of lens explants over the culture period, as cells elongated and multilayered (C,E). In the presence of UO126, the multilayering could be blocked with cells remaining as a monolayer on the lens capsule (D,F), similar to control explants (A). Abbreviations: ca, lens capsule. Scale bar: 20 µm.

 


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  		Fig. 7. UO126 blocks FGF-induced cell elongation in lens explants. Representative scanning electron micrographs of cells in explants cultured for 5 days in the absence of FGF2 (A,B) or with 100 ng/ml of FGF2 (C,D) in the presence of DMSO (A,C) or DMSO with UO126 (B,D). FGF2 induced the elongation of lens epithelial cells (C, arrows). In the presence of UO126, this elongation could be blocked (D), with cells demonstrating a similar morphology to cells in control explants (A,B). Scale bar: 5 µm.

 


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  		Fig. 8. UO126 blocks FGF-induced upregulation of filensin expression, which accompanies fibre differentiation. Representative micrographs of cells in lens explants cultured for 5 days in the absence of FGF2 (A-C) or with 100 ng/ml FGF2 (D-F) in the presence of DMSO (A,B,D,E) or DMSO with UO126 (C,F), immunolabelled for filensin (B,C,E,F) or counterstained with Hoechst dye (A,D). FGF2 induced a marked increase in filensin expression (E). In the presence of UO126, no reactivity was observed in response to FGF2 (F), with levels comparable with control explants (C). Scale bar: 50 µm.

 


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  		Fig. 9. UO126 does not block FGF-induced ß-crystallin expression, which accompanies fibre differentiation. Representative micrographs of cells in lens explants cultured for 5 days in the absence of FGF2 (A-C) or with 100 ng/ml FGF2 (D-F) in the presence of DMSO (A,B,D,E) or DMSO with UO126 (C,F), immunolabelled for ß-crystallin (B,C,E,F) or counterstained with Hoechst dye (A,D). FGF2 induces a marked increase in ß-crystallin expression (E) with little to no reactivity detected in control explants (B). In the presence of UO126, although the cells retain an epithelial morphology in response to FGF2, they express ß-crystallin (F). Scale bar: 40 µm.

 


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  		Fig. 10. Representative frozen sections of neonatal lenses counterstained with Hoechst dye (A) or immunolabelled for phospho-ERK (B) or phospho-p38 (C). Strong punctate nuclear reactivity for phospho-ERK was detected throughout the epithelial cells, extending into the transitional zone where cells are elongating into fibres (A,B). Fibre cells deeper in the cortex demonstrated a marked reduction in labelling with no reactivity apparent in these maturing cells (A,B, arrows). No labelling for phospho-p38 was detected in the lens (C). Abbreviations: le, lens epithelium; lf, lens fibres. Scale bar: 40 µm.

 

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© The Company of Biologists Ltd 2001