FGFR and PTEN signaling interact during lens development to regulate cell survival

Abstract

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K-AKT and RAS-RAF-MEK-ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells.

Keywords: Differentiation; FGF Receptor; Lens; PTEN; Survival.

Figure 1. Le-Cre efficiently deletes LoxP-flanked Pten and Fgfr2

RT-qPCR was used to detect Fgfr2 (A) and Pten (B) expression in E15.5 Le-Cre negative (Control), Pten^Δ/Δ Fgfr2^Δ/Δ, and (Pten/R2)^Δ/Δ lenses to determine the efficiency of the deletion of LoxP-flanked alleles. Lenses with LoxP-flanked Pten and the Le-Cre transgene (Pten^Δ/Δ) displayed similar expression levels of Fgfr2 transcript as control lenses, but expressed very little Pten transcript. LoxP-Flanked Fgfr2 lenses containing the Le-Cre transgene (Fgfr2^Δ/Δ) displayed a significant reduction in Fgfr2 transcripts and a significant increase in Pten transcripts. Lenses containing both LoxP -flanked Pten and Fgfr2 and the Le-Cre transgene (Pten/R2)^Δ/Δ contained almost no Fgfr2 or Pten transcripts. Error bars represent standard error of the mean (s.e.m.) with significance level indicated above the relevant bars.

Figure 2. Pten deletion rescues the lens size and elongation defects in Fgfr2^Δ/Δ lenses

Hematoxylin and Eosin staining was implemented to analyze the morphology and size of the four different genotypes being compared, Cre-negative controls (A, A’, E, E’, I); Pten^Δ/Δ (B, B’, F, F’ J); Fgfr2^Δ/Δ (C, C’, G, G’ K); and (Pten/R2)^Δ/Δ (D, D’, H, H’ L) at E12.5 (A–D, A’–D’), E15.5 (E–H, E’–H’), and E18.5 (I–L). A’–D’ and E’–H’ are higher magnifications of the boxed in regions of A–D and E-H respectively. Lens planar area measurements in mm² were taken using a Nikon TI-80 microscope in conjunction with their advanced research software at E15.5 and E18.5 (M). At E12.5 the posterior cells of Fgfr2^Δ/Δ lenses did not elongate (compare C to A, C’ to A’-white arrows). Deleting Pten restored the fiber cell elongation phenotype displayed in FGFR2-deficient lenses (compare D to C, D’ to C’-white arrows). At E15.5 and E18.5 Fgfr2^Δ/Δ lenses were significantly smaller than control lenses (compare G to E, K to I, M). Pten deletion rescued lens size in FGFR2-deficient lenses at E15.5 and E18.5 (compare H to G, L to K), but did not restore the eyelid closure phenotype present in FGFR2-deficient lenses (compare boxed regions in L and K to I). Pten deletion, by itself, did not visibly alter the size or morphology of the lens at any of the 3 stages compared (compare B, B’, F, F’ J to A, A’ E, E’, I, M). Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 100μm in AD; 20 μm in A’–D’ and E’–H’; 50 μm in E’H; 200 μm in (I–L).

Figure 3. Pten deletion restores lens cell survival in FGFR2-deficient lenses

TUNEL analysis was implemented on E12.5 (A–D) and E15.5 (E–H) lenses comparing Cre-negative controls (A, E), Pten^Δ/Δ (B, F) Fgfr2^Δ/Δ (C, G), and (Pten/R2)^Δ/Δ (D, H). I and J represent the apoptotic index at E12.5 and E15.5, respectively. At E12.5, the apoptotic index was calculated for the entire lens due to the apoptosis detected in both posterior and anterior lens cells, whereas the apoptotic index was only calculated for the epithelial cell layer at E15.5 as apoptosis was only detected in the epithelium. At E12.5 and E15.5 Fgfr2^Δ/Δ lenses displayed a significant increase in TUNEL positive cells as compared to control lenses (compare C to A, G to E, I), although the apoptotic index was higher at E12.5 than E15.5 (compare C to G). At both E12.5 and E15.5 deleting Pten rescued the cell survival phenotype associated with FGFR2 loss (compare D to C, H to G, I, J). Despite the rescue of apoptosis when Fgfr2^Δ/Δ lenses are compared to (Pten/R2)^Δ/Δ lenses, (Pten/R2)^Δ/Δ lenses continued to exhibit higher levels of apoptosis at E12.5 and E15.5 (compare D to A, H to E, I, J). Moreover, the apoptotic index in Pten^Δ/Δ lenses was increased at E12.5 in comparison to control lenses (compare B to A-white arrows; I). Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 100μm in AH. White arrows point to TUNEL positive foci.

Figure 4. Cell cycle analysis in Pten^Δ/Δ, Fgfr2^Δ/Δ, and (Pten/R2)^Δ/Δ lenses

BrdU incorporation assay was used on Cre-negative controls (A, A’, E), Pten^Δ/Δ (B, B’, F) Fgfr2^Δ/Δ (C, C’, G), and (Pten/R2)^Δ/Δ (D, D’, H) to assess the impact of deleting Pten, Fgfr2, and the combination of the two on lens epithelial cell proliferation and cell cycle withdrawal. By E12.5, control lenses failed to exhibit any BrdU incorporation in the differentiating fiber cells (A, A’). E12.5 Pten^Δ/Δ (B, B’) ,Fgfr2^{Δ/ Δ (}C, C’), and (Pten/R2)^Δ/Δ (D, D’) lenses displayed BrdU incorporation of nuclei in the fiber cell mass, although Fgfr2^Δ/Δ lenses contained the most proliferation in posterior lens cells (compare C’ to B’ and D’). Furthermore, overall proliferation was increased in E12.5 Fgfr2^Δ/Δ lenses, yet was not significantly increased in Pten^Δ/Δ or (Pten/R2)^Δ/Δ lenses at this stage (I). By E15.5 (E–H, J) lens epithelial proliferation was not altered in Pten^Δ/Δ (F, J) Fgfr2^Δ/Δ (G, J), or (Pten/R2)^Δ/Δ (H, J). A’–D’ represent boxed regions of A–D selected for higher magnification. Dashed white lines outline the edges of the lens and white arrows point towards posterior lens cells remaining prolific (A’–D’). Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 100 μm in A–D; 50 μm in A’–D’; 200 μm in E–H.

Figure 5. Pten deletion restores decreased β- and γ-crystallin expression to the FGFR2- deficient lenses at E12.5

Cre-negative control (A, E, I, M), Pten^Δ/Δ (B, F, J, N) , Fgfr2^Δ/Δ (C, G, K, O) , and (Pten/R2)^Δ/Δ (D, H, L, P) lenses were analyzed by immunohistochemistry at E12.5 (A–D, I–L), and E15.5 (E–H, M–P), to determine the expression of γ-crystallin (A–H) and β-crystallin (I–P). At E12.5 γ-crystallin was present in very few posterior cells of Fgfr2^Δ/Δ lenses (compare C to A). E12.5 (Pten/R2)^Δ/Δ lenses displayed an increase in γ-crystallin expression relative to Fgfr2^Δ/Δ lenses (compare D to C), but remained reduced in comparison to control lenses (compare D to A). By E15.5, Fgfr2^Δ/Δ lenses expressed γ-crystallin protein in all fiber cells (compare G to C). c-Maf transcript levels were not reduced in either Fgfr2^Δ/Δ or (Pten/R2)^Δ/Δ lenses (Q). Isolated Pten deletion did not alter γ-crystallin protein at E12.5 (compare B to A), or E15.5 (compare F to E). The quantification of c-Maf (Q) was standardized to Gapdh mRNA. Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 100 μm in A–D, I–L; 200 μm in E–H, M–P.

Figure 6. Fgfr2^Δ/Δ lenses exhibit reduced Aquaporin0 expression and a nuclear retention phenotype that Pten deletion fails to rescue

Cre-negative control (A, E), Pten^Δ/Δ (B, F) , Fgfr2^Δ/Δ (C, G) , and (Pten/R2)^Δ/Δ (D, H) lenses were analyzed for Aquaporin0 protein expression at E15.5 (A–D) , and for denucleation by TUNEL at E18.5 (E–H) . Both Fgfr2^Δ/Δ (compare C to A; J, K) and (Pten/R2)^Δ/Δ (Compare D to A; J, K) lenses experienced a reduced Aquaporin0 expression level. Additionally, Fgfr2^Δ/Δ (C) and (Pten/R2)^Δ/Δ (D) contained several heightened areas of Aquaporin0 expression in a circular pattern (C and D white arrows). Aquaporin0 expression in Pten^Δ/Δ lenses did not differ from controls (compare B to A; J, K). Western blot analysis was performed at E15.5 to confirm the reduced Aquaporin0 protein observed with FGFR2 deficiency (K). The western blot quantification was standardized to GAPDH protein. TUNEL analysis detected normal fiber cell DNA degradation in both control (E) and Pten deleted lenses (F). However DNA degradation in the central fiber cells was markedly reduced in both Fgfr2^Δ/Δ (G) and (Pten/R2)^Δ/Δ (H) lenses. Quantitative RT-PCR revealed a reduction in trancripts for DnaseIIβ at E16.5 (I). The quantification of DnaseIIβ was standardized to GADPH mRNA. Reduced transcript levels of DnaseIIβ were observed in Fgfr2^Δ/Δ and (Pten/R2)^Δ/Δ lenses (I). Pten^Δ/Δ lenses did not display defects in Aquaporin0 protein expression (J)/localization (B), nuclear removal (F), or DnaseIIβ expression (I). White arrows point towards heightened and abnormal expression of Aquaporin0 in C and D, and TUNEL foci in E, F, and H. Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 200 μm in A–D; 100 μm in E–H.

Figure 7. Deleting Pten restores pAKT and pERK1/2 in FGFR2-deficient lenses

E15.5 Cre-negative control, Pten^Δ/Δ , Fgfr2^Δ/Δ , and (Pten/R2)^Δ/Δ lenses were analyzed by western blot analysis to determine the impact of deleting Pten, Fgfr2, or both Pten and Fgfr2 on activation of ERK1/2 and AKT. Levels of pAKT were significantly reduced in Fgfr2^Δ/Δ lenses (A, B). Additionally deleting Pten with the Fgfr2 deletion raised the level of pAKT well beyond the levels of control lenses (A, B). As expected, Pten^Δ/Δ lenses experienced very high levels of pAKT (A, B). Interestingly, the total amount of AKT was reduced in both Pten^Δ/Δ lenses and (Pten/R2)^Δ/Δ lenses (A, C). Fgfr2^Δ/Δ lenses experienced a significant reduction in p-ERK (A, D), and additionally deleting Pten raised the level of pERK1/2 above that of control lenses (A, D). Pten^Δ/Δ lenses experienced modestly elevated levels of pERK1/2 (A, D). Total ERK levels remained unaltered in all of the examined genotypes (A, E). Total ERK1/2 and AKT were standardized to GAPDH while pERK1/2 and pAKT were normalized to total ERK1/2 and total AKT, respectively. Errors bars on the graphs represent s.e.m, with significance levels indicated above the relevant genotypes.

Figure 8. FGFR2 deficiency leads to increased activation of C-JUN and p53 and loss of MDM2 phosphorylation

E15.5 whole lenses of Cre-negative control, Pten^Δ/Δ , Fgfr2^Δ/Δ , and (Pten/R2)^Δ/Δ were analyzed by western blot analysis using antibodies to p-p53 (Ser 15), p-cJUN (Thr 63/Thr 73) and p-MDM2 (Ser 166). GAPDH was used as a loading control. Fgfr2^Δ/Δ lenses increased both phospho-p53 (A-top panel, B) and p-cJUN (A-3^rd panel, C) while decreasing the amount of p-MDM2 (A-5^th panel, D). The deletion of Pten lowered p-p53 in FGFR2-deficient lenses. Likewise deleting Pten returned cJUN activation and p-MDM2 to control levels (A-3^rd panel, C and A-5^th panel, D, respectively). Error bars on the graphs represent +/− s.e.m. p-p53, p-cJUN and p-MDM2 were standardized to GAPDH for quantification.