The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth

Abstract

Purpose: Signal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110β catalytic subunits of PI3K were conditionally deleted in the mouse lens.

Methods: Floxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice. Lenses of age-matched animals were dissected and photographed. Postnatal lenses were fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin. Cell proliferation was quantified by labeling S-phase cells in intact lenses with 5-ethynyl-2'-deoxyuridine. Protein kinase B (AKT) activation was examined by Western blotting.

Results: Lens-specific deletion of p110α resulted in a significant reduction of eye and lens size, without compromising lens clarity. Conditional knockout of p110β had no effect on lens size or clarity, and deletion of both the p110α and p110β subunits resulted in a phenotype that resembled the p110α single-knockout phenotype. Levels of activated AKT were decreased more in p110α- than in p110β-deficient lenses. A significant reduction in proliferating cells in the germinative zone was observed on postnatal day 0 in p110α knockout mice, which was temporally correlated with decreased lens volume.

Conclusions: These data suggest that the class 1A PI3K signaling pathway plays an important role in the regulation of lens size by influencing the extent and spatial location of cell proliferation in the perinatal period.

Figure 1

Deletion of p110α significantly reduced sizes of eyes and lenses. (A). There were no significant differences in overall body sizes among wild-type, p110α KO, p110β KO, and p110α/p110β double-KO animals up to 24 weeks of age (P > 0.05). (B) In contrast, eye mass was reduced 18% at 1 week and 22% at 12 weeks in p110α KO animals compared to that in wild-type littermates (P < 0.05). (C) Plotting lens volume versus age for all knockouts showed that p110α KO lenses were 27% smaller at 1 week and 26% smaller at 12 weeks than those of wild-type animals (P < 0.05). p110β KO lenses were similar to those of wild-type mice at all ages examined, and the size of p110α/p110β double-KO lenses resembled those of the p110α single-KOs. Data are mean ± SD; n = 11–46 animals of each genotype at each time point.

Figure 2

Loss of PI3K did not affect lens clarity. (A–D) Dissected wild-type lenses remained transparent for 1 to 24 weeks. (E–H) p110α KO. (I–L) p110β KO, and (M–P) p110α/p110β double-KO lenses also remained clear and free of cataracts throughout the time period examined, although p110α KO and p110α/p110β double-KO lenses were noticeably smaller than those of wild-type or p110β KO mice. Scale bar: (A): 1 mm.

Figure 3

PI3K knockout lenses lacked histological abnormalities. (A) On P7, sagittal sections through the central region of wild-type lenses appeared normal (200×). (B) p110α KO and (C) p110α/p110β double-KO lenses also lacked any structural anomalies, except they were smaller than those of wild-type mice. (D–F) Higher magnification (400×) views showed normal differentiation of lens fibers from equatorial epithelial cells in all three genotypes of lenses.

Figure 4

AKT signaling was reduced in PI3K KO lenses. (A) Wild-type lens epithelial cells expressed p110α, which was absent in KO mice. (B) Wild-type lenses also expressed p110β, which was lacking in lenses from KO mice. Levels of phosphorylated AKT were reduced in both p110α and p110β KO epithelial cells. Total AKT levels were similar in both wild-type and PI3K KO animals. (C) Independent blots (n = 3) were quantitated and plotted. Phospho-AKT-to-total AKT ratios were reduced 63% in p110α and 30% in p110β KO epithelial cells (P < 0.05). Data are mean ± SE and are derived from 2-week-old mice.

Figure 5

Distribution of proliferating cells is altered in p110α KO lenses. (A–F) Wild-type lenses displayed a characteristic pattern of EdU labeling between E14 and P2, with the greatest proliferation observed in the circumferential germinative zone near the lens equator. (G–J) Between E14 and E17, p110α KO lenses had a pattern of proliferation similar to that of wild-type littermates. (K) On P0, EdU staining in the circumferential germinative zone was greatly reduced in p110α KO mice. (L) On P2, the characteristic pattern of EdU labeling was restored in p110α KO lenses.

Figure 6

Quantification of germinative zone staining on P0 and P2. (A) On P0, p110α knockout lenses (red line) had peak fluorescent intensity values near the equator that were 41% lower than those in wild-type (black line [P < 0.05]). (B) By P2, there were clear peaks of EdU labeling near the equator of p110α-deficient lenses, and the maximum fluorescence was only 12% lower than that in wild-type (P > 0.05). Data are mean ± SE (gray lines) and aligned at the center of the lens. The diameters of p110α KO lenses were 11% to 12% smaller on P0 and P2. n = 6 lenses of each genotype at each time point.

Figure 7

Failure of germinative zone epithelial cells to proliferate on P0 in p110α knockout mice reduced lens volume. (A) Between E14 and E17, total EdU fluorescence levels for wild-type were similar to those for p110α KO lenses. On P0, EdU fluorescence was reduced 38% in p110α animals compared to that in wild-type mice. By P2, EdU fluorescence levels had partially recovered in p110α KO lenses, to a level 22% less than that in wild-type mice. (B) Calculation of lens volumes showed the greatest differences were observed on P0, when p110α KO lenses were 32% smaller than those of wild-type mice (P < 0.05). Data are mean ± SD. n = 8–23 lenses of each genotype at each time point.