Sprouty2-modulated Kras signaling rescues Shp2 deficiency during lens and lacrimal gland development

Abstract

Shp2/Ptpn11 tyrosine phosphatase is a general regulator of the RTK pathways. By genetic ablation, we demonstrate that Shp2 is required for lacrimal gland budding, lens cell proliferation, survival and differentiation. Shp2 deletion disrupted ERK signaling and cell cycle regulation, which could be partially compensated by activated Kras signaling, confirming that Ras signaling was the main downstream target of Shp2 in lens and lacrimal gland development. We also showed that Sprouty2, a general suppressor of Ras signaling, was regulated by Shp2 positively at the transcriptional level and negatively at the post-translational level. Only in the absence of Sprouty2 could activated Kras signaling robustly rescue the lens proliferation and lacrimal-gland-budding defects in the Shp2 mutants. We propose that the dynamic regulation of Sprouty by Shp2 might be important not only for modulating Ras signaling in lens and lacrimal gland development, but also for RTK signaling in general.

Fig. 1.

Shp2 is required for lens and lacrimal gland development. (A-D) The Le-Cre;Shp2^flox/flox mutant lens exhibited normal primary lens fiber cell elongation at E12.5 but reduced lens size at E14.5. (E-H) The anterior lens epithelial cells shifted posteriorly in the E15.5 Le-Cre;Shp2^flox/flox mutant lens (arrowheads), and no lacrimal gland was detected (arrows). (I,J) At P0, the Le-Cre;Shp2^flox/flox mutant lens was completely surrounded by epithelial cells (arrowheads), indicating a failure of lens epithelial cell differentiation. Scale bars: 100 μm. LE, lens epithelium; LF, lens fiber; LG, lacrimal gland.

Fig. 2.

Defective lens epithelial cell development in the Shp2 mutant lens. (A-H) Similar to wild type controls, the Le-Cre;Shp2^flox/flox mutant lens expressed Pax6 and Prox1 at E10.5, and α-, β- and γ-crystallins at E16.5. (I-L) The Pax6 and E-Cadherin positive lens epithelium encircled the Le-Cre;Shp2^flox/flox mutant lens, indicative of the failure of lens epithelial cells to differentiate into secondary lens fibers. (M-P) Cell proliferation and survival defects in the Le-Cre;Shp2^flox/flox mutant lens as shown by reduced BrdU incorporation and increased TUNEL staining. (Q) The ratio of BrdU-positive cells versus DAPI-positive cells decreased in the epithelial but increased in the fiber compartments of the E14.5 Le-Cre;Shp2^flox/flox lens (n=3) compared with the wild type (n=4) (*P<0.001). The dashed lines in M and N encircled the lens cells that were counted. (R) The percentage of TUNEL-positive cells increased in the E14.5 Le-Cre;Shp2^flox/flox mutant lens (n=5) compared with the wild type (n=3) (*P<0.0001). All lens cells within the dashed circle in O and P were counted. Scale bars: 100 μm.

Fig. 3.

Shp2 ablation disrupted ERK but not AKT signaling. (A-D) At E10.5 in the Shp2 mutant, both Shp2 mRNA and protein were lost in the lens vesicle (arrows) and the adjacent ectoderm destined to become lacrimal gland (arrowheads). (E-J) Phospho-ERK expression in the Shp2 mutant lens was preserved at E10.5, but abolished after E12.5 (arrows). (K-P) Shp2 deletion abolished the expression of the RTK responsive genes, Erm, Er81 and Pea3 in lens and lacrimal gland development. (Q) Western blot confirmed that phospho-ERK (pERK) but not phospho-AKT (pAKT) was lost in the E16.5 Shp2 mutant lens. Scale bars: 100 μm.

Fig. 4.

Kras activation partially compensated for the loss of Shp2. (A) The LSL-Kras^G12D allele can be activated when a LoxP sites-flanked transcriptional STOP cassette (LSL) is removed by the Le-Cre driver, which also inactivates the Shp2^flox allele in lens and lacrimal gland. This results in activated Kras signaling in the Shp2 mutant background. (B) Allele-specific genotyping revealed the loss of the LSL allele in the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutant lens but not in the tail, demonstrating the efficiency and specificity of the Le-Cre transgene in cleaving the STOP cassette. As a control, the Kras allele was unaffected. (C-E′) At E14.5, Kras activation led to increased lens size and slight bulging of lacrimal gland ectoderm (arrowheads). (F-H′) At E14.5, Kras^G12D mutation induced significant phospho-ERK expression in the lens (arrows), and to a lesser extent, in the lacrimal gland (arrowheads). (I-K′) RTK signaling response genes Erm and Pea3 were weakly but clearly induced in the lacrimal gland primordium by Kras activation (arrows). (L-Q) The Kras^G12D mutation also reversed the loss of Cyclin D1 and D3 expression in the Shp2 mutant lens, leading to increased phospho-Histone H3 (pHH3) and Ki67 expressions (arrows). (R-W) At E15.5, Cyclin D1, pHH3 and Ki67 expressions were observed in the small lacrimal gland bud in the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutants (highlighted by dashed lines). (X) Cell proliferation in the E15.5 lens and lacrimal gland as measured by the percentage of the Ki67- and pHH3-positive cells in the wild type (n=8), the Le-Cre;Shp2^flox/flox (n=6) and the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D (n=8) mutants (*P<0.001, **P<0.0001). Scale bars: 100 μm.

Fig. 5.

Shp2 controls Sprouty2 expression and tyrosine phosphorylation. (A-F) Sprouty2 mRNA expression (arrows, arrowheads) was lost in the Shp2 mutants but induced by the addition of the Kras^G12D allele. (G) The specificity of the Sprouty2 antibody was demonstrated in the western blot (IB) of the Spry2^KO/KO mutant lens. The same blot was also probed with the ERK antibody as a loading control. (H) Sprouty2 was immunoprecipitated (IP) from equal amounts of lens lysate from the newborn animals as shown by the β-actin western blots (Input). The western blots further confirmed the loss of Sprouty2 in the Le-Cre;Shp2^flox/flox mutants, the association of Shp2 with Sprouty2 in the wild-type controls and increased tyrosine phosphorylation of Sprouty2 in Le-Cre;Shp2^flox/flox;LSL-Kras^G12D compound mutants. As a control, no tyrosine phosphorylated Sprouty2 were detected in a non-specific IgG immunoprecipitate. Scale bars: 100 μm. HC, immunoglobulin heavy chain (50 kD); LC, immunoglobulin light chain (25 kD).

Fig. 6.

Sprouty2 ablation synergized with Kras activation to rescue the Shp2 mutant phenotype. (A,B) Western blot analysis with infrared fluorescence showed that the phospho-ERK (pERK) level in the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutant lens was lower than that in wild type or the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D;Spry2^flox/flox mutant. The relative pERK/ERK ratios were averaged from three independent sets of samples. (C-G) The lens phospho-ERK expression was lost in the Le-Cre;Shp2^flox/flox and the Le-Cre;Shp2^flox/flox;Spry2^flox/flox mutants, upregulated in the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutants, and fully recovered in the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D;Spry2^flox/flox mutants (arrows). (H-L) Sprouty2 deletion had no effect in the Le-Cre;Shp2^flox/flox mutant background, but when combined with the Kras^G12D mutation, it helped to restore the Shp2 lens growth and the normal lens epithelium cell migration pattern (arrows). (M-V) Similarly, the lacrimal gland phospho-ERK staining and outgrowth (arrows) was partially rescued by Kras activation alone and fully restored after additional Sprouty2 deletion. (W) Quantification of the lacrimal gland length and lens sizes at E15.5. [*P<0.001 for the Le-Cre;Shp2^flox/flox (n=5) or the Le-Cre;Shp2^flox/flox;Spry2^flox/flox mutants (n=7) compared with wild type (n=5) or the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutants (n=4); ** P<0.01 for the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D;Spry2^flox/flox mutants (n=4) compared with the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D mutants (n=5)]. Scale bars: 100 μm.

Fig. 7.

Model of Shp2-Ras-Sprouty2 signaling in lens and lacrimal gland development. In wild-type cells, Shp2 activates Ras-ERK signaling to induce Spry2 transcription but also suppresses Sprouty2 protein activity by tyrosine dephosphorylation, thus weakening the intensity of the Sprouty2-Ras negative feedback loop. In the Le-Cre;Shp2^flox/flox;LSL-Kras^G12D cells, the activated Kras^G12D mutant was still able to induce Spry2 transcription, but the Sprouty2 protein was hyperphosphorylated in the absence of Shp2 phosphatase, resulting in stronger suppression of Ras signaling.