Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation
Peng-Chieh Chen, … , Jonathan G. Seidman, Raju Kucherlapati
Peng-Chieh Chen, … , Jonathan G. Seidman, Raju Kucherlapati
Published November 1, 2010
Citation Information: J Clin Invest. 2010;120(12):4353-4365. https://doi.org/10.1172/JCI43910.
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Research Article Genetics

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation

Abstract

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%–15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated phenotypes, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS.

Authors

Peng-Chieh Chen, Hiroko Wakimoto, David Conner, Toshiyuki Araki, Tao Yuan, Amy Roberts, Christine E. Seidman, Roderick Bronson, Benjamin G. Neel, Jonathan G. Seidman, Raju Kucherlapati

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Figure 1

Generation of knock-in mice expressing Sos1EK.

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Generation of knock-in mice expressing Sos1EK. (A) Schematic represe...
(A) Schematic representation of unidirectional targeting of Sos1EK in exon 16. When Cre recombinase is present, inversions occur either between the wloxP sites or the mloxP sites at opposite orientations, generating loxP sites with identical orientation. Subsequently, Cre recombinase mediates the excision between wloxP or mloxP sites in the same orientation, resulting in a targeted exon bearing the E846K mutation (solid triangles, wloxP; open triangles, mloxP; ovals, FRT site). Exons are indicated by rectangles. Primer pairs indicated by red, blue, green, and purple arrows were used in long-range PCR to screen positive ES cell clones (see Methods and Supplemental Figure 6). (B) Results of PCR of 2 ES cell clones with (and 1 clone without) Ad-Cre infection, showing that the desired modification has been achieved, using primer pairs indicated by orange arrows in A. M, DNA marker. Numbers indicate the size of PCR bands in kilo-base pairs (kb). (C) Sos1EK activates Erk1/2 in 2 ES cell clones after Ad-Cre infection. ES cells were starved and stimulated with EGF (25 ng/ml) and analyzed by immunoblotting. (D) PCR results for genotyping the Sos1EK-targeted locus, using primer pairs indicated by brown arrows in A. (E) Sequencing results of the RT-PCR of WT and Sos1+/EK RNAs. The asterisk indicates the E846K mutation. (F) Immunoblotting showing equal amount of Sos1 proteins in tissues of all genotypes (1, WT; 2, Sos1+/EK; 3, Sos1EK/EK). (G) Kaplan-Meier survival curves of the mice. WT, n = 92; Sos1+/EK, n = 137; P = 0.02 versus WT. Sos1EK/EK, n = 4; P < 0.001 versus WT.