Mesenchymal expression of activated K-ras yields Noonan Syndrome-like bone defects that are rescued by mid-gestational MEK inhibition.

Abstract

Activating germline K-ras mutations cause Noonan syndrome (NS), which is characterized by several developmental deficits including cardiac defects, cognitive delays and skeletal abnormalities. NS patients have increased signaling through the MAPK pathway. To model NS skeletal defects and understand the effect of hyperactive K-ras signaling on normal limb development, we generated a mouse model in which activated K-rasG12D was expressed specifically in mesenchymal progenitors of the limb bud. These mice display short, abnormally mineralized long bones that phenocopy those of NS patients. This defect was first apparent at E14.5, and was characterized by a delay in bone collar formation. Coincident mutation of p53 had no effect on the K-rasG12D induced bone defect, arguing that it is does not result from senescence or apoptosis. Instead, our data revealed profound defects in the development of the committed osteoblasts; their appearance is delayed, concordant with the delay in bone collar formation, and they display an aberrant localization outside of the bone shaft. Additionally, we see growth plate defects including a reduction in the hypertrophic chondrocyte layer. Most importantly, we found that in utero delivery of a MEK inhibitor between E10.5 and E14.5 is sufficient to completely suppress the ability of activated K-ras to induce NS-like long bone defects in embryogenesis. These data define a critical point in mid-gestation in which elevated MAPK signaling impairs growth plate and bone collar formation and yield NS-like limb defects. Moreover, they offer insight into possible therapeutic strategies for skeletal defects in patients with Noonan Syndrome.\n\nSIGNIFICANCE STATEMENTNoonan syndrome is a genetic condition that is characterized by various developmental defects including skeletal abnormalities that lead to short stature. These patients carry mutations that activate Ras/MAPK signaling. We have generated a mouse model that recapitulates these Noonan Syndrome-like bone defects. Analysis of these animals establishes the developmental window in which bone formation goes awry, and reveals disruption of an early event that is critical for the longitudinal growth of bones. Additionally, we show that treatment with an inhibitor of Ras/MAPK signaling during this key developmental window is sufficient to completely suppress these Noonan Syndrome-like bone defects. This offers possible therapeutic strategies for skeletal defects in patients with Noonan Syndrome.