Aberrant Ras/MAPK signaling in skeletal development

Abstract

The Mitogen-activated protein kinase(MAPK) signaling pathway has been studied intensively in the context of neoplastic transformation. Other studies have focused on the roles of this pathway during development and have modeled syndromes, such as Noonan Syndrome (NS), that are caused by aberrant germline Ras/MAPK signaling. One hallmark of these developmental syndromes is a defect in the skeletal development of the patients. However, the in vivo role of MAPK signaling during bone development is still controversial, with some studies supporting a positive role of the MAPK pathway during this process, and others arguing for a suppressive role. To analyze in depth the bone defects caused by aberrant K-ras/MAPK signaling during development and to understand how bone mass is regulated via K-ras/MAPK control of osteoblast differentiation we have generated two novel mouse models. Specifically, to investigate how hyperactive K-ras affects skeletal development when expressed in a spatial and temporal specific manner we activated K-rasG12D at various stages during limb development, either in all mesenchymal lineages (Prx1-Cre;K-rasG12D mutants), or specifically in the osteoblasts (Osx1-Cre;K-rasG12D). The Prx1-Cre;K-rasG12D mutant mice display greatly shortened and thickened bones that mirror the defects found in patients with NS. We determined that this bone defect appears during embryogenesis around E14.5 and is distinguished by impairment in formation of the bone collar. Furthermore, we describe a treatment strategy that rescues the skeletal phenotype, and identify a narrow developmental time window, where in utero treatment with MEK inhibitor is sufficient to completely rescue the bone phenotype. The Osx1-Cre;K-rasG12D mutant mice allowed us to activate expression of hyperactive K-ras in the osteoblast population at different times during the development of the organism. Using this model, we defined two time windows when expression of active K-ras results in opposing bone phenotypes in the mutant mice. In the first time window (approximately between E11.5 and E14.5) induction of active K-rasG12D in osteoblasts precursors impairs terminal differentiation and leads to profound bone loss. In the second time window, activation of K-rasG12D at birth acts to promote osteoblast differentiation and consequently long bone mineralization. Taken together, our data has revealed a critical involvement of K-ras/MAPK signaling in osteoblast differentiation during skeletal development, in embryogenesis and after birth. They also show how the MAPK pathway can be modulated chemically to rescue the skeletal defects seen in a mouse model for NS. These findings yield insight into diseases of the bone, including both developmental syndromes caused by aberrant MAPK activation, such as NS, and diseases characterized by an imbalance in the bone mass, such as osteoporosis.