Dysregulated ECM remodeling proteins lead to aberrant osteogenesis of Costello syndrome iPSCs.

Costello syndrome (CS) is an autosomal dominant disorder caused by mutations in HRAS. Although CS patients have skeletal abnormalities, the role of mutated HRAS in bone development remains unclear. Here, we use CS induced pluripotent stem cells (iPSCs) undergoing osteogenic differentiation to investigate how dysregulation of extracellular matrix (ECM) remodeling proteins contributes to impaired osteogenesis. Although CS patient-derived iPSCs develop normally to produce mesenchymal stem cells (MSCs), the resulting CS MSCs show defective osteogenesis with reduced alkaline phosphatase activity and lower levels of bone mineralization. We found that hyperactivation of SMAD3 signaling during the osteogenic differentiation of CS MSCs leads to aberrant expression of ECM remodeling proteins such as MMP13, TIMP1, and TIMP2. CS MSCs undergoing osteogenic differentiation also show reduced β-catenin signaling. Knockdown of TIMPs permits normal differentiation of CS MSCs into osteoblasts and enhances β-catenin signaling in a RUNX2-independent manner. Thus, this study demonstrates that enhanced TIMP expression induced by hyperactivated SMAD3 signaling impairs the osteogenic development of CS MSCs via an inactivation of β-catenin signaling.