Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome

The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased cell growth in mutant osteoblasts compared with control cells. The mutation also caused increased alkaline phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associated with increased type I collagen expression. Mutant cells also showed increased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Tn ist mutant osteoblasts displayed a cell-autonomous reduction of osteocalcin mRNA expression in basal conditions and during osteogenesis. The data show that genetic deletion of Twist causing reduced Twist dosage increases cell gran th, collagen expression, and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossification induced by deletion of the bHLH Tn ist domain in Saethre-Chotzen syndrome.