This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.PROBLEM: Craniosynostosis is characterized by premature cranial suture fusion with a prevalence of 1 per 2500 live births. The clinical consequences are mild to severe craniofacial deformities, mental retardation, blindness and even death. Surgical management typically involves several extremely invasive procedures beginning with suturectomy of the synostosed sutures in infancy. These suturectomy sites often show rapid and extensive bone formation and eventually resynostosis. This requires multiple re-operations which dramatically increase patient morbidity and mortality. Transforming growth factor-3 (Tgf-beta3) is an important regulator of suture fusion. Abnormally low Tgf-beta3 has been noted in the cranial sutures of humans with familial, non-syndromic craniosynostosis. Additionally, studies have shown that increased exposure to Tgf-beta3 prevents cranial suture fusion both in vitro and in vivo. Exogenous application of Tgf-beta 3 cytokine has been shown to inhibit cranial suture fusion in wild type murine models and in craniosynostotic rabbits. It is necessary to establish the mechanism by which Tgf-beta3 rescues the cranial suture fusion before this can be considered for therapeutic applications.Tgf-beta3 induced suture maintenance is possibly mediated by upregulation of apoptosis and decreased levels of cell proliferation. Overexpression of pro-apoptotic Bax was seen in the sutural tissues rescued by the Tgf-beta3 and increased expression of Bcl-2 is seen in the untreated fusing or fused rat posterior frontal sutures. Mechanisms by which osteoblasts are directed toward apoptosis are not entirely clear and the signaling pathways by which Tgf-beta3 induce the programmed cell death remain largely unknown. Recent studies have shown the importance of canonical Wnt signaling in the control of osteoblast and osteocyte programmed cell death. The purpose of the proposed study is to determine the effects of Tgf-beta3 on the programmed cell death of rat calvarial osteoblasts and to elucidate the signaling pathways that mediate the control of apoptosis by Tgf-beta3 in rat calvarial osteoblasts. In the proposed study we will test the following HYPOTHESIS: Tgf-beta3 promotes apoptosis in rat calvarial osteoblasts by antagonizing canonical Wnt signaling via upregulation of Secreted frizzled related protein-1(sFRP-1). We will test this hypothesis with the following SPECIFIC AIMS: 1. Determine the effect of Tgf-beta3 on apoptosis of rat calvarial osteoblasts 2. Investigate the effect of Tgf-beta3 treatment on sFRP-1 expression in rat calvarial osteoblasts 3. Determine the ability of sFRP-1 to upregulate the rat osteoblast apoptosis via induction of one or more apoptotic genes and/or downregulation of one or more anti-apoptotic genes. 4. Investigate the effect of sFRP-1 modulation on canonical Wnt signaling. SIGNIFICANCE: Results from the proposed study will provide a molecular basis for Tgf-beta3 induced maintenance of suture patency. This will help to develop a cytokine-based adjunct therapy which could allow for earlier and less radical surgical interventions. This would decrease complications inherent in these high risk procedures, prevent post-surgical resynostosis, obviate the need for multiple surgeries, and greatly improve treatment outcomes for children with craniosynostosis.
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