TBX5 : the missing culprit gene in thalidomide toxicity -

Abstract

Holt-Oram syndrome (HOS) is a rare autosomal dominant disease associated mainly with upper limb malformation and congenital heart defects (CHD) caused by a haploinsufficiency of T-box transcription factor 5 (TBX5). Congenital heart disease (CHD) is a leading cause of death, with an incidence of approximately 6–8 in 1,000 live births. Only 13 percent of all CHD cases are thought to be inherited and the rest are sporadic in nature. Some CHD are caused by environmental factors and teratogens like thalidomide. Thalidomide was synthesized in 1957 and marked as a sedative drug that was used by pregnant women to prevent morning sickness but it caused severe malformations in the newborns similar to those detected in HOS patients, thus it was removed from the market in 1961. Previous studies showed that TBX5 transcription was reduced as a response to thalidomide detected by semi-quantitative RT-PCRs on RNA extracted from wing buds of chicken embryos.We aimed to investigate the effect of thalidomide on TBX5, suggesting an interaction between them as the in-silico docking prediction showed. We used the electric mobility shift assay (EMSA) to confirm that thalidomide decreases the binding affinity between TBX5 protein and consensus sequence of T-box. While thalidomide didn’t affect the cellular localization or the protein stability of TBX5 as indicated by immuno-fluorescence and western blot respectively. Suppressed expression activity of vascular endothelial growth factor (VEGF) and atrial natriuretic factor (ANF) promoter was obtained in the presence of thalidomide assessed by luciferase assay. While thalidomide was neither able to suppress the interaction of TBX5 with GATA4 presented by VEGF promoter expression, nor affected this interaction on the protein level as shown by co-immunoprecipitation assay. Thalidomide could significantly suppress cellular proliferation and migration of embryonal rhabdomyosarcoma cells as indicated by the MTT and wound healing assays respectively, but it did not affect the endogenous