Identificación y caracterización de las proteínas que interaccionan con SHOX y SHOX2

Zusammenfassung

SHOX encodes a transcription factor involved in skeletal l development. Alterations of SHOX and its regulatory elements have been reported in two skeletal dysplasias: Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD) as well as in a proportion of individuals with idiopathic short stature (ISS). We studied the clinical and molecular characteristics of 12 Spanish families with multiple members affected with LWD or LMD, who present the SHOX mutation p.A170P (c.508>GC) in heterozygosity or homozygosity, respectively. In all studied families, the p.A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the p.A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. In conclusion, we have identified p.A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals. SHOX2 is the human paralog of SHOX, presenting with a global homology of 83% and an identical homeodomain. Due to this similarity we set out to investigate if SHOX2 could also interact with SHOX interacting proteins. Using yeast two-hybrid assays and coimmunoprecipitation in human HEK293 cells, we demonstrated that SHOX2 interacts with SOX5, SOX6 and MAGED1 were identified. Subsequently we characterized the domains that participate in these interactions. The physiological importance of these interactions was investigated by studying the effect of SHOX2 on two SHOX transcriptional targets, ACAN and NPPB, important in skeletal growth. Our results show that SHOX2 can act as a cofactor and activate ACAN expression throught its interaction with trio SOX, whilst SHOX2 was able to activate directly the NPPB promoter. SHOX2 mutations were shown to reduce the activation of these genes. Finally, we observed the expression patterns of SHOX2, SHOX, SOX5, SOX6, SOX9 and MAGED1 in the human growth plate, in different gestational stages.