title: NPPB and ACAN, two novel SHOX2 transcription targets implicated in skeletal development.
abstract: SHOX and SHOX2 transcription factors are highly homologous, with even identical homeodomains. Genetic alterations in SHOX result in two skeletal dysplasias; Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), while no human genetic disease has been linked to date with SHOX2. SHOX2 is, though, involved in skeletal development, as shown by different knockout mice models. Due to the high homology between SHOX and SHOX2, and their functional redundancy during heart development, we postulated that SHOX2 might have the same transcriptional targets and cofactors as SHOX in limb development. We selected two SHOX transcription targets regulated by different mechanisms: 1) the natriuretic peptide precursor B gene (NPPB) involved in the endochondral ossification signalling and directly activated by SHOX; and 2) Aggrecan (ACAN), a major component of cartilage extracellular matrix, regulated by the cooperation of SHOX with the SOX trio (SOX5, SOX6 and SOX9) via the protein interaction between SOX5/SOX6 and SHOX. Using the luciferase assay we have demonstrated that SHOX2, like SHOX, regulates NPPB directly whilst activates ACAN via its cooperation with the SOX trio. Subsequently, we have identified and characterized the protein domains implicated in the SHOX2 dimerization and also its protein interaction with SOX5/SOX6 and SHOX using the yeast-two hybrid and co-immunoprecipitation assays. Immunohistochemistry of human fetal growth plates from different time points demonstrated that SHOX2 is coexpressed with SHOX and the members of the SOX trio. Despite these findings, no mutation was identified in SHOX2 in a cohort of 83 LWD patients with no known molecular defect, suggesting that SHOX2 alterations do not cause LWD. In conclusion, our work has identified the first cofactors and two new transcription targets of SHOX2 in limb development, and we hypothesize a time- and tissue-specific functional redundancy between SHOX and SHOX2.
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Aza-Carmona
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Veronica
authors.initials: V
authors.lastname: Barca-Tierno
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Hisado-Oliva
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Alberta
authors.initials: A
authors.lastname: Belinchón
authors.affiliation: Dept. Celular Biology, Immunology & Neurosciences, Facultad de Medicina, Universidad de Barcelona, Barcelona, Spain.
authors.forename: Darya
authors.initials: D
authors.lastname: Gorbenko-del Blanco
authors.affiliation: Department of Pathology, Hospital Universitario La Paz, Madrid, Spain.
authors.forename: Jose Ignacio
authors.initials: JI
authors.lastname: Rodriguez
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Benito-Sanz
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Campos-Barros
authors.affiliation: Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain ; Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto Carlos III, Madrid, Spain.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Heath
mesh_terms: D053674:Aggrecans / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics* / Q000378:metabolism; D001846:Bone Development / Q000235:genetics; D002460:Cell Line; D015331:Cohort Studies; D006130:Growth Disorders / Q000235:genetics; D006132:Growth Plate / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D020097:Natriuretic Peptide, Brain / Q000235:genetics* / Q000378:metabolism; D010009:Osteochondrodysplasias / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D055503:Protein Multimerization; D055747:SOX Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: C508319:ACAN protein, human; D053674:Aggrecans; D018398:Homeodomain Proteins; D020033:Protein Isoforms; C484664:SHOX2 protein, human; D055747:SOX Transcription Factors; D020097:Natriuretic Peptide, Brain; D009320:Atrial Natriuretic Factor