SHOX interacts with the chondrogenic transcription factors SOX5 and SOX6 to activate the aggrecan enhancer

Abstract

SHOX (short stature homeobox-containing gene) encodes a transcription factor implicated in skeletal development. SHOX haploinsufficiency has been demonstrated in Leri-Weill dyschondrosteosis (LWD), a skeletal dysplasia associated with disproportionate short stature, as well as in a variable proportion of cases with idiopathic short stature (ISS). In order to gain insight into the SHOX signalling pathways, we performed a yeast two-hybrid screen to identify SHOX-interacting proteins. Two transcription factors, SOX5 and SOX6, were identified. Co-immunoprecipitation assays confirmed the existence of the SHOX-SOX5 and SHOX-SOX6 interactions in human cells, whereas immunohistochemical studies demonstrated the coexpression of these proteins in 18- and 32-week human fetal growth plates. The SHOX homeodomain and the SOX6 HMG domain were shown to be implicated in the SHOX-SOX6 interaction. Moreover, different SHOX missense mutations, identified in LWD and ISS patients, disrupted this interaction. The physiological importance of these interactions was investigated by studying the effect of SHOX on a transcriptional target of the SOX trio, Agc1, which encodes one of the main components of cartilage, aggrecan. Our results show that SHOX cooperates with SOX5/SOX6 and SOX9 in the activation of the upstream Agc1 enhancer and that SHOX mutations affect this activation. In conclusion, we have identified SOX5 and SOX6 as the first two SHOX-interacting proteins and have shown that this interaction regulates aggrecan expression, an essential factor in chondrogenesis and skeletal development.

Negative Regulation of Human Growth Hormone Gene Expression by Insulin Is Dependent on Hypoxia-inducible Factor Binding in Primary Non-tumor Pituitary Cells

“…54001) (35). Briefly, after treatment, cells were suspended in hypotonic buffer (20 mM HEPES, pH 7.5, 5 mM sodium fluoride, 10 M sodium molybdate, and 0.1 mM EDTA) containing phosphatase inhibitors and incubated on ice for 15 min.…”

Evidence for a Circadian Effect on the Reduction of Human Growth Hormone Gene Expression in Response to Excess Caloric Intake

“…Nuclear Protein Extraction and Detection-The EpiSeeker nuclear extraction kit was used according to the manufacturer's instructions (Abcam; ab113474) (40). Briefly, the cells were suspended in hypotonic buffer containing dithiothreitol and proteinase inhibitors on ice for 15 min.…”

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia

“…The preparations were incubated overnight at 41C with a rabbit polyclonal SHOX antibody. 20 The cells were then washed with PBS-T and incubated with the secondary antibody, Alexa fluor 488 goat anti-rabbit IgG (Commercial Molecular Probes, Invitrogen). The cells were subsequently washed again with PBS-T, counterstained with DAPI (Sigma-Aldrich) and mounted with Mowiol 4-88 (Sigma-Aldrich).…”

“…Subsequently, histological analysis was undertaken as previously described. 20 Controls for specific antibody staining were performed by (1) using preimmune serum from the rabbit immunized against the SHOX peptide, at a dilution of 1:1000; (2) replacing the primary antibody with PBS; (3) using a rabbit polyclonal IgG isotype control (Ab27472, Abcam, Cambridge, UK) at a dilution of 1:50 and (4) staining sections of a normal adult colon with the SHOX antibody. Sections were examined using a DM5500B fluorescent microscope (Leica), and photos were taken using a DFC360FX digital camera (Leica) and visualized using the LA software (Leica).…”