Effets de l'estradiol et du chargement mécanique sur la régulation de la POC5 et du récepteur ADGRG7 dans la scoliose idiopathique

Scoliosis is a complex three-dimensional deformity of the spine, with 1.5–3% prevalence in the general population. The most commonly known type of scoliosis is idiopathic scoliosis (IS), including adolescent idiopathic scoliosis (AIS) affecting principally girls during puberty. The etiology is largely unknown, but clinical observations revealed the role of hereditary and rapid growth in the development of this condition. There exists strong evidence that there is a genetic component to the disease. More recently, several genes were suspected to cause or contribute to IS. Our group identified gene variants of POC5 centriolar protein in a large French family with multiple members affected with IS. In the same family, we suspected the involvement of ADGRG7 (an orphan receptor that belongs to the Adhesion G protein-coupled receptors) gene mutation in the pathogenicity of IS.  In the present work, we focused on elucidating the role of wild type (wt) and mutant (mut) POC5 proteins (in vitro and in vivo) as well as the regulation of POC5 and ADGRG7 by estradiol (E2), with the goal to test whether these genes could be functionally connected with scoliosis.  To investigate the role of POC5 gene in IS, we overexpressed mutant POC5 in cell lines by transient transfection (in vitro study) and created a loss-of-function model in zebrafish (in vivo study). The role of POC5 was investigated by: 1) mass spectroscopy analysis and co-immunoprecipitation to identify differences in binding partners between the wt POC5 and mut POC5 proteins; 2) immunolocalization of wt and mut POC5 proteins at the cellular level; 3) histology and immunohistochemistry performed on tissues from wt (control) and scoliotic (poc5 mut) zebrafish. Our work identified several interacting partners with POC5, and documented functional connections with respect to cilia and centrosome dysfunction. A number of ciliary proteins were identified to be interacting with wt POC5 but not mut POC5 like CEP290, RAB11, CKAP5, Annexin 2 and Septin 9. At the cellular level, localization and co-localisation of wt POC5 and mut POC5 protein with alpha acetylated tubulin (cilia marker), confirmed the consequence of the mutation on subcellular location with respect to cilium length and staining intensity. In vivo, several defects in the retina of zebrafish and inner ear were identified in mutpoc5 zebrafish compared to wt zebrafish. Finally, using different markers for retinal layers and alpha acetylated tubulin, the defects were localized in the outer segment layer and cones of the retina.   To further investigate the possible roles of POC5 and ADGRG7 in IS, we examined how  POC5 and ADGRG7 are regulated at the transcriptional level. Human osteoblasts derived from control and AIS patients were used as a cell model, and the expression of POC5 and ADGRG7 protein was monitored upon E2 stimulation. The promoter region of the human POC5 and ADGRG7 gene was then cloned and analyzed for functional cis-elements mediating effects of E2. Deletion analysis of the ADGRG7 promoter indicates that the SP1 site in the 474bp fragment is required for both basal activity and hormone-induced activation, and mutations of the binding sites within this region result in the loss of transactivation. Further results from chromatin immunoprecipitation (ChIP) assay showed that SP1 and ESRα bind to ADGRG7 promoter. Our results suggest that the regulation of ADGRG7 expression by E2 is due to the association of ESRα and SP1 proteins to ADGRG7 promoter. The same experimental strategy was applied for studying the POC5 regulation by E2. Deletion analysis and ChIP confirmed the regulation of POC5 through ESRα. Through promoter studies, qPCR, and western blot and (ChIP) assay, this study clarifies how POC5 and ADGRG7 are regulated by E2.  Another aspect of this project was to study the differential effects of mechanical stress on wt and mut POC5 expressing cells, including human osteoblasts. Cells were exposed to mechanical stress for different time points and then different signalling pathways (including ERK, p38, NFκB) were tested. Our results show that there is difference in the response of control normal cells and cells overexpressing the mut POC5. The effects of loading including the signalling pathways were found to be initiated through TRPV4 which is a stretch-activated Ca2+- permeable channel and localizes to the primary cilium and plasma membrane.  The importance of this work is that it covers several factors that contribute to the pathogenesis of AIS. Based on our findings, AIS is a complex multifactorial disease where POC5, cilia, E2 and mechanical load intreplay in the pathogenesis of the disease. The significance of this work is that it puts the basics for understanding the molecular mechanisms that are implicated in AIS.

Keyphrases: Estradiol (E2), Cilia, Adolescent Idiopatic Scoliosis, POC5, Estrogen receptor, Mechanical stress, ADGRG7, TRPV4, E2