Exploration des mécanismes moléculaires de l'inhibition de la thromboxane synthase au cours de la différenciation et la minéralisation des ostéoblastes, et étude de la physiopathologie du variant pathogène d'IFITM5 dans l'ostéogenèse imparfaite de ty

Bone remodeling and repair involve coordinated osteoblast and osteoclast activity. The latter ensure bone resorption while osteoblasts play an anabolic role by producing a collagen-rich extracellular matrix and facilitating its mineralization by forming hydroxyapatite, the primary inorganic component providing bone rigidity and structural support. My thesis works focus on the effects of thromboxane A synthase (TBXAS) inhibition on the differentiation and mineralisation of osteoblasts, and on the pathophysiology of osteogenesis imperfecta type V ( OI type V). In the first part of the study, two selective inhibitors of TBXAS, ozagrel and picotamide were used based on the previous findings that ozagrel treatment reduced the receptor activator of nuclear factor kappa-B ligand (RANKL) expression and increased osteoprotegerin (OPG), thus favoring bone formation by limiting osteoclast differentiation. Firstly, we showed that ozagrel and picotamide treatments lead to increased mineralisation of mouse and human osteoblasts. To elucidate the molecular mechanisms underlying these effects, bulk RNA sequencing was performed on differentiating primary osteoblasts obtained from wild-type (WT) mouse calvaria treated with TBXAS inhibitors. Transcriptomic analysis revealed that TBXAS inhibition shifted cyclooxygenase (COX) metabolism towards prostaglandin (PG) synthesis. Among prostaglandins, PGE2 is well known to mediate mechanical cues in osteoblasts, promoting bone formation and strength, especially during bone remodeling and repair. Differentially expressed genes (DEGs) showed significant upregulation of the genes encoding mature osteoblast markers such as osteocalcin and periostin (Postn). Further analysis identified active cyclic AMP/GMP, Rho/ROCK, and MAP kinase signaling pathways. Moreover, angiogenic factors such as platelet-derived growth factor subunit B (Pdgfb) and vascular endothelial growth factor A (Vegfa) were significantly upregulated, indicating a positive effect of the treatment on angiogenesis, a crucial process for effective bone repair. RT-qPCR validation in differentiating mouse and human osteoblasts confirmed significant upregulation of osteoblast differentiation marker genes: COL1A1 (collagen type I), RUNX2 (runt-related transcription factor 2), POSTN (periostin), and BGLAP (osteocalcin) along with PG transporter and receptor genes such as Slco2a1, Abcc4, and PTGER2. These findings highlight the anabolic potential of TBXAS inhibitors, which could be exploited for treating bone fragility disorders like osteoporosis and OI. The second part of this study focused on OI type V, characterised by recurrent fractures, skeletal deformities and hyperplastic callus formation. These are attributed primarily to a recurrent gain-of-function mutation in the interferon-induced transmembrane protein 5 (IFITM5) gene with molecular mechanisms involved remain unknown. To study the underlying pathomechanisms, bulk RNA sequencing was performed on primary proliferative osteoblasts derived from bone biopsies of OI type V patients compared with controls. The analysis of DEGs revealed dysregulation of calcium signaling affecting calcineurin and phosphoinositide 3-kinase regulated pathways. This dysregulation resulted in the significant upregulation of RUNX2, known to be the master regulator of bone growth, and myocyte enhancer factor 2 (MEF2C), important for endochondral ossification. In addition to that downregulation of their known repressors histone deacetylase 4 and 5 (HDAC4 & HDAC5) point towards increase in their transcriptional activity. These findings align with elevated alkaline phosphatase activity and accelerated mineralization in patient osteoblast cultures, consistent with the hyperplastic phenotype observed in OI type V. In conclusion, this thesis work provides novel insights into the molecular pathways underlying TBXAS inhibition and physiopathology of IFITM5 pathogenic variant in osteoblasts.