Targeting the Gαi1/3–Gab1 Axis Enhances BMP2-Mediated Bone Regeneration

Background Bone morphogenetic protein 2 (BMP2) is a pivotal cytokine for skeletal formation, homeostasis, and regeneration. While its signaling through serine/threonine kinase receptors (BMPRI/II) and downstream pathways (PI3K-Akt-mTOR, Erk, Smad) is known to drive osteogenesis, the full mechanistic picture remains incomplete. Our prior work identified Gαi1 and Gαi3, inhibitory G protein alpha subunits, as key mediators for several receptor tyrosine kinase (RTK) signaling pathways. Their potential role in BMP2 signaling and bone formation, however, was undefined. Methods We employed a multi-modal approach combining clinical observation, genetic mouse models, and molecular/cellular techniques. Bone marrow mesenchymal stem cells (BMSCs) from young and elderly subjects, as well as from wild-type (WT) and Gαi1/3 double knockout (DKO) mice, were analyzed. In vitro , BMP2-induced signaling and osteogenic differentiation were assessed in BMSCs, MC3T3-E1 osteoblasts, and mouse embryonic fibroblasts (MEFs) following genetic manipulation (knockout, knockdown, overexpression) of Gαi1/3. In vivo effects were evaluated in Gαi1/3 DKO mice and a femoral fracture model using mice with BMSC-specific Gαi1/3 knockdown (Gαi1/3-CKD), treated with or without BMP2. Mechanistic studies involved co-immunoprecipitation, molecular docking, and analysis of the adaptor protein Gab1. Results Gαi1/3 expression was reduced in bone tissue and BMSCs from aged/osteoporotic subjects and mice. Gαi1/3 DKO mice exhibited delayed skeletal development, reduced bone mass, and impaired bone formation. In vitro , genetic ablation of Gαi1/3, but not Gαi2, abolished BMP2-induced activation of downstream Akt, Erk, and Smad pathways in MEFs, MC3T3-E1 cells, and BMSCs, and severely blunted BMP2-stimulated osteogenic differentiation and mineralization. Conversely, Gαi1/3 overexpression enhanced these effects. In the fracture model, BMP2 failed to promote healing in Gαi1/3-CKD mice. Mechanistically, Gαi1/3 were essential for BMP2-induced phosphorylation of the scaffolding protein Gab1. Gαi1/3 physically associated with BMP2 receptors (BMPR1A/B, BMPR2) and Gab1, facilitating this complex formation. Disrupting this interaction via dominant-negative Gαi1/3 mutants impaired downstream signaling and osteogenesis. Conclusions Our findings establish the Gαi1/3-Gab1 signaling axis as a critical and novel component of BMP2 receptor (serine/threonine kinase) signal transduction, essential for BMP2-induced osteogenesis both in vitro and in vivo . This work expands the functional repertoire of Gαi proteins beyond GPCRs and RTKs and identifies Gαi1/3 as potential therapeutic targets for enhancing bone formation and treating bone disorders like fractures and osteoporosis.