Fibrous dysplasia (FD) is a bone mesenchymal stromal cells (BMSCs)-derived disorder caused bygene mutation, characterized by excessive fibrous tissue proliferation in bone and the formation of immature trabecular bone. Although impaired osteogenesis of BMSCs is central to FD pathogenesis, the underlying mechanism remains largely elusive. Here we demonstrate that hyperactivation of the cAMP-PRKA/PKA signaling axis disrupts mitochondrial homeostasis through impaired mitophagy, ultimately leading to diminished amorphous calcium phosphate (ACP) secretion and consequent mineralization failure in FD. Mechanistically, in FD BMSCs, PRKA activation inhibits DNM1L/DRP1 recruitment to mitochondria through phosphorylation at S637, thereby suppressing mitochondrial fission. Consequently, excessive mitochondrial fusion leads to an elevated mitochondrial membrane potential, impaired mitophagy, and diminished ACP release. Collectively, our findings reveal a novel signaling nexus linking cAMP-PRKA signaling, mitochondrial dynamics, and biomineralization processes in FD pathogenesis, providing critical insights into the molecular basis of this disorder.: ACP: amorphous calcium phosphate; ALPL: alkaline phosphatase, biomineralization associated; Baf A1: bafilomycin A1; BGLAP/osteocalcin: bone gamma-carboxylglutamate protein; BMD: bone mineral density; BMSCs: bone marrow stromal cells; BV/TV: bone volume/tissue volume; cAMP: cyclic adenosine monophosphate; COL1: collagen type I; COX4/COXIV: cytochrome c oxidase subunit 4; CsA: cyclosporin A; CT: computerized tomography; Cyto: cytoplasm; DAPI: diamino phenylindole; DEPs: differentially expressed proteins; DNM1L/DRP1: dynamin 1 like; ECL: electrochemiluminescence; ECM: extracellular matrix; FBS: fetal bovine serum; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; FD: fibrous dysplasia; GNAS/guanine nucleotide-binding protein alpha-stimulating activity peptide: GNAS complex locus; GTPase: guanosine triphosphatase; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; Lyso: lysosome; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi: mitochondrial division inhibitor 1; MEMα: minimum essential medium α; MFN1: mitofusin 1; MFN2: mitofusin 2; Mito: mitochondria; MMP: mitochondrial membrane potential; MOI: multiplicity of infection; MV: matrix vesicle; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PHEX: phosphate regulating endopeptidase X-Linked; PHOSPHO1: phosphoethanolamine/phosphocholine phosphatase 1; PINK1: PTEN induced kinase 1; PRKA/protein kinase A: protein kinase cAMP-activated; PVDF: polyvinylidene fluoride; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: scanning electron microscopy; SOST: sclerostin; SP7/Osterix: Sp7 transcription factor; TA9: tyrphostin A9; Tb.N: trabecular. number; Tb.Sp: trabecular. separation; Tb.Th: trabecular. thickness; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine; UA: urolithin A. GNAS Abbreviation
