Endothelial Fam20a Loss Drives Pulp Calcification Via Calcium-Dysregulated Paracrine

To elucidate the molecular mechanism of FAM20A deficiency in driving pathological Pulp Calcification (PC) and identify novel therapeutic targets for Enamel Renal Syndrome (ERS). FAM20A expression dynamics under mechanical stress and calcium stimulation were analyzed via RT-qPCR, Western blot, and immunofluorescence. Endothelial FAM20A-knockdown models were generated to assess calcium homeostasis and ectopic mineralization using calcium imaging and osteogenic induction. A HUVEC-hDPSCs coculture system combined with ALP/ARS staining and odonto/osteogenic marker assays revealed FAM20A's role in endothelial-mesenchymal crosstalk. Conditioned medium and pathway validation identified key secreted factors. Endothelial-specific Fam20a knockout mice were analyzed by micro-CT and histology to evaluate spatiotemporal calcification patterns. Mechanical stress and calcium upregulated FAM20A expression and secretion. While FAM20A silencing disrupted calcium homeostasis, it did not directly induce endothelial mineralization. Mechanistically, calcium dysregulation triggered endothelial BMP2/4/7 oversecretion, which activated Smad1/5/8, MAPK, and PI3K/AKT pathways in hDPSCs, enhancing their odonto/osteogenic differentiation. In vivo, endothelial Fam20a ablation caused pulp chamber malformation, progressive root canal stenosis, and spatiotemporally defined ectopic calcification. This study uncovers a "calcium dysregulation - paracrine signaling - ectopic mineralization" axis driven by FAM20A deficiency, establishing its pivotal role in endothelial-pulp mesenchymal crosstalk. We propose BMP signaling as a therapeutic target for ERS, offering a paradigm for cross-lineage regulation in pathological calcification.