Heme-Mediated Danger Signaling Suppresses Osteogenesis Through TLR4-Dependent Inhibition of β-Catenin Signaling

Extracellular heme is a damage-associated molecular pattern (DAMP) released during hemolysis, hemorrhage, and tissue injury. Although its pro-inflammatory and cytotoxic effects are well established, its impact on osteoblast differentiation and bone formation remains incompletely understood. Here, we investigated the effects of extracellular heme on osteoblast differentiation and defined the signaling pathways involved. In MC3T3-E1 pre-osteoblasts, heme suppressed alkaline phosphatase (ALP) activity, calcium deposition, matrix mineralization, and expression of osteogenic markers, including Runx2 and Sp7 at day 3, Col1a1 and Alpl at day 7, and Bsp and Bglap at day 10, without affecting cell viability. Mechanistically, heme reduced AKT phosphorylation and inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, consistent with increased GSK-3β activity. This was accompanied by decreased accumulation of transcriptionally active β-catenin, impaired nuclear β-catenin localization, and suppression of canonical Wnt target genes (Axin2, Lef1, Ccnd1, c-Myc). Pharmacological inhibition of GSK-3β partially restored β-catenin signaling and osteogenic differentiation, whereas inhibition of Toll-like receptor 4 (TLR4) attenuated heme-induced suppression of the AKT/GSK-3β pathway. Combined inhibition of TLR4 and GSK-3β produced the most pronounced restoration of osteogenesis in the presence of heme. Consistent with the in vitro findings, maternal heme administration during pregnancy significantly reduced ALP activity in multiple fetal craniofacial ossification centers in vivo. Collectively, these findings demonstrate that extracellular heme suppresses osteogenesis and supports a model in which TLR4-associated dysregulation of the AKT/GSK-3β/β-catenin axis contributes to impaired osteogenic differentiation.