Ceramide-PKCζ signaling axis mediates burn-enhanced ectopic calcification during Achilles tendon healing

Burn injury frequently leads to heterotopic ossification (HO) during tendon healing, but the underlying molecular mechanisms remain poorly understood. This study investigated the role of the ceramide-protein kinase C zeta (PKCζ) signaling axis in burn-enhanced ectopic calcification during Achilles tendon healing and evaluated the therapeutic potential of PKCζ inhibition. A combined burn injury and Achilles tendon puncture model was established in C57BL/6 mice, with sham control, tendon puncture, and burn combined tendon puncture groups. Micro-computed tomography analysis revealed significantly increased bone volume at the calcaneal insertion site in burn-injured animals. Both total PKCζ and phosphorylated PKCζ expression were markedly elevated in the burn combined injury group. In vitro, C2-ceramide treatment significantly enhanced PKCζ activation and promoted osteogenic differentiation of Achilles tendon stem cells, as evidenced by increased alkaline phosphatase activity, calcium deposition, and upregulation of runt-related transcription factor 2 and osteopontin expression. Co-treatment with 2-acetyl-1,3-cyclopentanedione (ACPD), an atypical PKC (aPKC) inhibitor, effectively blocked ceramide-induced PKCζ activation and osteogenic differentiation. In vivo administration of the aPKC inhibitor significantly reduced ectopic calcification (bone volume on micro-CT) and decreased expression of PKCζ and osteogenic markers. These findings suggest that burn injury enhances ectopic calcification during Achilles tendon healing through activation of the ceramide-PKCζ signaling pathway, promoting aberrant osteogenic differentiation of tendon stem cells. Atypical PKC inhibition represents a potential therapeutic strategy for preventing burn-associated ectopic calcification.