HIF-1α deficiency impairs mandibular condylar osteochondral maturation and triggers progenitor hyperproliferation via dysregulated mTOR-cytoophidia dynamics

Mandibular condylar cartilage is a secondary cartilage with articular and growth functions. The role of hypoxia-inducible factor-1α (HIF-1α) in Osterix (Osx)-lineage cells during condylar development remains unclear. We investigated their developmental contribution and the function of HIF-1α in this lineage.

Genetic lineage tracing was performed using Osx-Cre; Rosa26-tdTomato mice. HIF-1α conditional knockout mice (HIF-1α^flox/flox; Osx-Cre) were generated to examine the role of HIF-1α in Osx-lineage cells. Histological, molecular, and micro-computed tomography analyses were used to assess condylar development. In vivo and in vitro approaches were employed to investigate chondrocyte proliferation, differentiation, and the underlying mechanisms.

Lineage tracing identified tdTomato⁺ Osx-lineage cells in condylar cartilage from E14.5 and demonstrated a developmental shift from chondrogenic toward osteogenic compartments. The proportion of tdTomato⁺ cells associated with COL II-positive cartilage decreased from 76.41% at E14.5 to 51.81% at 2 weeks (mean difference, -24.60 percentage points; 95% CI, -35.42 to -13.79), whereas the proportion associated with OCN-positive osteoblasts increased from 42.45% at 4 weeks to 76.62% at 6 weeks (mean difference, 34.17 percentage points; 95% CI, 25.66 to 42.67; n=4 mice per stage). Compared with controls, knockout mice showed lower type II collagen-positive cartilage area at 8 weeks (group means, 4.78% versus 44.73%; mean difference, -39.95 percentage points; 95% CI, -43.39 to -36.52), lower subchondral bone volume fraction at 4 weeks (35.88% versus 39.01%; mean difference, -3.14 percentage points; 95% CI, -5.12 to -1.15), and greater cartilage EdU labeling at 3 weeks (13.86% versus 3.47%; mean difference, 10.39 percentage points; 95% CI, 9.01 to 11.78; n = 4 mice per group for each comparison). In ATDC5 cells, HIF-1α knockdown increased mTOR-S6K1 signaling and altered cytoophidia organization. Rapamycin reduced EdU labeling in HIF-1α-knockdown cells (group means, 24.65% versus 34.92%; mean difference, -10.27 percentage points; 95% CI, -15.36 to -5.17; n = 3 independent experiments per group).

HIF-1α signaling in Osx-lineage progenitors is essential for the establishment and maturation of the mandibular condylar osteochondral unit. Beyond its established role in skeletal differentiation, HIF-1α appears to regulate progenitor proliferative behavior through mTOR-associated cytoophidia dynamics, suggesting a metabolic mechanism underlying mandibular condyle morphogenesis.