Inhibition of Transforming Growth Factor β1/Smad3 Signaling Decreases Hypoxia‐Inducible Factor‐1α Protein Stability by Inducing Prolyl Hydroxylase 2 Expression in Human Periodontal Ligament Cells

Background: Hypoxia‐inducible factor‐1α (HIF‐1α), the α subunit of the heterodimeric transcription factor HIF‐1, maintains oxygen homeostasis by regulating gene expression. Under normoxic conditions, HIF‐1α expression is maintained at low steady‐state levels by the critical oxygen sensor prolyl hydroxylase 2 (PHD2). Transforming growth factor β1 (TGF‐β1) activates Smad3 signaling and contributes to HIF‐1α stabilization under normoxic conditions. In chronic periodontitis, HIF‐1α is expressed highly in gingival fibroblasts and upregulates inflammatory factor transcription, which promotes periodontal inflammation. Here, the authors investigated the effect of TGF‐β1/Smad3 signaling and its blockade by the specific inhibitor of Smad3 (SIS3) on HIF‐1α expression and stability in human periodontal ligament cells. Methods: The authors investigated the effect of TGF‐β1 on HIF‐1α protein stability using cycloheximide. Furthermore, they analyzed HIF‐1α expression, PHD2 expression, and Smad3 phosphorylation following TGF‐β1 stimulation in the presence or absence of SIS3. Results: The half‐life of HIF‐1α was prolonged in TGF‐β1–treated cells. TGF‐β1 treatment induced HIF‐1α gene expression and enhanced HIF‐1α protein stability while decreasing PHD2 expression and activating Smad3 phosphorylation. Notably, HIF‐1α protein expression was not detectable prior to TGF‐β1 stimulation. Furthermore, SIS3 treatment abrogated Smad3 phosphorylation, impaired TGF‐β1–induced HIF‐1α gene expression and protein stability, and stimulated TGF‐β1–mediated PHD2 inhibition. Conclusion: These results demonstrate that HIF‐1α transcription and protein synthesis are controlled by TGF‐β1/Smad3 signaling, whereas HIF‐1α protein stability is controlled by PHD2, which is regulated by TGF‐β1/Smad3 signaling.