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

Abstract

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. 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. 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. 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.