HIF-1α drives the transcription of NOG to inhibit osteogenic differentiation of periodontal ligament stem cells in response to hypoxia.

Osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is limited in hypoxia, and HIF-1α is key to the response to hypoxia. However, its mechanisms remain largely unknown. This study discovered an osteogenesis-related gene sensitive to hypoxia in PDLSCs, and investigated the molecular mechanisms between HIF-1α and the gene. NOG, a gene that negatively regulates osteogenesis, was discovered by RNA-seq. Under normoxic conditions, HIF-1α overexpression led to enhanced expression of NOG/Noggin and inhibited the expression of osteogenesis-related genes, while inhibition of HIF-1α reversed this effect. The expression of HIF-1α, NOG/Noggin and the osteogenesis-related genes were detected by qRT-PCR or Western blot. Mechanistically, we verified that HIF-1α binds to the hypoxia response element (-1505 to -1502) in the promotor of NOG to enhance secretion of Noggin by chromatin immunoprecipitation and a dual-luciferase reporter assay. IHC staining findings in an animal model verified that Noggin-associated osteogenic differentiation was inhibited in hypoxia. NOG displayed a concordant relationship with HIF-1α, and secreted more with increasing of HIF-1α. Hypoxia stabilized HIF-1α, which bound to the HRE (-1505 to -1502) of the NOG promotor to enhance NOG transcription resulted in inhibiting osteogenic differentiation of PDLSCs. This study offers a promising therapy for periodontitis.

Transforming growth factor-ß1 and hypoxia inducible factor-1α synergistically inhibit the osteogenesis of periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) exhibit potential for osteogenesis in vitro and in vivo and are a candidate cell type for periodontal regeneration for the treatment of periodontitis. However, periodontitis is accompanied by hypoxia, and it is not clear how hypoxia affects the osteogenesis of PDLSCs. In this study, we found that the expression of hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1) is enhanced in the osteogenesis of PDLSCs under nonhypoxic conditions. TGF-ß1 can induce the stabilization of HIF-1α through the phosphorylation of mothers against decapentaplegic homolog 3 (Smad3) in PDLSCs, and in turn, HIF-1α inhibits the mRNA and protein expression of TGF-ß1 and inhibits the phosphorylation of Smad3 in PDLSCs. In addition, both HIF-1α and TGF-ß1 reduce the expression of crucial osteogenic gene runt-related transcription factor 2 (RUNX2) and the mineralization of PDLSCs in normoxia. In conclusion, our results showed that TGF-ß1 can induce the stabilization of HIF-1α in PDLSCs under nonhypoxic conditions, that HIF-1α can negatively regulate the TGF-ß1/Smad3 signal pathway in PDLSCs, and that TGF-ß1 and HIF-1α can synergistically inhibit the osteogenesis of PDLSCs.

Hypoxia Mediates Runt-Related Transcription Factor 2 Expression via Induction of Vascular Endothelial Growth Factor in Periodontal Ligament Stem Cells.

Periodontitis is characterized by the loss of periodontal tissues, especially alveolar bone. Common therapies cannot satisfactorily recover lost alveolar bone. Periodontal ligament stem cells (PDLSCs) possess the capacity of self-renewal and multilineage differentiation and are likely to recover lost alveolar bone. In addition, periodontitis is accompanied by hypoxia, and hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor in the response to hypoxia. Thus, we aimed to ascertain how hypoxia affects runt-related transcription factor 2 (RUNX2), a key osteogenic marker, in the osteogenesis of PDLSCs. In this study, we found that hypoxia enhanced the protein expression of HIF-1α, vascular endothelial growth factor (VEGF), and RUNX2 ex vivo and in situ. VEGF is a target gene of HIF-1α, and the increased expression of VEGF and RUNX2 proteins was enhanced by cobalt chloride (CoCl2, 100 µmol/L), an agonist of HIF-1α, and suppressed by 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1, 10 µmol/L), an antagonist of HIF-1α. In addition, VEGF could regulate the expression of RUNX2, as RUNX2 expression was enhanced by human VEGF (hVEGF165) and suppressed by VEGF siRNA. In addition, knocking down VEGF could decrease the expression of osteogenesis-related genes, i.e., RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1), and hypoxia could enhance the expression of ALP, COL1, and osteocalcin (OCN) in the early stage of osteogenesis of PDLSCs. Taken together, our results showed that hypoxia could mediate the expression of RUNX2 in PDLSCs via HIF-1α-induced VEGF and play a positive role in the early stage of osteogenesis of PDLSCs.