（ 様 式 ３ ）論文

要旨Hif-1α Expression is essential for BMP9-mediated Osteoblast Differentiation
through the induction of a Glycolytic Enzyme, PDK1Muhammad Subhan AmirOBJECTIVE. Tissue engineering of bone has received much interest, as bone is one of the tissues
with the highest regenerative potential in human body. Bone healing or regeneration involves anintricate network of molecules including bone morphogenetic proteins (BMPs). Recent reports
showed that BMP-9 has the strongest osteoinductive potential among BMP members, and it may
be considered as a useful cytokine for bone regeneration therapy. In recent years, the influence ofoxygen tension on bone functions has become a major research focus. Changes in oxygen
concentrations act as a signal that regulates expression of various hypoxia-responsive genes
through Hypoxia inducible factor (Hif) pathway. Hif-1α is a well-established hypoxia-responsive
transcription factor inducing the gene expression of many angiogenic and glycolytic proteins.
Notably, Hif-1α often coordinates various developmental processes of the human body, including
skeletal development. However, it is not clear if Hif-1α exerts any effect on BMP9-induced
osteogenic differentiation. In this study, we aimed to investigate the functional roles of Hif-1α inthe signaling pathway of BMP9-induced osteoblast differentiation.

METHODS. MC3T3-E1 cells (a mouse osteoblast cell line) were stimulated with recombinant
BMP9 or BMP2 for the time course analysis of Hif-1 protein expression. We then stimulated
MC3T3-E1 cells with BMP9 under normoxic or hypoxic (2% O2) condition with or without
Chrysin (PHD activator) or NSC697923 (Ubc13 inhibitor). Hif-1α and PDK1 gene knockdown
was performed by transfecting MC3T3-E1 cells with Hif-1α -or PDK1-specific siRNA. Protein
and mRNA expression levels of Hif-1, glycolytic, angiogenic and osteogenic genes were analyzed
by Western blotting and real-time PCR, respectively.

RESULTS. Hif-1α protein expression was significantly induced by both BMP9 and BMP2 in
MC3T3-E1 cells within 2 hours. Combination of BMP9 and hypoxia resulted in synergistic
increase of Hif-1α protein level. Although Chrysin inhibited both BMP9- and hypoxia-induced Hif1α protein expressions, NSC697923 failed to inhibit BMP9-induced HIF1α protein expression.
When Hif-1α expression was knocked down by siRNA, the mRNA expression level of a glycolytic
enzymes, PDK1and LDHa, but not that of ALP, Runx2, Osterix, or VEGF, was significantlyinhibited in BMP9-stimulated MC3T3-E1 cells. We then assessed the roles of Hif-1a and PDK1 inBMP9-mediated osteoblast differentiation by transducing Hif-1a-specific, PDK-1 specific, orcontrol siRNA into MC3T3-E1 cells, followed by BMP9 stimulation for 3 days to induce
osteoblastic differentiation. Significant decrease of ALP mRNA level, but not that of Runx2 orOsterix, was detected with both Hif-1 and PDK1 siRNA treatments, indicating that Hif-1 andPDK-1 play important roles in BMP9-induced osteoblast differentiation in a manner independentof Runx2 or Osterix.
DISCUSSION. Here, we have revealed for the first time that the protein expression of Hif-1α is
rapidly induced by osteogenic BMPs under normoxic condition. The Hif-1α induction mechanismby BMP9 seems to differ from that by hypoxia. Unexpectedly, Hif-1α expression is essential forthe induction of glycolytic enzymes, PDK1 and LDHa, but not that of an angiogenic cytokine
VEGF, in BMP9-stimulated osteoblasts. Being consistent with previous reports that increased
glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that Hif-1αexpression is important for BMP9-mediated osteoblast differentiation through the induction ofPDK1.