Hypoxia induces BMP-2 expression via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts.
It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP-2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP-2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG-63, hFOB and bone marrow stromal cells M2-10B4. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF-1 protein. The binding of HIF-1alpha to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia-induced HIF-1alpha activation and subsequent BMP-2 up-regulation. Taken together, our results provide evidence that hypoxia enhances BMP-2 expression in osteoblasts by an HIF-1alpha-dependent mechanism involving the activation of ILK/Akt and mTOR pathways.
Graduate Institute of Sports and Health, National Changhua University of Education, Changhua County, Taiwan.
Wen-Pei
WP
Tseng
Shu-Ning
SN
Yang
Chih-Ho
CH
Lai
Chih-Hsin
CH
Tang
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D015854:Up-Regulation; D000099200:Scaffold Protein ILK
D055396:Bone Morphogenetic Protein 2; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C525718:BMP2 protein, human; C546842:MTOR protein, human; C546843:mTOR protein, mouse