Hypoxic downregulation of cellular proliferation and loss of phenotype stability in human osteoblasts is mediated by HIF-1α.
Both, skeletal development and fracture healing depend on an orchestrated sequence of cellular growth and differentiation processes. Regional changes in tissue oxygen tension were proposed as key regulators of osteoblast proliferation and phenotype. Hypoxia results in the stabilization of hypoxia-inducible factor-1α (HIF-1α), thus influencing expression of a multitude of genes required for cellular adaptation. In the present study we dissected the effects of HIF-1α on cellular proliferation and gene expression of primary human osteoblasts. Primary human osteoblasts were studied by transfecting siRNA and plasmids coding for human HIF-1α. Gene expression was analyzed by western blot and quantitative PCR. Functional assays were performed to study HIF-1α function, i.e. proliferation and cell cycle analysis. As previously reported exposure to hypoxia led to a stabilization of HIF-1α on protein level and resulted in reduced rates of proliferation and osteocalcin expression. Furthermore, the expression of the proproliferative gene survivin was significantly reduced (p < 0.01). Knock down of HIF-1α attenuated hypoxic downregulation of proliferation (p < 0.05), and osteocalcin (p < 0.05) as well as survivin (p < 0.05) expression significantly. Importantly, the isolated overexpression of HIF-1α impaired proliferative activity and led to significantly reduced rates of expression of osteocalcin (p < 0.05) and survivin (p < 0.01). The present study shows that HIF-1α might reduce proliferation and survivin expression in primary human osteoblasts independently from cellular hypoxia. Furthermore, HIF-1α promoted the loss of the characteristic osteoblastic marker, osteocalcin in vitro. These findings underline the important role of HIF-1α in bone physiology and pathophysiology. Modulating HIF-1α function in hypoxic environments could be of value for future therapeutic approaches.
Department of Orthopedic Surgery, University of Regensburg, Asklepios Klinikum Bad Abbach, Bad Abbach, Germany. p.lechler@asklepios.com
Philipp
P
Lechler
Silvan M
SM
Klein
Lukas
L
Prantl
Carsten
C
Englert
Tobias
T
Renkawitz
Joachim
J
Grifka
D000368:Aged; D000369:Aged, 80 and over; D002455:Cell Division / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051034:Inhibitor of Apoptosis Proteins / Q000096:biosynthesis / Q000235:genetics; D008875:Middle Aged; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D010100:Oxygen / Q000494:pharmacology; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051034:Inhibitor of Apoptosis Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D000077022:Survivin; D015675:Osteocalcin; D010100:Oxygen