TY - JOUR
T1 - Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways
AU - Mizuno, Shiro
AU - Bogaard, Herman J
AU - Voelkel, Norbert F
AU - Umeda, Yukihiro
AU - Kadowaki, Maiko
AU - Ameshima, Shingo
AU - Miyamori, Isamu
AU - Ishizaki, Takeshi
PY - 2009/3/6
Y1 - 2009/3/6
N2 - BACKGROUND: Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear.METHODS: Normal human lung fibroblasts (NHLF) were cultured in a hypoxic chamber or exposed to desferroxamine (DFX). DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis.RESULTS: DNA synthesis was increased by moderate hypoxia (2% oxygen) but was decreased by severe hypoxia (0.1% oxygen) and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection.CONCLUSION: These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.
AB - BACKGROUND: Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear.METHODS: Normal human lung fibroblasts (NHLF) were cultured in a hypoxic chamber or exposed to desferroxamine (DFX). DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis.RESULTS: DNA synthesis was increased by moderate hypoxia (2% oxygen) but was decreased by severe hypoxia (0.1% oxygen) and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection.CONCLUSION: These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.
KW - Cell Cycle
KW - Cell Hypoxia
KW - Cell Proliferation/drug effects
KW - Cells, Cultured
KW - Cyclin-Dependent Kinase Inhibitor p21/metabolism
KW - Cyclin-Dependent Kinase Inhibitor p27
KW - DNA Replication
KW - Deferoxamine/pharmacology
KW - Fibroblasts/drug effects
KW - Humans
KW - Intracellular Signaling Peptides and Proteins/metabolism
KW - Iron Chelating Agents/pharmacology
KW - Lung/drug effects
KW - Oxygen/metabolism
KW - RNA Interference
KW - RNA, Small Interfering/metabolism
KW - Signal Transduction/drug effects
KW - Transfection
KW - Tumor Suppressor Protein p53/genetics
U2 - 10.1186/1465-9921-10-17
DO - 10.1186/1465-9921-10-17
M3 - Article
C2 - 19267931
VL - 10
SP - 17
JO - Respiratory research
JF - Respiratory research
SN - 1465-9921
ER -