Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways.
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.


CONCLUSIONS
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.
Mizuno
Shiro
S
Third Department of Internal Medicine, University of Fukui, Yoshida-gun, Fukui, Japan. shirotan@qf6.so-net.ne.jp
Bogaard
Herman J
HJ
Voelkel
Norbert F
NF
Umeda
Yukihiro
Y
Kadowaki
Maiko
M
Ameshima
Shingo
S
Miyamori
Isamu
I
Ishizaki
Takeshi
T
D002453:Cell Cycle; D015687:Cell Hypoxia; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004261:DNA Replication; D003676:Deferoxamine / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007502:Iron Chelating Agents / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D047908:Intracellular Signaling Peptides and Proteins; D007502:Iron Chelating Agents; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003676:Deferoxamine; D010100:Oxygen