Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3–6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.
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The involvement of HIF isoforms varies in different cellular mechanisms associated with proliferative responses. Our inhibitory study demonstrated that a HIF-2α inhibitor, but not a HIF-1α inhibitor, decreased the hypoxia-mediated proliferation in HPFs, suggesting that hypoxia-mediated pulmonary fibroblast proliferation is HIF-2α-dependent. This finding was supported by knockdown experiments in which the knockdown of HIF-2α in IPF fibroblasts reduced cell proliferation under hypoxic conditions (3% O2)11. Furthermore, pulmonary arterial fibroblast proliferation is decreased by the knockdown of HIF-2α, but not HIF-1α, under moderate hypoxic conditions (1% O2)56. Interestingly, HIF-2α promotes cell proliferation in renal-cell adenocarcinoma cells (WT8, 786 –O RCC), whereas HIF-1α inhibits the proliferation of HCT116 colon carcinoma cells at 0.5% O29.