Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells.
Hepatocyte growth factor (HGF), a multifunctional cytokine of mesenchymal origin, activates the DNA binding of hypoxia inducible factor-1 (HIF-1) in the HepG2 cell line: the activated complex contained the inducible alpha subunit. An increased expression of HIF-1alpha (mRNA and nuclear protein levels) was observed. To investigate the molecular basis of the HIF-1 response under this non-hypoxic condition, we evaluated first the expression of putative target genes. We found a time-dependent increase in steady-state mRNA levels of heme oxygenase and urokinase plasminogen activator at 4 h, followed by that of urokinase receptor at 10 h. The enhanced expression of these genes might confer the invasive phenotype, since HGF is a proliferative and scatter factor. Second, we examined some aspects of HIF-1 activity regulation in HGF-treated cells with the following findings: (i) the activation of HIF-1 DNA binding was prevented by proteasome blockade, probably because stabilization of the cytosolic alpha-subunit protein level is not sufficient to generate a functional form: also under these conditions nuclear protein level of HIF-1alpha did not increase; (ii) N-acetylcysteine, a free radical scavenger, strongly decreased HIF-1 activation suggesting a role of reactive oxygen species in this process; (iii) the thiol reducing agent dithiothreitol was ineffective. Third, consistent with these data, N-acetylcysteine reduced the stimulatory effect of HGF on stress kinase activities, while p42/44 mitogen activated kinase (MAPK) was unmodified, suggesting an involvement of c-Jun-N-terminal kinase (JNK) and p38 MAPK in HIF-1 activation. Finally, LY 294002 induced the blockade of phosphatidylinositol 3-kinase (PI3K), one of the principal transducers of HGF/Met receptor signalling, prevented the enhancement of HIF-1 DNA binding and JNK activity, but the inhibition of p42/44 MAPK phosphorylation with PD 98059 was ineffective. In conclusion, we suggest that HGF triggers a signal transduction cascade involving PI3K and ultimately activates HIF-1.
Tacchini
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Institute of General Pathology and CNR Center for Research on Cell Pathology, University of Milan, via L.Mangiagalli, 31-20133 Milan, Italy.
Dansi
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Matteucci
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Desiderio
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D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000502:physiology; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics; D017228:Hepatocyte Growth Factor / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology; D009097:Multienzyme Complexes / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010084:Oxidation-Reduction; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics; D055293:Receptors, Urokinase Plasminogen Activator; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014157:Transcription Factors; D017228:Hepatocyte Growth Factor; D006419:Heme Oxygenase (Decyclizing); D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex