Ligand-independent tyrosine kinase signalling in RTH 149 trout hepatoma cells: comparison among heavy metals and pro-oxidants.
Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg(2+) and Cu(2+)in the microM range, and H(2)O(2) in the mM range, induced tyrosine phosphorylation. The effect of Cu(2+)was prevented by pre-incubation with genistein, while those of Hg(2+)and H(2)O(2) were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H(2)O(2), Hg(2+), and Cu(2+) plus low H(2)O(2). Cell pre-incubation with p38 inhibitors indicated that ERK activation by H(2)O(2) is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H(2)O(2) only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects.
Burlando
Bruno
B
Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale Amedeo Avogadro, Corso Borsalino 54, 15100 Alessandria, Italy. burlando@unipmn.it
Magnelli
Valeria
V
Panfoli
Isabella
I
Berti
Elena
E
Viarengo
Aldo
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003300:Copper / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019833:Genistein / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008628:Mercury / Q000494:pharmacology; D019216:Metals, Heavy / Q000494:pharmacology*; D008894:Milk Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D007093:Imidazoles; D008024:Ligands; D019216:Metals, Heavy; D008894:Milk Proteins; D016877:Oxidants; D011725:Pyridines; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D003300:Copper; D006861:Hydrogen Peroxide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D008628:Mercury; C093642:SB 203580; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole