Distinct contributions of JNK and p38 to chromium cytotoxicity and inhibition of murine embryonic stem cell differentiation.
BACKGROUND
Potassium dichromate [Cr(VI)] is a widespread environmental toxicant responsible for increased risk of several human diseases. Cr(VI) exposure leads to activation of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK)1/2, p38, and extracellular-signal regulated kinase (ERK)1/2.


OBJECTIVE
We evaluated the contribution of MAPKs to Cr(VI) toxicity.


METHODS
Phosphorylation of MAPKs and their downstream effectors was evaluated by Western immunoblotting; reactive oxygen species were measured by DCFDA (5',6'-chloromethyl-2'-7'-dichlorofluorescin diacetate) labeling and flow cytometry, and glutathione and glutathione disulfide levels were determined by monochrome graphic spectroflurometer. Cytotoxicity was assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation. Embryoid body (EB) differentiation was evaluated by contracting cardiomyocyte formation, and real-time polymerase chain reaction (RT-PCR) was used for cardiomyocyte-specific and stem-cell-specific gene expression.


RESULTS
Acute treatment of mouse embryonic stem (ES) cells with 50 microM Cr(VI) induced the rapid phosphorylation of JNK, p38, and ERK and their respective downstream transcription factors, c-JUN, activating transcription factor-2, and ELK1. MAPK activation and cytotoxicity induction were partially blocked by pretreatment with the antioxidant N-acetyl cysteine. Ablation of the upstream MAP kinase kinase (MAP2K7) in ES cells prevented JNK activation, whereas ablation of MAP2K4 prevented both JNK and p38 activation. Using specific MAPK inhibitors and MAP2K4- and MAP2K7-deficient ES cells, we showed that JNK reduced acute Cr(VI) cytotoxicity, p38 potentiated it, and ERK had no effect. At low submicromolar concentrations, Cr(VI) caused MAP2K4/7-dependent JNK activation and MAP2K4-dependent p38 activation and strongly inhibited contracting cardiomyocyte development in wild-type ES cells, but much less so in Map2k7((-/-)) cells.


CONCLUSIONS
Each MAPK distinctly contributes to chromium toxicity. Whereas JNK prevents and p38 promotes acute cytotoxicity, JNK contributes to optimal inhibition of ES cell differentiation by chromium.
Chen
Liang
L
Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Ovesen
Jerald L
JL
Puga
Alvaro
A
Xia
Ying
Y
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D002857:Chromium / Q000633:toxicity*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D004785:Environmental Pollutants / Q000633:toxicity*; D004789:Enzyme Activation / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000502:physiology*; D048688:MAP Kinase Kinase 7 / Q000235:genetics / Q000502:physiology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000502:physiology*
D004785:Environmental Pollutants; D002857:Chromium; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; C482570:Map2k7 protein, mouse