TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression.
BACKGROUND
Although the pathogenic relevance of transforming growth factor-beta (TGF-beta) to glomerular sclerosis has been established, the intracellular mechanisms by which TGF-beta induces extracellular matrix accumulation are not fully understood. We examined whether the mitogen-activated protein (MAP) kinase pathway is involved in TGF-beta1-induced collagen expression by cultured human mesangial cells.


METHODS
The activation of MAP kinase pathways by TGF-beta1 was assessed by immunoblot with anti-phospho-ERK or -JNK antibodies and by transfection of plasmids expressing pathway-specific transcription activators fused to the DNA-binding domain of GAL4, as well as a GAL4 response element-luciferase reporter gene. The role of MAP kinase was assessed using biochemical inhibitors and transiently expressed dominant negative mutant constructs. The effects on TGF-beta1-induced alpha1(I) collagen expression were evaluated by Northern blot and by activation of a transiently transfected alpha1(I) promoter-luciferase reporter construct.


RESULTS
ERK and JNK phosphorylation occurred 30 minutes and one hour, respectively, after TGF-beta1 treatment. A biochemical blockade of the ERK pathway inhibited TGF-beta1-induced alpha1(I) collagen expression. A dominant negative mutant of ERK1 but not of JNK decreased alpha1(I) gene promoter activation. Activation of the TGF-beta-responsive p3TP-Lux construct was partially inhibited by cotransfection of an ERK1 dominant negative mutant.


CONCLUSIONS
These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.
Hayashida
T
T
Department of Pediatrics, Chilren's Memorial Institute for Education, Northwestern University Medical School, Chicago, Illinois, USA. hayashida@nwu.edu
Poncelet
A C
AC
Hubchak
S C
SC
Schnaper
H W
HW
D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051785:Smad Proteins; D015534:Trans-Activators / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D003094:Collagen; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases