The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.
BACKGROUND
Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells.


METHODS
Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs.


RESULTS
Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant.


CONCLUSIONS
Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.
Poncelet
A C
AC
Department of Pediatrics and Children's Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, Illinois, USA. anne-c@nwu.edu
de Caestecker
M P
MP
Schnaper
H W
HW
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D005920:Glomerular Mesangium / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008156:Luciferases; D016296:Mutagenesis / Q000502:physiology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D012694:Serine; D003094:Collagen; D008156:Luciferases