Involvement of HB-EGF and EGF receptor transactivation in TGF-beta-mediated fibronectin expression in mesangial cells.
BACKGROUND
Gq-coupled receptors are known to transactivate epidermal growth factor receptor (EGFR) via the Ca2+ and PKC pathways to phosphorylate extracellular signal-regulated kinase (ERK).


METHODS
We studied the involvement of EGFR in transforming growth factor-beta (TGF-beta)-mediated fibronectin (FN) expression using glomerular mesangial cells.


RESULTS
TGF-beta up-regulated FN mRNA accumulation in a time- and dose-dependent manner, which was completely inhibited by phosphatidylcholine-phospholipase C (PC-PLC) inhibitor and PKC inhibitors (calphostin-C and staurosporin). The EGFR inhibitor AG1478 completely abolished TGF-beta-mediated FN expression. ERK inactivation by PD98059, and p38MAPK inhibitor SB203580 also showed significant inhibitory effects. Addition of neutralizing anti-heparin-binding EGF-like growth factor (HB-EGF) antibody, pretreatment with heparin and the metalloproteinase (MMP) inhibitor batimastat blocked FN expression. In mesangial cells stably transfected with a chimera containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by TGF-beta (2.1-fold at 0.5 min) and reached a 3.7-fold increase at two minutes, which was abolished by calphostin-C or batimastat. TGF-beta phosphorylated EGFR, ERK and p38MAPK in a PKC- and MMP-dependent manner. Smad2 phosphorylation by TGF-beta was not affected by AG1478, and HB-EGF did not activate Smad2. FN mRNA stability was not affected by TGF-beta. Cycloheximde did not interfere with TGF-beta-mediated FN expression.


CONCLUSIONS
The present study demonstrated that HB-EGF processed and released via PC-PLC-PKC signaling is an intermediate molecule for TGF-beta-mediated EGFR transactivation, and subsequent activation of ERK and p38MAPK is involved in FN expression via transcriptional regulation without requiring new protein synthesis.
Uchiyama-Tanaka
Yoko
Y
Department of Medicine II, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan.
Matsubara
Hiroaki
H
Mori
Yasukiyo
Y
Kosaki
Atsushi
A
Kishimoto
Noriko
N
Amano
Katsuya
K
Higashiyama
Shigeki
S
Iwasaka
Toshiji
T
D000818:Animals; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases
D005353:Fibronectins; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C