Hormones – Cytokines – Signaling
Kidney International (2002) 62, 799–808; doi:10.1046/j.1523-1755.2002.00537.x
Involvement of HB-EGF and EGF receptor transactivation in TGF-
–mediated fibronectin expression in mesangial cells
Yoko Uchiyama-Tanaka, Hiroaki Matsubara, Yasukiyo Mori, Atsushi Kosaki, Noriko Kishimoto, Katsuya Amano, Shigeki Higashiyama and Toshiji Iwasaka
Department of Medicine II, Kansai Medical University, and Department of Biochemistry, School of Allied Health Science, Osaka University Faculty of Medicine, Osaka, Japan
Correspondence: Hiroaki Matsubara, M.D., Department of Medicine II, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan. E-mail: matsubah@takii.kmu.ac.jp
Received 19 September 2001; Revised 23 April 2002; Accepted 24 April 2002.
Abstract
Involvement of HB-EGF and EGF receptortransactivation in TGF-
–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-
(TGF-)–mediated fibronectin (FN) expression using glomerular mesangial cells.
Results
TGF- 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-–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- (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- phosphorylated EGFR, ERK and p38MAPK in a PKC- and MMP-dependent manner. Smad2 phosphorylation by TGF- was not affected by AG1478, and HB-EGF did not activate Smad2. FN mRNA stability was not affected by TGF-. Cycloheximde did not interfere with TGF-–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-–mediated EGFR transactivation, and subsequent activation of ERK and p38MAPK is involved in FN expression via transcriptional regulation without requiring new protein synthesis.
Keywords:
heparin-binding EGF-like growth factor, cell signaling, glomerular mesangial cells, transcriptional regulation, glomerular sclerosis, extracellular matrix
Abbreviations:
ALP, alkaline phosphatase; ATP, adenosine 5'-triphosphate; BSA, bovine serum albumin; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparin binding-epidermal-like growth factor; MAPK, mitogen-activated protein kinase; MEK, ERK kinase, MMP, matrix metalloproteinase; MMP-2, 72-kD gelatinase A; MMP-9, 92-kD gelatinase B; PC-PLC, phosphotidylcholine-phospholipase C; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; TGF-, transforming growth factor-
