Angiotensin II signaling and HB-EGF shedding via metalloproteinase in glomerular mesangial cells

Background. Angiotensin II (Ang II) has been implicated in the development of glomerulosclerosis by stimulating fibronectin (FN) synthesis. The proces sing and release of heparin binding-endothelin growth factor (HB-EGF) are a ctivated by protein kinase C (PKC) and Ca2+ signaling. We studied the roles of HB-EGF and endothelial growth factor (EGF) receptor (EGFR) in Ang II-in duced FN expression using mesangial cells. Methods. Mesangial cells were prepared from mouse kidneys by the explant me thod and cells were used at passages 4 and 5. Results. Ang II stimulated FN mRNA levels dose-dependently with a maximal i ncrease (3.4-fold) after 12 hours of incubation. This action was completely inhibited by PKC inhibitors and slightly blocked by Ca2+ chelating agents. FN mRNA accumulation by Ang II was abolished by tyrosine kinase inhibitors , a specific inhibitor for EGFR (AG1478) and extracellular signal-regulated kinase (ERK) inactivation. Addition of neutralizing anti-HB-EGF antibody, as well as pretreatment with heparin or the metalloproteinase inhibitor bat imastat abolished induction of FN expression by Ang II. In mesangial cells stably transfected with a chimeric construct containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly inc reased by Ang II (1.7-fold at 0.5 min) and reached a 4.1-fold increase at t wo minutes. Ang II phosphorylated EGFR (maximal at 2 min) and ERK (maximal at 8 min) in a PKC- and metalloproteinase-dependent manner. Ang II stimulat ed the expression and release of transforming growth factor-beta (TGF-beta) via EGFR-mediated signaling, and the released TGF-beta also contributed to Ang II-mediated FN expression via EGFR transactivation. Conclusions. Ang II-mediated FN expression was regulated by autocrine effec ts of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk betwe en Ang II and growth factor receptor signaling pathways.