Transactivation of the epidermal growth factor receptor by angiotensin II in glomerular podocytes.
OBJECTIVE
Activation of angiotensin II (ANG2) receptors stimulates extracellular signal-regulated kinases (ERKs) that, in some cell systems, are mediated by transactivating the epidermal growth factor (EGF) receptor (EGFR) through mechanisms involving matrix metalloprotease (MMP)-stimulated processing of heparin-binding EGF (HB-EGF) from its precursor.


METHODS
The signaling pathways linked to ANG2-dependent ERK activation were determined in an immortalized mouse podocyte cell line by monitoring ANG2-stimulated phosphorylation of ERK1/2.


RESULTS
ANG2 induced transient ERK phosphorylation that was maximal at 5 min and then rapidly dissipated. ANG2-dependent ERK activation was inhibited by: (1) the type-1 ANG2-selective antagonist losartan; (2) the type-2 ANG2-selective antagonist PD123319; (3) an inhibitor of MMP2/9; (4) the EGFR kinase inhibitor AG1478, and (5) the HB-EGF antagonists CRM197 and heparin. ANG2-dependent ERK activation was mediated by both protein kinase C (PKC)- and calcium-dependent mechanisms and was associated with tyrosine phosphorylation of EGFR. To determine if ANG2-dependent HB-EGF release could act in a paracrine fashion on adjacent cells, HEK293 cells were stably transfected with green fluorescent protein-tagged ERK2 (GFP-ERK2). In stably transfected HEK293 cells, EGF stimulated phosphorylation of endogenous ERK1/2 as well as GFP-ERK2. In contrast, ANG2 had no effect on ERK phosphorylation in stably transfected HEK293 cells. When podocytes were co-cultured with stably transfected HEK293 cells, however, treatment with ANG2 rapidly stimulated GFP-ERK2 phosphorylation. Both the MMP2/9 inhibitor and AG1478 attenuated ANG2-dependent phosphorylation of GFP-ERK2 in the co-culture system.


CONCLUSIONS
These data indicate that ERK activation is induced by ANG2 in podocytes by mechanisms involving ANG2-dependent release of HB-EGF which, in turn, may act in an autocrine and paracrine fashion to stimulate ERK activity.
Division of Nephrology, Department of Medicine, Duke University and Durham VA Medical Centers, Durham, NC 27710, USA.
Patrick J
PJ
Flannery
Robert F
RF
Spurney
D000804:Angiotensin II / Q000494:pharmacology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D057912:Angiotensin II Type 2 Receptor Blockers; D000818:Animals; D002460:Cell Line; D018920:Coculture Techniques; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D019808:Losartan / Q000494:pharmacology; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D050199:Podocytes / Q000187:drug effects* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation
D047228:Angiotensin II Type 1 Receptor Blockers; D057912:Angiotensin II Type 2 Receptor Blockers; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; C073402:PD 123319; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D045726:Metalloproteases; D019808:Losartan