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.
Flannery
Patrick J
PJ
Division of Nephrology, Department of Medicine, Duke University and Durham VA Medical Centers, Durham, NC 27710, USA.
Spurney
Robert F
RF
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