Angiotensin II regulation of TGF-beta in murine mesangial cells involves both PI3 kinase and MAP kinase.
BACKGROUND
Chronic activation of the angiotensin II (AngII) type 1 receptor (AT-1) is a central event in the development of chronic kidney disease (CKD), in part through enhanced expression of TGF-beta, and AT-1 receptor blockade inhibits the progression to CKD in a variety of disease states. The AT-1 receptor is a heptahelical Gaq/11-coupled receptor that initiates phospholipase C activity and release of intracellular calcium; recent data suggest that the AT-1 receptor can also activate the epidermal growth factor receptor (EGFR), although the roles of specific EGF-mediated signaling cascades in AT-1 effects on mesangial cell biology are uncertain. We hypothesized that 2 EGFR-activated pathways, PI3 kinase and MAP kinase, are stimulated by the AT-1 receptor and, in part, regulate the effects of AngII on TGF-beta1 levels in mesangial cells.


METHODS
We examined the effects of AT-1 receptor activation on EGFR, PI3 kinase, and MAP kinase activation in murine mesangial cells. Upon achieving 60-80% confluence, the medium was changed to low-serum for 48 hr and cells were exposed to either the AT-1 receptor blocker, losartan, the EGFR blocker, AG1478, or control medium, and then stimulated with AngII. Similar experiments were performed using LY294002 and U0126, specific inhibitors of PI3 kinase and MEK, respectively. Total cellular protein lysates and RNA were isolated. Activation of the receptors and pathways was evaluated by immunoblotting and levels of TGF-beta mRNA were measured using real-time quantitative RT-PCR.


RESULTS
AngII induced autophosphorylation of EGFR (pY1068) and activated Akt and ERK, downstream targets of PI3 kinase and MAP kinase, respectively. AngII-mediated EGFR autophosphorylation was inhibited by losartan and AG1478. AG1478 also inhibited both basal and AngII-mediated activation of Akt and ERK. Finally, AngII-mediated increase in TGF-beta mRNA was inhibited by losartan, AG1478, LY249002, and U0126.


CONCLUSIONS
Stimulation of the AT-1 receptor in murine mesangial cells results in activation of the EGF receptor with subsequent signaling through PI3 kinase and MAP kinase, thereby regulating TGF-beta mRNA levels. These data suggest that AT-1 receptor signaling pathways through EGFR may serve as a therapeutic target to inhibit the development of CKD.
Department of Internal Medicine, Washington Hospital Center, and Medstar Research Institute, Washington, DC, USA. perlmaa@mail.rockefeller.edu
Alan
A
Perlman
Loredo M
LM
Lawsin
Padmaja
P
Kolachana
Motoyasu
M
Saji
Jack
J
Moore
Mathew D
MD
Ringel
D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015151:Immunoblotting; D019808:Losartan / Q000494:pharmacology; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
D000804:Angiotensin II; D002070:Butadienes; D002867:Chromones; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D019808:Losartan; D020929:Mitogen-Activated Protein Kinase Kinases; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger; D044140:Receptor, Angiotensin, Type 1; D016212:Transforming Growth Factor beta; D020032:Tyrphostins; C113580:U 0126; C101044:RTKI cpd; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one