EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway.
The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.
Chiu
Terence
T
Department of Medicine, School of Medicine, CURE, Digestive Diseases Research Center, Molecular Biology Institute, University of California, Los Angeles, California, USA.
Santiskulvong
Chintda
C
Rozengurt
Enrique
E
D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D066246:ErbB Receptors / Q000502:physiology*; D020868:Gene Silencing; D007413:Intestinal Mucosa / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000502:physiology; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D015533:Transcriptional Activation / Q000502:physiology*
D000804:Angiotensin II; D004247:DNA; D066246:ErbB Receptors; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases