Arsenite activation of P13K/AKT cell survival pathway is mediated by p38 in cultured human keratinocytes.
BACKGROUND
Arsenic has been considered as a carcinogen. Recently the issue of arsenic in drinking water raised an unprecedented social concern on human health, and yet the molecular mechanisms through which arsenic induces cancer remain unknown. Activation of cell survival pathway leading to the activation of eNOS has been associated with various types of cancer. The objective of this study was to investigate the pathway leading to the activation of eNOS in response to arsenite using human keratinocytes.


METHODS
Cultured keratinocytes (HaCat cells) were exposed to arsenite with or without pretreatment of various inhibitors. Western blot analysis was performed to determine the activation of p38, AKT, eNOS. EGFR tyrosine phosphorylation was detected by immunoprecipitation and Western blot analysis. pNPP assay was used to measure phosphatase activity in cell lysate. FACS analysis was performed for the determination of generation of reactive oxygen species.


RESULTS
Arsenite induced the activation of AKT at both Ser473 and Thr308, and its downstream effector eNOS in cultured human keratinocytes. Arsenite also induced phosphorylation of p38. PI-3-kinase inhibitors, Wortmannin and LY294002 inhibited arsenite-induced phosphorylation of AKT and eNOS but had no effect on phosphorylation of p38. Interestingly, however, SB203580, a known p38 inhibitor, completely inhibited arsenite-induced phosphorylation of AKT and eNOS. Arsenite induced generation of reactive oxygen species and inactivated phosphatase activity, but did not activate EGF receptor tyrosine phosphorylation.


CONCLUSIONS
Collectively, our data indicate that arsenite induces activation of AKT and eNOS, via PI-3-kinase and p38 pathway, likely bypassing the activation of EGF receptor in cultured human keratinocytes.
Souza
K
K
Department of Biology, Providence College, and Roger Williams Medical Center, Providence, RI, USA.
Maddock
D A
DA
Zhang
Q
Q
Chen
J
J
Chiu
C
C
Mehta
S
S
Wan
Y
Y
D018053:Arsenites / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D015603:Keratinocytes / Q000473:pathology / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013723:Teratogens / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases
D018053:Arsenites; D066246:ErbB Receptors; D006861:Hydrogen Peroxide; D020928:Mitogen-Activated Protein Kinases; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D017382:Reactive Oxygen Species; D013723:Teratogens; D048051:p38 Mitogen-Activated Protein Kinases; C494918:AKT1 protein, human; C015001:arsenite