Activator protein 1 activation following hypoosmotic stress in HepG2 cells is actin cytoskeleton dependent.
BACKGROUND
Following hypoosmotic stress-induced cell volume change, the actin cytoskeleton reorganizes itself. The role of this reorganization in the activation of the phosphatidylinositol 3-OH-kinase/protein kinase B/activator protein 1 (PI-3-K/PKB/AP-1) proliferative signaling cascade is unknown. Focal adhesion kinase (FAK) participates in the cytoskeleton-based activation of PI-3-K. We hypothesized that hypoosmotic stress-induced activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation.


METHODS
HepG2 cells were incubated for 1 h with or without 20 microM cytochalasin D, an actin disrupter, and were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. Tumor necrosis factor alpha (1.4 nM) and medium alone served as positive and negative controls, respectively. Western blots measured cytoplasmic phosphorylated or total FAK and PKB. EMSAs measured nuclear AP-1. All experiments were performed in triplicate.


RESULTS
Exposure to hypoosmotic stress resulted in activation of the following signaling messengers in a sequential fashion: (1) phosphorylation of FAK occurred by 2 min, (2) phosphorylation of PKB occurred by 10 min, (3) nuclear translocation of AP-1 occurred by 30 min. All three signaling events were abolished when these cells were pretreated with cytochalasin D.


CONCLUSIONS
Actin reorganization following hypoosmotic stress is essential for the FAK-mediated activation of the PI-3-K/PKB/AP-1 proliferative cascade. These data delineate a possible mechanism by which the cell swelling-induced cytoskeletal changes can initiate proliferative signal transduction in human liver cancer.
Kim
R D
RD
Department of Surgery, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Darling
C E
CE
Roth
T P
TP
Ricciardi
R
R
Chari
R S
RS
D000199:Actins / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D008113:Liver Neoplasms; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009997:Osmotic Pressure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000199:Actins; D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D018808:Transcription Factor AP-1; D015638:Cytochalasin D; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt