Lene Wierød

Cytoplasmic retention of peroxide‐activated ERK provides survival in primary cultures of rat hepatocytes

Reactive oxygen species (ROS) are implicated in tissue damage causing primary hepatic dysfunction following ischemia/reperfusion injury and during inflammatory liver diseases. A potential role of extracellular signal‐regulated kinase (ERK) as a mediator of survival signals during oxidative stress was investigated in primary cultures of hepatocytes exposed to ROS. Hydrogen peroxide (H2O2) induced a dose‐dependent activation of ERK, which was dependent on MEK activation. The ERK activation pattern was transient compared with the ERK activation seen after stimulation with epidermal growth factor (EGF). Nuclear accumulation of ERK was found after EGF stimulation, but not after H2O2 exposure. A slow import/rapid export mechanism was excluded through the use of leptomycin B, an inhibitor of nuclear export sequence–dependent nuclear export. Reduced survival of hepatocytes during ROS exposure was observed when ERK activation was inhibited. Ribosomal S6 kinase (RSK), a cytoplasmic ERK substrate involved in cell survival, was activated and located in the nucleus of H2O2‐exposed hepatocytes. The activation was abolished when ERK was inhibited with U0126. In conclusion, our results indicate that activity of ERK in the cytoplasm is important for survival during oxidative stress in hepatocytes and that RSK is activated downstream of ERK. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:200–207.)

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF.

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.

MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1‐activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2‐activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1‐mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline‐rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21‐activated kinase phosphorylates S298 and thus enhances the MEK1‐ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion‐dependent manner.—Skarpen, E., Flinder, L. I., Rosseland, C. M., Ørstavik, S., Wierød, L., Pedersen Oksvold, M., Skålhegg, B. S., Huitfeldt, H. S. MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments. FASEB J. 22, 466–476 (2008)

EGF‐induced ERK‐activation downstream of FAK requires rac1‐NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox‐sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1‐NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1‐NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAKs ability to act as a scaffold for EGF‐mediated signaling, including activation of ERK. Accordingly, we found that EGF‐induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK‐regulated S910‐site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading. J. Cell. Physiol. 226: 2267–2278, 2011.

Distinct functions of H-Ras and K-Ras in proliferation and survival of primary hepatocytes due to selective activation of ERK and PI3K

Ras proteins mediate signals both via extracellular signal‐regulated kinase 1 and 2 (ERK), and phosphoinositide 3‐kinase (PI3K). These signals are key events in cell protection and compensatory cell growth after exposure to cell damaging and pro‐apoptotic stimuli, thus maintaining homeostasis. By transfection techniques, we found that both H‐Ras and K‐Ras were expressed and appeared functionally active in primary hepatocytes. We compared the ability of H‐Ras and K‐Ras homologues to preferentially activate one of the two pathways, thereby differentially controlling cell survival and growth. We found that ectopic expression of dominant negative (DN) H‐RasN17, but not DN K‐RasN17, efficiently inhibited both phosphorylation and translocation of ERK to the nuclear compartment, which are prerequisites for cell cycle progression. Furthermore, ectopic expression of constitutive active (CA) H‐RasV12, but not CA K‐RasV12, potentiated EGF‐induced proliferation. We also found that expression of CA mutants of either H‐Ras or K‐Ras protected hepatocytes from transforming growth factor‐β1 (TGF‐β1)‐induced apoptosis. However, H‐Ras‐induced survival was mediated by ERK/RSK as well as by PI3K, whereas K‐Ras‐induced survival was mediated by PI3K only. In conclusion, H‐Ras and K‐Ras had differential functions in proliferation and survival of primary hepatocytes. H‐Ras was the major mediator of ERK‐induced proliferation and survival, whereas H‐Ras and K‐Ras both mediated PI3K‐induced survival. J. Cell. Physiol. 215: 818–826, 2008.

Activation of the p53–p21 Cip1 pathway is required for CDK2 activation and S-phase entry in primary rat hepatocytes

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative ‘brake’ to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21Cip1 in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53V143A) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21Cip1 was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21Cip1 expression.

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes

Abstract.  Introduction/Objectives: Cell cycle progression is driven by the coordinated regulation of cyclin‐dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G1 phase. It is an on‐going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. Methods and results: In this study, we have explored the role of CDK4 activity during G1 progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1‐induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2‐Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. Conclusions: Thus, epidermal growth factor‐induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin‐CDK complexes in G1 phase.

RAF‐targeted therapy for hepatocellular carcinoma in the regenerating liver

Post‐operative liver regeneration may contribute to tumor recurrence. There is a theoretical need for an adjuvant therapy that can suppress tumor growth without adversely affecting post‐operative liver regeneration.

EGF Activates Autocrine TGFα to Induce Prolonged EGF Receptor Signaling and Hepatocyte Proliferation

Background/Aims: EGF receptor is a main participant in the regulation of liver regeneration. In primary hepatocyte cultures, EGF or TGFα binding to EGF receptor activates Erk1/2 and PI3K pathways, induces cyclin D1 and thus initiates DNA synthesis. We have explored mechanisms by which prolonged EGF receptor activation induces hepatocyte proliferation. Methods: EGF receptor activation, as well as Erk1/2 and PI3K signaling were explored in EGF-stimulated primary hepatocyte cultures by Western blotting and immunocytochemistry. TGFα release to the medium was quantified by ELISA. Effects of a neutralizing antibody to TGFα on EGF receptor signaling and proliferation were explored. Results: Inhibitors of PI3K or Erk1/2 inhibited cyclin D1 expression and G1 progression when added 12 hours after EGF stimulation, whereas depletion of EGF from the medium at this time point did not. ELISA demonstrated that EGF induced TGFα release to the medium. Cyclin D1 induction and cellular proliferation were efficiently inhibited when a neutralizing antibody to TGFα was added to the medium. This also occurred when the antibody was added 12 hours after EGF stimulation. Conclusion: Sustained EGF receptor activity and signaling through both Erk1/2 and PI3K pathways were necessary for proliferation. This was achieved by EGF activation of autocrine TGFα.

Studies of the autoinhibitory segment comprising residues 31–60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and is internalized as a complex with the LDLR. In the acidic milieu of the sorting endosome, PCSK9 remains bound to the LDLR and prevents the LDLR from folding over itself to adopt a closed conformation. As a consequence, the LDLR fails to recycle back to the cell membrane. Even though it is the catalytic domain of PCSK9 that interacts with the LDLR at the cell surface, the structurally disordered segment consisting of residues 31–60 and which is rich in acidic residues, has a negative effect both on autocatalytic cleavage and on the activity of PCSK9 towards the LDLR. Thus, this unstructured segment represents an autoinhibitory domain of PCSK9. One may speculate that post-translational modifications within residues 31–60 may affect the inhibitory activity of this segment, and represent a mechanism for fine-tuning the activity of PCSK9 towards the LDLR. Our data indicate that the inhibitory effect of this unstructured segment results from an interaction with basic residues of the catalytic domain of PCSK9. Mutations in the catalytic domain which involve charged residues, could therefore be gain-of-function mutations by affecting the positioning of this segment.