Morten P. Oksvold

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.)

Expression of B-Cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells

PURPOSE Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation. METHODS Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81. FINDINGS Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity. IMPLICATIONS Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.

Immunocytochemical Localization of Shc and Activated EGF Receptor in Early Endosomes After EGF Stimulation of HeLa Cells

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.

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.

Serine mutations that abrogate ligand-induced ubiquitination and internalization of the EGF receptor do not affect c-Cbl association with the receptor

In the present study, we examined EGF-induced internalization, degradation and trafficking of the epidermal growth factor receptor (EGFR) mutated at serines 1046, 1047, 1057 and 1142 located in its cytoplasmic carboxy-terminal region. We found the serine-mutated EGFR to be inhibited in EGF-induced internalization and degradation in NIH3T3 cells. We therefore tested the hypothesis that these mutations affect ligand-induced c-Cbl association with the receptor, leading to inhibited receptor ubiquitination. EGF was unable to induce ubiquitination of the serine-mutated EGFR, yet EGF-induced phosphorylation of the c-Cbl-binding site at tyrosine 1045, and c-Cbl-EGFR association, was unaffected. To compare the relevance of these serine residues with tyrosine 1045 in their regulation of c-Cbl binding and receptor ubiquitination, we analysed an EGFR mutated at tyrosine 1045 (Y1045F). EGF-induced c-Cbl-EGFR binding was partially inhibited, and receptor ubiquitination was abrogated in cells expressing Y1045F-EGFR. In contrast, ligand-induced internalization and degradation of the Y1045F mutant was similar to that of wild-type EGFR. Together, our data indicate that the serine residues and tyrosine 1045 are essential for EGF-induced receptor ubiquitination, but only the serine residues are critical for EGFR internalization and degradation.

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)

TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

BackgroundCytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.ResultsTGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.ConclusionsWe suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.

Altered regulation of EGF receptor signaling following a partial hepatectomy.

We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre‐replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC‐regulated T654‐site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654‐phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF‐mediated EGFR autophosphorylation, altered ligand‐induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR‐Ras‐Raf‐ERK pathway. Further, we found that both TPA and a PH enhanced EGF‐induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase‐activity. This may be a pre‐replicative competence event that increases growth factor effects during G1 progression.

Magnetic bead-based isolation of exosomes.

Exosomes are here defined as extracellular vesicles (EVs) in the approximate size range of 30-100 nm in diameter, and are observed in most body fluids containing typical exosomal markers such as CD9, CD63, and CD81. Potential subpopulations of exosomes can be captured by targeting these markers using magnetic beads. Magnetic beads are versatile tools for exosome isolation and downstream analysis. Here, we describe the workflow of immuno magnetic isolation and analysis of exosomes by flow cytometry, Western immunoblotting, and electron microscopy.

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.