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Medical Sciences	100%

Selected Abstracts

PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival

HEPATOLOGY, Issue 5 2002
Alexandre Coutant
Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal,regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication. [source]

RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo

INTERNATIONAL JOURNAL OF CANCER, Issue 6 2010
Luc Gailhouste
Abstract The mitogen-activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock-down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation. [source]

Phosphorylated Map Kinase (ERK1, ERK2) Expression is Associated with Early Tau Deposition in Neurones and Glial Cells, but not with Increased Nuclear DNA Vulnerability and Cell Death, in Alzheimer Disease, Pick's Disease, Progressive Supranuclear Palsy and Corticobasal Degeneration

BRAIN PATHOLOGY, Issue 2 2001
I. Ferrer
Abnormal tau phosphorylation and deposition in neurones and glial cells is one of the major features in tau pathies. The present study examines the involvement of the Ras/MEK/ERK pathway of tau phosphorylation in Alzheimer disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), by Western blotting, single and double-labelling immunohistochemistry, and p21Ras activation assay. Since this pathway is also activated in several paradigms of cell death and cell survival, activated ERK expression is also analysed with double-labelling immunohistochemistry and in situ end-labelling of nuclear DNA fragmentation to visualise activated ERK in cells with increased nuclear DNA vulnerability. The MEK1 antibody recognises one band of 45 kD that identifies phosphorylation-independent MEK1, whose expression levels are not modified in diseased brains. The ERK antibody recognises one band of 42 kD corresponding to the molecular weight of phosphorylation-independent ERK2; the expression levels, as well as the immunoreactivity of ERK in individual cells, is not changed in AD, PiD, PSP and CBD. The antibody MAPK-P distinguishes two bands of 44 kD and 42 kD that detect phosphorylated ERK1 and ERK2. MAPK-P expression levels, as seen with Western blotting, are markedly increased in AD, PiD, PSP and CBD. Moreover, immunohistochemistry discloses granular precipitates in the cytoplasm of neurones in AD, mainly in a subpopulation of neurones exhibiting early tau deposition, whereas neurones with developed neurofibrillary tangles are less commonly immunostained. MAPK-P also decorates neurones with Pick bodies in PiD, early tau deposition in neurones in PSP and CBD, and cortical achromatic neurones in CBD. In addition, strong MAPK-P immunoreactivity is found in large numbers of tau -positive glial cells in PSP and CBD, as seen with double-labelling immunohistochemistry. Yet no co-localisation of enhanced phosphorylated ERK immunoreactivity and nuclear DNA fragmentation is found in AD, PiD, PSP and CBD. Finally, activated Ras expression levels are increased in AD cases when compared with controls. These results demonstrate increased phosphorylated (active) ERK expression in association with early tau deposition in neurones and glial cells in taupathies, and suggest activated Ras as the upstream activator of the MEK/ERK pathway of tau phosphorylation in AD. [source]

Inhibitors of the PI3-kinase/Akt pathway induce mitotic catastrophe in non-small cell lung cancer cells

INTERNATIONAL JOURNAL OF CANCER, Issue 5 2006
Therese H Hemström
Abstract Non-small cell lung cancer cells (NSCLC) are more resistant to anticancer treatment as compared with other types of cancer cells. Recently (Hemström et al., Exp Cell Res 2005;305:200,13) we showed that apoptosis of U1810 NSCLC cells induced by the staurosporine analog PKC 412 correlated with inhibition of Akt and ERK1/2, suggesting the involvement of these kinases in cell survival. Here we investigated the contribution of the PI3-kinase/Akt and MEK/ERK pathways to survival of NSCLC cells. The two signaling pathways were studied by using different combinations of the PI3-kinase inhibitors LY-294002 and wortmannin, the Akt activator Ro 31-8220, the MEK inhibitor PD 98059 and PKC 412. PI3-kinase inhibitors induced apoptosis-like death in U1810 cells. H157 cells in general were relatively resistant to PI3 kinase/Akt inhibitors yet these compounds sensitized cells to the DNA-damaging drug VP-16, while Ro 31-8220 could not. PD 98059 only had a sensitizing effect on H157 cells when combined with PI3-kinase inhibition and VP-16. Morphological data indicated that LY-294002 and PKC 412 induced cell death at anaphase and metaphase, respectively, suggesting death by mitotic catastrophe. Analyzes of cells blocked in G2/M-phase by nocodazol revealed that LY-294002 increased, while PKC 412 decreased histone H3 phosphorylation, suggesting that LY-294002 allowed, while PKC 412 inhibited cells to leave M-phase. Flow cytometric analysis of cell cycle distribution demonstrated that LY-294002 allowed cells to leave G2/M phase, while PKC 412 inhibited cytokinesis, resulting in formation of multinucleated cells. These results indicate that sensitization of NSCLC cells by PI3-kinase inhibition involves interplay between cell cycle regulation, mitotic catastrophe and apoptosis. © 2006 Wiley-Liss, Inc. [source]

Oncostatin M,induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: An implication for the pathogenesis of arthritis

ARTHRITIS & RHEUMATISM, Issue 5 2009
Sang-Heng Kok
Objective To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. Methods The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. Results Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA,protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. Conclusion These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis. [source]