MicroRNAs (miRNAs) have been reported to be associated with the development of cancers. However, the function of miRNAs in human hepatocellular carcinoma (HCC) remains largely undefined. Here we found that overexpression of miR-10a promoted the migration and invasion of QGY-7703 and HepG2 cells in vitro but suppressed metastasis in vivo. Cell adhesion assays showed that miR-10a suppressed HCC cell-matrix adhesion, which could explain the results of the in vivo animal experiments. The Eph tyrosine kinase receptor, EphA4, was identified as the direct and functional target gene of miR-10a. Knockdown of EphA4 phenocopied the effect of miR-10a and ectopic expression of EphA4 restored the effect of miR-10a on migration, invasion, and adhesion in HCC cells. We further demonstrated that miR-10a and EphA4 regulated the epithelial-mesenchymal transition process and the b1-integrin pathway to affect cell invasion and adhesion. Conclusion: Our findings highlight the importance of miR-10a in regulating the metastatic properties of HCC by directly targeting EphA4 and may provide new insights into the pathogenesis of HCC. (HEPATOLOGY 2013;57:667-677)

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) ␤ receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short ␣-helix followed by a three-stranded ␤-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second ␤ strands and along one face of the ␤-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WWlike domain. Receptor tyrosine kinases (RTKs)1 are transmembrane proteins that regulate numerous aspects of cell physiology including proliferation and survival. Binding of a soluble ligand to the extracellular domain of these receptors typically induces receptor dimerization and trans-phosphorylation of the cytoplasmic catalytic domain. This tyrosine phosphorylation stimulates the intrinsic tyrosine kinase activity of the receptor and generates binding sites for signaling proteins containing SH2 domains. Although ligand-induced dimerization is an important trigger of receptor activation, receptor activity is also subject to additional levels of regulation. For example, the cytoplasmic juxtamembrane region of receptor tyrosine kinases, which is located between the transmembrane domain and the kinase domain, has been implicated in regulation of receptor enzymatic activity (e.g. see Ref.

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.Oxidative stress can contribute to neuronal toxicity and has been implicated in both acute injury and chronic neuropathological conditions (1, 2). Many in vitro models have been used to examine the mechanistic basis for neuronal cell death induced by oxidative stress. For example, oxidative toxicity can be induced by glutamate treatment in the HT22 mouse hippocampal cell line (3-5) and immature primary embryonic rat cortical neuron cultures (4, 6, 7). In these models, glutamate treatment leads to glutathione (GSH) 2 depletion and subsequent accumulation of reactive oxygen species (ROS) (8). Many intracellular second messenger pathways are required for oxidative toxicity in HT22 cells including arachidonic acid metabolites, cyclic GMP (cGMP), and calcium (8). In addition, signaling kinases, such as mitogen-activated protein kinases (MAPK), are activated upon glutamate-induced oxidative stress in HT22 cells and primary neurons and are likely to affect targets that either limit or promote oxidative toxicity. Extracellular signal-regulated kinases-1/2 (ERK1/2) has been implicated in glutamate-induced neuronal oxidative toxicity based upon the neuroprotective effects of U0126, a specific inhibitor of the ERK1/2-activating kinase, MEK1/2 (5). U0126 is also effective at reducing brain injury following focal ischemia in rodents suggesting that ERK1/2 may also promote neur...

Abstract. Genipin, an active constituent of Gardenia fruit, has been reported to show an antitumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G 1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.