MLK3 phosphorylation by ERK1/2 is required for oxidative stress-induced invasion of colorectal cancer cells.

Mixed lineage kinase 3 (MLK3) functions in migration and/or invasion of several human cancers; however, the role of MLK3 in colorectal cancer (CRC) invasion is unknown. MLK3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) which activates MAPK pathways through either kinase-dependent or -independent mechanisms. Human colorectal tumors display increased levels of reactive oxygen species (ROS) or oxidative stress. ROS, such as H2O2, are important for carcinogenesis and activate MAPK signaling pathways. In human colorectal carcinoma (HCT116) cells treated with H2O2, extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated and MLK3 exhibited reduced electrophoretic mobility (shift) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was eliminated by phosphatase treatment. Pretreatment with the ROS scavenger N-acetyl-L-cysteine, the ERK1/2 inhibitor UO126, or ERK1/2 siRNA knockdown blocked the H2O2-induced shift of MLK3, while MLK3 inhibition with Cep1347 did not. In co-immunoprecipitation experiments performed on H2O2-treated HCT116 cells, endogenous MLK3 associated with endogenous ERK1/2 and B-Raf. Active ERK1 phosphorylated kinase dead FLAG-MLK3 in vitro, whereas ERK1 phosphorylation of kinase dead FLAG-MLK3-S705A-S758A was reduced. Both MLK3 siRNA knockdown and FLAG-MLK3-S705A-S758A expression decreased ERK1/2 activation in H2O2-treated cells. Prolonged H2O2 treatment activated ERK1/2 and promoted invasion of colon cancer cells, which was attenuated by MLK3 siRNA knockdown. Furthermore, S705A-S758A-FLAG-MLK3 demonstrated decreased oxidative-stress induced colon cancer cell invasion, but increased interaction with GST-B-Raf as compared with wild-type-FLAG-MLK3 in H2O2-treated cells. These results suggest oxidative stress stimulates an ERK1/2-dependent phosphorylation of MLK3 on Ser705 and Ser758, which promotes MLK3-dependent B-Raf and ERK1/2 activation; this positive feedback loop enhances the invasion of colon cancer cells.

MLK4ß functions as a negative regulator of MAPK signaling and cell invasion.

Mixed lineage kinase (MLK) 4, or MLK4, is a member of the MLK family of mitogen-activated protein kinase kinase kinases (MAP3Ks). Typically, MAP3Ks function to activate the mitogen-activated protein kinase (MAPK)-signaling pathways and regulate different cellular responses. However, here we report that MLK4ß, unlike the other MLKs, negatively regulates the activities of the MAPKs, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and the MAP2Ks, MEK3 and 6. Our results show that MLK4ß inhibits sorbitol- and tumor necrosis factor-induced activation of p38. Furthermore, MLK4ß interacts with another MLK family member, MLK3, in HCT116 cells. Exogenous expression of MLK4ß inhibits activation of MLK3 and also blocks matrix metalloproteinase-9 gelatinase activity and invasion in SKOV3 ovarian cancer cells. Collectively, our data establish MLK4ß as a novel suppressor of MLK3 activation, MAPK signaling and cell invasion.

Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content.

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.

NMDA agonists and antagonists as probes of glutamatergic dysfunction and pharmacotherapies in neuropsychiatric disorders.

Antagonists of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors and agonists of the glycine-B coagonist site of these receptors have been important tools for characterizing the contributions of NMDA receptor pathophysiology to a large number of neuropsychiatric conditions and for treating these conditions. Among these disorders are Alzheimer's disease, chronic pain syndromes, epilepsy, schizophrenia, Parkinson's disease, Huntington's disease, addiction disorders, major depression, and anxiety disorders. This review will examine pathophysiological and therapeutic hypotheses generated or supported by clinical studies employing NMDA antagonists and glycine-B agonists and partial agonists. It will also consider ethical issues related to human psychopharmacological studies employing glutamatergic probes.

5-HT2 receptor regulation of extracellular GABA levels in the prefrontal cortex.

Monoamines, including both dopamine and serotonin, synapse onto prefrontal cortical interneurons. Dopamine has been shown to activate these GABAergic interneurons, but there are no direct data on the effects of serotonin on GABA release in the prefrontal cortex. We, therefore, examined the effects of the 5-HT2a/c agonist 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) on extracellular GABA levels in the prefrontal cortex of the rat. Local infusions of DOI dose-dependently increased cortical extracellular GABA levels. In addition, systemic DOI administration resulted in Fos protein expression in glutamic acid decarboxylase67-immunoreactive interneurons of the prefrontal cortex. These data indicate that serotonin, operating through a 5-HT2 receptor, acutely activates GABAergic interneurons in the prefrontal cortex. These data further suggest that there may be convergent regulation of interneurons by dopamine and serotonin in the prefrontal cortex.

The NMDA antagonist model for schizophrenia: promise and pitfalls.

Drug models have been extensively used to study the pathophysiology of schizophrenia. While they provide good insight into the neurobiology of this disorder, they have several shortcomings, which if known, help in the interpretation of results. In this paper we will discuss these shortcomings in general, and in relation to the N-methyl D-aspartate antagonist model for schizophrenia. This model has recently received a great deal of attention since both phencyclidine and the structurally related drug ketamine, produce symptoms that extend beyond psychosis per se to include other symptoms associated with schizophrenia. In fact, subanesthetic doses of ketamine in healthy individuals produce not only paranoia and perceptual alterations but also thought disorder, negative symptoms, cognitive deficits, as well as impairment on a number of electrophysiologic tests known to be abnormal in schizophrenia. These effects of ketamine will be discussed with a particular emphasis on implications for the pathophysiology and therapeutics of this disorder.