Gene expression profiling of pituitary melanotrope cells during their physiological activation

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Interest in BDNF as an activity-dependent modulator of neuronal structure and function in the adult brain has intensified in recent years. Localization of BDNF-TrkB to glutamate synapses makes this system attractive as a dynamic, activity-dependent regulator of excitatory transmission and plasticity. Despite individual breakthroughs, an integrated understanding of BDNF function in synaptic plasticity is lacking. Here, we attempt to distill current knowledge of the molecular mechanisms and function of BDNF in LTP. BDNF activates distinct mechanisms to regulate the induction, early maintenance, and late maintenance phases of LTP. Evidence from genetic and pharmacological approaches is reviewed and tabulated. The specific contribution of BDNF depends on the stimulus pattern used to induce LTP, which impacts the duration and perhaps the subcellular site of BDNF release. Particular attention is given to the role of BDNF as a trigger for protein synthesis-dependent late phase LTP--a process referred to as synaptic consolidation. Recent experiments suggest that BDNF activates synaptic consolidation through transcription and rapid dendritic trafficking of mRNA encoded by the immediate early gene, Arc. A model is proposed in which BDNF signaling at glutamate synapses drives the translation of newly transported (Arc) and locally stored (i.e., alphaCaMKII) mRNA in dendrites. In this model BDNF tags synapses for mRNA capture, while Arc translation defines a critical window for synaptic consolidation. The biochemical mechanisms by which BDNF regulates local translation are also discussed. Elucidation of these mechanisms should shed light on a range of adaptive brain responses including memory and mood resilience.

Although dendritic growth and differentiation are critical for the proper development and function of neocortex, the molecular signals that regulate these processes are largely unknown. The potential role of neurotrophins was tested by treating slices of developing visual cortex with NGF, BDNF, NT-3, or NT-4 and by subsequently visualizing the dendrites of pyramidal neurons using particle-mediated gene transfer. Specific neurotrophins increased the length and complexity of dendrites of defined cell populations. Basal dendrites of neurons in each cortical layer responded most strongly to a single neurotrophin: neurons in layer 4 to BDNF and neurons in layers 5 and 6 to NT-4. In contrast, apical dendrites responded to a range of neurotrophins. On both apical and basal dendrites, the effects of the TrkB receptor ligands, BDNF and NT-4, were distinct. The spectrum of neurotrophic actions and the laminar specificity of these actions implicate endogenous neurotrophins as regulatory signals in the development of specific dendritic patterns in mammalian neocortex.