Downstream Transcriptional Responses Research Articles

Changes in the Proteome after NeuronalZif268Overexpression

Long-lasting forms of brain plasticity are a cellular basis for long-term memory, and their disturbance underlies pathological conditions such as dementia and cognitive impairment. Neuronal plasticity is a complex process that utilizes molecular cascades in the cytoplasm and the nucleus and involves numerous transcription factors, in particular, immediate early genes (IEGs). The signaling cascades that control IEGs are fairly well described, but the downstream transcriptional response is poorly understood, especially its late components. Here, we investigated the response induced by the IEG Zif268 in the adult brain in relation to long-term memory. Using a mouse model with increased neuronal expression of Zif268 that leads to improved memory, we identified an ensemble of proteins regulated by Zif268 expression and differentiated between direct and indirect targets based on the presence of a consensus binding motif in their promoter. We show that Zif268 regulates numerous substrates with diverse biological functions including protein modification and degradation (proteasome-core complex), phosphorylation, cell division, sensory perception, metabolism, and metal ion transport. The results provide a comprehensive and quantitative data set characterizing the Zif268-dependent proteome in the adult mouse brain and offers biologically important new insight into activity-dependent pathways downstream of IEGs.

Extracellular Matrix-Induced Gene Expression in Human Breast Cancer Cells

Extracellular matrix (ECM) molecules modify gene expression through attachment-dependent (focal adhesion-related) integrin receptor signaling. It was previously unknown whether the same molecules acting as soluble peptides could generate signal cascades without the associated mechanical anchoring, a condition that may be encountered during matrix remodeling and degradation and relevant to invasion and metastatic processes. In the current study, the role of ECM ligand-regulated gene expression through this attachment-independent process was examined. It was observed that fibronectin, laminin, and collagen type I and II induce Smad2 activation in MCF-10A and MCF-7 cells. This activation is not caused by transforming growth factor (TGF)-beta ligand contamination or autocrine TGF involvement and is 3- to 5-fold less robust than the TGF-beta1 ligand. The resulting nuclear translocation of Smad4 in response to ECM ligand indicates downstream transcriptional responses occurring. Coimmunoprecipitation experiments determined that collagen type II and laminin act through interaction with integrin alpha(2)beta(1) receptor complex. The ECM ligand-induced Smad activation (termed signaling crosstalk) resulted in cell type and ligand-specific transcriptional changes, which are distinct from the TGF-beta ligand-induced responses. These findings show that cell-matrix communication is more complex than previously thought. Soluble ECM peptides drive transcriptional regulation through corresponding adhesion and non-attachment-related processes. The resultant gene expressional patterns correlate with pathway activity and not by the extent of Smad activation. These results extend the complexity and the existing paradigms of ECM-cell communication to ECM ligand regulation without the necessity of mechanical coupling.

Effects of the MEK inhibitor, SL-327, on rewarding, motor- and cellular-activating effects of D-amphetamine and SKF-82958, and their augmentation by food restriction in rat.

Food restriction (FR) enhances learned and unlearned behavioral responses to drugs of abuse and increases D-1 dopamine (DA) receptor-mediated activation of extracellular signal-regulated kinases (ERK) 1/2 MAP kinase in nucleus accumbens (NAc). While a role has been established for ERK signaling in drug-mediated associative learning, it is not clear whether ERK regulates unconditioned behavioral effects of abused drugs. The purpose of this study was to determine whether blockade of ERK signaling, using the brain-penetrant MEK inhibitor, SL-327, decreases behavioral or NAc cellular responses to acute drug treatment and their augmentation by FR. Separate experiments assessed the effects of SL-327 (50 mg/kg, intraperitoneally) on (1) the reward-potentiating effect of D-amphetamine in an intracranial self-stimulation protocol, (2) the locomotor-activating effect of the D-1 agonist, SKF-82958, and (3) Fos-immunostaining induced in the NAc by SKF-82958. FR rats displayed enhanced responses to drug treatment on all measures. SL-327 had no effect on sensitivity to rewarding brain stimulation or the reward-potentiating effect of D-amphetamine. The MEK inhibitor, U0126, microinjected into the NAc was also without effect. The locomotor-activating effect of SKF-82958 was unaffected by SL-327. In contrast, SL-327 decreased NAc Fos-immunostaining and abolished the difference between feeding groups. These results support the conclusion that ERK signaling does not mediate unlearned behavioral responses to drug treatment. However, the upregulation of ERK and downstream transcriptional responses to acute drug treatment may underlie the reported enhancement of reward-related learning in FR subjects.

Genome‐wide identification of touch‐ and darkness‐regulated Arabidopsis genes: a focus on calmodulin‐like and XTH genes

We sought to gain insight into functions potentially altered by mechanostimulation and investigate the relationship between touch and darkness responses. Microarrays and quantitative RT-PCR were conducted to identify genes and analyze behaviors of calmodulin-like (CML) and xyloglucan endotransglucosylase/hydrolase (XTH) genes. Strikingly, 589 genes had touch-inducible expression; 171 had reduced expression. Darkness increased expression of 461 genes and decreased expression of 72 genes. Over half of the touch-inducible genes resembled the TCH genes in that they were also up-regulated by darkness; 67% of those darkness-inducible were also touch inducible. Expression of 12 CMLs and four XTHs was elevated by touch; three XTHs had reduced expression. In darkness-treated plants, 10 CMLs and nine XTHs had increased expression and one XTH was repressed. Over 2.5% of total genes were touch-inducible. Many were also darkness up-regulated, consistent with the hypothesis that these stimuli have partially overlapping signal transduction pathways. Regulated gene identities suggest that calcium and kinase signaling, wall modification, disease resistance and downstream transcriptional responses may be altered in response to mechanostimulation or darkness.