Anti-CREB (I127) Antibody (A25106)

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is abundantly expressed in the brain and is critical for the normal function of synapses. cAMP response element binding protein (CREB) is a transcription factor which initiates the expression of proteins that related to the regulation of synaptic plasticity and memory function. Studies have shown that UCH-L1 can influence the expression and activity of CREB, but the underlying mechanisms remain unclear. In this study, we used UCH-L1 inhibitor LDN to treat mice hippocampal slices and found that UCH-L1 inhibition caused the dephosphorylation of CREB at Ser133 site. Meanwhile, hyperphosphorylation of microtubule-associated protein tau; increased expression of synaptic protein components of PSD-95 and synapsin-1, and decreased activity of tyrosine kinase Fyn were observed after UCH-L1 inhibition. Moreover, all these alternations have an influence on the normal function of N-methyl-D-aspartate (NMDA) receptor NR2B subunit which is likely to result in the dephosphorylation of CREB. We also found that LDN treatment mediated protein kinase A (PKA) deactivation was involved in the dephosphorylation of CREB. Thus, our study introduces a novel possible mechanism for elaborating the effects of UCH-L1 inhibition on the CREB activity and the implicated signaling pathways.

Nicorandil, an ATP-sensitive potassium (KATP) channel opener, is known to have protective effects on ischemic injury in heart and brain. One of the most important protective mechanisms is the anti-apoptotic effect on cardiomyocytes and neurons. This study explored the anti-apoptotic effect of nicorandil against neurotoxicity in SH-SY5Y cells overexpressing the Swedish mutant APP (APPsw) and the possible mechanisms involved. We used SH-SY5Y cells transiently transfected with APPsw as a cellular model of Alzheimer's disease. Cells were treated with nicorandil (0.1, 0.5, 1 mM) for 24 h with and without glibenclamide (10 μM), a KATP channel inhibitor. The cells were then collected for MTT, apoptosis assay, and Western blot. In addition, we also investigated the potential involvement of the PI3K/Akt pathway in nicorandil-mediated neuroprotection of APPsw cells. Our results showed that nicorandil dose-dependently increased cell viability and reduced the rate of apoptosis as measured by MTT assay and annexin V/PI staining. Western blot showed that nicorandil could upregulate Bcl-2 levels and downregulate Bax and caspase-3 expression. Further studies showed that nicorandil increased the levels of phospho-Akt and upregulated element-binding protein activity by PI3K activation. Applying a PI3K inhibitor, LY294002 blocked the protection. All these findings suggest that nicorandil might be a potential treatment option for Alzheimer's disease.

Picroside II, an iridoid glucoside found in the root of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae), has been demonstrated to reduce apoptosis in neuronal cells and other cell types. However, whether picroside II has a protective effect against cardiomyocyte apoptosis is poorly understood. In the present study, we investigated the cardioprotective role of picroside II and the underlying mechanisms in hypoxia/reoxygenation-induced cardiomyocyte apoptosis. The pretreatment with picroside II markedly attenuated hypoxia/reoxygenation-induced cell damage dose-dependently, which was evident by the increased cell viability and the corresponding decrease in lactate dehydrogenase release (LDH). The pretreatment with picroside II inhibited apoptosis confirmed by Annexin V-FITC staining, Hoechst 33258 nuclear staining and by assessment of caspase-3 activity. In addition, we found that picroside II not only increased the expression of Bcl-2, while decreasing Bax expression, but also augmented Akt and cAMP response element-binding protein (CREB) phosphorylation and ultimately inhibited hypoxia/reoxygenation-induced apoptosis. Furthermore, the protective effects of picroside II were abrogated by pretreatment of the cells with wortmannin or LY294002, a specific PI3K inhibitor. The present study suggests that picroside II inhibits hypoxia/reoxygenation-induced apoptosis in cardiomyocytes by activating the PI3K/Akt and CREB pathways and modulating expression of Bcl-2 and Bax.

An experiment was performed to observe the changes in Raf-1 kinase/mitogen-activated protein kinase ERK (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in cultured hippocampal neurons and its correlation with neurons apoptosis induced by intracellular zinc depletion. Cultured hippocampal neurons were exposed to a cell membrane-permeant zinc chelator TPEN (2 µM), and to TPEN plus zinc sulfate (5 µM) for 24 h. Cultures were then processed to detect neuronal viability by the methyl thiazolyl tetrazolium assay, while apoptosis rate was simultaneously observed by the flow cytometric analysis. Caspase-3, Raf-1, pMEK, pERK1/2, and pCREB protein levels were examined by Western blot assays. The viability in TPEN-incubated neurons was notably decreased, apoptosis rate and expression of caspase-3 significantly increased compared to untreated controls. The significant down-regulation of Raf/MEK/ERK signaling pathway and expression of pCREB were decreased in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced alterations described. The results demonstrated zinc-modulated apoptosis and the expression of Raf/MEK/ERK at the protein level in hippocampal neurons. It is possible that zinc depletion-induced apoptosis in cultured hippocampal neurons may be relevant to the changes of Raf/MEK/ERK signaling pathway.

We described a prospective application of ligand-based virtual screening program SHAFTS to discover novel inhibitors for p90 ribosomal S6 protein kinase 2 (RSK2). Taking the putative 3D conformations of two weakly binding RSK2 NTKD inhibitors as query templates, SHAFTS was used to perform 3D similarity based virtual screening because of a lack of crystal structure of RSK2 protein, thus leading to the identification of several novel scaffolds that would have been missed by conventional 2D fingerprint methods. The most potent hit compounds show low micromolar inhibitory activities against RSK2. In particular, one of the hit compounds exhibits potent antimigration activity against the MDA-MB-231 tumor cell. The results exemplified SHAFTS' application in active enrichment and scaffold hopping, which is of general interest for lead identification in drug discovery endeavors and also provides novel scaffolds that lay the foundation for uncovering new RSK2 regulatory mechanisms.

CONTEXT:

Hydrosalpinx impairs endometrial receptivity and embryo implantation. However, the exact underlying mechanism remains elusive.

OBJECTIVE:

This study aimed to explore how an miR-133b-mediated mechanism controls endometrial receptivity and embryo attachment in the endometrium of women with hydrosalpinx.

DESIGN, SETTING, PATIENTS, AND INTERVENTIONS:

Ishikawa cells were treated with hydrosalpinx fluid (HF) from infertile patients and cultured for in vitro analysis. The attachment rates of BeWo spheroids and mouse embryos to Ishikawa cells were assayed.

PRIMARY OUTCOME MEASURE:

miR-133b, serum and glucocorticoid-regulated kinase 1 (SGK1), and homeobox A10 (HOXA10) expression levels were evaluated by quantitative real-time PCR and Western blot assays.

RESULTS:

The expression of miR-133b and HOXA10 was significantly down-regulated, whereas the miR-133b target gene SGK1 was up-regulated in mid-secretory endometrial tissues of women with hydrosalpinx and in HF-treated Ishikawa cells. Moreover, hydrosalpinx inhibited miR-133b expression through the activation of nuclear factor κB/p65, and SGK1 decreased miR-133b-induced HOXA10 expression by phosphorylating cAMP responsive element binding protein in Ishikawa cells. Our results also showed that miR-133b and HOXA10 contributed to BeWo spheroid adhesiveness, whereas SGK1 inhibited BeWo spheroid attachment to Ishikawa cells. Importantly, miR-133b overexpression reversed the HF-mediated impairment of embryo attachment in vitro.

CONCLUSIONS:

miR-133b directly targets SGK1 to reverse the hydrosalpinx-induced down-regulation of HOXA10 and to attenuate the impairment of embryo attachment in vitro.