Phragmunis a suppresses glioblastoma through the regulation of MCL1-FBXW7 by blocking ELK1-SRF complex-dependent transcription.

Glioblastoma (GBM) is a highly aggressive brain tumor. During screening work, we found a new compound named phragmunis A (PGA), which is derived from the fruitbody of Trogia venenata, exhibits a potential cytotoxic effect on patient-derived recurrent GBM cells and temozolomide (TMZ)-resistant cell lines. The present study was designed to investigate the potential molecular mechanism of the anti-glioma effects of PGA in vitro and in vivo. Studies investigating the mechanism revealed that PGA diminished the binding efficiency of ETS family of transcription factor (ELK1) and Serum response factor (SRF), and suppressed ELK1-SRF complex-dependent transcription, which decreased the transcriptional levels of downstream genes Early growth response protein 1 (EGR1)-Polycomb ring finger (BMI1), thus inducing the imbalanced regulation between Myeloid cell leukaemia-1 (MCL1) and F-Box and WD repeat domain containing 7 (FBXW7). Finally, orthotopic xenograft models were established to confirm the anti-glioma effect of PGA on tumour growth. We showed, for the first time, that the cytotoxic effects of PGA occurred by inducing MCL1 inhibition and FBXW7 activation by blocking ELK1-SRF complex-dependent transcription. The blockage of ELK1-mediated transcription resulted in the suppression of EGR1-BMI1, which led to the upregulation of FBXW7 expression and downregulation of MCL1. These findings suggested that PGA could be a therapeutic drug candidate for the treatment of recurrent GBM by targeting the ELK1-SRF complex.

Xinhuang Tablets Improve Intestinal Barrier Function via Regulating Epithelial Tight Junctions in Dextran Sulfate Sodium-Induced Ulcerative Colitis Mice.

Intestinal mucosal barrier dysfunction is involved in the pathogenesis of inflammatory bowel disease, including ulcerative colitis (UC). Xinhuang tablets (XHTs) have been prescribed for several kinds of inflammatory diseases, including UC, whereas its possible underlying molecular mechanisms had never been explored. Mouse model of UC was constructed by DSS treatment and followed by XHT treatment. Disease activity index, histopathological of colonic tissue, tumor necrosis factor-alpha (TNF-α), and serum amyloid A (SAA) levels in serum were further assessed. The underlying mechanism was further explored by determination of the expression of epithelial tight junction-related protein. XHT administration ameliorated dextran sulfate sodium (DSS)-induced clinical symptoms, colonic histological injury, and decreased the circulating levels of TNF-α and SAA. Moreover, XHT treatment significantly increased the protein levels of zona occludens (ZO)-1, whereas decreased the levels of phosphorylation of Elk-1. In conclusion, this study confirmed the therapeutic effects of XHT treatment on UC in a DSS-induced mouse model, and indicated that by increasing expression of epithelial tight junctions and decreasing phosphorylation of Elk-1 might be one of the underlying mechanisms of XHT treatment on UC.

l-Theanine attenuates neointimal hyperplasia via suppression of vascular smooth muscle cell phenotypic modulation.

Neointimal hyperplasia is a prominent pathological phenomenon in the process of stent restenosis. Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play major pathological processes involved in the development of restenosis. l-Theanine, one of the major amino acid components in green tea, has been reported to improve vascular function. Here we display the effects of l-theanine on neointima formation and the underlying mechanism. In the rat carotid-artery balloon-injury model, l-theanine greatly inhibited neointima formation and prevented VSMCs from a contractile phenotype switching to a synthetic phenotype. In vitro study showed that l-theanine significantly inhibited PDGF-BB-induced VSMC proliferation and migration, which was comparable with the effect of l-theanine on AngII-induced VSMC proliferation and migration. Western blot analysis demonstrated that l-theanine suppressed PDGF-BB and AngII-induced reduction of SMA and SM22α and increment of OPN, suggesting that l-theanine inhibited the transformation of VSMCs from contractile to the synthetic phenotype. Further experiments showed that l-theanine exhibits potential preventive effects on neointimal hyperplasia and related vascular remodeling via inhibition of phosphorylation of Elk-1 and activation of MAPK1. The present study provides the new experimental evidence that l-theanine has potential clinical application as an anti-restenosis agent for the prevention of restenosis.

Targeted overexpression of the long noncoding RNA ODSM can regulate osteoblast function in vitro and in vivo.

Ameliorating bone loss caused by mechanical unloading is a substantial clinical challenge, and the role of noncoding RNAs in this process has attracted increasing attention. In this study, we found that the long noncoding RNA osteoblast differentiation-related lncRNA under simulated microgravity (lncRNA ODSM) could inhibit osteoblast apoptosis and promote osteoblast mineralization in vitro. The increased expression level of the lncRNA ODSM partially reduced apoptosis and promoted differentiation in MC3T3-E1 cells under microgravity unloading conditions, and the effect was partially dependent on miR-139-3p. LncRNA ODSM supplementation in hindlimb-unloaded mice caused a decrease in the number of apoptotic cells in bone tissue and an increase in osteoblast activity. Furthermore, targeted overexpression of the lncRNA ODSM in osteoblasts partially reversed bone loss induced by mechanical unloading at the microstructural and biomechanical levels. These findings are the first to suggest the potential value of the lncRNA ODSM in osteoporosis therapy and the treatment of pathological osteopenia.

Total Flavonoid Extract from Hawthorn (Crataegus pinnatifida) Improves Inflammatory Cytokines-Evoked Epithelial Barrier Deficit.

BACKGROUND Intestinal epithelial barrier dysfunction is involved in the development and pathogenesis of intestinal diseases, such as irritable bowel syndrome, inflammatory bowel disease, and celiac disease. This study was performed to evaluate the ability of total flavonoid extract from hawthorn (TFH) to improve TNF-alpha-evoked intestinal epithelial barrier deficit. MATERIAL AND METHODS Caco-2 cells monolayers were exposed to TNF-alpha in different concentrations of TFH. Intestinal epithelial barrier function was evaluated using epithelial permeability and transepithelial electrical resistance (TER). RESULTS Our findings showed that TFH alleviated the increase of paracellular permeability and the decline of transepithelial electrical resistance (TER) evoked by TNF-alpha. Additionally, 24-h pre-incubation with TFH inhibited TNF-alpha-evoked secretion of pro-inflammatory factors (IL-6, IL-8, MCP-1, and IL-1ß). Furthermore, TFH inhibited TNF-alpha-evoked overexpression of pMLC and MLCK and alleviated breakdown of TJs protein (ZO-1 and occludin). The activations of Elk-1 and NFkappaBp65 were inhibited by TFH pre-incubation. CONCLUSIONS TFH can alleviate TNF-alpha-evoked intestinal epithelial barrier deficit via the NFkappaBp65-mediated MLCK-MLC signaling pathway.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Transcriptional activation of miR-320a by ATF2, ELK1 and YY1 induces cancer cell apoptosis under ionizing radiation conditions.

MicroRNAs (miRNAs or miRs) play important roles in numerous cellular processes, including development, proliferation, tumorigenesis and apoptosis. It has been reported that miRNA expression is induced by ionizing radiation (IR) in cancer cells. However, the underlying molecular mechanisms are not yet fully understood. In this study, endogenous miR­320a and its primary precursor (pri­miR­320a) were assayed by reverse transcription­quantitative PCR (RT­qPCR). Luciferase activities were measured using a dual­luciferase reporter assay system. Western blot analysis was used to determine the protein expressions of upstream and downstream genes of miR­320a. Cell apoptosis was evaluated by Annexin V apoptosis assay and cell proliferation was measured using the trypan blue exclusion method. The results revealed that miR­320a expression increased linearly with the IR dose and treatment duration. Three transcription factors, activating transcription factor 2 (ATF2), ETS transcription factor (ELK1) and YY1 transcription factor (YY1), were activated by p38 mitogen­activated protein kinase (MAPK) and mitogen­activated protein kinase 8 (JNK) and by upregulated miR­320a expression under IR conditions. In addition, it was identified that X­linked inhibitor of apoptosis (XIAP) was an miR­320a target gene during the IR response. By targeting XIAP, miR­320a induced apoptosis and inhibited the proliferation of the cancer cells. On the whole, the results of this study demonstrated that miRNA­320a, regulated by the p38 MAPK/JNK pathway, enhanced the radiosensitivity of cancer cells by inhibiting XIAP and this may thus prove to be a potential therapeutic approach with which to overcome radioresistance in cancer treatment.

Diallyl disulphide inhibits apolipoprotein(a) expression in HepG2 cells through the MEK1-ERK1/2-ELK-1 pathway.

BACKGROUND: Lipoprotein(a) [LP(a)] is implicated as a common and independent risk factor for cardiovascular diseases. The therapeutic options currently available for reducing plasma LP(a) concentrations are limited. Diallyl disulphide (DADS), the main component of garlic, regulates lipid metabolism in hepatocytes and adipocytes through ERK1/2 signalling. This study aimed to assess the effect of DADS on apolipoprotein(a) [apo(a)] in HepG2 cells. We also determined the effects of DADS on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms. METHODS: We examined the role of DADS on apo(a) expression in HepG2 cells by treating cell with different concentrations of DADS (10, 20, 40 and 80 µg/mL) for 24 h or treating cells with 40 µg/mL DADS for 0, 6, 12, 24 and 48 h. Then we used quantitative real-time PCR to analysis apo(a) mRNA levels, used Western blot to analysis apo(a) protein levels and used enzyme-linked immunosorbent assay to test apo(a) secreted levels. To farther determined the role of DADS, we applied Transfection of small interfering RNA to knockdown ELK-1levels and applied PD98059, a specific inhibitor of ERK1/2, to block ERK1/2 signal. RESULTS: The results show DADS inhibited apo(a) at both the mRNA and protein levels in HepG2 cells in a dose-dependent manner. DADS-mediated inhibition of apoa(a) expression in HepG2 cells was attenuated when the cells were cultured in medium containing PD98059 (ERK1/2 inhibitor) or were transfected with siRNAs against MEK1 or ELK-1. Overexpression of apo(a) yielded similar results. CONCLUSIONS: This study reveals that DADS can downregulate apo(a) expression in a dose-dependent manner via the MEK-ERK12-ELK-1 pathway.

Prion formation correlates with activation of translation-regulating protein 4E-BP and neuronal transcription factor Elk1.

Cellular mechanisms play a role in conversion of the normal prion protein PrP(C) to the disease-associated protein PrP(Sc). The cells provide not only PrP(C), but also still largely undefined factors required for efficient prion replication. Previously, we have observed that interference with ERK and p38-JNK MAP kinase pathways has opposing effects on the formation of prions indicating that the process is regulated by a balance in intracellualar signaling pathways. In order to obtain a "flow-chart" of such pathways, we here studied the activation of MEK/ERK and mTORC1 downstream targets in relation to PrP(Sc) accumulation in GT1-1 cells infected with the RML or 22L prion strains. We show that inhibition of mTORC1 with rapamycin causes a reduction of PrP(Sc) accumulation at similar low levels as seen when the interaction between the translation initiation factors eIF4E and eIF4G downstream mTORC1 is inhibited using 4EGI-1. No effect is seen following the inhibition of molecules (S6K1 and Mnk1) that links MEK/ERK signaling to mTORC1-mediated control of translation. Instead, stimulation (high [KCl] or [serum]) or inhibition (MEK-inhibitor) of prion formation is associated with increased or decreased phosphorylation of the neuronal transcription factor Elk1, respectively. This study shows that prion formation can be modulated by translational initiating factors, and suggests that MEK/ERK signaling plays a role in the conversion of PrP(C) to PrP(Sc) via an Elk1-mediated transcriptional control. Altogether, our studies indicate that prion protein conversion is under the control of intracellular signals, which hypothetically, under certain conditions may elicit irreversible responses leading to progressive neurodegenerative diseases.

Herbal formula HMC05 prevents human aortic smooth muscle cell migration and proliferation by inhibiting the ERK1/2 MAPK signaling cascade.

HMC05 is a formulation derived from eight medicinal herbs, and prevents neointima formation by inhibition of the mitogen-activated protein kinase (MAPK) pathway with induction of heat shock protein 27 expression. In this study, we investigated the influence of HMC05 regulation on the MAPK/extracellular signal-regulated kinase (ERK) 1/2 signaling cascade in the inhibition of the migration and proliferation of human aortic smooth muscle cells (HASMCs). The inhibitory effects of HMC05 (25, 50, and 100 µg/ml) on tumor necrosis factor-alpha (TNF-α; 0 or 100 ng/ml)-induced HASMC migration and proliferation were investigated by wound migration and proliferation assays, Western blotting and reverse transcription-polymerase chain reaction. HMC05 completely inhibited TNF-α-induced HASMC migration and proliferation. HMC05 prevented TNF-α receptor 1-mediated phosphorylation of signal transduction molecules involved in MAPK signaling cascades such as MEK1/2, ERK1/2, Elk-1 transcription factor and p90 kDa ribosomal S6 kinase. The expression of matrix metalloproteinase, a modulator of vascular smooth muscle cell proliferation and migration, was inhibited by HMCO5 treatment, as was TNF-α-induced mRNA expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1. HMC05 disruption of the MEK/ERK/Elk-1 and p90RSK pathways prevents HASMC migration and proliferation.

Downregulation of CuZn-superoxide dismutase contributes to beta-adrenergic receptor-mediated oxidative stress in the heart.

OBJECTIVE: Sustained beta-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown. METHODS AND RESULTS: Adult male Wistar rats were implanted with osmotic minipumps to infuse either l-isoproterenol (ISO, 25 microg/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and 3) augmentation of oxidative stress, with a approximately 3-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (SOD) enzyme activity (30%), protein (40%), and mRNA (60%), without changes in Mn-SOD, catalase, or glutathione peroxidase. Elk-1 and YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn-SOD expression, respectively. ISO-treated hearts exhibited a 3-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn-SOD gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a beta1-AR antagonist, but not by ICI118,551, a beta2-AR antagonist. CuZn-SOD downregulation paralleled the increase in ROS, and were similarly blocked by beta1- but not beta2-AR blockade. There were no changes in CuZn-SOD mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn-SOD mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn-SOD siRNA transfection of H9c2 cardiomyocytes to suppress CuZn-SOD protein by approximately 40-50% (analogous to the in vivo changes) induced cellular apoptosis. CONCLUSIONS: Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta1-AR, thereby contributing to beta-AR-mediated oxidative stress.

Identification of inhibitors of the kinase activity of oncogenic V600E BRAF in an enzyme cascade high-throughput screen.

The Cancer Genome Project has identified several oncogenic mutations in BRAF that represent important opportunities for cancer drug discovery. The V600E BRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of V600E BRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 +/- 0.12. A series of 3,5, di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 microM) in a V600E BRAF assay and represent a potential starting point for future drug discovery and development.