During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Here, we provide evidences that natural product derivative 3-azido Withaferin A (3-AWA) abrogated EMT and invasion by modulating β-catenin localization and its transcriptional activity in the prostate as well as in breast cancer cells. This study, for the first time, reveals 3-AWA treatment consistently sequestered nuclear β-catenin and augmented its cytoplasmic pool as evidenced by reducing β-catenin transcriptional activity in these cells. Moreover, 3-AWA treatment triggered robust induction of pro-apoptotic intracellular Par-4, attenuated Akt activity and rescued Phospho-GSK3β (by Akt) to promote β-catenin destabilization. Further, our in vitro studies demonstrate that 3-AWA treatment amplified E-cadherin expression along with sharp downregulation of c-Myc and cyclin D1 proteins. Strikingly, endogenous Par-4 knock down by siRNA underscored 3-AWA mediated inhibition of nuclear β-catenin was Par-4 dependent and suppression of Par-4 activity, either by Bcl-2 or by Ras transfection, restored the nuclear β-catenin level suggesting Par-4 mediated β-catenin regulation was not promiscuous. In vivo results further demonstrated that 3-AWA was effective inhibitor of tumor growth and immunohistochemical studies indicated that increased expression of total β-catenin and decreased expression of phospho-β-catenin and Par-4 in breast cancer tissues as compared to normal breast tissue suggesting Par-4 and β-catenin proteins are mutually regulated and inversely co-related in normal as well as cancer condition. Thus, strategic regulation of intracellular Par-4 by 3-AWA in diverse cancers could be an effective tool to control cancer cell metastasis. Conclusively, this report puts forward a novel approach of controlling deregulated β-catenin signaling by 3-AWA induced Par-4 protein.

The molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. In the present study, pharmacological inhibitors were deployed to investigate potential processes by which the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors modulates Tau phosphorylation in rat hippocampal slices. Our results demonstrated that Tau phosphorylation at Ser199-202 residues was decreased in NMDA-treated hippocampal slices, an effect that was not reproduced at Ser262 and Ser404 epitopes. NMDA-induced reduction of Tau phosphorylation at Ser199-202 was further promoted when NR2A-containing receptors were pharmacologically isolated and were completely abrogated by the NR2A receptor antagonist NVP-AAM077. Compared with nontreated slices, we observed that NMDA receptor activation was reflected in high Ser9 and low Tyr216 phosphorylation of glycogen synthase kinase-3 beta (GSK3β), suggesting that NMDA receptor activation might diminish Tau phosphorylation via a pathway involving GSK3β inhibition. Accordingly, we found that GSK3β inactivation by a protein kinase C- (PKC-) dependent mechanism is involved in the NMDA-induced reduction of Tau phosphorylation at Ser199-202 epitopes. Taken together, these data indicate that NR2A receptor activation may be important in limiting Tau phosphorylation by a PKC/GSK3β pathway and strengthen the idea that these receptors might act as an important molecular device counteracting neuronal cell death mechanisms in various pathological conditions.

Leptin (Lep), an adipose-derived hormone, exerts very important functions in the body mainly on energy storage and availability. The physiological effects of Lep controlling the body weight and suppressing appetite are mediated by the long form of Lep receptor in the hypothalamus. Lep receptor activates several downstream molecules involved in key pathways related to cell survival such as STAT3, PI3K, MAPK, AMPK, CDK5 and GSK3β. Collectively, these pathways act in a coordinated manner and form a network that is fully involved in Lep physiological response. Although the major interest in Lep is related to its role in the regulation of energy balance, and since resistance to Lep affects is the primary risk factor for obesity, the interest on their effects on brain cognition and neuroprotection is increasing. Thus, Lep and Lep mimetic compounds now await and deserve systematic exploration as the orchestrator of protective responses in the nervous system. Moreover, Lep might promote the activation of a cognitive process that may retard or even partially reverse selected aspects of Alzheimer's disease or ageing memory loss.

Melusin is a muscle-specific protein which interacts with β1 integrin cytoplasmic domain and acts as chaperone protein. Its overexpression induces improved resistance to cardiac overload delaying left ventricle dilation and reducing the occurrence of heart failure. Here, we investigated possible protective effect of melusin overexpression against acute ischemia/reperfusion (I/R) injury with or without Postconditioning cardioprotective maneuvers. Melusin transgenic (Mel-TG) mice hearts were subjected to 30-min global ischemia followed by 60-min reperfusion. Interestingly, infarct size was reduced in Mel-TG mice hearts compared to wild-type (WT) hearts (40.3 ± 3.5 % Mel-TG vs. 59.5 ± 3.8 % WT hearts; n = 11 animals/group; P < 0.05). The melusin protective effect was also demonstrated by measuring LDH release, which was 50 % lower in Mel-TG compared to WT. Mel-TG hearts had a higher baseline level of AKT, ERK1/2 and GSK3β phosphorylation, and displayed increased phospho-kinases level after I/R compared to WT mice. Post-ischemic Mel-TG hearts displayed also increased levels of the anti-apoptotic factor phospho-BAD. Importantly, pharmacological inhibition of PI3K/AKT (Wortmannin) and ERK1/2 (U0126) pathways abrogated the melusin protective effect. Notably, HSP90, a chaperone known to protect heart from I/R injury, showed high levels of expression in the heart of Mel-TG mice suggesting a possible collaboration of this molecule with AKT/ERK/GSK3β pathways in the melusin-induced protection. Postconditioning, known to activate AKT/ERK/GSK3β pathways, significantly reduced IS and LDH release in WT hearts, but had no additive protective effects in Mel-TG hearts. These findings implicate melusin as an enhancer of AKT and ERK pathways and as a novel player in cardioprotection from I/R injury.

Tyrosine kinase inhibitors (TKIs) against EGFR and c-Met are initially effective when administered individually or in combination to non-small cell lung cancer (NSCLC) patients. However, the overall efficacies of TKIs are limited due to the development of drug resistance. Therefore, it is important to elucidate mechanisms of EGFR and c-Met TKI resistance in order to develop more effective therapies. Model NSCLC cell lines H1975 and H2170 were used to study the similarities and differences in mechanisms of EGFR/c-Met TKI resistance. H1975 cells are positive for the T790M EGFR mutation, which confers resistance to current EGFR TKI therapies, while H2170 cells are EGFR wild-type. Previously, H2170 cells were made resistant to the EGFR TKI erlotinib and the c-Met TKI SU11274 by exposure to progressively increasing concentrations of TKIs. In H2170 and H1975 TKI-resistant cells, key Wnt and mTOR proteins were found to be differentially modulated. Wnt signaling transducer, active β-catenin was upregulated in TKI-resistant H2170 cells when compared to parental cells. GATA-6, a transcriptional activator of Wnt, was also found to be upregulated in resistant H2170 cells. In H2170 erlotinib resistant cells, upregulation of inactive GSK3β (p-GSK3β) was observed, indicating activation of Wnt and mTOR pathways which are otherwise inhibited by its active form. However, in H1975 cells, Wnt modulators such as active β-catenin, GATA-6 and p-GSK3β were downregulated. Additional results from MTT cell viability assays demonstrated that H1975 cell proliferation was not significantly decreased after Wnt inhibition by XAV939, but combination treatment with everolimus (mTOR inhibitor) and erlotinib resulted in synergistic cell growth inhibition. Thus, in H2170 cells and H1975 cells, simultaneous inhibition of key Wnt or mTOR pathway proteins in addition to EGFR and c-Met may be a promising strategy for overcoming EGFR and c-Met TKI resistance in NSCLC patients.

Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.

Hepatocellular carcinoma (HCC) is one of the common malignant cancer worldwide, and its molecular pathogenesis remains elusive and recently long non-coding RNAs (lncRNAs) have been reported that play divergent roles in HCC tumorigenesis and development. In current study, we found a lncRNA, CRNDE is more commonly up-regulated in HCC malignant tissues and associated with poor clinical outcomes. Furthermore, both loss- and gain-functions assays revealed that CRNDE promotes HCC cell proliferation and growth in vitro and in vivo. In addition, we found that CRNDE regulates PI3K/Akt/GSK3β-Wnt/β-catenin axis to exert its oncogenic role in HCC cell proliferation and growth. In conclusion, our findings enlarged our knowledge in the roles of lncRNAs in the progression of HCC and CRNDE as a lncRNA could be a novel target for HCC treatment.

Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

Hyperphosphorylation of tau is closely associated with its aggregation by as yet undefined mechanisms. We attempted herein to further investigate the interrelationships between tau aggregation and phosphorylation by inhibition and activation of cdk5 and GSK3β in cells expressing normal tau and a mutant form of tau (3PO-tau), which generates intracellular aggregates while retaining microtubule-binding capacity). Aggregates were routinely observed in cells expressing 3PO-tau, but never in cells expressing normal tau, whether or not cdk5 or GSK3β was overexpressed. In addition, in cells expressing 3PO-tau, both the percentage of cells with aggregates, as well as the size of aggregates, was increased following overexpression of cdk5 or GSK3β, decreased following treatment with pharmacological agents (roscovitine and lithium) active against these kinases, and increased following treatment with the phosphatase inhibitor okadaic acid. These findings collectively indicate that phosphorylation potentiates aggregation in the presence of one or more key tau mutations. These findings confirm and extend prior studies in which overexpression of the cdk5 activator p35, or GSK3β, induced phosphorylation, mislocalization and/or aggregation of tau.

BACKGROUND

Curcuma kwangsiensis S. G. Lee & C. F. Liang (Guangxi ezhu, in Chinese) belongs to the Zingiberaceae family, has been used as a traditionally Chinese medicine nearly 2000 year. Curcumol is one of the guaiane-type sesquiterpenoid hemiketal isolated from medicine plant Curcuma kwangsiensis S. G. Lee & C. F. Liang, which has been reported possesses anti-cancer effects. Our previous study found that the most contribution to inhibit nasopharyngeal carcinoma cell growth was curcumol.

OBJECTIVE

To assess the effect of curcumol on cell cycle arrest against human colon cancer cells (CRC) cells (LoVo and SW480) and explore its mechanism in vitro and in vivo.

METHODS

Curcumol was dissolved in absolute ethyl alcohol. The concentration of absolute ethyl alcohol in the control group or in experimental samples was always 1/500 (v/v) of the final medium volume. LoVo and SW480 cells were treated with different concentrations of curcumol (0, 53, 106, 212 and 424μM). And then the cell cycle of each group was examined by flow cytometry. The protein levels of PI3K, p-Akt, cyclin D1, cyclin E, CDK2, CDK4 and GSK3β were determined by Western blot. The mRNA expression of PI3K, Akt, cyclin D1, CDK4, P27, p21, and P16 in the treated cells were analyzed by real-time RT-PCR. In addition, the antitumor activity of curcumol was evaluated in nude mice bearing orthotopic tumor implants.

RESULTS

Curcumol induced cell cycle arrest in G1/S phase. RT-qPCR and Western blot data showed that curcumol enhanced the expression of GSK3β, P27, p21 and P16, and decreased the levels of PI3K, phosphorylated Akt (p-Akt), cyclin D1, CDK4, cyclin E and CDK2. Furthermore, curcumol induced reactive oxygen species (ROS) generation in LoVo cells, and ROS scavenger N-acetylcysteine (NAC) significantly reversed curcumol-induced cell growth inhibition. Besides, curcumol also prevented the growth of human colon cancer cells xenografts in nude mouse, accompanied by the reduction of PI3K, Akt, cyclin D1, CDK4, cycln E and significant increase of GSK3β.

CONCLUSIONS

Curcumol caused cell cycle arrest at the G0/G1 phase by ROS production and Akt/ GSK3β/cyclin D1 pathways inactivation, indicating the potential of curcumol in the prevention of colon cancer carcinogenesis.

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process by which a fully differentiated epithelial cell acquires mesenchymal traits, and therefore, mesenchymal abilities such as motility and invasiveness. It is a pivotal physiological process involved in embryogenesis (Type 1 EMT) and in wound healing and tissue remodeling (Type 2 EMT), which, some authors claim, but there are still some controversies, has also been co-opted by tumor cells to increase their malignant potential (Type 3 EMT). Many biomarkers of Type 3 EMT have been characterized and classified into functional categories (i.e., extracellular proteins, cell surface molecules, cytoskeletal markers, transcriptional factors, and, recently, micro RNAs). The extra and intracellular signals that lead to EMT are only starting to be understood, but there is a consensus that Ras and TGF-beta signaling must converge with NF-κB in order to achieve a full EMT. The most classical experimental model is the induction of EMT by TGF-beta in cultures of epithelial cells. Other pathways involving GSK3b, and Wnt/beta-catenin, are also implicated. Ultimately, every EMT-inducing pathway will activate any of the E-cadherin transcriptional repressors (ZEB1, ZEB2, Twist, Snail or Slug). Although in the pre-clinical setting, EMT has also been related to an accelerated tumor progression and to an increased resistance to conventional chemotherapy. In this sense, several groups are beginning to use EMT as a predictive marker of response to treatment. Finally, two chemicals targeting TGF-beta are in clinical trials and many laboratories have initiated studies to use other EMT-related molecules as a therapeutic target for the cancer patient with some modest, but encouraging results.

G protein-coupled Receptor Kinase 6 (GRK6) belongs to a family of kinases that phosphorylate GPCRs. GRK6 levels were found to be altered in Parkinson's Disease (PD) and D(2) dopamine receptors are supersensitive in mice lacking GRK6 (GRK6-KO mice). To understand how GRK6 modulates the behavioral manifestations of dopamine deficiency and responses to L-DOPA, we used three approaches to model PD in GRK6-KO mice: 1) the cataleptic response to haloperidol; 2) introducing GRK6 mutation to an acute model of absolute dopamine deficiency, DDD mice; 3) hemiparkinsonian 6-OHDA model. Furthermore, dopamine-related striatal signaling was analyzed by assessing the phosphorylation of AKT/GSK3β and ERK1/2. GRK6 deficiency reduced cataleptic behavior, potentiated the acute effect of L-DOPA in DDD mice, reduced rotational behavior in hemi-parkinsonian mice, and reduced abnormal involuntary movements induced by chronic L-DOPA. These data indicate that approaches to regulate GRK6 activity could be useful in modulating both therapeutic and side-effects of L-DOPA.

In the heart the most prominent cardiac gap junction protein is connexin43 (Cx43). Increased Cx43 expression has been identified in cardiac hypertrophy and may contribute to arrhythmias. Besides acute effects on gap junction channel function, chronic regulation of Cx43 expression can affect intercellular communication. Since both cyclic mechanical stretch (CMS) and catecholamines play an important role in cardiac physiology and pathophysiology, we wanted to elucidate whether a prolonged β- or α-adrenoceptor stimulation may modulate the effects of CMS on Cx43 expression. Neonatal rat cardiomyocytes were cultured on flexible 6-well plates. Thereafter, cells were kept static without any treatment or stimulated with 0.1μmol/L isoprenaline or phenylephrine for 24h without or with additional CMS (1Hz; 10% elongation). Isoprenaline and phenylephrine given alone significantly increased Cx43-protein and -mRNA level. Also CMS resulted in a significant Cx43-protein and -mRNA up-regulation. The combined treatment of the cells with either isoprenaline or phenylephrine and stretch also resulted in an up-regulation of Cx43-protein and -mRNA, which did not exceed those of stretch, isoprenaline or phenylephrine alone. However, while CMS reduced the Cx43-protein/mRNA ratio, adrenergic stimulation increased Cx43-protein/mRNA ratio. While isoprenaline and phenylephrine increased Cx43-phosphorylation, additional CMS significantly reduced P-Cx43/Cx43 ratio. For further investigation of the underlying signal transduction pathway, we examined the phosphorylated forms of ERK1/2, GSK3β and AKT and could demonstrate that these protein kinases are also significantly up-regulated following stretch or adrenoceptor stimulation. Again the combined treatment of cardiomyocytes with CMS and isoprenaline or phenylephrine had no additive effects. Thus, the combination of α- or β-adrenoceptor stimulation and CMS up-regulates Cx43 expression and leads to phosphorylation of ERK1/2 and AKT (=activation) and of GSK3β (=inactivation). There were no significant additive effects compared to CMS or adrenergic stimulation alone indicating a possible ceiling effect. However, CMS and adrenergic stimulation differentially affected Cx43-protein/mRNA ratio and Cx43-phosphorylation.

Unraveling the biochemical and genetic alterations that control the aggregation of protein tau is crucial to understand the etiology of tau-related neurodegenerative disorders. We expressed wild type and six clinical frontotemporal dementia with parkinsonism (FTDP) mutants of human protein tau in wild-type yeast cells and cells lacking Mds1 or Pho85, the respective orthologues of the tau kinases GSK3β and cdk5. We compared tau phosphorylation with the levels of sarkosyl-insoluble tau (SinT), as a measure for tau aggregation. The deficiency of Pho85 enhanced significantly the phosphorylation of serine-409 (S409) in all tau mutants, which coincided with marked increases in SinT levels. FTDP mutants tau-P301L and tau-R406W were least phosphorylated at S409 and produced the lowest levels of SinT, indicating that S409 phosphorylation is a direct determinant for tau aggregation. This finding was substantiated by the synthetic tau-S409A mutant that failed to produce significant amounts of SinT, while its pseudophosphorylated counterpart tau-S409E yielded SinT levels higher than or comparable to wild-type tau. Furthermore, S409 phosphorylation reduced the binding of protein tau to preformed microtubules. The highest SinT levels were found in yeast cells subjected to oxidative stress and with mitochondrial dysfunction. Under these conditions, the aggregation of tau was enhanced although the protein is less phosphorylated, suggesting that additional mechanisms are involved. Our results validate yeast as a prime model to identify the genetic and biochemical factors that contribute to the pathophysiology of human tau.

Cutaneous squamous cell carcinomas (SCCs) are commonly diagnosed skin cancers that may progress to invasiveness in the absence of early intervention. Using a murine model of SCC, we have previously demonstrated that activation of the Rho-associated kinase (ROCK) signaling pathway promotes rapid progression of pre-neoplastic lesions to invasive SCC. Herein we demonstrate that in human cutaneous SCC, ROCK signaling is increasingly up-regulated with tumor progression in both tumor cells and cells of the tumor microenvironment and is accompanied by key tumor-promoting changes in the extracellular matrix protein composition. The mechanotransduction pathway mediated by integrin signaling through FAK, GSK3β, and the transcription coactivator β-catenin is also progressively activated in human cutaneous SCC. Our observations indicate that ROCK activation is a tumor promoter in human cutaneous SCC and acts via mechanotransduction of signals to β-catenin. Our experiments raise the possibility that inhibition of ROCK signaling could be a useful therapeutic approach to halt cutaneous SCC progression by reducing the signal flux through this pathway to physiologic levels, thereby normalizing the extracellular matrix composition.

Hydrogen sulfide (H2S) is a toxic gas now being recognized as an endogenous signaling molecule in multiple organ systems, in particular, the cardiovascular system. H2S is known to regulate cardiac function and protect against ischemic injury. However, little information is available regarding the effect of H2S on cardiac function in insulin resistance. This study was designed to examine the impact of H2S supplementation on cardiac function using an Akt2 knockout model of insulin resistance. Wild-type and Akt2 knockout mice were treated with NaHS (50 μM·kg(-1)·day(-1) ip for 10 days) prior to evaluation of echocardiographic, cardiomyocyte contractile, and intracellular Ca(2+) properties, apoptosis, and mitochondrial damage. Our results revealed that Akt2 ablation led to overtly enlarged ventricular end-systolic diameter, reduced myocardial and cardiomyocyte contractile function, and disrupted intracellular Ca(2+) homeostasis and apoptosis, the effects of which were ameliorated by H2S. Furthermore, Akt2 knockout displayed upregulated apoptotic protein markers (Bax, caspase-3, caspase-9, and caspace-12) and mitochondrial damage (reduced aconitase activity and NAD(+), elevated cytochrome-c release from mitochondria) along with reduced phosphorylation of PTEN, Akt, and GSK3β in the absence of changes in pan protein expression, the effects of which were abolished or significantly ameliorated by H2S treatment. In vitro data revealed that H2S-induced beneficial effect against Akt2 ablation was obliterated by mitochondrial uncoupling. Taken together, our findings suggest the H2S may reconcile Akt2 knockout-induced myocardial contractile defect and intracellular Ca(2+) mishandling, possibly via attenuation of mitochondrial injury and apoptosis.

Integrin-linked kinase (ILK) is a major structural adaptor protein governing signaling complex formation and cytoskeletal dynamics. Here, through the use of conditional knock-out mice, we demonstrate a requirement for ILK in oligodendrocyte differentiation and axonal myelination in vivo. In conjunction, ILK-deficient primary oligodendrocytes are defined by a failure in process extension and an inability to form myelin membrane upon axonal contact. Surprisingly, phosphorylation of the canonical downstream targets Akt and GSK3β is unaffected following ILK loss. Rather, the defects are due in part to actin cytoskeleton dysregulation with a correspondent increase in active RhoA levels. Morphological rescue is possible following Rho kinase inhibition in an oligodendrocyte subset. Furthermore, phenotypic severity correlates with environmental complexity; oligodendrocytes are severely malformed in vitro (a relatively simple environment), but undergo phenotypic recovery in the context of the whole animal. Together, our work demonstrates ILK as necessary for normal oligodendrocyte development, reinforces its role as a bridge between the actin cytoskeleton and cell membrane, and highlights the overarching compensatory capacity of oligodendrocytes in response to cellular milieu.

BACKGROUND

Although dilated cardiomyopathy (DCM) is a leading cause of heart failure (HF), the mechanism underlying DCM is not well understood. Previously, it has been demonstrated that an integrative analysis of gene expression and protein-protein interaction (PPI) networks can provide insights into the molecular mechanisms of various diseases. In this study we develop a systems approach by linking public available gene expression data on ischemic dilated cardiomyopathy (ICM), a main pathological form of DCM, with data from a layered PPI network. We propose that the use of a layered PPI network, as opposed to a traditional PPI network, provides unique insights into the mechanism of DCM.

METHODS

Four Cytoscape plugins including BionetBuilder, NetworkAnalyzer, Cerebral and GenePro were used to establish the layered PPI network, which was based upon validated subcellular protein localization data retrieved from the HRPD and Entrez Gene databases. The DAVID function annotation clustering tool was used for gene ontology (GO) analysis.

RESULTS

The assembled layered PPI network was divided into four layers: extracellular, plasma membrane, cytoplasm and nucleus. The characteristics of the gene expression pattern of the four layers were compared. In the extracellular and plasma membrane layers, there were more proteins encoded by down-regulated genes than by up-regulated genes, but in the other two layers, the opposite trend was found. GO analysis established that proteins encoded by up-regulated genes, reflecting significantly over-represented biological processes, were mainly located in the nucleus and cytoplasm layers, while proteins encoded by down-regulated genes were mainly located in the extracellular and plasma membrane layers. The PPI network analysis revealed that the Janus family tyrosine kinase-signal transducer and activator of transcription (Jak-STAT) signaling pathway might play an important role in the development of ICM and could be exploited as a therapeutic target of ICM. In addition, glycogen synthase kinase 3 beta (GSK3B) may also be a potential candidate target, but more evidence is required.

CONCLUSIONS

This study illustrated that by incorporating subcellular localization information into a PPI network based analysis, one can derive greater insights into the mechanisms underlying ICM.

Despite compelling evidence supporting key roles for glycogen synthase kinase 3β (GSK3β), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3β activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK(ATP) channels through GSK3β, and this GSK3β signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3β-S9A mice; (ii) Cx43 and GSK3β proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3β inhibition results in mitoK(ATP) opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK(ATP) channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide (43)GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK(ATP)-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3β transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK(ATP) channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

Renal cell carcinoma (RCC) is the most common malignancy in the kidney. Antiangiogenic targeted therapies inhibit the progression of RCC, but have limited impacts on invasion or metastasis of tumor cells. Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth/survival, cell-cycle progression, epithelial-mesenchymal transition (EMT), invasion/migration, and angiogenesis. However, the role of ILK in RCC has not been evaluated. We investigated the role of ILK on cancer progression and metastasis and the therapeutic potential of ILK inhibition in RCC. Our investigation reveals that ILK is expressed at a low level in normal cells and low-stage RCC cells and is highly expressed in advanced and metastatic cells. Caki-1, a metastatic RCC cell line, showed higher expression of molecular EMT markers, including Snail and Zeb1, but decreased activity of GSK3β. Knockdown of ILK using small interference (si)-ILK minimally inhibited tumor proliferation and cell-cycle progression was not significantly affected. However, ILK knockdown suppressed the formation of stress fibers and focal adhesions and impeded phenotypic EMT markers, including cell migration and invasion, in Caki-1 and UMRC-3 cells. Finally, in vivo knockdown of ILK suppressed the progression, invasion, and metastasis of primary RCC in nude mice by downregulation of EMT markers (Snail, Zeb1, vimentin, and E-cadherin). Our results show that ILK may be essential for invasion and metastasis in RCC and regulates vimentin and E-cadherin expression by regulating the EMT-related transcription factors Snail and Zeb1. These results suggest that ILK may be a potential target in RCC.

Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

Phthalates are environmental hormone-like molecules that are associated with breast cancer risk and are involved in metastasis, a process that requires the epithelial-mesenchymal transition (EMT). However, few studies have addressed the potential effects of phthalates on stem cells. Here we tested the hypothesis that phthalates such as butyl benzyl phthalate and di-n-butyl phthalate induce EMT in R2d cells, a stem cell-derived human breast epithelial cell line that is responsive to estradiol for tumor development. We observed that phthalates induced EMT as evidenced by morphological changes concomitant with increased expression of mesenchymal markers and decreased expression of epithelial markers. Molecular mechanism studies revealed that histone deacetylase 6 (HDAC6) is required for phthalate-induced cell migration and invasion during EMT in vitro and metastasis into the lungs of nude mice. We also constructed a series of mutant HDAC6 promoter fragments and found that the transcription factor AP-2a plays a novel role in regulating the HDAC6 promoter. Furthermore, phthalates stimulated estrogen receptors and triggered the downstream EGFR-PKA signaling cascade, leading to increased expression of AP-2a in the nucleus. We also observed that phthalates increased expression of the PP1/HDAC6 complex and caused Akt activation and GSK3β inactivation, leading to transcriptional activation of vimentin through the β-catenin-TCF-4/LEF1 pathway. Understanding the signaling cascades of phthalates that activate EMT through HDAC6 in breast epithelial stem cells provides the identification of novel therapeutic target for human breast cancer.