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.

Therapeutic concentrations of lithium salts inhibit glycogen synthase kinase 3 beta (GSK3β) and phosphoinositide (PI) signaling suggesting that abnormal activation of these pathways could be a factor in the pathophysiology of bipolar disorder (BD). Involvement of these pathways is also supported by recent genome-wide association studies (GWASs). One way investigators have investigated the molecular basis of BD and the therapeutic action of lithium is by microarray expression studies, since both GSK3β- and PI-mediated signal transduction pathways are coupled to transcriptional activation and inhibition. However, expression profiling has some limitations and investigators cannot use the approach to analyze fetal brain tissue, arguably the most relevant biological structure related to the development of genetically based psychiatric disorders. To address these shortcomings, the authors have taken a novel approach using chromatin immunoprecipitation-enriched material annealed to microarrays (ChIP-chip) targeting genes in fetal brain tissue bound by β-catenin, a transcription factor that is directly regulated by GSK3β. The promoters for 640 genes were found to be bound by β-catenin, many of which are known schizophrenia (SZ), autism spectrum disorder (ASD), and BD candidates, including CACNA1B, NRNG, SNAP29, FGFR1, PCDH9, and nine others identified in recently published GWASs and genome-wide searches for copy number variants (CNVs). The findings suggest that seemingly disparate candidate genes for SZ and BD can be incorporated into a common molecular network revolving around GSK3β/β-catenin signaling. In addition, the finding that a putative lithium-responsive pathway may influence a subgroup of SZ and ASD candidate genes could have therapeutic implications.

Selenium deficiency is known to increase cancer risk by so far unclear mechanisms. Selenium exerts its biological effects via selenocysteine as an integral part of selenoproteins. Certain selenoproteins have redox properties, thereby providing a tool to regulate hydroperoxide-mediated signaling. Selenium deficiency does not only reduce synthesis of selenoproteins but also affects the expression of other proteins and even pathways. A moderate Se deficiency activates the Nrf2 and the Wnt pathways. The link between both pathways appears to be GSK3β which in the active state prepares Nrf2 as well as β-catenin, the key player in Wnt signaling, for ubiquitination and proteasomal degradation, thus silencing their transcriptional activity. Upon stimulation by Wnt signals, GSK3β becomes inactivated and transcription factors are stabilized. Many intermediate steps in both pathways can be modulated by hydroperoxides, making them predestined to be regulated by selenoproteins. Oxidation sensors are (i) Keap1 which keeps Nrf2 in the cytosol unless it is modified by hydroperoxides/electrophiles and (ii) nucleoredoxin (Nrx) which is associated with disheveled (Dvl). NOX1-derived H2O2 oxidizes Nrx leading to the liberation of Dvl and the activation of Wnt signaling. Selenium deficiency can support oxidation of both sensors and activate both pathways. The consequences are dual: while the Keap1/Nrf2 system is generally believed to protect against oxidative stress, diverse xenobiotics, inflammation, and carcinogenesis, the Wnt response is considered rather a risky one in these respects. However, not only healthy cells but also malignant ones benefit from intact Keap1/Nrf2 signaling, making a dysregulated hydroperoxide signaling a plausible explanation for the increased cancer risk in selenium deficiency.

OBJECTIVE

The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity.

METHODS

Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression.

RESULTS

We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity.

CONCLUSIONS

Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3β inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81(WT) cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents.

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3β. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences.

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

Aberrant ion channel function has been heralded as a main underlying mechanism driving epilepsy and its symptoms. However, it has become increasingly clear that treatment strategies targeting voltage-gated sodium or calcium channels merely mask the symptoms of epilepsy without providing disease-modifying benefits. Ion channel function is likely only one important cog in a highly complex machine. Gross morphological changes, such as reactive sprouting and outgrowth, may also play a role in epileptogenesis. Mechanisms responsible for these changes are not well-understood. Here we investigate the potential involvement of the neurite outgrowth-promoting molecule collapsin response mediator protein 2 (CRMP2). CRMP2 activity, in this respect, is regulated by phosphorylation state, where phosphorylation by a variety of kinases, including glycogen synthase kinase 3 β (GSK3β) renders it inactive. Phosphorylation (inactivation) of CRMP2 was decreased at two distinct phases following traumatic brain injury (TBI). While reduced CRMP2 phosphorylation during the early phase was attributed to the inactivation of GSK3β, the sustained decrease in CRMP2 phosphorylation in the late phase appeared to be independent of GSK3β activity. Instead, the reduction in GSK3β-phosphorylated CRMP2 was attributed to a loss of priming by cyclin-dependent kinase 5 (CDK5), which allows for subsequent phosphorylation by GSK3β. Based on the observation that the proportion of active CRMP2 is increased for up to 4 weeks following TBI, it was hypothesized that it may drive neurite outgrowth, and therefore, circuit reorganization during this time. Therefore, a novel small-molecule tool was used to target CRMP2 in an attempt to determine its importance in mossy fiber sprouting following TBI. In this report, we demonstrate novel differential regulation of CRMP2 phosphorylation by GSK3β and CDK5 following TBI.

Bioactive molecules and stem cell-based regenerative engineering is emerging a promising approach for regenerating tissues. Autotaxin (ATX) is a key enzyme that regulates lysophosphatidic acid (LPA) levels in biological fluids, which exerts a wide range of cellular functions. However, the biological role of ATX in human umbilical cord blood-derived mesenchymal stem cells (hMSCs) migration remains to be fully elucidated. In this study, we observed that hMSCs, which were stimulated with LPA, accelerated wound healing, and LPA increased the migration of hMSCs into a wound site in a mouse skin wound healing model. In an experiment to investigate the effect of LPA on hMSC migration, ATX and LPA increased hMSC migration in a dose-dependent manner, and LPA receptor 1/3 siRNA transfections inhibited the ATX-induced cell migration. Furthermore, LPA increased Ca(2+) influx and PKC phosphorylation, which were blocked by Gαi and Gαq knockdown as well as by Ptx pretreatment. LPA increased GSK3β phosphorylation and β-catenin activation. LPA induced the cytosol to nuclear translocation of β-catenin, which was inhibited by PKC inhibitors. LPA stimulated the binding of β-catenin on the E-box located in the promoter of the CDH-1 gene and decreased CDH-1 promoter activity. In addition, the ATX and LPA-induced increase in hMSC migration was blocked by β-catenin siRNA transfection. LPA-induced PKC phosphorylation is also involved in Rac1 and CDC42 activation, and Rac1 and CDC42 knockdown abolished LPA-induced F-actin reorganization. In conclusion, ATX/LPA stimulates the migration of hMSCs through LPAR1/3-dependent E-cadherin reduction and cytoskeletal rearrangement via PKC/GSK3β/β-catenin and PKC/Rho GTPase pathways.

Effective treatment and/or prevention strategies for neonatal persistent pulmonary hypertension of the newborn (PPHN) have been an important topic in neonatal medicine. However, mechanisms of impaired pulmonary vascular structure in hypoxia-induced PPHN are poorly understood and consequently limit the development of effective treatment. In this study, we aimed to explore the molecular signaling cascades in the lungs of a PPHN animal model and used primary cultured rat pulmonary microvascular endothelial cells to analyze the physiological benefits of ghrelin during the pathogenesis of PPHN. Randomly selected newborn rats were exposed to hypoxia (10-12%) or room air and received daily s.c. injections of ghrelin (150 μg/kg) or saline. After 2 weeks, pulmonary hemodynamics and morphometry were assessed in the rats. Compared with the control, hypoxia increased pulmonary arterial pressure, right ventricle (RV) hypertrophy, and arteriolar wall thickness. Ghrelin treatment reduced both the magnitude of PH and the RV/(left ventricle+septum (Sep)) weight ratio. Ghrelin protected neonatal rats from hypoxia-induced PH via the upregulation of phosphorylation of glycogen synthase kinase 3β (p-GSK3β)/β-catenin signaling and associated with β-catenin translocation to the nucleus in the presence of growth hormone secretagogue receptor-1a. Our findings suggest that s.c. administration of ghrelin improved PH and attenuated pulmonary vascular remodeling after PPHN. These beneficial effects may be mediated by the regulation of p-GSK3β/β-catenin expression. We propose ghrelin as a novel potential therapeutic agent for PPHN.

Glycogen synthase kinase 3 (GSK3), a prominent enzyme in carbohydrate metabolism, also has a major role in brain function. It is physiologically regulated by the kinase Akt, which phosphorylates GSK3 to inhibit catalytic activity. Inositol hexakisphosphate-1 (IP6K1) generates the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7), which physiologically inhibits Akt leading to enhanced GSK3 activity. We report that IP6K1 binds and stimulates GSK3 enzymatic activity in a non-catalytic fashion. Physiological relevance is evident in the inhibition of GSK3 activity in the brains of IP6K1-deleted mice. Behavioral alterations of IP6K1 knockout mice resemble those of GSK3 mutants. Accordingly, modulation of IP6K1-GSK3β interaction may exert beneficial effects in psychiatric disorders involving GSK3.

OBJECTIVE

Insulin resistance has been reported in up to 70% of women with polycystic ovary syndrome (PCOS). Physiologic and genetic data currently implicate post-insulin receptor signaling defects in substrates such as glycogen synthase kinase 3beta (GSK3beta). The AKT2 gene was chosen as a candidate for PCOS because its product affects glucose metabolism and mitogenic signaling, interacts with GSK3beta, and mediates cell survival in the ovary.

METHODS

Subjects were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham, and control subjects were recruited from the surrounding community; 287 white women with PCOS and 187 white control subjects were genotyped for four single nucleotide polymorphisms (SNPs) in AKT2. Genotyping took place at Cedars-Sinai Medical Center in Los Angeles. SNPs and haplotypes were tested for association with PCOS risk and phenotypic markers of PCOS.

RESULTS

Minor allele carriers of SNPs rs3730051 and rs8100018 had increased odds of PCOS (odds ratio [OR] 2.2, P = 0.004, and 2.4, P = 0.001, respectively). The haplotype T-G-C-T was significantly associated with PCOS (OR 2.0, P = 0.01). Carriers of the risk haplotypes for both AKT2 and GSK3B had a further increased odds of PCOS (OR 3.1, P = 0.005).

CONCLUSIONS

These data suggest that polymorphisms in two components of the insulin signaling pathway, AKT2 and GSK3B, are associated with PCOS. The presence of multiple lesions in a single pathway may confer increased risk.

Transmembrane protein 88 (TMEM88) is a transmembrane protein that plays a crucial role in regulating human stem cell differentiation and embryonic development. However, its expression and clinicopathologic significance in human neoplasms is unclear. In this study, the expression and subcellular localizations of TMEM88 were assessed in 214 cases of non-small cell lung cancer (NSCLC). Notably, TMEM88 was highly expressed in the cytosol of ∼60% NSCLC specimens examined. Higher expression of cytosolic TMEM88 in NSCLC correlated significantly with poor differentiation, high TNM stage, lymph node metastasis, and inferior survival. In NSCLC cells displaying membrane-localized TMEM88, we observed an inhibition of canonical Wnt signaling due to interactions of TMEM88 with the Wnt pathway factor Dishevelled (DVLS). In contrast, NSCLC cells with cytosol-localized TMEM88 lacked effects on Wnt signaling. Cytosolic interactions of TMEM88 and DVLS increased the expression of phosphorylated, active forms of p38, GSK3β (Thr390), and Snail, thereby reducing the expression of the tight junction-associated proteins ZO-1 and occludin, effects associated with enhanced invasive and metastatic cell characters. Importantly, attenuating the expression of cytosolic TMEM88 reduced metastatic prowess in xenograft models. Overall, our findings show how mislocalization of TMEM88 to the cytosol in NSCLC cells ablates its Wnt pathway regulatory properties, thereby promoting invasion and metastasis by activating the p38-GSK3β-Snail signaling pathway.

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down-regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease.

Peroxisome proliferator activated receptor γ (PPARγ) has been closely involved in the process of cardiovascular diseases. This study was to investigate whether pioglitazone (PIO), a PPARγ agonist, could protect against pressure overload-induced cardiac hypertrophy. Mice were orally given PIO (2.5 mg/kg) from 1 week after aortic banding and continuing for 7 weeks. The morphological examination and biochemical analysis were used to evaluate the effects of PIO. Neonatal rat ventricular cardiomyocytes were also used to verify the protection of PIO against hypertrophy in vitro. The results in our study demonstrated that PIO remarkably inhibited hypertrophic response induced by aortic banding in vivo. Besides, PIO also suppressed cardiac fibrosis in vivo. PIO treatment also inhibited the activation of protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β) and mitogen-activated protein kinase (MAPK) in the heart. In addition, PIO alleviated angiotensin II-induced hypertrophic response in vitro. In conclusion, PIO could inhibit cardiac hypertrophy via attenuation of AKT/GSK3β and MAPK pathways.

DAX1 is well known for its fundamental role in several types of cancer, while its biological role in cervical cancer remains largely unexplored. The expression of DAX1 in cervical carcinoma tissue was examined using immunohistochemistry and western blot. The effects of DAX1 silencing on the cell growth, tumor formation, and CSC (cancer stem cell) characteristics were also investigated. DAX1 expressed a gradual increase from normal cervix to high-grade squamous intraepithelial lesions, and consequently to cervical cancer. Silence of DAX1 significantly inhibited the cell growth, tumorigenicity, and tumorsphere formation. Furthermore, the TOP/FOP-Flash reporter assay revealed that Wnt/β-catenin pathway was significantly inactivated in DAX1-silenced cervical cancer cells with the downregulation of Wnt/β-catenin targeting genes, including cyclinD1 and c-myc. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assay confirmed that DAX1 transcriptionally repressed glycogen synthase kinase 3β (GSK3β), an inhibitor of the Wnt/β-catenin pathway, by physically interacting with -666~-444 motif on the GSK3β promoter. Additionally, the blockage of GSK3β by CHIR-99021 resulted in a significant increase of CSC characteristics induced by the silence of DAX1. Our data demonstrated that DAX1 is overexpressed in cervical cancer, and that it promotes cell growth and tumorigenicity through activating Wnt/β-catenin pathway mediated by GSK3β.

The circadian clock orchestrates diverse physiological processes critical for health and disease. CREB, hepatocyte specific (CREBH) is a liver-enriched, endoplasmic reticulum (ER)-tethered transcription factor known to regulate the hepatic acute phase response and energy homeostasis under stress conditions. We demonstrate that CREBH is regulated by the circadian clock and functions as a circadian regulator of hepatic lipid metabolism. Proteolytic activation of CREBH in the liver exhibits typical circadian rhythmicity controlled by the core clock oscillator BMAL1 and AKT/glycogen synthase kinase 3β (GSK3β) signaling pathway. GSK3β-mediated phosphorylation of CREBH modulates the association between CREBH and the coat protein complex II transport vesicle and thus controls the ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian manner. Functionally, CREBH regulates circadian expression of the key genes involved in triglyceride (TG) and fatty acid (FA) metabolism and is required to maintain circadian amplitudes of blood TG and FA in mice. During the circadian cycle, CREBH rhythmically regulates and interacts with the hepatic nuclear receptors peroxisome proliferator-activated receptor α and liver X receptor α as well as with the circadian oscillation activator DBP and the repressor E4BP4 to modulate CREBH transcriptional activities. In conclusion, these studies reveal that CREBH functions as a circadian-regulated liver transcriptional regulator that integrates energy metabolism with circadian rhythm.