Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3β phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G^-csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytochrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3β inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3β inhibitors to induce cardiac differentiation. GSK3β inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes.

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3β. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3β in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3β to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.

Transplanted neural stem and progenitor cells (NSCs) produce mostly astrocytes in injured spinal cords. Lithium stimulates neurogenesis by inhibiting GSK3b (glycogen synthetase kinase 3-beta) and increasing WNT/beta catenin. Lithium suppresses astrogliogenesis but the mechanisms were unclear. We cultured NSCs from subventricular zone of neonatal rats and showed that lithium reduced NSC production of astrocytes as well as proliferation of glia restricted progenitor (GRP) cells. Lithium strongly inhibited STAT3 (signal transducer and activator of transcription 3) activation, a messenger system known to promote astrogliogenesis and cancer. Lithium abolished STAT3 activation and astrogliogenesis induced by a STAT3 agonist AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), suggesting that lithium suppresses astrogliogenesis by inhibiting STAT3. GSK3β inhibition either by a specific GSK3β inhibitor SB216763 or overexpression of GID5-6 (GSK3β Interaction Domain aa380 to 404) did not suppress astrogliogenesis and GRP proliferation. GSK3β inhibition also did not suppress STAT3 activation. Together, these results indicate that lithium inhibits astrogliogenesis through non-GSK3β-mediated inhibition of STAT. Lithium may increase efficacy of NSC transplants by increasing neurogenesis and reducing astrogliogenesis. Our results also may explain the strong safety record of lithium treatment of manic depression. Millions of people take high-dose (>1 gram/day) lithium carbonate for a lifetime. GSK3b inhibition increases WNT/beta catenin, associated with colon and other cancers. STAT3 inhibition may reduce risk for cancer.

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3β phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity.

BACKGROUND

Proteins of the insulin signaling pathway are needed for cell proliferation and development and glucose homeostasis. It is not known whether insulin signalling markers (Foxo1, Gsk3β) can be correlated with the expression on PI3K-Akt-mTOR pathway, which are needed for cell survival and maintenance of glucose homeostasis. In the present study, we studied the expression of Foxo1, Gsk3β and PI3K-Akt-mTOR in the brain of streptozotocin-induced type 2 diabetes mellitus Wistar rats.

METHODS

The study was performed both in vitro (RIN5F cells) and in vivo (male Wistar rats). Gene expression of Nf-kB, IkB, Bax, Bcl-2 and Pdx1 gene was studied invitro by qRT-PCR in RIN5F cells. In STZ (65 mg/kg i.p.)-induced type 2 DM Wistar rats, blood glucose and insulin levels, iNOS, Foxo1, NF-κB, pGsk3β and PPAR-γ1 levels along with PI3k-Akt-mTOR were measured in brain tissue.

RESULTS

RIN5F cells treated with STZ showed increase in the expression of NF-kB and Bax and decrease in IkB, Bcl-2 and PDX1. Brain tissue of STZ-induced type 2 DM animals showed a significant reduction in secondary messengers of insulin signalling (Foxo1) (P < 0.001) and Gsk3β (P < 0.01) and a significant alteration in the expression of phosphorylated-Akt (P < 0.001) mTOR (P < 0.01) and PI3K.

CONCLUSIONS

These results suggest that STZ induces pancreatic β cell apoptosis by enhancing inflammation. Significant alterations in the expression brain insulin signaling and cell survival pathways seen in brain of STZ-treated animals implies that alterations neuronal apoptosis may have a role in altered glucose homeostasis seen in type 2 DM that may also explain the increased incidence of cognitive dysfunction seen in them.

We investigated the effects of an aqueous alcohol extract of Rhodiola rosea (R. rosea) and its hydrolysate on melanin synthesis and the mechanisms mediating the activity. The ratio of tyrosol to salidroside was 2.3 in hydroalcoholic extract, and 51.0 in hydrolysate. We found that R. rosea extract and its hydrolysate inhibited melanin synthesis and tyrosinase activity in mouse melanoma cells (B16F0 cells). R. rosea extract also inhibited gene and protein expression of melanocortin 1 receptor (MC1R) and inhibited c-AMP response element binding protein (CREB) phosphorylation, suppressed the activation of AKT and glycogen synthase kinase-3 beta (GSK3β), and inhibited the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related protein 1 (TRP-1). R. rosea hydrolysate inhibited the phosphorylation of CREB, the activation of AKT and GSK3β, and the expression of MITF and tyrosinase. Our results suggest that R. rosea extract is a novel tyrosinase inhibitor and that it exerts its effects by regulating the CREB/MITF/tyrosinase pathway in B16F0. Further in vivo studies are needed to determine the effectiveness of R. rosea extract as a skin whitening agent.

Estrogen and androgen receptors (ER and AR) play key roles in breast and prostate cancers, respectively, where they regulate the transcription of large arrays of genes. The activities of ER and AR are controlled by large networks of protein kinases and transcriptional coregulators, including Ddx5 and its highly related paralog Ddx17. The Ddx5 and Ddx17 RNA helicases are also splicing regulators. Here, we report that Ddx5 and Ddx17 are master regulators of the estrogen- and androgen-signaling pathways by controlling transcription and splicing both upstream and downstream of the receptors. First, Ddx5 and Ddx17 are required downstream of ER and AR for the transcriptional and splicing regulation of a large number of steroid hormone target genes. Second, Ddx5 and Ddx17 act upstream of ER and AR by controlling the expression, at the splicing level, of several key regulators of ER and AR activities. Of particular interest, we demonstrate that Ddx5 and Ddx17 control alternative splicing of the GSK3β kinase, which impacts on both ER and AR protein stability. We also provide a freely available online resource which gives information regarding splicing variants of genes involved in the estrogen- and androgen-signaling pathways.

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.

Parkinson's disease (PD) is a pathological condition characterized by a progressive neurodegeneration of dopaminergic neurons with the consequent reduction of dopamine content in the substantia nigra. The neurotoxin 6-hydroxydopamine (6-OHDA) is widely used to mimic the neuropathology of PD in both in vivo and in vitro experimental models. We found that, as expected, in dopaminergic human SH-SY5Y neuroblastoma cells the toxin reduced cell viability causing programmed cell death as assessed by an increase in DNA fragmentation. We also examined, in these cells, the activation/inactivation of several pro and anti apoptotic signaling pathways by 6-OHDA including p-38 kinase (p-38), c-Jun N-terminal kinase (JNK), protein kinase B (also known as Akt), glycogen synthase kinase-3β (GSK3β), and Bcl-2 protein. Guanine-based purines, exert neuroprotective effects and we previously reported that guanosine activates cell survival pathways including PI3K/Akt/PKB signaling in different kinds of cells including glia and neuroblastoma cells. In the present study we found that guanosine (300 µM) protected SH-SY5Y neuroblastoma cells when they were exposed to 6-OHDA, promoting their survival. Guanosine reduced the 6-OHDA mediated activation of p-38 and JNK. Moreover the nucleoside potentiated the early increase in the phosphorylation of the anti-apoptotic kinase Akt and the increase in the expression of the anti-apoptotic Bcl-2 protein induced by 6-OHDA. In summary our results show that guanosine results to be neuroprotective in a recognized in vitro model of PD thus suggesting that it could represent a new potential pharmacological tool to be studied in the therapeutic approach to PD.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. It arises from loss of intestinal epithelial homeostasis and hyperproliferation of the crypt epithelium. In order to further understand the pathogenesis of CRC it is important to further understand the factors regulating intestinal epithelial proliferation and more specifically, regulation of the intestinal epithelial stem cell compartment. Here, we investigated the role of the RNA binding protein RBM3 in stem cell homeostasis in colorectal cancers. Using a doxycycline (Dox) inducible RBM3 overexpressing cell lines HCT 116 and DLD-1, we measured changes in side population (SP) cells that have high xenobiotic efflux capacity and increased capacity for self-renewal. In both cell lines, RBM3 induction showed significant increases in the percentage of side population cells. Additionally, we observed increases in spheroid formation and in cells expressing DCLK1, LGR5 and CD44Hi . As the Wnt/β-catenin signaling pathway is important for both physiologic and cancer stem cells, we next investigated the effects of RBM3 overexpression on β-catenin activity. RBM3 overexpression increased levels of nuclear β-catenin as well as TCF/LEF transcriptional activity. In addition, there was inactivation of GSK3β leading to decreased β-catenin phosphorylation. Pharmacologic inhibition of GSK3β using (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO) also recapitulates the RBM3 induced β-catenin activity. In conclusion, we see that RNA binding protein RBM3 induces stemness in colorectal cancer cells through a mechanism involving suppression of GSK3β activity thereby enhancing β-catenin signaling. © 2015 Wiley Periodicals, Inc.

To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTEN(mu) cells, as evidenced by enhanced phosphorylation of mTOR (Ser2481), AKT (Ser473) and glycogen synthase kinase 3 beta (GSK3β) (Ser9) as compared with PTEN(wt) cells. In addition, PTEN(wt) cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTEN(wt) cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTEN(wt) cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3β. Thus, we established the reciprocal activation of mTORC2 and GSK3β in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTEN(mu) and PTEN(wt) cells. It inhibited both mTORC1/2 and AKT completely in PTEN(mu) cells, whereas it inhibited only mTORC1 in PTEN(wt) cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTEN(wt) cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTEN(mu) cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM.

BACKGROUND

The developmental transcription factor Grainyhead-like 3 (GRHL3) plays a critical tumor suppressor role in the mammalian epidermis through direct regulation of PTEN and the PI3K/AKT/mTOR signaling pathway. GRHL3 is highly expressed in all tissues derived from the surface ectoderm, including the oral cavity, raising a question about its potential role in suppression of head and neck squamous cell carcinoma (HNSCC).

METHODS

We explored the tumor suppressor role of Grhl3 in HNSCC using a conditional knockout (Grhl3 (∆/-) /K14Cre (+) ) mouse line (n = 26) exposed to an oral chemical carcinogen. We defined the proto-oncogenic pathway activated in the HNSCC derived from these mice and assessed it in primary human HNSCC samples, normal oral epithelial cell lines carrying shRNA to GRHL3, and human HNSCC cell lines. Data were analyzed with two-sided chi square and Student's t tests.

RESULTS

Deletion of Grhl3 in oral epithelium in mice did not perturb PTEN/PI3K/AKT/mTOR signaling, but instead evoked loss of GSK3B expression, resulting in stabilization and accumulation of c-MYC and aggressive HNSCC. This molecular signature was also evident in a subset of primary human HNSCC and HNSCC cell lines. Loss of Gsk3b in mice, independent of Grhl3, predisposed to chemical-induced HNSCC. Restoration of GSK3B expression blocked proliferation of normal oral epithelial cell lines carrying shRNA to GRHL3 (cell no., Day 8: Scramble ctl, 616±21.8 x 10(3) vs GRHL3-kd, 1194±44 X 10(3), P < .001; GRHL3-kd vs GRHL3-kd + GSK3B, 800±98.84 X 10(3), P = .003) and human HNSCC cells.

CONCLUSIONS

We defined a novel molecular signature in mammalian HNSCC, suggesting new treatment strategies targeting the GRHL3/GSK3B/c-MYC proto-oncogenic network.

In pancreatic β-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3β (GSK3β) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3β on its inactivating phosphorylation site Ser(9). Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser(9) phosphorylation of GSK3β. Accordingly, GSK3β Ser(9) phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KD GSK3β protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3β, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3β-mediated PDX-1 protein degradation in pancreatic β-cells.

Glycogen synthase kinase 3β (GSK3β) is phosphorylated and inactivated by the phosphoinositide 3 kinase PI3K/Akt pathway. Activation of Akt phosphorylates GSK3β preventing phosphorylation of cyclin D1 which leads to accumulation and nuclear localisation of cyclin D1, activation of CDK4/6 and cell cycle progression. The CCND1 gene found at chromosome 11q13 has been shown to be amplified in approximately 15 % of breast cancers. Cyclin D1, the product of the CCND1 gene, is one of the most commonly overexpressed proteins in breast cancer. Protein expression for GSK3β, phosphorylated-GSK3β (p-GSK3β), cyclin D1 and gene expression of CCND1 were examined in tissue microarrays of 1,686 patients from the Edinburgh Breast Conservation Series. High GSK3β expression was associated with reduced distant relapse-free survival (DRFS), while no association between p-GSK3β and breast cancer-specific survival was seen. CCND1 amplification is also associated with poor DRFS. On the contrary, cyclin D1 overexpression is associated with an increase in DRFS. Multivariate analysis was performed. We suggest that analysis of both GSK3β and cyclin D1 expressions can be considered as a marker of good prognosis in early breast cancer.

Neurological disorders such as Alzheimer's disease, stroke and epilepsy are currently marred by the lack of effective treatments to prevent neuronal death. Erroneous cell cycle reentry (CCR) is hypothesized to have a causative role in neurodegeneration. We show that forcing S-phase reentry in cultured hippocampal neurons is sufficient to induce neurodegeneration. We found that kainic-acid treatment in vivo induces erroneous CCR and neuronal death through a Notch-dependent mechanism. Ablating Notch signaling in neurons provides neuroprotection against kainic acid-induced neuronal death. We further show that kainic-acid treatment activates Notch signaling, which increases the bioavailability of CyclinD1 through Akt/GSK3β pathway, leading to aberrant CCR via activation of CyclinD1-Rb-E2F1 axis. In addition, pharmacological blockade of this pathway at critical steps is sufficient to confer resistance to kainic acid-induced neurotoxicity in mice. Taken together, our results demonstrate that excitotoxicity leads to neuronal death in a Notch-dependent manner through erroneous CCR.

Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2), an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride)-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC) biophysical properties. This led to the identification of (S)-lacosamide ((S)-LCM), a stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S)-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R)-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S)-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are perfunctory for neurite outgrowth. Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

Malignant glioma is an aggressive brain cancer that responds poorly to chemotherapy. However, the molecular mechanism underlying the development of chemoresistance in glioma is not well-understood. In this study, we show that long non-coding RNA AC023115.3 is induced by cisplatin in human glioblastoma cells and that elevated AC023115.3 promotes cisplatin-induced apoptosis by inhibiting autophagy. Further mechanistic studies revealed that AC023115.3 acts as a competing endogenous RNA for miR-26a and attenuates the inhibitory effect of miR-26a on GSK3β, a proline-directed serine-threonine kinase that promotes the degradation of Mcl1, leading to an increase in GSK3β and a decrease in autophagy. Additionally, we discovered that AC023115.3 improves chemosensitivity of glioma cells to cisplatin by regulating the miR-26a-GSK3β-Mcl1 pathway. Thus, these data indicate that the AC023115.3-miR-26a-GSK3β signalling axis plays an important role in reducing the chemoresistance of glioma.

BACKGROUND

Memories return to a labile state following their retrieval and must undergo a process of reconsolidation to be maintained. Thus, disruption of cocaine reward memories by interference with reconsolidation may be therapeutically beneficial in the treatment of cocaine addiction.

OBJECTIVE

The objectives were to elucidate the signaling pathway involved in reconsolidation of cocaine reward memory and to test whether targeting this pathway could disrupt cocaine-associated contextual memory.

METHODS

Using a mouse model of conditioned place preference, regulation of the activity of glycogen synthase kinase-3 (GSK3), mammalian target of Rapamycin complex 1 (mTORC1), P70S6K, β-catenin, and the upstream signaling molecule Akt, was studied in cortico-limbic-striatal circuitry after re-exposure to an environment previously paired with cocaine.

RESULTS

Levels of phosporylated Akt-Thr308, GSK3α-Ser21, GSK3β-Ser9, mTORC1, and P70S6K were reduced in the nucleus accumbens and hippocampus 10 min after the reactivation of cocaine cue memories. Levels of pAkt and pGSK3 were also reduced in the prefrontal cortex. Since reduced phosphorylation of GSK3 indicates heightened enzyme activity, the effect of a selective GSK3 inhibitor, SB216763, on reconsolidation was tested. Administration of SB216763 immediately after exposure to an environment previously paired with cocaine abrogated a previously established place preference, suggesting that GSK3 inhibition interfered with reconsolidation of cocaine-associated reward memories.

CONCLUSIONS

These findings suggest that the Akt/GSK3/mTORC1 signaling pathway in the nucleus accumbens, hippocampus, and/or prefrontal cortex is critically involved in the reconsolidation of cocaine contextual reward memory. Inhibition of GSK3 activity during memory retrieval can erase an established cocaine place preference.

Fipronil (FPN) is a phenylpyrazole insecticide acted on insect gamma-aminobutyric acid (GABA) receptors. Although action of FPN is restricted on insect neuronal or muscular transmitter system, a few studies have assessed the effects of this neurotoxicant on neuronal cell death. To determine the mechanisms underlying FPN-induced neuronal cell death, we investigated whether reactive oxygen species (ROS) plays a role in FPN-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. FPN was cytotoxic to these cells and its cytotoxicity showed a concentration-dependent manner. Additionally, FPN treatment significantly decreased the tyrosine hydroxylase (TH) expression without change of glutamic acid decarboxylase 65 (GAD65) expression. FPN-induced dopaminergic cell death involved in increase of ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FPN treatment, dopamine (DA) levels decreased significantly in both cell and culture media, and oxidative effects of DA were blocked by NAC pretreatment. We showed that cell death in response to FPN was due to apoptosis since FPN increased cytochrome c release into the cytosol and activated caspase-3. It also led to nuclear accumulation of p53 and reduced the level of Bcl-2 protein in a concentration-dependent manner. Additionally, FPN altered the level of Akt/glycogen synthase kinase-3 (GSK3β) phosphorylation. FPN reduced the Akt phosphorylation on Ser473, and in parallel with the inactivation of Akt, phosphorylation of GSK3β on Ser9 which inactivates GSK3β, decreased after treatment with FPN. Furthermore, inhibition of the GSK3β signal protected the cell against FPN-induced cell death. These results suggest that regulation of GSK3β activity may control the apoptosis induced by FPN-induced oxidative stress associated with neuronal cell death.

Magnolol is isolated from the herb Magnolia officinalis, which has been demonstrated to exert pharmacological effects. Our aim was to investigate whether magnolol is able to act as an anti-inflammatory agent that brings about neuroprotection using a global ischemic stroke model and to determine the mechanisms involved. Rats were treated with and without magnolol after ischemia reperfusion brain injury by occlusion of the two common carotid arteries. The inflammatory cytokine production in serum and the volume of infarction in the brain were measured. The proteins present in the brains obtained from the stroke animal model (SAM) and control animal groups with and without magnolol treatment were compared. Magnolol reduces the total infarcted volume by 15% and 30% at dosages of 10 and 30mg/kg, respectively, compared to the untreated SAM group. The levels of acute inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 were attenuated by magnolol. Magnolol was also able to suppress the production of nitrotyrosine, 4-hydroxy-2-nonenal (4-HNE), inducible NO synthase (iNOS), various phosphorylated p38 mitogen-activated protein kinases and various C/EBP homologues. Furthermore, this modulation of ischemia injury factors in the SAM model group treated with magnolol seems to result from a suppression of reactive oxygen species production and the upregulation of p-Akt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings confirm the anti-oxidative properties of magnolol, including the inhibition of ischemic injury to neurons; this protective effect seems to involve changes in the in vivo activity of Akt, GSK3β and NF-κB.

Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on β-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of β-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for β-catenin processing. The β-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3β were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that β-catenin fragments were translocated to the nucleus. The accumulation of β-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the β-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of β-catenin and disassociation of the β-catenin destruction complex by gingipain-dependent proteolytic processing. β-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.

The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer cells and suggest that patients׳ hormonal status through this activity may play a significant role in some therapeutic outcomes.