GSK-3beta Phosphorylation Research Articles

Role of CK1 in GSK3β-mediated phosphorylation and degradation of Snail

The epithelial to mesenchymal transition (EMT) that occurs during embryonic development has begun to attract attention as a potential mechanism for tumor cell metastasis. Snail is a well-known Zn-finger transcription factor that promotes EMT by repressing E-cadherin expression. It is known that Snail is phosphorylated by GSK3beta and degraded by beta-TrCP-mediated ubiquitination. Here we described another protein kinase, CK1, whose phosphorylation of Snail is required for the subsequent GSK3beta phosphorylation. Specific inhibition or depletion of CK1varepsilon inhibits the phosphorylation and degradation of Snail and promotes cell migration, suggesting a central role of CK1varepsilon in the EMT process. Furthermore, our study uncovered distinct roles and steps of Snail phosphorylation by CK1varepsilon and GSK3beta. Taken together, we identified CK1varepsilon as a new component of the Snail-mediated EMT process, providing insight into the mechanism of human cancer metastasis.

Connective tissue growth factor (CTGF) transactivates nuclear factor of activated T‐cells (NFAT) in cells of the osteoblastic lineage

Connective tissue growth factor (CTGF), a member of the Cyr 61, CTGF, Nov (CCN) family of proteins, regulates multiple cellular functions. Overexpression of CTGF in vivo causes osteopenia, but in vitro CTGF can induce osteoblastogenesis. To investigate mechanisms involved in the effects of CTGF on osteoblastic cell differentiation, we examined whether CTGF modifies the activity of nuclear factor of activated T-cells (NFATc) 1, a transcription factor that cooperates with osterix in the formation of new bone. CTGF increased the transactivation of a transiently transfected reporter construct, where 9 NFAT binding sites direct the expression of luciferase (9xNFAT-Luc) and the activity of the Regulators of calcineurin 1 exon 4 (Rcan1.4) promoter, an NFAT target gene. We postulated that CTGF could modify the phosphorylation of NFAT by regulating glycogen synthase kinase 3beta (GSK3beta). CTGF increased the mRNA levels of Protein kinase cyclic guanosine monophosphate (cGMP) dependent type II (Prkg2), the gene encoding for cGMP dependent protein kinase II (cGKII) which phosphorylates GSK3beta. Accordingly, CTGF induced GSK3beta phosphorylation and decreased the active pool of GSK3beta, a kinase that phosphorylates NFAT and leads to its nuclear export. As a consequence, CTGF favored the nuclear localization of NFATc1. Downregulation of PRKG2 by RNA interference reversed the effect of CTGF on the transactivation of the 9xNFAT reporter construct and the Rcan 1.4 promoter, confirming the role of cGKII in the activation of NFAT by CTGF. In conclusion, CTGF enhances NFAT signaling through the induction of cGKII and the phosphorylation of GSK3beta.

Beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells.

Preconditioning Promotes Survival and Angiomyogenic Potential of Mesenchymal Stem Cells in the Infarcted Heart via NF-κB Signaling

We proposed that pharmacological manipulation of mesenchymal stem cells (MSCs) with diazoxide enhanced their survival and regenerative potential via NFkappaB regulation. MSCs preconditioned ((PC)MSCs) with diazoxide and later subjected to oxidant stress with 100 micromol/L H(2)O(2) either immediately or after 24 h exhibited higher survival (p < 0.01 vs nonpreconditioned MSCs; (Non-PC)MSCs) with concomitantly increased phosphorylation of PI3K, Akt, GSK3beta (cytoplasmic), and NF-kappaB (p65) (nuclear). Akt kinase activity was determined as a function of GSK3beta activity. Pretreatment of (PC)MSCs with Wortmannin (Wt), NEMO-binding domain (NBD), or NF-kappaB (p50) siRNA abolished NF-kappaB (p65) activity. Preconditioning increased NF-kappaB-dependent elevation of secretable growth factors associated with their paracrine effects. Inhibition of PI3K activity with Wt reduced (PC)MSCs viability at both early and 24 h time-points. However, inhibition of NF-kappaB reduced viability of (PC)MSCs only at 24 h time-point. For in vivo studies, DMEM without cells (group-1) or containing 1 x 10(6) male (Non-PC)MSCs (group-2), (PC)MSCs (group-3), (PC)MSCs pretreated with Wortmannin (group-4) or NF-kappaB decoy (group-5) were transplanted in a female rat model of acute myocardial infarction. Group-3 showed highest cell survival and growth factor expression, increased angiomyogenesis, and functional improvement. We conclude that activation of NF-kappaB by preconditioning promoted (PC)MSCs survival and angiomyogenic potential in the infarcted heart.

Homologous posttranscriptional regulation of insulin-like growth factor-I receptor level via glycogen synthase kinase-3β and mammalian target of rapamycin in adrenal chromaffin cells: Effect on tau phosphorylation

In cultured bovine adrenal chromaffin cells, approximately 24 h-treatment with insulin-like growth factor-I (IGF-I) decreased cell surface (125)I-IGF-I binding capacity and IGF-I receptor protein level by approximately 64% (EC(50) = 5.0 nM; t(1/2) = approximately 7 h). IGF-I-induced IGF-I receptor decrease was abolished by LY294002 (phosphoinositide 3-kinase inhibitor) and partially attenuated by rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]). SB216763 (an inhibitor of glycogen synthase kinase-3 [GSK-3]) down-regulated IGF-I receptor, which was further decreased by IGF-I. IGF-I increased inhibitory Ser(9)-phosphorylation of GSK-3beta and stimulatory Ser(2448)-phosphorylation of mTOR. l-leucine increased phosphorylation of mTOR (but not GSK-3beta), and down-regulated IGF-I receptor, both events being abolished by rapamycin. IGF-I-induced IGF-I receptor decrease was not prevented by proteolysis inhibitors. Pulse-label with [(35)S]methionine/cysteine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that SB216763 or L-leucine retarded synthesis of IGF-I receptor and its precursor molecule. SB216763 (but not l-leucine) destabilized IGF-I receptor mRNA and decreased its level, without changing IGF-I receptor gene transcription. In SB216763-treated cells, IGF-I-induced Tyr-autophosphorylation of IGF-I receptor was decreased by 36%, compared to nontreated cells. IGF-I attenuated constitutive Ser(396)-phosphorylation of tau by 30% in nontreated cells, but not in SB216763-treated cells. IGF-I-induced down-regulations of (125)I-IGF-I binding and IGF-I receptor, as well as IGF-I-induced phosphorylations of GSK-3beta and mTOR were restored to the control levels of nontreated cells after washout of IGF-I (10 nM for 12 h)-treated cells. Thus, IGF-I down-regulated functional IGF-I receptor via GSK-3beta inhibition and mTOR activation; constitutive activity of GSK-3beta maintained IGF-I receptor level in nonstimulated cells.

Modulation of Microvascular Smooth Muscle Adhesion and Mechanotransduction by Integrin-Linked Kinase

In this study, we investigated the involvement of integrin-linked kinase (ILK) in the adhesion of arteriolar vascular smooth muscle cells (VSMC) to fibronectin (FN) and in the mechano-responsiveness of VSMC focal adhesions (FA). ILK was visualized in VSMC by expressing EGFP-ILK and it was knocked down using ILK-shRNA constructs. Atomic force microscopy (AFM) was used to characterize VSMC interactions with FN, VSMC stiffness and to apply and measure forces at a VSMC single FA site. ILK was localized to FA and silencing ILK promoted cell spreading, enhanced cell adhesion, reduced cell proliferation and reduced downstream phosphorylation of GSK-3beta and PKB/Akt. AFM studies demonstrated that silencing ILK enhanced alpha5beta1 integrin adhesion to FN and enhanced VSMC contraction in response to a pulling force applied at the level of a single FN-FA site. ILK functions in arteriolar VSMC appear linked to multiple signaling pathways and processes that inhibit cell spreading, cell adhesion, FA formation, adhesion to FN and the mechano-responsiveness of FN-FA sites.

AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition.

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.

ERα signaling through slug regulates E-cadherin and EMT

The ERalpha signaling pathway is one of the most important and most studied pathways in human breast cancer, yet numerous questions still exist such as how hormonally responsive cancers progress to a more aggressive and hormonally independent phenotype. We have noted that human breast cancers exhibit a strong direct correlation between ERalpha and E-cadherin expression by immunohistochemistry, suggesting that ERalpha signaling might regulate E-cadherin and implying that this regulation might influence epithelial-mesenchymal transition (EMT) and tumor progression. To investigate this hypothesis and the mechanisms behind it, we studied the effects of ERalpha signaling in ERalpha-transfected ERalpha-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERalpha knockdown in naturally expressing ERalpha-positive lines, MCF-7 and T47D. When ERalpha was overexpressed in the ERalpha-negative lines, 17beta-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERalpha was knocked down in the ERalpha-positive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERalpha signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERalpha, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site estrogen response elements (EREs); indirectly by phosphorylation and inactivation of GSK-3beta through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3beta inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERalpha and E-cadherin immunoreactivity. Our findings indicate that ERalpha signaling through slug regulates E-cadherin and EMT.

Insulin like growth factor-1 prevents 1-mentyl-4-phenylphyridinium-induced apoptosis in PC12 cells through activation of glycogen synthase kinase-3beta

Dopaminergic neurons are lost mainly through apoptosis in Parkinson's disease. Insulin like growth factor-1 (IGF-1) inhibits apoptosis in a wide variety of tissues. Here we have shown that IGF-1 protects PC12 cells from toxic effects of 1-methyl-4-phenylpyridiniumion (MPP +). Treatment of PC12 cells with recombinant human IGF-1 significantly decreased apoptosis caused by MPP + as measured by acridine orange/ethidium bromide staining. IGF-1 treatment induced sustained phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) as shown by western blot analysis. The anti-apoptotic effect of IGF-1 was abrogated by LY294002, which indirectly inhibits phosphorylation of GSK-3beta. Lithium chloride (LiCl), a known inhibitor of GSK-3beta, also blocked MPP +-induced apoptosis. Finally, although IGF-1 enhanced phosphorylation of extracellular signal-regulated kinases ERK1 and 2 (ERK1/2), PD98059, a specific inhibitor of ERK1/2, did not alter the survival effect of IGF-1. Thus, our findings indicate that IGF-1 protects PC12 cells exposed to MPP + from apoptosis via the GSK-3beta signaling pathway.

Neural Wiskott-Aldrich syndrome protein modulates Wnt signaling and is required for hair follicle cycling in mice

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Ischemic post-conditioning reduces infarct size of the in vivo rat heart: role of PI3-K, mTOR, GSK-3β, and apoptosis

Post-conditioning by repetitive cycles of reperfusion/ischemia after prolonged ischemia protects the heart from infarction. The objectives of this study were: Are kinases (PI3-kinase, mTOR, and GSK-3beta) involved in the signaling pathway of post-conditioning? Does post-conditioning result in a diminished necrosis or apoptosis? In open chest rats the infarct size was determined after 30 min of regional ischemia and 30 min of reperfusion using propidium iodide and microspheres. Post-conditioning was performed by three cycles of 30 s reperfusion and reocclusion each, immediately upon reperfusion. PI3-kinase and mTOR were blocked using wortmannin (0.6 mg/kg) or rapamycin (0.25 mg/kg), respectively. The phosphorylation of GSK-3beta and p70S6K was determined with phospho-specific antibodies. TUNEL staining and detection of apoptosis-inducing factor (AIF) were used for the determination of apoptosis. Control hearts had an infarct size of 49 +/- 3%, while post-conditioning significantly reduced it to 29 +/- 3% (P < 0.01). Wortmannin as well as rapamycin completely blocked the infarct size reduction of post-conditioning (51 +/- 2% and 54 +/- 5%, respectively). Western blot analysis revealed that post-conditioning increased the phosphorylation of GSK-3beta by 2.3 times (P < 0.01), and this increase could be blocked by wortmannin, a PI3-kinase inhibitor. Although rapamycin blocked the infarct size reduction, phosphorylation of p70S6K was not increased in post-conditioned hearts. After 2 h of reperfusion, the post-conditioned hearts had significantly fewer TUNEL-positive nuclei (35 %) compared to control hearts (53%; P < 0.001). AIF was equally reduced in post-conditioned rat hearts (P < 0.05 vs. control). Infarct size reduction by ischemic post-conditioning of the in vivo rat heart is PI3-kinase dependent and involves mTOR. Furthermore, GSK-3beta, which is thought to be a regulator of the mPTP, is part of the signaling pathway of post-conditioning. Finally, apoptosis was inhibited by post-conditioning, which was shown by two independent methods. The role of apoptosis and/or autophagy in post-conditioning has to be further elucidated to find therapeutic targets to protect the heart from the consequences of acute myocardial infarction.

Ghrelin Modulates Insulin Sensitivity and Tau Phosphorylation in High Glucose-Induced Hippocampal Neurons

Ghrelin, a 28-amino acid brain-gut peptide expressed in periphery tissues and the central nervous system, has been demonstrated to increase insulin sensitivity in adipocytes. Recent data have indicated that insulin resistance exists in the brain and is related to Alzheimer's Disease (AD). The aim of this study was to investigate whether ghrelin increased high glucose-induced hippocampal neuron insulin sensitivity, and further modulated tau phosphorylation. Hippocampal neurons were cultured in concentrations of 25 mM and 75 mM glucose. The effect of ghrelin on hippocampal neuronal insulin sensitivity was detected by [(3)H]-2-deoxy-D-glucose uptake. The expression of Akt, glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation was determined via Western blotting. Culturation in 75 mM glucose resulted in decreased neuronal glucose uptake and an increase in the level of tau phosphorylation at Ser 199. In neurons treated with ghrelin for 1 h, neuronal glucose uptake was increased and tau hyperphosphorylation was improved. Ghrelin activated Akt and GSK-3beta phosphorylation, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin eliminated ghrelin's effect on neuronal glucose uptake and tau phosphorylation. This study demonstrated that ghrelin increased insulin-stimulated neuronal glucose uptake in 25 mM or 75 mM glucose, raised insulin sensitivity, improved insulin resistance and decreased tau abnormal phosphorylation via the PI3-K/Akt-GSK pathway. Ghrelin is a potential new medicine in the treatment of AD.

Pharmacological Aspects of the Enzastaurin-Pemetrexed Combination in Non-Small Cell Lung Cancer (NSCLC)

Conventional regimens have limited impact against NSCLC. Current research is focusing on multiple pathways as potential targets, and this review describes pharmacological aspects underlying the combination of the PKCbeta-inhibitor enzastaurin with the multitargeted antifolate pemetrexed. Pemetrexed is commonly used, alone or combined with platinum compounds, in NSCLC treatment, and ongoing studies are evaluating its target, thymidylate synthase (TS), as predictor of drug activity. Enzastaurin is a biological targeted agent being actively investigated against different tumors as single agent or in combination. All the downstream events following PKCbeta inhibition by enzastaurin are not completely known, and assays to evaluate possible biomarkers, such as expression of PKC, VEGF and GSK3beta, in tissues and/or in blood samples, are being developed. Enzastaurin-pemetrexed combination was synergistic in preclinical models, including NSCLC cells, where enzastaurin reduced phosphoCdc25C, resulting in G2/M-checkpoint abrogation, and Akt and GSK3beta; phosphorylation, favoring apoptosis induction in pemetrexed-damaged cells. Enzastaurin also significantly reduced VEGF secretion and pemetrexed-induced upregulation of TS expression, possibly via E2F-1 reduction, while the combination decreased TS activity. Similarly, the accumulation of deoxyuridine (a marker of TS inhibition) and the reduction of GSK3beta phosphorylation were detectable in clinical samples from a phase-Ib trial of pemetrexed-enzastaurin combination. In conclusion, the favorable toxicity profile and the multiple effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on proteins involved in pemetrexed activity, provide experimental basis for future studies on enzastaurin-pemetrexed combination and their possible pharmacodynamic markers in NSCLC patients.

Abstract C65: Enhanced antitumor effect by the combination of a PI3K inhibitor ZSTK474 with an mTOR inhibitor rapamycin

Abstract PI3K is a promising molecular target for the therapy of cancer. Various PI3K inhibitors are now under development. We developed a novel PI3K inhibitor ZSTK474 and demonstrated its potent antitumor efficacy. ZSTK474 is highly specific to PI3K and does not inhibit other kinases including mTOR that is a down stream component of PI3K pathway. The purpose of this study was to examine whether dual inhibition of PI3K and mTOR further improved antitumor efficacy. To assess the effect of the combination of ZSTK474 and rapamycin on prostate cancer PC-3 cells and lung cancer A549 cells in vitro, we used the isobologram method. The combination of ZSTK474 and rapamycin showed a supra-additive effect (synergism) on both PC-3 and A549. We then evaluated the efficacy of the combination of ZSTK474 with an mTOR inhibitor rapamycin against the xenografts of PC-3 and A549. Suboptimal doses of ZSTK474 and rapamycin were determined as 100 mg/kg/day (p.o., from day 0 to day 14, except days 3 and 10) and 0.1 mg/kg/day (i.p., from day 0 to day 5 except day 3), respectively. Then, the combination of the two drugs was compared to the single drug treatments. The combination of ZSTK474 and rapamycin showed almost complete inhibition of the tumor growth whereas each single drug treatment showed partial inhibition. The synergy in the toxicity was not observed. We examined the effects of the combination on the PI3K/Akt downstream molecules in the PC-3 tumor tissues by immunohistochemical technique. The single treatment of ZSTK474 reduced the phosphorylation of Akt (Ser473), GSK3-beta and 4E-BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1. The single treatment of rapamycin reduced the phosphorylation of 4E-BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1, but did not reduce the phosphorylation of Akt (Ser473) or that of GSK3-beta. The combination of ZSTK474 and rapamycin reduced the phosphorylation of Akt, GSK3-beta, 4E-BP1 and S6, and the expression of Cyclin D1. These results confirmed the therapeutic efficacy of the combination of ZSTK474 and rapamycin against PC-3 xenografts. Therefore, the combination of PI3K inhibitor and mTOR inhibitor may be effective for cancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C65.

Identification of an emerin-beta-catenin complex in the heart important for intercalated disc architecture and beta-catenin localisation.

How mutations in the protein emerin lead to the cardiomyopathy associated with X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) is unclear. We identified emerin at the adherens junction of the intercalated disc, where it co-localised with the catenin family of proteins. Emerin bound to wild type beta-catenin both in vivo and in vitro. Mutating the GSK3beta phosphorylation sites on beta-catenin abolished this binding. Wild type but not mutant forms of emerin associated with X-EDMD were able to reduce beta-catenin protein levels. Cardiomyocytes from emerin-null mice hearts exhibited erroneous beta-catenin distribution and intercalated disc architecture. Treatment of wild type cardiomyocytes with phenylephrine, which inactivates GSK3beta, redistributed emerin and beta-catenin. Emerin was identified as a direct target of GSK3beta activity since exogenous expression of GSK3beta reduced emerin levels at the nuclear envelope. We propose that perturbation to or total loss of the emerin-beta-catenin complex compromises both intercalated disc function and beta-catenin signalling in cardiomyocytes.

Brain-derived neurotrophic factor prevents phencyclidine-induced apoptosis in developing brain by parallel activation of both the ERK and PI-3K/Akt pathways

Phencyclidine is an N-methyl d-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain-derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration-dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3beta activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3beta at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3beta inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3beta and activation of CREB.

Downregulation of survival signalling pathways and increased apoptosis in the transition of pressure overload‐induced cardiac hypertrophy to heart failure

1. Transition from compensated left ventricular (LV) hypertrophy to decompensated heart failure was characterized using a pressure-overload induced model to elucidate the temporal relationship between cardiomyocyte apoptosis and survival signalling in this transition. 2. Mice were subjected to transverse aortic constriction (TAC) or sham operation for 1-16 weeks and were studied by echocardiography, catheterization and histology. Relevant gene expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, Akt and glycogen synthase kinase (GSK)-3beta were determined. 3. Transverse aortic constriction resulted in myocyte hypertrophy and fibrosis from Week 4 and a progressive increase in left ventricular (LV) dimensions and wall thicknesses with maintained contractile function by Week 12. However, a sharp decline in contractile function and elevated LV end-diastolic pressure from 12 to 16 weeks were observed after TAC, indicating functional decompensation. 4. Following TAC, mRNA levels of atrial natriuretic peptide, B-type natriuretic peptide, beta-myosin heavy chain (MHC) and transforming growth factor-beta1 were increased time dependently, whereas mRNA expression of alpha-MHC, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a and Bcl-2 were decreased. The ratio of Bcl-2/Bax was decreased and this was consistent with progressively increased myocyte apoptosis demonstrated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling staining. Phosphorylation of ERK1/2 was increased by Week 4, but decreased thereafter. Levels of phosphorylated Akt declined from Week 8, whereas GSK3beta phosphorylation increased from 1 to 8 weeks, then decreased from Week 12 after TAC. 5. In conclusion, TAC resulted in early concentric and late eccentric hypertrophy with eventual development of LV dysfunction. This transition was temporally associated with a progressive increase in cell size, fibrosis and myocyte apoptosis. Downregulation of ERK1/2, Akt and GSK3beta and enhanced cardiomyocyte apoptosis are implicated as important mechanisms in the transition from compensated hypertrophy to heart failure.

Role of the low‐density lipoprotein receptor‐related protein‐1 in regulation of chondrocyte differentiation

The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/beta-catenin (betacat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCzeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1.

Calcineurin dephosphorylates glycogen synthase kinase‐3 beta at serine‐9 in neuroblast‐derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3beta (GSK-3beta). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1-401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3beta, and an increase in the phosphorylation of tau. K(m) of calcineurin for a GSK-3beta phosphopeptide was 469.3 microM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3beta were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3beta. We conclude that in vitro, calcineurin can dephosphorylate GSK-3beta at Ser-9 and form a stable complex with GSK-3beta, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3betain vivo.

Endogenous IGF-I and αVβ3 Integrin Ligands Regulate Increased Smooth Muscle Hyperplasia in Stricturing Crohn's Disease

Insulin-like growth factor-I (IGF-I) regulates human intestinal smooth muscle growth by stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented when alphaVbeta3 integrin is occupied by its ligands, fibronectin and vitronectin. Increased IGF-I expression and muscle cell hyperplasia are features of stricturing Crohn's disease (CD); however, the role of IGF-I in stricture formation is unknown. The aim was to identify the functional role of endogenous IGF-I and alphaVbeta3 integrin ligands in regulating muscle cell hyperplasia in stricturing CD. Smooth muscle cells were isolated from muscularis propria of stricturing CD or normal margins. Quantitative polymerase chain reaction, immunoblot analysis, and enzyme-linked immunosorbent assay were used to measure fibronectin, vitronectin, alphaVbeta3 integrin, and IGF-I levels. Activation of the IGF-I receptor, Erk1/2, p70S6 kinase, and GSK-3beta was measured by immunoblot. Proliferation was quantified by Ki67 immunostaining and [(3)H]thymidine incorporation. Apoptosis was measured from caspase-3 cleavage and nucleosome accumulation. IGF-I, vitronectin, and fibronectin RNA and protein levels were increased 1.8- to 3.4-fold in muscle cells from strictures over normal margins. Basal IGF-I receptor phosphorylation was increased 320% in strictured over normal muscle, and basal Erk1/2, p70S6 kinase, and GSK-3beta phosphorylation were increased 205%-292% in strictures. In muscle cells from strictures, Ki67 immunoreactivity and [(3)H]thymidine incorporation were increased and apoptosis was decreased compared with normal margins. Antagonists of the IGF-I receptor or alphaVbeta3 integrin reversed these changes. Smooth muscle cell hyperplasia in stricturing CD is regulated by increased endogenous IGF-I and alphaVbeta3 integrin ligands that regulate augmented proliferation and diminished apoptosis.