Increase In GSK-3β Phosphorylation Research Articles

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

The AMPK and AKT/GSK3β pathways are involved in recombinant proteins fibroblast growth factor 1 (rFGF1 and rFGF1a) improving glycolipid metabolism in rainbow trout (Oncorhynchus mykiss) fed a high carbohydrate diet

Fibroblast growth factor 1 (FGF1) regulates vertebrate cell growth, proliferation and differentiation, and energy metabolism. In this study, we cloned rainbow trout (Oncorhynchus mykiss) fgf1 and fgf1a, prepared their recombinant proteins (rFGF1 and rFGF1a), and described the molecular mechanisms by which they improve glycolipid metabolism in carnivorous fish. A 31-d feeding trial was conducted to investigate whether they could enhance glycolipid metabolism in rainbow trout on high-carbohydrate diets (HCD). A total of 720 rainbow trout (8.9 ± 0.5 g) were equally divided into 4 groups: the chow diet (CD) group injected with PBS, the HCD group injected with PBS, the HCD group injected with rFGF1 (400 ng/g body weight), and the HCD group injected with rFGF1a (400 ng/g body weight). The results showed that short-term HCD had a significant positive effect on the specific growth rate (SGR) of rainbow trout (P < 0.05). However, it led to an increase in crude fat, serum triglyceride (TG) and glucose content, as well as serum glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) contents (P < 0.05), suggesting a negative health effect of HCD. Nevertheless, rFGF1 and rFGF1a showed beneficial therapeutic effects. They significantly reduced the crude fat content of the liver, serum TG, GOT, and GPT contents caused by HCD (P < 0.05). The upregulation in atgl, hsl, and acc2 mRNAs implied the promotion of TG catabolism. Moreover, rFGF1 and rFGF1a contributed to promoting lipolysis by activating the AMPK pathway and reducing lipid accumulation in the liver caused by HCD. In addition, the rFGF1 and rFGF1a-treated groups significantly reduced serum glucose levels and elevated hepatic glycogen content under HCD, and increased glucose uptake by hepatocytes. We observed a decrease in mRNA levels for pepck, g6pase, and pygl, along with an increase in mRNA levels for gys, glut2, and gk in the liver. Furthermore, these proteins regulated hepatic gluconeogenesis and glycogen synthesis by increasing the phosphorylation level of AKT, ultimately leading to an increase in GSK3β phosphorylation. In conclusion, this study demonstrates that rFGF1 and rFGF1a can enhance lipolysis and glucose utilization in rainbow trout by activating the AMPK pathway and AKT/GSK3β axis.

OBG-like ATPase 1 inhibition attenuates angiotensin II-induced hypertrophic response in human ventricular myocytes via GSK-3beta/beta-catenin signalling.

Obg-like ATPase 1 (OLA1) that possesses both GTP and ATP hydrolyzing activities has been shown to be involved in translational regulation of cancer cell growth and survival. Also, GSK3β signalling has been implicated in cardiac development and disease. However, the role of OLA1 in pathological cardiac hypertrophy is unknown. We sought to understand the mechanism by which OLA1 regulates GSK3β-β-Catenin signalling and its functional significance in angiotensin-II (ANG II)-induced cardiac hypertrophic response. OLA1 function and its endogenous interaction with GSK3β/β-catenin signalling in cultured human ventricular cardiomyocytes (AC16 cells) and mouse hearts (in vivo) was evaluated with/without ANG II-stimulated hypertrophic response. ANG II administration in mice increases myocardial OLA1 protein expression with a corresponding increase in GSK3β phosphorylation and decrease in β-Catenin phosphorylation. Cultured cardiomyocytes treated with ANG II show endogenous interaction between OLA1 and GSK3β, nuclear accumulation of β-Catenin and significant increase in cell size and expression of hypertrophic marker genes such as atrial natriuretic factor (ANF; NPPA) and β-myosin heavy chain (MYH7). Intriguingly, OLA1 inhibition attenuates the above hypertrophic response in cardiomyocytes. Taken together, our data suggest that OLA1 plays a detrimental role in hypertrophic response via GSK3β/β-catenin signalling. Translation strategies to target OLA1 might potentially limit the underlying molecular derangements leading to left ventricular dysfunction in patients with maladaptive cardiac hypertrophy.

Microcystin-Leucine-Arginine Induces Tau Pathology Through Bα Degradation via Protein Phosphatase 2A Demethylation and Associated Glycogen Synthase Kinase-3β Phosphorylation

Microcystin-leucine-arginine (MC-LR) has been implicated as a potential environmental factor in Alzheimer's disease because of its potent inhibition of protein phosphatase 2A (PP2A) activity, but experimental evidence to support its detailed neurotoxic effects and their underlying mechanisms has been lacking. The present study investigated the role of PP2A catalytic subunit (PP2Ac) demethylation and its link with glycogen synthase kinase-3β (GSK)-3β in tau hyperphosphorylation induced by MC-LR. The results showed that MC-LR treatment significantly increased demethylation of PP2Ac, with a concomitant increase in GSK-3β phosphorylation at Ser9 resulting in elevated tau hyperphosphorylation at PP2A-favorable sites in SH-SY5Y cells and rat hippocampus. Coimmunoprecipitation experiments showed that MC-LR treatment dissociated PP2Ac from Bα, making it incompetent in binding tau, thus causing tau hyperphosphorylation. Moreover, we found that inhibition of PP2A resulted in an increase in phosphorylation of GSK-3β at Ser9 and a decrease in GSK-3β activity, which further promoted demethylation of PP2Ac induced by MC-LR. These findings suggest a scenario in which MC-LR-mediated demethylation of PP2Ac is associated with GSK-3β phosphorylation at Ser9 and contributes to dissociation of Bα from PP2Ac, which would result in Bα degradation and disruption of PP2A/Bα-tau interactions, thus promoting tau hyperphosphorylation and paired helical filaments-tau accumulation and, consequently, axonal degeneration and cell death.

Abstract 419: OLA1 Regulates GSK3β/eIF2-alpha Signaling Axis in Angiotensin II-treated Human Ventricular Cardiomyocytes

Pathological cardiac hypertrophy in response to stress or disease such as hypertension, myocardial infarction, valvular disease or neurohomonal dysregulation often result in heart failure. Cardiac hypertrophy is associated with changes in key molecular features including protein synthesis, sarcomeric organization, fibrogenesis, cell death and energy metabolism. OBG like ATPase 1 (OLA1) belongs to the translation factor-related (TRAFAC) class, Obg family, and YchF subfamily of P-loop GTPases that play a crucial role in the regulation of diverse cellular processes including protein translation, intracellular transport, signal transduction and growth. Recent study has shown that OLA1 functions as an intrinsic regulator in cellular stress responses, such as oxidative stress and heat shock. However, its role in cardiac hypertrophy is unknown so far. Here, we investigated the effect of OLA1 deficiency on angiotensin II (ANG II)-induced hypertrophic signaling response in immortalized human ventricular cardiomyocytes (hCM) and in heart tissue from OLA1 knockout mice. We observed that ANG II-induced increase in cardiomyocyte cell size was lower in OLA1 knockdown hCM cells as compared to control transfected cells. Interestingly, OLA1 knockdown inhibits the above effect and increases phosphorylation of GSK3β and eIF2α (inactive state). Furthermore, OLA1 homozygous knockout mice showed increase in phosphorylation of GSK3β. Taken together, these findings show that OLA1 deficiency decreases ANG II-induced hypertrophy signaling response in ventricular cardiomyocytes. This study provides the first evidence for the role of OLA1 in ANG II-induced hypertrophic response of human ventricular cardiomyocytes.

Effects of Acute and Chronic Electroconvulsive Shocks on Glycogen Synthase Kinase 3β Level and Phosphorylation in Mice

Glycogen synthase kinase 3β (GSK-3β) is recently proposed as a novel target in the treatment of mood disorders. Recent evidence has suggested that acute and chronic administration of antidepressants led to inhibition of GSK-3β via phosphorylation of Serine9 residue. Acute electroconvulsive shock (ECS) has been reported to increase GSK-3β phosphorylation transiently. In this study, the changes in the level of GSK-3β and its phoshorylated form (phospho-Ser9-GSK-3β) following chronic ECS (cECS) were investigated in mice. Mice were given daily ECS via bilateral corneal electrodes for 10 consecutive days in the chronic group and a single ECS in the acute group. Electrodes were applied without stimulation in corresponding sham groups. Immunoblotting for GSK-3β and phospho-Ser9-GSK-3β was performed with the frontal cortex and hippocampus samples, extracted 10 minutes after single ECS, and 24 hours after the last ECS in the chronic group. The optical densities of the bands obtained were compared between the active treatment and sham groups for each condition separately. The level of phospho-Ser9-GSK-3β was not different following chronic ECS, but significantly higher following acute ECS, compared with the corresponding sham group, in the hippocampus and frontal cortex. The level of GSK-3β was similar to sham following both acute and chronic ECS, in both regions. The transient increase in GSK-3β phosphorylation observed following acute ECS in the mice hippocampus and frontal cortex was not found to persist 24 hours following chronic ECS. The mechanism of action of ECS does not seem to involve persistent change in the level and phosphorylation of GSK-3β.

Propofol may protect PC12 cells from β-amyloid25-35 induced apoptosis through the GSK-3β signaling pathway

Background There are two major pathological hallmarks of Alzheimer's disease. One is the progressive accumulation of beta-amyloid (Aβ) in the form of senile plaques; the other is hyperphosphorylated tau, causing neuronal apoptosis. Some inhalation anesthetics, such as isoflurane and desflurane, have been suggested to induce Aβ accumulation and cause AD-like neuropathogenesis. Whether intravenous anesthetics have similar effects is still unclear. We therefore set out to determine the relationship between propofol and AD-like pathogenesis. Methods PC12 cells were cultured in serum-free medium for 12 hours prior to drug treatment. Various concentrations from 5 μmol/L to 80 μmol/L of aggregated Aβ25-35 were added to determine a proper concentration for further study. After exposure to 10 μmol/L Aβ25-35 alone or with 20 μmol/L propofol for 6 hours, PC12 cell viability was determined by MTT assay. Western blotting and immunocytochemical staining were performed to observe the protein expression of the Bcl-2 family, tau phosphorylation at different sites, and tau protein kinases and phosphatases. Results Aβ25-35 induced a decrease in PC12 cell viability in a dose-dependent manner. Exposure to 10 μmol/L Aβ25-35 for 6 hours resulted in the mild cell survival, accompanied by a decline in Bcl-2, and an increase in phosphorylation of GSK-3β and tau at different sites. Compared with the Aβ25-35 group, cells treated with propofol alone showed no significant difference, while cells co-incubated with propofol and Aβ25-35 showed a significantly higher survival rate (P &lt;0.01 or P &lt;0.05). Tau phosphorylation at Ser396, Ser404 and Thr231 and the level of GSK-3β in PC12 cells increased after exposure to 10 μmol/L Aβ25-35. Co-incubation with propofol attenuated cellular apoptosis by inhibiting tau phosphorylation. Conclusions These data indicate that propofol may protect PC12 cells from Aβ25-35-induced apoptosis and tau hyperphosphorylation through the GSK-3β pathway, therefore it may be a safer anesthesia for AD and elderly patients.

Helium-induced cardioprotection of healthy and hypertensive rat myocardium in vivo

Helium protects healthy myocardium against ischemia/reperfusion injury by early and late preconditioning (EPC, LPC) and postconditioning (PostC). We investigated helium-induced PostC of the hypertensive heart and enhancement by addition of LPC and EPC. We also investigated involvement of signaling kinases glycogen synthase kinase 3 beta (GSK-3β) and protein kinase C-epsilon (PKC-ε). To assess myocardial cell damage, we performed infarct size measurements in healthy Wistar Kyoto (WKY rats, n=8–9) and Spontaneous Hypertensive rats (SHR, n=8–9) subjected to 25min ischemia and 120min reperfusion. Rats inhaled 70% helium for 15min after index ischemia (PostC), combined with 15min helium 24h prior to index ischemia (LPC+PostC), a triple intervention with additional 3 short cycles of 5min helium inhalation shortly before ischemia (EPC+LPC+PostC), or no further treatment. In WKY rats, PostC reduced infarct size from 46±2% (mean±S.E.M) in the control group to 29±2%. LPC+PostC or EPC+LPC+PostC reduced infarct sizes to a similar extent (30±3% and 32±2% respectively). In SHR, EPC+LPC+PostC reduced infarct size from 53±3% in control to 39±3%, while PostC or LPC+PostC alone were not protective; infarct size 48±4% and 44±4%, respectively. Neither PostC in WKY rats nor EPC+LPC+PostC in SHR was associated with an increase in phosphorylation of GSK-3β and PKC-ε after 15min of reperfusion. Concluding, a triple intervention of helium conditioning results in cardioprotection in SHR, whereas a single intervention does not. In WKY rats, the triple intervention does not further augment protection. Helium conditioning is not associated with a mechanism involving GSK-3β and PKC-ε.

If you can’t beat me, join me: collaborating oncogenes circumventing senescence and causing cancer

death. Signaling routes commonly altered in cancer include the RAS-MAP Kinase and PI3 Kinase (PI3K) pathways. Activation of the first can take place at many levels, most frequently through mutations in members of the RAS and RAF families, while also further upstream receptor tyrosine kinases can be activated by mutation or amplification. PI3K pathway activation occurs largely through mutations in its catalytic subunit PIK3CA or amplification of AKT family members. Additionally, the Phosphatase and Tensin Homolog (PTEN), a phosphatase negatively regulating the PI3K pathway by dephosphorylating the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), is inactivated in several tumor types by different mechanisms including mutation, deletion, and epigenetic silencing. Perhaps somewhat counter intuitively, although PI3K is an established downstream effector of RAS in oncogenic transformation, simultaneous mutations in RAS and in the PIK3CA ⁄ PTEN ⁄AKT pathway do occur in human cancer. Consistently, in several mouse models, they have been shown to cooperate in accelerating tumorigenesis. While it is reasonably well understood why mutant RAS and deficiencies in the pRB or p53 pathways collaborate in cancer, it is less clear what the mechanistic basis is for activating mutations at two points in a presumed single signaling pathway, like RAS and PI3K. Kennedy et al. recently reported on a molecular interplay model in which the second oncogene, acting downstream, counteracts the senescence-inducing effect of the first (Kennedy et al.,

Lithium Fails to Protect Dopaminergic Neurons in the 6-OHDA Model of Parkinson’s Disease

Lithium has been used for the treatment of bipolar mood disorder and is shown to have neuroprotective properties. Since lithium inhibits the activity of glycogen synthase kinase 3 (GSK3) which is implicated in various human diseases, particularly neurodegenerative diseases, the therapeutic potential of lithium receives great attention. Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by the pathological loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Intranigral injection of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) causes selective and progressive degeneration of dopaminergic neurons in SNpc, and is a commonly used animal model of PD. The current study was designated to determine whether lithium is effective in alleviating 6-OHDA-induced neurodegeneration in the SNpc of rats. We demonstrated that chronic subcutaneous administration of lithium inhibited GSK3 activity in the SNpc, which was evident by an increase in phosphorylation of GSK3β at serine 9, cyclin D1 expression, and a decrease in tau phosphorylation. 6-OHDA did not affect GSK3 activity in the SNpc. Moreover, lithium was unable to alleviate 6-OHDA-induced degeneration of SNpc dopaminergic neurons. The results suggest that GSK3 is minimally involved in the neurodegeneration in the rat 6-OHDA model of PD.

008 Spontaneous cardiac hypertrophy and identification of novel hypertrophic pathways in a genetic mouse model that emulates human diabetes

Animal models are key in the exploration of the pathophysiological mechanisms and complications of diabetes mellitus (DM). Most current models have considerable limitations in that they do not faithfully replicate human forms of DM. Recently, a novel human relevant mouse model of diabetes (GENA 348) was identified through the Medical Research Council, Harwell, UK mouse mutagenesis programme in which a point mutation in the glucokinase gene results in severely impaired glucokinase function and significant hyperglycaemia. Similar mutations in the glucokinase gene are known to underlie Maturity Onset Diabetes of the Young Type 2 (MODY 2) in humans. We studied the GENA 348 mouse to determine whether it expresses a cardiac phenotype to provide insight into the pathophysiological mechanisms underlying the development of diabetic cardiomyopathy. Fifteen wild type (WT) controls and eight homozygote mutant (HO) mice had serial echocardiography performed at 3 and 6 months. At 3 months no evidence of cardiac hypertrophy or contractile dysfunction was demonstrated in HO compared to WT mice. By 6 months of age, echo demonstrated development of cardiac hypertrophy in GENA 348 HO mice characterised by a 16% increase in left ventricular mass/body weight (wt 4.28±0.15 vs HO 4.95±0.26, p&lt;0.05), a 25% increase in dPW/dD (WT 23.3±1.0 vs HO 29.1±1.1, p&lt;0.01) and an 18% increase in left ventricular relative wall thickness (WT 0.45±0.01 vs HO 0.53±0.02, p&lt;0.05). No differences in systolic function were observed although significant diastolic dysfunction was also evident with a 31% reduction in the E:A ratio (WT 2.65±0.2 vs HO 1.84±0.19, p&lt;0.05) and a 34% increase in the IVRT (WT 15.2±1.1 ms vs HO 20.5±0.37 ms, p&lt;0.01). Histological staining illustrated cellular hypertrophy with a 60% increase in cardiomyocyte size (WT 238±14 μm² vs HO 383±26 μm², p&lt;0.001). Hypertrophic pathways were examined through western blot analysis, a 140% increase in Akt phosphorylation, a key mediator of cardiac hypertrophy and a 65% increase in GSK3β phosphorylation one of its key regulators was observed. We also demonstrate that advanced glycation end products (AGE) were elevated by 86% in the serum of mutant GENA 348 mice. Collectively, these data indicate GENA 348 mutant mice develop a cardiac phenotype including hypertrophy and diastolic dysfunction similar to the clinical manifestations of diabetic cardiomyopathy. Furthermore, a novel hypertrophic pathway has been identified in which AGE stimulation through GSK3β leads to activation of the intracellular Akt pathway. GENA 348 may thus provide a valuable, human-relevant tool for studying the molecular determinants of DM related cardiovascular complications.

ZM336372 Induces Apoptosis Associated with Phosphorylation of GSK-3β in Pancreatic Adenocarcinoma Cell Lines

ZM336372 is small molecule tyrosine kinase modulator. It has been shown to inhibit glycogen synthase kinase-3beta (GSK-3beta) through phosphorylation of GSK-3beta at Ser 9. GSK-3beta has previously been shown to mediate cell survival in pancreatic cancer cells. Here we determine the effects of ZM336372 on GSK-3beta phosphorylation, apoptosis, and growth in pancreatic adenocarcinoma cell lines. Panc-1 and MiaPaCa-2 cells were treated with ZM336372 or lithium chloride (LiCl) and compared with solvent control. The effects on proliferation for each cell line were determined using the MTT assay. Western blot analysis was performed to examine the effects of treatment on the phosphorylation of GSK-3beta. In addition, western blot was utilized to examine the cleavage of poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. A dose-dependent increase in phosphorylation of GSK-3beta was observed after treatment with both ZM336372 and LiCl. Growth inhibition due to treatment with ZM336372 and LiCl also occurred in a dose-dependent fashion. An increase in cleaved PARP was demonstrated after treatment with both agents, as was seen previously with GSK-3beta inhibition in pancreatic adenocarcinoma cells. This is the first description of growth inhibition and apoptosis in pancreatic cancer cells related to GSK-3beta inhibition through treatment with ZM336372.

Phosphorylation of glycogen synthase kinase-3β in relation to diapause processing in the silkworm, Bombyx mori

Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25 °C for 15 days and then transferred to 5 °C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5 °C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs.

Involvement of FrzA/sFRP-1 and the Wnt/Frizzled Pathway in Ischemic Preconditioning

Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3beta via the Akt/PI3-Kinase pathway during ischemic preconditioning (PC) has been shown to be cardioprotective. As FrzA/sFRP-1, a secreted antagonist of the Wnt/Frizzled pathway, is expressed in the heart and is able to decrease the phosphorylation of GSK-3beta in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (alpha-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3beta phosphorylation as well as protein kinase C (PKC) epsilon activation in transgenic mice after PC as compared with littermates. Phospho-Akt (P-Akt), phospho-JNK, or the cytoplasmic beta-catenin levels were not modified, phospho-p38 (P-p38) was slightly increased in transgenic mice after PC as compared with littermates. FrzA transgenic mice displayed a larger infarct size and a greater worsening of cardiac function compared with littermates. All these differences were reversed by the addition of doxycycline. This study demonstrates for the first time that disruption of a beta-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3beta and reverses the benefit of preconditioning.

Wnt/β-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy

Beta-catenin is a transcriptional activator shown to regulate the embryonic, postnatal, and oncogenic growth of many tissues. In most research to date, beta-catenin activation has been the unique downstream function of the Wnt signaling pathway. However, in the heart, a Wnt-independent mechanism involving Akt-mediated phosphorylation of glycogen synthase kinase (GSK)-3beta was recently shown to activate beta-catenin and regulate cardiomyocyte growth. In this study, results have identified the activation of the Wnt/beta-catenin pathway during hypertrophy of mechanically overloaded skeletal muscle. Significant increases in beta-catenin were determined during skeletal muscle hypertrophy. In addition, the Wnt receptor, mFrizzled (mFzd)-1, the signaling mediator disheveled-1, and the transcriptional co-activator, lymphocyte enhancement factor (Lef)-1, are all increased during hypertrophy of the overloaded mouse plantaris muscle. Experiments also determined an increased association between GSK-3beta and the inhibitory frequently rearranged in advanced T cell-1 protein with no increase in GSK-3beta phosphorylation (Ser9). Finally, skeletal muscle overload resulted in increased nuclear beta-catenin/Lef-1 expression and induction of the transcriptional targets c-Myc, cyclin D1, and paired-like homeodomain transcription factor 2. Thus this study provides the first evidence that the Wnt signaling pathway induces beta-catenin/Lef-1 activation of growth-control genes during overload induced skeletal muscle hypertrophy.

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor.

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and Gö 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.