Glycogen Synthase Kinase-3β Activity Research Articles

Gimap5-dependent inactivation of GSK3β is required for CD4+ T cell homeostasis and prevention of immune pathology

Abstract GTPase of immunity-associated protein 5 (Gimap5) has been linked with lymphocyte survival, autoimmunity and colitis, but its mechanisms of action are unclear. Here we show that Gimap5 is essential for inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction as well as NFATc1 nuclear import, thereby reducing the frequency of CD4+ T cells that complete cell cycle. Additionally, Gimap5 facilitates phosphorylation of GSK3β at Ser389 and its nuclear translocation. Impairment in the latter steps of GSK3β regulation by Gimap5 is associated with increased DNA damage and reduced survival of activated CD4+ T cells. Importantly, pharmacological targeting and genetic deletion of GSK3β in mice can override Gimap5-deficiency in CD4+ T cells, limiting DNA damage and enhancing T cell proliferation and survival. Additionally, this ameliorates immune-mediated pathology in vivo caused by loss of Gimap5. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation. T cells from this patient exhibit impaired proliferation, but can be rescued in vitro with GSK3 inhibitors. Given the restricted expression of Gimap5 in lymphocytes, we propose that its control of GSK3b activity is an important checkpoint in lymphocyte proliferation and function.

Wnt3a ligand facilitates autophagy in hippocampal neurons by modulating a novel GSK-3\u03b2-AMPK axis

BackgroundIn the adult central nervous system (CNS), Wnt signaling regulates dendritic structure and synaptic plasticity. The Wnt signaling pathway can be divided into the canonical (β-catenin-dependent) and non-canonical pathways. In the canonical pathway, the binding of canonical ligands such as Wnt3a to the Frizzled receptor induces inactivation of glycogen synthase kinase-3β (GSK-3β), which stabilizes β-catenin and allows its translocation to the nucleus. However, to date, few studies have focused on β-catenin-independent Wnt signaling or explained the underlying mechanisms connecting Wnt signaling to cellular energy metabolism. A recent study demonstrated negative regulation of 5′ adenosine monophosphate-activated protein kinase (AMPK), a major target of GSK-3β that regulates cellular metabolism under diverse conditions. Mainly based on these observations, we evaluated whether Wnt3a ligand modulates autophagy by regulating the GSK-3β/AMPK axis.MethodsCultured primary hippocampal neurons and slices of the CA1 region of rat hippocampus were used. GSK-3β inhibition, AMPK activation, PP2Ac expression, and LC3 processing were examined by western blotting. Autophagic compartments were studied using the CYTO-ID® fluorescent probe, and mature autophagosomes were observed via transmission electron microscopy (TEM).ResultsWnt3a ligand, acting through the Frizzled receptor, promotes the rapid activation of AMPK by inactivating GSK-3β. Biochemical analysis of downstream targets indicated that Wnt3a ligand modulates autophagy in hippocampal neurons.ConclusionsOur results revealed new aspects of Wnt signaling in neuronal metabolism. First, AMPK is an additional target downstream of the Wnt cascade, suggesting a molecular mechanism for the metabolic effects previously observed for Wnt signaling. Second, this mechanism is independent of β-catenin, suggesting a relevant role for non-genomic activity of the Wnt pathway in cellular metabolism. Finally, these results have new implications regarding the role of Wnt signaling in the modulation of autophagy in neurons, with a possible role in the removal of accumulated intracellular proteins.

Follistatin-Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β-Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase-3β Activity.

The fight against vascular disease requires functional endothelial cells (ECs) which could be provided by differentiation of induced Pluripotent Stem Cells (iPS Cells) in great numbers for use in the clinic. However, the great promise of the generated ECs (iPS‐ECs) in therapy is often restricted due to the challenge in iPS‐ECs preserving their phenotype and function. We identified that Follistatin‐Like 3 (FSTL3) is highly expressed in iPS‐ECs, and, as such, we sought to clarify its possible role in retaining and improving iPS‐ECs function and phenotype, which are crucial in increasing the cells’ potential as a therapeutic tool. We overexpressed FSTL3 in iPS‐ECs and found that FSTL3 could induce and enhance endothelial features by facilitating β‐catenin nuclear translocation through inhibition of glycogen synthase kinase‐3β activity and induction of Endothelin‐1. The angiogenic potential of FSTL3 was also confirmed both in vitro and in vivo. When iPS‐ECs overexpressing FSTL3 were subcutaneously injected in in vivo angiogenic model or intramuscularly injected in a hind limb ischemia NOD.CB17‐Prkdcscid/NcrCrl SCID mice model, FSTL3 significantly induced angiogenesis and blood flow recovery, respectively. This study, for the first time, demonstrates that FSTL3 can greatly enhance the function and maturity of iPS‐ECs. It advances our understanding of iPS‐ECs and identifies a novel pathway that can be applied in cell therapy. These findings could therefore help improve efficiency and generation of therapeutically relevant numbers of ECs for use in patient‐specific cell‐based therapies. In addition, it can be particularly useful toward the treatment of vascular diseases instigated by EC dysfunction. Stem Cells 2018;36:1033–1044

Estrogen-related receptor alpha is involved in Alzheimer's disease-like pathology

Estrogen-related receptor alpha (ERRα) is a transcriptional factor associated with mitochondrial biogenesis and energy metabolism. However, little is known about the role of ERRα in Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, ERRα protein and mRNA were decreased in a region- and age-dependent manner. In HEK293 cells that stably express human full-length β-amyloid precursor protein (APP), overexpression of ERRα inhibited the amyloidogenic processing of APP and consequently reduced Aβ1–40/1–42 level. ERRα overexpression also attenuated Tau phosphorylation at selective sites, with the concomitant reduction of glycogen synthase kinase 3β (GSK3β) activity. Interestingly, alterations of APP processing and Tau phosphorylation induced by hydrogen peroxide were reversed by ERRα overexpression in HEK/APP cells. These results indicated that ERRα plays a functional role in AD pathology. By attenuating both amyloidogenesis and Tau phosphorylation, ERRα may serve as a potential therapeutic target for AD.

Toosendanin inhibits adipogenesis by activating Wnt/\u03b2-catenin signaling

Toosendanin (TSN), a triterpenoid extracted from Melia toosendan, has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, and anti-arthritic activities. However, its anti-adipogenic effect remains unknown. Here, we found that TSN dose-dependently attenuated lipid accumulation in preadipocytes 3T3-L1 as evidenced by Oil Red O staining. TSN also significantly downregulated mRNA and protein levels of adipocytokines (adiponectin and leptin), CCAAT/enhancer binding proteins α (C/EBP-α), peroxisome proliferator-activated receptor γ (PPAR-γ), fatty acid synthase, and acetyl-CoA carboxylase in adipocytes. To understand the mechanism, we observed that TSN effectively activated Wnt/β-catenin pathway, in which TSN increased low density lipoprotein receptor related protein 6, disheveled 2, β-catenin, and cyclin D1 expression levels, while it inactivated glycogen synthase kinase 3β by enhancing its phosphorylation. Moreover, TSN reduced weight of gonadal white fat and serum triacylglycerol (TAG) content in high-fat diet (HFD)-fed mice. Interestingly, the in vivo studies also demonstrated that TSN promoted the expression of β-catenin, but accordingly repressed C/EBP-α and PPAR-γ expression in HFD-induced mice. Overall, TSN is capable of inhibiting the lipogenesis of adipocytes by activating the Wnt/β-catenin pathway, suggesting potential application of TSN as a natural anti-obesity agent.

Cigarette Smoke Extract Enhances IL-17A-Induced IL-8 Production via Up-Regulation of IL-17R in Human Bronchial Epithelial Cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine mainly derived from T helper 17 cells and is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) has been considered as a primary risk factor of COPD. However, the interaction between CS and IL-17A and the underlying molecular mechanisms have not been clarified. In the current study, we investigated the effects of cigarette smoke extract (CSE) on IL-17A-induced IL-8 production in human bronchial epithelial cells, and sought to identify the underlying molecular mechanisms. IL-8 production was significantly enhanced following treatment with both IL-17A and CSE, while treatment with either IL-17A or CSE alone caused only a slight increase in IL-8 production. CSE increased the transcription of IL-17RA/RC and surface membrane expression of IL-17R, which was suppressed by an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway (LY294002). CSE caused inactivation of glycogen synthase kinase-3β (GSK-3β) via the PI3K/Akt pathway. Blockade of GSK-3β inactivation by overexpression of constitutively active GSK-3β (S9A) completely suppressed the CSE-induced up-regulation of IL-17R expression and the CSE-induced enhancement of IL-8 secretion. In conclusion, inactivation of GSK-3β via the PI3K/Akt pathway mediates CSE-induced up-regulation of IL-17R, which contributes to the enhancement of IL-17A-induced IL-8 production.

Glycogen synthase kinase-3β activity and cognitive functioning in patients with bipolar I disorder

Cognitive deficits are common in patients with bipolar disorder (BD) in remission and may be associated with glycogen synthase kinase-3 (GSK-3) activity, which is inhibited by lithium. GSK-3 may be a relevant treatment target for interventions tailored at cognitive disturbances in BD but the relation between GSK-3 activity, cognition and lithium treatment is unknown. We therefore investigated the possible association between GSK-3 activity and cognition and whether lithium treatment moderates this association in patients with BD. In a prospective 6–12 month follow-up study, GSK- 3β activity in peripheral blood mononuclear cells was measured concurrently with cognitive performance assessed using a comprehensive test battery in 27 patients with BD-I in early and late remission following a manic or mixed episode. The GSK-3β activity, measured as serine-9 phosphorylated GSK-3β (pGSK-3β) and the GSK-3β ratio (serine-9-pGSK-3β /total GSK-3β), was negatively associated with sustained attention (p = 0.009 and p = 0.042, respectively), but not with other cognitive domains or global cognition. A crossover interaction between lithium treatment and the GSK activity was observed, indicating that lower pGSK-3β levels (p = 0.015) and GSK ratio (p = 0.010) were associated with better global cognition in lithium users whereas the opposite association was observed in non-lithium treated patients. Findings were not statistically significant after Bonferroni correction. In conclusion, cognitive functioning may be associated with GSK-3 activity in patients with BD-I and lithium treatment may modulate this relationship. Future studies in larger sample sizes are warranted to confirm these associations.

Pinocembrin from Penthorum chinense Pursh suppresses hepatic stellate cells activation through a unified SIRT3-TGF-β-Smad signaling pathway

The inactivation of hepatic stellate cells (HSCs) has been verified to be an effective therapeutic strategy for treatment of liver fibrosis. Penthorum chinense Pursh has been widely used to protect liver in China; while, the role of P. chinense Pursh in treatment of liver fibrosis is still unexplored. In the current study, the aqueous extract of P. chinense Pursh (PCE) was found to suppress the expressions of fibrotic markers, including collagen I and α-smooth muscle actin (α-SMA), in human HSCs (LX-2); and its major active constituent, pinocembrin (PIN), was discovered to inhibit the expressions of fibrotic markers in LX-2 cells and rat HSCs (HSC-T6). Further study indicated that PIN suppressed the activation of LX-2 and HSC-T6 cells through elevating the expression and activity of silent mating type information regulation 2 homolog 3 (SIRT3). Via SIRT3, PIN activated superoxide dismutase 2 (SOD2), to alleviate the accumulation of reactive oxygen species (ROS) and inhibit phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling, resulting in decreased production of transforming growth factor-β (TGF-β) and nuclear translocation of the transcription factor Sma- and Mad-related proteins (Smad). Furthermore, PIN activated glycogen synthase kinase 3β (GSK3β) through SIRT3, to enhance Smad protein degradation. Taken together, PCE and PIN were identified as potential anti-fibrotic agents, which might be well developed as a candidate for treatment of liver fibrosis.

Gimap5-dependent inactivation of GSK3\u03b2 is required for CD4+ T cell homeostasis and prevention of immune pathology

GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, autoimmunity, and colitis, but its mechanisms of action are unclear. Here, we show that Gimap5 is essential for the inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction and NFATc1 nuclear import, thereby limiting productive CD4+ T cell proliferation. Additionally, Gimap5 facilitates Ser389 phosphorylation and nuclear translocation of GSK3β, thereby limiting DNA damage in CD4+ T cells. Importantly, pharmacological inhibition and genetic targeting of GSK3β can override Gimap5 deficiency in CD4+ T cells and ameliorates immunopathology in mice. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation in vitro that can be rescued with GSK3 inhibitors. Given that the expression of Gimap5 is lymphocyte-restricted, we propose that its control of GSK3β is an important checkpoint in lymphocyte proliferation.

Cathepsin L promotes ionizing radiation-induced U251 glioma cell migration and invasion through regulating the GSK-3β/CUX1 pathway

Cathepsin L (CTSL) is a lysosomal cysteine protease overexpressed and secreted by tumor cells. Our previous study found that CTSL was involved in ionizing radiation (IR)-induced epithelial-mesenchymal transition (EMT) and the increase of glioma invasion and migration. However, the mechanisms by which CTSL promoted this IR-induced glioma migration and invasion remained unclear. In this study, we demonstrated that IR reduced glycogen synthase kinase-3β (GSK-3β) activity, via the CTSL-mediated phosphorylation of its serine-9 residue, in U251 cells. Moreover, inhibition of p-GSK-3βSer9 in overexpressing CTSL cells attenuated EMT and decreased the expression of snail, an EMT-related transcription factor. As a result, U251 cell migration and invasion was inhibited compared to over-CTSL cells. Alternatively, when CTSL was activated by IR or exogenously overexpressed, CTSL promoted EMT by processing homeobox protein cut-like1 (CUX1) to produce the physiologically active p110 isoform. In brief, this study revealed that IR-induced EMT as well as migration and invasion of glioma cells are mediated by CTSL through the Akt/GSK-3β/snail and CUX1 pathways. Consequently, this research also led to the identification of a potential novel target for therapeutic intervention of glioma.

Effect of tibolone pretreatment on kinases and phosphatases that regulate the expression and phosphorylation of Tau in the hippocampus of rats exposed to ozone.

Oxidative stress (OS) is a key process in the development of many neurodegenerative diseases, memory disorders, and other pathological processes related to aging. Tibolone (TIB), a synthetic hormone used as a treatment for menopausal symptoms, decreases lipoperoxidation levels, prevents memory impairment and learning disability caused by ozone (O3) exposure. However, it is not clear if TIB could prevent the increase in phosphorylation induced by oxidative stress of the microtubule-associated protein Tau. In this study, the effects of TIB at different times of administration on the phosphorylation of Tau, the activation of glycogen synthase kinase-3β (GSK3β), and the inactivation of Akt and phosphatases PP2A and PTEN induced by O3 exposure were assessed in adult male Wistar rats. Rats were divided into 10 groups: control group (ozone-free air plus vehicle [C]), control + TIB group (ozone-free air plus TIB 1 mg/kg [C + TIB]); 7, 15, 30, and 60 days of ozone exposure groups [O3] and 7, 15, 30, and 60 days of TIB 1 mg/kg before ozone exposure groups [O3 + TIB]. The effects of O3 exposure and TIB administration were assessed by western blot analysis of total and phosphorylated Tau, GSK3β, Akt, PP2A, and PTEN proteins and oxidative stress marker nitrotyrosine, and superoxide dismutase activity and lipid peroxidation of malondialdehyde by two different spectrophotometric methods (Marklund and TBARS, respectively). We observed that O3 exposure increases Tau phosphorylation, which is correlated with decreased PP2A and PTEN protein levels, diminished Akt protein levels, and increased GSK3β protein levels in the hippocampus of adult male rats. The effects of O3 exposure were prevented by the long-term treatment (over 15 days) with TIB. Malondialdehyde and nitrotyrosine levels increased from 15 to 60 days of exposure to O3 in comparison to C group, and superoxide dismutase activity decreased. Furthermore, TIB administration limited the changes induced by O3 exposure. Our results suggest a beneficial use of hormone replacement therapy with TIB to prevent neurodegeneration caused by O3 exposure in rats.

In vivo and ex vivo analyses of amyloid toxicity in the Tc1 mouse model of Down syndrome.

Rationale:The prevalence of Alzheimer’s disease is increased in people with Down syndrome. The pathology appears much earlier than in the general population, suggesting a predisposition to develop Alzheimer’s disease. Down syndrome results from trisomy of human chromosome 21, leading to overexpression of possible Alzheimer’s disease candidate genes, such as amyloid precursor protein gene. To better understand how the Down syndrome context results in increased vulnerability to Alzheimer’s disease, we analysed amyloid-β [25-35] peptide toxicity in the Tc1 mouse model of Down syndrome, in which ~75% of protein coding genes are functionally trisomic but, importantly, not amyloid precursor protein.Results:Intracerebroventricular injection of oligomeric amyloid-β [25-35] peptide in three-month-old wildtype mice induced learning deficits, oxidative stress, synaptic marker alterations, activation of glycogen synthase kinase-3β, inhibition of protein kinase B (AKT), and apoptotic pathways as compared to scrambled peptide-treated wildtype mice. Scrambled peptide-treated Tc1 mice presented high levels of toxicity markers as compared to wildtype mice. Amyloid-β [25-35] peptide injection in Tc1 mice induced significant learning deficits and enhanced glycogen synthase kinase-3β activity in the cortex and expression of apoptotic markers in the hippocampus and cortex. Interestingly, several markers, including oxidative stress, synaptic markers, glycogen synthase kinase-3β activity in the hippocampus and AKT activity in the hippocampus and cortex, were unaffected by amyloid-β [25-35] peptide injection in Tc1 mice.Conclusions:Tc1 mice present several toxicity markers similar to those observed in amyloid-β [25-35] peptide-treated wildtype mice, suggesting that developmental modifications in these mice modify their response to amyloid peptide. However, amyloid toxicity led to severe memory deficits in this Down syndrome mouse model.

Metformin Inhibits the Development of L-DOPA-Induced Dyskinesia in a Murine Model of Parkinson's Disease.

Metformin is a medication that is widely prescribed for the management of type 2 diabetes. In addition to its anti-diabetic uses, metformin has been proposed as a therapeutically effective drug candidate in various central nervous system disorders, including Parkinson's disease (PD). PD is characterized by severe movement defects and is commonly treated with the dopamine (DA) precursor 3,4-dihydroxyphenyl-l-alanine (L-DOPA). However, prolonged use of L-DOPA can lead to the development of L-DOPA-induced dyskinesia (LID). Here, we hypothesized that metformin co-treatment would improve LID in the 6-hydroxydopamine (6-OHDA)-lesioned mouse model of PD. Metformin did not interfere the pharmacotherapeutic effects of L-DOPA in the cylinder test. Furthermore, metformin co-treatment with L-DOPA attenuated the development of LID in unilaterally 6-OHDA-lesioned mice. Metformin showed a long-lasting effect on axial, limb, and orofacial abnormal involuntary movement scores for up to 20days after treatment initiation. Interestingly, persistent enhancement of the mammalian target of rapamycin, dopamine D1 receptor, and extracellular signaling-regulated kinase 1/2 signaling was maintained in the DA-denervated striatum during metformin treatment. Metformin globally normalized the increased glycogen synthase kinase 3β activity induced by chronic treatment of L-DOPA in a manner associated with Akt activation in unilaterally 6-OHDA-lesioned mice. These findings suggest that metformin may have therapeutic potential for the suppression or management of L-DOPA-induced motor complications in patients with PD.

Effects of estrogen on glycogen synthase kinase-3β activity

Objective To investigate the effects of 17β-estradiol (E2) on glycogen synthase kinase-3β (GSK-3β) activity and the possible related mechanisms. Methods E2 was added to rat primary cortical neurons and its effect on cell viability was measured by methyl thiazol tetrazolium (MTT) assays; its effect on GSK-3 activity was detected directedly. The phosphorylated (9Ser) and non-phosphorylated GSK-3β expression after drug treatment was detected by Western blotting. The effects of estrogen receptor blocker and various kinase inhibitors on GSK-3 phosphorylation and GSK-3 activity were also measured. Results As compared with control group, in a concentration range of 1, 10, 100, 200 and 500 nmol/L, E2 didn’t significantly decrease cell viability in rat primary cortical neurons (the corresponding cell viability was 98.4%, 107.5%, 105.8%, 95.6% and 82.3% respectively). At concentrations of 1, 10 and 100 nmol/L, E2 dose-dependently decreased GSK-3 activity (to 85.6%, 78.5% and 65.4% of control group respectively). Western blotting indicated that E2 also dose-dependently increased GSK-3β (9Ser) phosphorylation without significantly affecting non-phosphorylated GSK-3β expression. The effect of E2 on GSK-3β phosphorylation could be partially blocked by estrogen receptor antagonist ICI-182780. The GSK-3β phosphorylation induced by E2 could be partially reversed by mitogen-activated protein kinase (MAPK) inhibitor PD98059 and protein kinase C (PKC) inhibitor GF109203X, but not by an Akt inhibitor Akt-I. The GSK-3 activity was decreased to 65.4% (P=0.000) of control group after E2 administration, and this effect could be partially reversed by ICI, PD98059, GF109203X administration, but not by Akt-I. These data were in accordance with those of Western blotting. Conclusion E2 can decrease GSK-3 activity as indicated by increased GSK-3β (9Ser) phosphorylation in an estrogen receptor-dependent manner, and the effect of E2 on GSK-3 phosphorylation may be mediated through MAPK and PKC signaling pathways. Key words: Estrogens; Glycogen syntheses kinase-3β; Alzheimer’s disease

Effects of cannabidiol interactions with Wnt/β-catenin pathway and PPARγ on oxidative stress and neuroinflammation in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease, in which the primary etiology remains unknown. AD presents amyloid beta (Aβ) protein aggregation and neurofibrillary plaque deposits. AD shows oxidative stress and chronic inflammation. In AD, canonical Wingless-Int (Wnt)/β-catenin pathway is downregulated, whereas peroxisome proliferator-activated receptor γ (PPARγ) is increased. Downregulation of Wnt/β-catenin, through activation of glycogen synthase kinase-3β (GSK-3β) by Aβ, and inactivation of phosphatidylinositol 3-kinase/Akt signaling involve oxidative stress in AD. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid from Cannabis sativa plant. In PC12 cells, Aβ-induced tau protein hyperphosphorylation is inhibited by CBD. This inhibition is associated with a downregulation of p-GSK-3β, an inhibitor of Wnt pathway. CBD may also increase Wnt/β-catenin by stimulation of PPARγ, inhibition of Aβ and ubiquitination of amyloid precursor protein. CBD attenuates oxidative stress and diminishes mitochondrial dysfunction and reactive oxygen species generation. CBD suppresses, through activation of PPARγ, pro-inflammatory signaling and may be a potential new candidate for AD therapy.

Cyclin D2 is a critical mediator of exercise-induced cardiac hypertrophy.

A number of signaling pathways underlying pathological cardiac hypertrophy have been identified. However, few studies have probed the functional significance of these signaling pathways in the context of exercise or physiological pathways. Exercise studies were performed on females from six different genetic mouse models that have been shown to exhibit alterations in pathological cardiac adaptation and hypertrophy. These include mice expressing constitutively active glycogen synthase kinase-3β (GSK-3βS9A), an inhibitor of CaMK II (AC3-I), both GSK-3βS9A and AC3-I (GSK-3βS9A/AC3-I), constitutively active Akt (myrAkt), mice deficient in MAPK/ERK kinase kinase-1 (MEKK1-/-), and mice deficient in cyclin D2 (cyclin D2-/-). Voluntary wheel running performance was similar to NTG littermates for five of the mouse lines. Exercise induced significant cardiac growth in all mouse models except the cyclin D2-/- mice. Cardiac function was not impacted in the cyclin D2-/- mice and studies using a phospho-antibody array identified six proteins with increased phosphorylation (greater than 150%) and nine proteins with decreased phosphorylation (greater than 33% decrease) in the hearts of exercised cyclin D2-/- mice compared to exercised NTG littermate controls. Our results demonstrate that unlike the other hypertrophic signaling molecules tested here, cyclin D2 is an important regulator of both pathologic and physiological hypertrophy. Impact statement This research is relevant as the hypertrophic signaling pathways tested here have only been characterized for their role in pathological hypertrophy, and not in the context of exercise or physiological hypertrophy. By using the same transgenic mouse lines utilized in previous studies, our findings provide a novel and important understanding for the role of these signaling pathways in physiological hypertrophy. We found that alterations in the signaling pathways tested here had no impact on exercise performance. Exercise induced cardiac growth in all of the transgenic mice except for the mice deficient in cyclin D2. In the cyclin D2 null mice, cardiac function was not impacted even though the hypertrophic response was blunted and a number of signaling pathways are differentially regulated by exercise. These data provide the field with an understanding that cyclin D2 is a key mediator of physiological hypertrophy.

DCP-LA, a New Strategy for Alzheimer's Disease Therapy

Alzheimer’s disease (AD) is characterized by extensive deposition of amyloid β (Aβ) and formation of neurofibrillary tangles (NFTs) consisting of hyperphosphorylated Tau. So far, a variety of AD drugs targeting Aβ have been developed, but ended in failure. A recent focus on AD therapy, therefore, is development of Tau-targeted drugs. Aβ activates glycogen synthase kinase-3β (GSK-3β), that plays a central role in Tau phosphorylation, responsible for NFT formation. The linoleic acid derivative DCP-LA has been developed as a promising drug for AD therapy. DCP-LA serves as a selective activator of PKCe and a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B). DCP-LA restrains Tau phosphorylation efficiently due to PKCe-mediated direct inactivation of GSK-3β, to PKCe/Akt-mediated inactivation of GSK-3β, and to receptor tyrosine kinase/insulin receptor substrate 1/phosphoinositide 3-kinase/3-phosphoinositide-dependent protein kinase 1/Akt-mediated inactivation of GSK-3β in association with PTP1B inhibition. Moreover, DCP-LA ameliorates spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. Consequently, combination of PKCβ activation and PTP1B inhibition must be an innovative strategy for AD therapy.

Self-assembled N-cadherin mimetic peptide hydrogels promote the chondrogenesis of mesenchymal stem cells through inhibition of canonical Wnt/β-catenin signaling.

N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37°C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.

Polygonatum Sibiricum Polysaccharide Promotes Osteoblastic Differentiation Through the ERK/GSK-3β/β-Catenin Signaling Pathway In Vitro.

Natural compound polygonatum sibiricum polysaccharide (PSP) has a variety of biological actions such as reducing blood fat, antitumor and antioxidant activities, and enhancing immune function. Our previous study has revealed that PSP can promote osteoblastic (OB) differentiation in bone mesenchymal stem cells by increasing nuclear accumulation of β-catenin. This study was designed to investigate that PSP can upregulate nuclear β-catenin, which was prevented by inhibiting the glycogen synthase kinase 3β (GSK-3β) activity, by effectively activating Wnt signaling independent of low-density lipoprotein receptor-related protein 5 (LRP5). We showed that PSP reduced the level of GSK-3β, which phosphorylates and destabilizes nuclear β-catenin through extracellular signal-regulated kinase (ERK) signaling pathway. Taken together, these findings demonstrate that PSP promotes OB differentiation and mineralization in vitro through the ERK/GSK-3β/β-catenin signaling pathways. This study may aid in the development of a therapeutic approach utilizing PSP for the enhancement of bone health and prevention of osteoporosis.

Ethanol-induced damage to the developing spinal cord: The involvement of CCR2 signaling

Ethanol exposure during development causes fetal alcohol spectrum disorders (FASD). A large body of evidence shows that ethanol produces multiple abnormalities in the developing central nervous system (CNS), such as smaller brain size, reduced volume of cerebral white matter, permanent loss of neurons, and alterations in synaptogenesis and myelinogenesis. The effects of ethanol on the developing spinal cord, however, receive little attention and remain unclear. We used a third trimester equivalent mouse model to investigate the effect of ethanol on the developing spinal cord. Ethanol caused apoptosis and neurodegeneration in the dorsal horn neurons of mice of early postnatal days, which was accompanied by glial activation, macrophage infiltration, and increased expression of CCR2, a receptor for monocyte chemoattractant protein 1 (MCP-1). Ethanol-induced neuronal death during development resulted in permanent loss of spinal cord neurons in adult mice. Ethanol stimulated endoplasmic reticulum (ER) stress and oxidative stress, and activated glycogen synthase kinase 3β (GSK3β) and c-Jun N-terminal kinase (JNK) pathways. Knocking out MCP-1 or CCR2 made mice resistant to ethanol-induced apoptosis, ER stress, glial activation, and activation of GSK3β and JNK. CCR2 knock out offered much better protection against ethanol-induced damage to the spinal cord. Thus, developmental ethanol exposure caused permanent loss of spinal cord neurons and CCR2 signaling played an important role in ethanol neurotoxicity.