Pioglitazone inhibits advanced glycation end product-induced matrix metalloproteinases and apoptosis by suppressing the activation of MAPK and NF-κB

Apoptosis and degeneration coming mainly from chondrocytes are important mechanisms in the onset and progression of osteoarthritis. Specifically, advanced glycation end products (AGEs) play an important role in the pathogenesis of osteoarthritis. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist has a protective effect on cartilage. This study aims to evaluate the effect of pioglitazone on AGEs-induced chondrocyte apoptosis and degeneration and their underlying mechanism. The in vitro study shows that AGEs induce cleavage of caspase-3 and PARP, up-regulate MMP-13 expression, enhance chondrocyte apoptosis and down-regulate PPARγ expression in human primary chondrocytes, which is reversed by pioglitazone. Furthermore, AGEs activate phosphorylation of Erk, JNK, and p38, and pioglitazone reverses AGEs-induced phosphorylation of Erk and p38. AGEs-induced degradation of IκBα and translocation of nuclear NF-κB p65 is reversed by pioglitazone. Pretreatment of chondrocytes with SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and BAY-11-7082 (NF-κB inhibitor) inhibit AGEs-induced apoptosis and degeneration. In vivo experiments suggest that pioglitazone reverses AGEs-induced cartilage degeneration and apoptosis in a mouse model, as demonstrated by HE and Safranin O staining, immunohistochemical analyses of Type II collagen (Col II), metalloproteinases (MMPs) and caspase-3. These findings suggest that pioglitazone, a PPARγ agonist, inhibits AGEs-induced chondrocytes apoptosis and degeneration via suppressing the activation of MAPK and NF-κB.     

Tangeretin inhibits streptozotocin-induced cell apoptosis via regulating NF-κB pathway in INS-1 cells

Oxidative stress is considered to play an important role in inducing the pancreatic β-cells apoptosis and promoting the development of diabetes mellitus. Tangeretin is a plant-derived flavonoid that retains antidiabetic effects. However, the role of tangeretin in streptozotocin (STZ)-induced β-cell apoptosis remains unclear. In this study, we aimed to examine the effects of tangeretin on STZ-induced cell apoptosis and the underlying mechanisms implicated in vitro. Our results showed that tangeretin improved the cell viability in STZ-induced INS-1 cells. Tangeretin reduced the increase of apoptosis ratio and revered the altered expressions of Bax and Bcl-2 caused by STZ induction. Furthermore, the impairment of insulin secretion ability as well as a reduction in messenger RNA levels of insulin 1 and 2 was significantly attenuated by tangeretin in STZ-induced INS-1 cells. Moreover, tangeretin resulted in a significant decrease in reactive oxygen species content, accompanied by an evident increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase. Mechanistic studies further revealed that tangeretin inhibited the NF-κB pathway in STZ-induced INS-1 cells. These data indicated that tangeretin improved the cell apoptosis induced by STZ in INS-1 cells, which might be partly due to its antioxidant potential. Furthermore, NF-κB was found to be involved in the protective effect of tangeretin. Collectively, the results indicated that tangeretin could be used as a therapeutic approach for diabetes mellitus treatment.     

Liquiritin attenuates advanced glycation end products-induced endothelial dysfunction via RAGE/NF-κB pathway in human umbilical vein endothelial cells

Advanced glycation end products (AGEs)-induced vasculopathy, including oxidative stress, inflammation and apoptosis responses, contributes to the high morbidity and mortality of coronary artery diseases in diabetic patients. The present study was conducted to evaluate the protective activity of liquiritin (Liq) on AGEs-induced endothelial dysfunction and explore its underlying mechanisms. After pretreatment with Liq, a significant reduction in AGEs-induced apoptosis, as well as reactive oxygen species generation and malondialdehyde level in human umbilical vein endothelial cells (HUVECs) were observed via acridine orange/ethidium bromide fluorescence staining test. Notably, Liq also significantly increased AGEs-reduced superoxide dismutase activity. Furthermore, the pretreatment with receptor for advanced glycation end products (RAGE)-antibody or Liq remarkably down-regulated TGF-beta1 and RAGE protein expressions and significantly blocked NF-κB activation which were proved by immunocytochemistry or immunofluorescence assays. These results indicated that Liq held potential for the protection on AGEs-induced endothelial dysfunction via RAGE/NF-κB pathway in HUVECs and might be a promising agent for the treatment of vasculopathy in diabetic patients.     

Synthetic resveratrol aliphatic acid inhibits TLR2-mediated apoptosis and an involvement of Akt/GSK3β pathway

As resveratrol derivatives, resveratrol aliphatic acids were synthesized in our laboratory. Previously, we reported the improved pharmaceutical properties of the compounds compared to resveratrol, including better solubility in water and much tighter binding with human serum albumin. Here, we investigate the role of resveratrol aliphatic acids in Toll-like receptor 2 (TLR2)-mediated apoptosis. We showed that resveratrol aliphatic acid (R6A) significantly inhibits the expression of TLR2. In addition, overexpression of TLR2 in HEK293 cells caused a significant decrease in apoptosis after R6A treatment. Moreover, inhibition of TLR2 by R6A decreases serum deprivation-reduced the levels of phosphorylated Akt and phosphorylated glycogen synthase kinase 3β (GSK3β). Our study thus demonstrates that the resveratrol aliphatic acid inhibits cell apoptosis through TLR2 by the involvement of Akt/GSK3β pathway.     

microRNA-448 inhibits the progression of non-small–cell lung cancer through regulating IRS2

Recently, microRNA-448 (miR-448) has been reported to be a tumor-associated miRNA in many human cancers. In this study, we investigated the function of miR-448 in non-small–cell lung cancer (NSCLC) progression and confirmed the relationship between miR-448 and insulin receptor substrates 2 (IRS2). First, downregulation of miR-448 and upregulation of IRS2 were detected in NSCLC using the quantitative real-time polymerase chain reaction (qRT-PCR) assay. Furthermore, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay showed that miR-448 inhibited cell viability in NSCLC. Transwell and Western blot assays indicated that the upregulation of miR-448 inhibited cell metastasis and epithelial-to-mesenchymal transition (EMT) in NSCLC. And it was found that overexpression of miR-448 reduced the adhesion of A549 cells to HUVEC cells using the adhesion assay. Furthermore, the dual luciferase assay indicated that miR-448 directly targeted IRS2 in NSCLC. In addition, it was found that IRS2 silencing had an inhibitory effect on the progression of NSCLC, and the upregulation of IRS2 partially impaired the inhibitory effect of miR-448 in NSCLC. Briefly, overexpression of miR-448 inhibited cell proliferation, metastasis, and EMT by suppressing IRS2 expression in NSCLC.     

DRG2 knockdown induces Golgi fragmentation via GSK3β phosphorylation and microtubule stabilization

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3β (GSK3β)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3β. Tau, a target of GSK3β, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3β phosphorylation, and microtubule stability.     

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

Background

Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established.

Scope of Review

EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells.

Major Conclusions

Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways.

General significance

These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.     

Grape seed procyanidin extract ameliorates lead-induced liver injury via miRNA153 and AKT/GSK-3β/Fyn-mediated Nrf2 activation

Lead-induced hepatotoxicity is characterized by an extensive oxidative stress. Grape seed procyanidin extract (GSPE) possesses abundant biological activities. Herein, we investigated the protective role of GSPE against lead-induced liver injury and determined the potential molecular mechanisms. In vivo, rats were treated with/without lead acetate (PbAc) (0.05%, w/v) in the presence/absence of GSPE (200 mg/kg). In vitro, hepatocytes were pretreated with/without GSPE (100 μg/ml) in the presence/absence of PbAc (100 μM). PbAc administration to rats resulted in anemia, liver dysfunction, lead accumulation in the bone and liver, oxidative stress, DNA damage and apoptosis. GSPE significantly attenuated these adverse effects, except lead accumulation in liver. GSPE also decreased the expression of miRNA153 and increased the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and levels of its downstream protein, and protein kinase B (AKT) phosphorylation in PbAc-induced liver injury. In primary hepatocytes treated with PbAc, GSPE increased hepatocyte viability and decreased lactate dehydrogenase release and reactive oxygen species levels. Dietary GSPE attenuated PbAc-induced liver injury in rats via an integrated mechanism associated with the miRNA153 and AKT/glycogen synthase kinase 3 beta/Fyn-mediated Nrf2 activation.     

GSK-3β inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway

Molecular Biology Reports - Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this...     

Unfractionated heparin inhibits lipopolysaccharide-induced inflammatory response through blocking p38 MAPK and NF-κB activation on endothelial cell

Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are glycosaminoglycans that are largely used as anti-thrombotic drugs. While the mechanisms of their anticoagulant actions in blood have been extensively studied, their effects on the inflammation of the endothelium are still under investigation since the endothelium plays a central role in sepsis. Furthermore, UFH is much cheaper than LMWH. The aim of this study was to determine how UFH regulates lipopolysaccharide (LPS)-induced inflammatory response on endothelial cells in vitro, and define the role of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in mediating this effect. Human pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.01U/ml-10U/ml), prior to stimulation with LPS (10μg/ml). Markers of systemic inflammation and endothelial activation were assessed. Interleukin (IL)-1β, IL-6, E-selectin, intercellular adhesion molecule (ICAM)-1 release were subsequently measured at 2h, 6h and 12h. Phosphorylation of p38 MAPK at 2h, 6h and nuclear translocation of the proinflammatory NF-κB at 2h were assessed. In HPMEC, UFH significantly attenuated LPS-induced production of IL-1β, IL-6, E-selectin and ICAM-1, as well as phosphorylation of p38 MAPK and NF-κB translocation, especially in 10U/ml. In conclusion, UFH at high dose significantly protects against endothelial-cell-mediated immune response. The inhibition of p38 MAPK and NF-κB activation certainly represents one of the mechanisms by which UFH exerts its anti-inflammatory effect.     

Caffeine inhibits cell proliferation and regulates PKA/GSK3β pathways in U87MG human glioma cells

Caffeine is the most commonly ingested methylxanthine and has anti-cancer effects in several types of cancer. In this study, we examined the anti-cancer effects of caffeine on gliomas, both in vitro and in vivo. In vitro, caffeine treatment reduced glioma cell proliferation through G(0)/G(1)-phase cell cycle arrest by suppressing Rb phosphorylation. In addition, caffeine induced apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Caffeine also phosphorylated serine 9 of glycogen synthase kinase 3 beta (GSK3β). Pretreatment with H89, a pharmacological inhibitor of protein kinase A (PKA), was able to antagonize caffeine-induced GSK3β(ser9) phosphorylation, suggesting that the mechanism might involve a cAMP-dependent PKA-dependent pathway. In vivo, caffeine-treated tumors exhibited reduced proliferation and increased apoptosis compared with vehicle-treated tumors. These results suggest that caffeine induces cell cycle arrest and caspase-dependent cell death in glioma cells, supporting its potential use in chemotherapeutic options for malignant gliomas.     

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3β of Wnt/β-catenin signaling pathway

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3′ UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active β-catenin, which is the downstream effector of GSK3β in the Wnt/β-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β-catenin signaling pathway.     

Cardiac-specific Traf2 overexpression enhances cardiac hypertrophy through activating AKT/GSK3β signaling

Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor-associated factor 2 (Traf2); however, Traf2 signaling in the adult mammalian cardiac hypertrophy is not fully understood. This study was aimed to identify the effect of Traf2 on cardiac hypertrophy and the underlying mechanisms. A significant up-regulation of Traf2 expression was observed in mice failing hearts. To further investigate the role of Traf2 in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of Traf2 function and cardiac-specific Traf2-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, Traf2 positively regulated angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [³H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by constriction of transverse aortic (TAC) in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Traf2 overexpression in the heart remarkably enhanced cardiac hypertrophy, left ventricular dysfunction in mice in response to TAC. Further analysis of the signaling pathway in vitro and in vivo suggested that these adverse effects of Traf2 were associated with the activation of AKT/glycogen synthase kinase 3β (GSK3β). The present study demonstrates that Traf2 serves as a novel mediator that enhanced cardiac hypertrophy by activating AKT/GSK3β signaling.     

PI3K/Akt-dependent phosphorylation of GSK3β and activation of RhoA regulate Wnt5a-induced gastric cancer cell migration

Wnt5a, a non-transforming Wnt family member, plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in gastric cancer progression remains poorly defined. In this study, we found that Wnt5a dose-dependently stimulated the migration of human gastric cancer cells (SGC-7901), with the maximal effect at 100 ng/mL, via enhancing phosphorylation of PI3K/Akt and GSK3β and activating RhoA. Pharmaceutical inhibition of PI3K with LY294002 or Akt siRNA significantly decreased Wnt5a-induced GSK3β phosphorylation and consequently cell migration. Additionally, GSK3β siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Analogously, pre-treatment with LiCl, which induced phosphorylation of GSK3β at Ser9, increased Wnt5a-induced cell migration. Finally, ectopic expression of dominant negative RhoA (N19) suppressed Wnt5a-induced cell migration. Taken together, we demonstrated for the first time that Wnt5a promoted gastric cancer cell migration via the PI3K/Akt/GSK3β/RhoA signaling pathway. These findings could provide a rationale for designing new therapy targeting gastric cancer metastasis.     

Tau phosphorylation and neuronal apoptosis induced by the blockade of PP2A preferentially involve GSK3β

Overactivation of GSK3β (glycogen synthase kinase-3β) and downregulation of PP2A (protein phosphatase-2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation in Alzheimer’s disease (AD). GSK3β and PP2A signaling pathways were reported to be interconnected. Targeting tau kinases was suggested to represent a therapeutic strategy for AD. Here, tau phosphorylation and neuronal apoptosis were induced in cortical cultured neurons by the inhibition of PP2A by okadaic acid (OKA). In this in vitro model of ‘tau pathology’ and neurodegeneration, we tested whether GSK3β and other tau kinases including DYRK1A and CDK5 were implicated. Our results show that the inhibitors of GSK3β, lithium and 6-BIO (6-bromoindirubin-3′-oxime), prevented OKA-induced tau phosphorylation and neuronal apoptosis. The implication of GSK3β in these OKA-induced effects was confirmed by its silencing by hairpin siRNA. By contrast, inhibition of DYRK1A (dual-specificity tyrosine-phosphorylation regulated kinase-1A) and CDK5 (cyclin-dependent kinase-5) reversed OKA-induced tau phosphorylation at certain sites but failed to prevent neuronal apoptosis. These results indicate that OKA-induced effects, especially neuronal apoptosis, are preferentially mediated by GSK3β. Furthermore, since chronic exposure to lithium and 6-BIO might be deleterious for neurons, we tested the effect of a new 6-BIO derivative, 6-BIBEO (6-bromoindirubin-3′-(2-bromoethyl)-oxime), which is much less cytotoxic and more selectively inhibits GSK3β compared to lithium and 6-BIO. We show that 6-BIBEO efficiently reversed OKA-induced tau phosphorylation and neuronal apoptosis. It will be interesting to test neuroprotection by 6-BIBEO in an in vivo model of tau pathology and neurodegeneration.     

Substance P inhibits high urea-induced apoptosis through the AKT/GSK-3β pathway in human corneal epithelial cells

To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.