Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin α_IIbβ₃ activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.     

Leptin,Acting at Central Level,Increases FGF21 Expression in White Adipose Tissue via PPARβ/δ

The altered function of adipose tissue can result in obesity, insulin resistance, and its metabolic complications. Leptin, acting on the central nervous system, modifies the composition and function of adipose tissue. To date, the molecular changes that occur in epididymal white adipose tissue (eWAT) during chronic leptin treatment are not fully understood. Herein we aimed to address whether PPARβ/δ could mediate the metabolic actions induced by leptin in eWAT. To this end, male 3-month-old Wistar rats, infused intracerebroventricularly (icv) with leptin (0.2 μg/day) for 7 days, were daily co-treated intraperitoneally (ip) without or with the specific PPARβ/δ receptor antagonist GSK0660 (1 mg/kg/day). In parallel, we also administered GSK0660 to control rats fed ad libitum without leptin infusion. Leptin, acting at central level, prevented the starvation-induced increase in circulating levels of FGF21, while induced markedly the endogenous expression of FGF21 and browning markers of eWAT. Interestingly, GSK0660 abolished the anorectic effects induced by icv leptin leading to increased visceral fat mass and reduced browning capacity. In addition, the pharmacological inhibition of PPARβ/δ alters the immunomodulatory actions of central leptin on eWAT. In summary, our results demonstrate that PPARβ/δ is involved in the up-regulation of FGF21 expression induced by leptin in visceral adipose tissue.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

Cannabidiol and the Canonical WNT/β-Catenin Pathway in Glaucoma

Glaucoma is a progressive neurodegenerative disease which constitutes the main frequent cause of irreversible blindness. Recent findings have shown that oxidative stress, inflammation and glutamatergic pathway play key roles in the causes of glaucoma. Recent studies have shown a down regulation of the WNT/β-catenin pathway in glaucoma, associated with overactivation of the GSK-3β signaling. WNT/β-catenin pathway is mainly associated with oxidative stress, inflammation and glutamatergic pathway. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid derived from Cannabis sativa plant which possesses many therapeutic properties across a range of neuropsychiatric disorders. Since few years, CBD presents an increased interest as a possible drug in anxiolytic disorders. CBD administration is associated with increase of the WNT/β-catenin pathway and decrease of the GSK-3β activity. CBD has a lower affinity for CB1 but can act through other signaling in glaucoma, including the WNT/β-catenin pathway. CBD downregulates GSK3-β activity, an inhibitor of WNT/β-catenin pathway. Moreover, CBD was reported to suppress pro-inflammatory signaling and neuroinflammation, oxidative stress and glutamatergic pathway. Thus, this review focuses on the potential effects of cannabidiol, as a potential therapeutic strategy, on glaucoma and some of the presumed mechanisms by which this phytocannabinoid provides its possible benefit properties through the WNT/β-catenin pathway.     

GSK3β Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.     

Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.     

Inhibition of β-Catenin Activity Abolishes LKB1 Loss-Driven Pancreatic Cystadenoma in Mice

Pancreatic cancer (PC) is the seventh leading cause of cancer death worldwide, and remains one of our most recalcitrant and dismal diseases. In contrast to many other malignancies, there has not been a significant improvement in patient survival over the past decade. Despite advances in our understanding of the genetic alterations associated with this disease, an incomplete understanding of the underlying biology and lack of suitable animal models have hampered efforts to develop more effective therapies. LKB1 is a tumor suppressor that functions as a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), which is an important mediator in the regulation of cell growth and epithelial polarity pathways. LKB1 is mutated in a significant number of Peutz–Jeghers syndrome (PJS) patients and in a small proportion of sporadic cancers, including PC; however, little is known about how LKB1 loss contributes to PC development. Here, we report that a reduction in Wnt/β-catenin activity is associated with LKB1 tumor-suppressive properties in PC. Remarkably, in vivo functional analyses of β-catenin in the Pdx-1-Cre LKB1^L/L β-catenin^L/L mouse model compared to LKB1 loss-driven cystadenoma demonstrate that the loss of β-catenin impairs cystadenoma development in the pancreas of Pdx-1Cre LKB1^L/L mice and dramatically restores the normal development and functions of the pancreas. This study further determined the in vivo and in vitro therapeutic efficacy of the β-catenin inhibitor FH535 in suppressing LKB1 loss-driven cystadenoma and reducing PC progression that delineates the potential roles of Wnt/β-catenin signaling in PC harboring LKB1 deficiency.     

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

NBM-BMX,an HDAC8 Inhibitor,Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the β-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.     

Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation,Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.     

Role of Receptor Protein Tyrosine Phosphatase β/ζ in Neuron–Microglia Communication in a Cellular Model of Parkinson’s Disease

Pleiotrophin (PTN) is a neurotrophic factor that regulates glial responses in animal models of different types of central nervous system (CNS) injuries. PTN is upregulated in the brain in different pathologies characterized by exacerbated neuroinflammation, including Parkinson’s disease. PTN is an endogenous inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ, which is abundantly expressed in the CNS. Using a specific inhibitor of RPTPβ/ζ (MY10), we aimed to assess whether the PTN/RPTPβ/ζ axis is involved in neuronal and glial injury induced by the toxin MPP+. Treatment with the RPTPβ/ζ inhibitor MY10 alone decreased the viability of both SH-SY5Y neuroblastoma cells and BV2 microglial cultures, suggesting that normal RPTPβ/ζ function is involved in neuronal and microglial viability. We observed that PTN partially decreased the cytotoxicity induced by MPP+ in SH-SY5Y cells underpinning the neuroprotective function of PTN. However, MY10 did not seem to modulate the SH-SY5Y cell loss induced by MPP+. Interestingly, we observed that media from SH-SY5Y cells treated with MPP+ and MY10 decreases microglial viability but may elicit a neuroprotective response of microglia by upregulating Ptn expression. The data suggest a neurotrophic role of microglia in response to neuronal injury through upregulation of Ptn levels.     

Crosstalk between β-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, β-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of β-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in β-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting β-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shβ-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as β-catenin knockdown. When investigating the effect of CCL2 on β-catenin activity, we found that CCL2 modulates components of the Wnt/β-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological β-catenin inhibitor MSAB reduces active β-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that β-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between β-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.     

Eugenol β-Amino/β-Alkoxy Alcohols with Selective Anticancer Activity

Eugenol, 4-allyl-2-methoxyphenol, is the main constituent of clove essential oil and has demonstrated relevant biological activity, namely anticancer activity. Aiming to increase this activity, we synthesized a series of eugenol β-amino alcohol and β-alkoxy alcohol derivatives, which were then tested against two human cancer cell lines, namely gastric adenocarcinoma cells (AGS) and lung adenocarcinoma cells (A549). An initial screening was performed to identify the most cytotoxic compounds. The results demonstrated that three β-amino alcohol derivatives had anticancer activity that justified subsequent studies, having been shown to trigger apoptosis. Importantly, the most potent molecules displayed no appreciable toxicity towards human noncancer cells. Structure-activity relationships show that changes in eugenol structure led to enhanced cytotoxic activity and can contribute to the future design of more potent and selective drugs.