Phosphorylation Of Glycogen Synthase Kinase-3β Research Articles

Angelica sinensis polysaccharide suppresses the Wnt/β-catenin-mediated malignant biological behaviors of breast cancer cells via the miR-3187-3p/PCDH10 axis

Breast cancer (BC) is one of the most common malignant tumors in women. Angelica sinensis polysaccharide (ASP) is one of the main components extracted from the traditional Chinese medicine Angelica sinensis. Research has shown that ASP affects the progression of various cancers by regulating miRNA expression. This study aimed to explore the specific molecular mechanism by which ASP regulates BC progression through miR-3187-3p. After the overexpression or knockdown of miR-3187-3p and PDCH10 in BC cells, the proliferation, migration, invasion, and phenotype of BC cells were evaluated after ASP treatment. Bioinformatics software was used to predict the target genes of miR-3187-3p, and luciferase gene reporter experiments reconfirmed the targeted binding relationship. Subcutaneous tumor formation experiments were conducted in nude mice after the injection of BC cells. Western blot and Ki-67 immunostaining were performed on the tumor tissues. The results indicate that ASP can significantly inhibit the proliferation, migration, and invasion of BC cells. ASP can inhibit the expression of miR-3187-3p in BC cells and upregulate the expression of PDCH10 by inhibiting miR-3187-3p. A regulatory relationship exists between miR-3187-3p and PDCH10. ASP can inhibit the expression of β-catenin and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) proteins through miR-3187-3p/PDCH10 and prevent the occurrence of malignant biological behavior in BC. Overall, this study revealed the potential mechanism by which ASP inhibits the BC process. ASP mediates the Wnt/β-catenin signaling pathway by affecting the miR-3187-3p/PDCH10 molecular axis, thereby inhibiting the proliferation, migration, invasion, and other malignant biological behaviors of BC cells.

Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3β and FOXO1 phosphorylation.

Establishment of a prognostic risk model for osteosarcoma and mechanistic investigation.

Objective: To investigate the immune mechanism of osteosarcoma (OS)-specific markers to mitigate bone destruction in the aggressive OS, prone to recurrence and metastasis. Methods: Gene expression patterns from the Gene Expression Omnibus (GEO) database (GSE126209) were analyzed using weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) analysis, least absolute shrinkage and selection operator (LASSO) modeling, and survival analysis to identify charged multivesicular body protein 4C (CHMP4C). Subsequently, its role in regulating the immune system and immune cell infiltration was explored. CHMP4C expression and signaling molecules in OS were assessed in osteosarcoma cell lines (MG63, U2OS, HOS) and hFOB1.19 cells using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The impact of CHMP4C upregulation and interference on OS-related signaling molecules in MG63 cells was studied. Functional validation of CHMP4C in MG63 OS cells was confirmed through cell counting Kit-8 (CCK-8), transwell, and colony formation assays. In vivo experiments were conducted using Specific Pathogen Free (SPF)-grade male BALB/C nude mice for OS xenograft studies. Results: Based on the gene expression profiles analysis of six osteosarcoma samples and six normal tissue samples, we identified 1,511 upregulated DEGs and 5,678 downregulated DEGs in normal tissue samples. A significant positive correlation between the "yellow-green" module and OS was found through WGCNA analysis. Expression levels of CHMP4C, phosphorylated Glycogen Synthase Kinase 3β (p-GSK3β), and β-catenin were notably higher in U2OS, HOS, and MG63 OS cells than in hFOB1.19 human osteoblasts. Overexpressing CHMP4C in MG63 OS cells upregulated CHMP4C, p-GSK3β, and β-catenin while downregulating GSK3β, leading to increased proliferation and migration of MG63 cells. Conversely, interrupting CHMP4C had the opposite effect. High expression of CHMP4C significantly accelerated the growth of OS in nude mice, resulting in substantial upregulation of CHMP4C, p-GSK3β, and β-catenin expression and suppression of Glycogen Synthase Kinase 3β (GSK3β) expression in OS tissues. Conclusion: CHMP4C may serve as a specific immunomodulatory gene for OS. Its activation of the Wnt/β-catenin signaling pathway, mainly by increasing the phosphorylation echelon of GSK3β, promotes the invasion and spread of OS.

Cardiomyocyte Adaptation to Exercise: K+ Channels, Contractility and Ischemic Injury.

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.

Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Anti-Proliferative Effects of Lidocaine as an Autophagy Inducer in Bladder Cancer via Intravesical Instillation: In Vitro and Xenograft Mouse Model Experiments.

Lidocaine exerts potential anti-tumor effects on various cancer cell lines, and its intravesical instillation is considered safer than intravenous administration for bladder cancer. However, the mechanisms underlying its anti-tumor effects have not been fully elucidated. Here, we aimed to elucidate the anti-tumor molecular mechanisms of lidocaine in bladder cancer cells and a xenograft model to substantiate the efficacy of its intravesical administration. We investigated the anti-proliferative and autophagyinducing activities of lidocaine in Nara Bladder Tumor No. 2 (NBT-II) rat bladder carcinoma cells using cell viability, flow cytometry, a wound healing assay, and western blotting. We also established a xenograft mouse model of bladder cancer, and cancer growth was examined using in vivo bioluminescence imaging. Lidocaine decreased cell viability, induced G0/G1 phase cell cycle arrest, and inhibited cell migration partially via glycogen synthase kinase (GSK) 3β phosphorylation. Moreover, a combination of lidocaine and SB216763 (a GSK3β inhibitor) suppressed autophagy-related protein expression. Bafilomycin-A1 with lidocaine significantly enhanced microtubule-associated protein 1A/1B-light chain (LC3B) expression; however, it decreased LC3B expression in combination with 3-methyladenine compared to lidocaine alone. In the xenograft mouse model, the bladder cancer volume was reduced by lidocaine. Overall, lidocaine exerts anti-proliferative effects on bladder cancer via an autophagy-inducing mechanism.

Metallothionein Alleviates Glutathione Depletion–Induced Oxidative Cardiomyopathy through CISD1-Dependent Regulation of Ferroptosis in Murine Hearts

This study was designed to discern the effect of heavy scavenger metallothionein on glutathione (GSH) deprivation-evoked cardiac anomalies and mechanisms involved with an emphasis on ferroptosis. Wild-type and cardiac metallothionein transgenic mice received GSH synthase inhibitor buthionine sulfoximine (BSO; 30 mmol/L in drinking water) for 14 days before assessment of myocardial morphology and function. BSO evoked cardiac remodeling and contractile anomalies, including cardiac hypertrophy, interstitial fibrosis, enlarged left ventricular chambers, deranged ejection fraction, fraction shortening, cardiomyocyte contractile capacity, intracellular Ca2+ handling, sarcoplasmic reticulum Ca2+ reuptake, loss of mitochondrial integrity (mitochondrial swelling, loss of aconitase activity), mitochondrial energy deficit, carbonyl damage, lipid peroxidation, ferroptosis, and apoptosis. Metallothionein itself did not affect myocardial morphology and function, although it mitigated BSO-provoked myocardial anomalies, loss of mitochondrial integrity and energy, and ferroptosis. Immunoblotting revealed down-regulated sarco(endo)plasmic reticulum Ca2+-ATPase 2a, glutathione peroxidase 4, the ferroptosis-suppressing iron-sulfur domain 1 (CISD1), and mitochondrial regulating glycogen synthase kinase-3β phosphorylation with elevated p53, myosin heavy chain-β isozyme, IκB phosphorylation, and SLC7A11 as well as unchanged SLC39A1, SLC1A5, and ferroptosis-suppressing protein 1 following BSO challenge, all of which, except glutamine transporter SLC7A11 and p53, were abrogated by metallothionein. Inhibition of CISD1 using pioglitazone nullified GSH-offered benefit against BSO-induced cardiomyocyte ferroptosis and contractile and intracellular Ca2+ derangement. Taken together, these findings support a regulatory modality for CISD1 in the impedance of ferroptosis in metallothionein-offered protection against GSH depletion-evoked cardiac aberration.

2,4'-Dihydroxybenzophenone Exerts Bone Formation and Antiosteoporotic Activity by Stimulating the β-Catenin Signaling Pathway.

2,4'-Dihydroxybenzophenone (DHP) is an organic compound derived from Garcinia xanthochymus, but there have been no reports on its biochemical functions and bioavailability. In this study, we evaluated whether DHP affects osteoblast differentiation and activation in MC3T3-E1 preosteoblast cells, as well as antiosteoporotic activity in zebrafish larvae. Nontoxic concentrations of DHP-treated MC3T3-E1 preosteoblast cells increased alkaline phosphatase (ALP) activation and mineralization in a concentration-dependent manner, accompanied by higher expression of osteoblast-specific markers, including Runt-related transcription factor 2 (RUNX2), osterix, and ALP. Consistent with the data in MC3T3-E1 preosteoblast cells, DHP upregulated osteoblast-specific marker genes in zebrafish larvae and simultaneously enhanced vertebral formation. We also revealed that DHP increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 and the total expression of β-catenin in the cytosol and markedly increased the localization of β-catenin into the nucleus. Furthermore, DHP restored the prednisolone (PDS)-induced marked decrease in ALP activity and mineralization, as well as osteoblast-specific marker expression. In PDS-treated zebrafish, DHP also alleviated PDS-induced osteoporosis by restoring vertebral formation and osteoblast-related gene expression. Taken together, these results suggest that DHP is a potential osteoanabolic candidate for treating osteoporosis by stimulating osteoblast differentiation.

Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3β (GSK3β), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3β pathways, and mediates the G1/S transition to control cell proliferation.

Parthenolide inhibits the proliferation and migration of cervical cancer cells via FAK/GSK3β pathway.

Cervical cancer (CC) ranks as the fourth most prevalent malignancy among women worldwide, necessitating effective therapeutic interventions to mitigate its detrimental impact on both physical and mental health. Parthenolide (PTL), a natural product of the sesquiterpene lactone derived from Feverfew leaves, has exhibited promising anti-tumor properties in previous studies; however, its precise effects and underlying molecular mechanisms in CC remain elusive. In this work, we investigated the effect of PTL on the proliferation and migration of CC cells. Western blot analysis and Reverse transcription‑quantitative PCR were used for mechanistic elucidation. Our findings indicated that PTL substantially inhibited the proliferation of HeLa and SiHa CC cell lines in a dose- and time-dependent manner. Moreover, PTL significantly suppressed the migration of CC cells by down-regulating the expression of vascular endothelial growth factor (VEGF), metastasis-associated protein 1 (MTA1), and transforming growth factor-β1 (TGF-β1). Mechanistically, PTL blocked the phosphorylation of focal adhesion kinase (FAK) and glycogen synthase kinase-3β (GSK3β) induced by epidermal growth factor (EGF). Further investigations revealed that PTL suppressed the proliferation of CC cells by inhibiting the EGF-mediated phosphorylation of the FAK/GSK3β signaling pathway. Taken together, the present in vitro results suggest that PTL may inhibit the proliferation and migration of CC cells through down-regulating the FAK/GSK3β signaling pathway, providing new insights for the application of PTL in the treatment of CC.

CXADR promote epithelial–mesenchymal transition in endometriosis by modulating AKT/GSK-3β signaling

ABSTRACT Endometriosis is a benign high prevalent disease exhibiting malignant features. However, the underlying pathogenesis and key molecules of endometriosis remain unclear. By integrating and analysis of existing expression profile datasets, we identified coxsackie and adenovirus receptor (CXADR), as a novel key gene in endometriosis. Based on the results of immunohistochemistry (IHC), we confirmed significant down-regulation of CXADR in ectopic endometrial tissues obtained from women with endometriosis compared with healthy controls. Further in vitro investigation indicated that CXADR regulated the stability and function of the phosphatases and AKT inhibitors PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2) and PTEN (phosphatase and tensin homolog). Loss of CXADR led to phosphorylation of AKT and glycogen synthase kinase-3β (GSK-3β), which resulted in stabilization of an epithelial–mesenchymal transition (EMT) factor, SNAIL1 (snail family transcriptional repressor 1). Therefore, EMT processs was induced, and the proliferation, migration and invasion of Ishikawa cells were enhanced. Over-expression of CXADR showed opposite effects. These findings suggest a previously undefined role of AKT/GSK-3β signaling axis in regulating EMT and reveal the involvement of a CXADR-induced EMT, in pathogenic progression of endometriosis.

Low Molecular Weight Collagen Peptide (LMWCP) Promotes Hair Growth by Activating the Wnt/GSK-3β/β-Catenin Signaling Pathway.

Low molecular weight collagen peptide (LMWCP) is a collagen hydrolysate derived from fish. We investigated the effects of LMWCP on hair growth using human dermal papilla cells (hDPCs), human hair follicles (hHFs), patch assay, and telogenic C57BL/6 mice, while also examining the underlying mechanisms of its action. LMWCP promoted proliferation and mitochondrial potential, and the secretion of hair growth-related factors, such as EGF, HB-EGF, FGF-4, and FGF-6 in hDPCs. Patch assay showed that LMWCP increased the neogeneration of new HFs in a dose-dependent manner. This result correlated with an increase in the expression of dermal papilla (DP) signature genes such as, ALPL, SHH, FGF7, and BMP-2. LMWCP upregulated phosphorylation of glycogen synthase kinase-3β (GSK-3β) and β-catenin, and nuclear translocation of β-catenin, and it increased the expression of Wnt3a, LEF1, VEGF, ALP, and β-catenin. LMWCP promoted the growth of hHFs and increased the expression of β-catenin and VEGF. Oral administration of LMWCP to mice significantly stimulated hair growth. The expression of Wnt3a, β-catenin, PCNA, Cyclin D1, and VEGF was also elevated in the back skin of the mice. Furthermore, LMWCP increased the expression of cytokeratin and Keratin Type I and II. Collectively, these findings demonstrate that LMWCP has the potential to increase hair growth via activating the Wnt/β-catenin signaling pathway.

Adipose-Derived Stem Cell Exosomes Antagonize the Inhibitory Effect of Dihydrotestosterone on Hair Follicle Growth by Activating Wnt/β-Catenin Pathway.

The most prevalent type of alopecia is androgenetic alopecia (AGA), which has a high prevalence but no effective treatment. Elevated dihydrotestosterone (DHT) level in the balding area was usually thought to be critical in the pathophysiology of AGA. The canonical Wnt/β-catenin signaling pathway plays a key role in promoting hair follicle development and sustaining the hair follicle cycle. Adipose-derived stem cell exosomes (ADSC-Exos) are widely used in the field of regenerative medicine due to the advantages of being cell free and immune privileged. Still, few studies have reported the therapeutic effect on hair disorders. As a result, we sought to understand how ADSC-Exos affected hair growth and explore the possibility that ADSC-Exos could counteract the hair-growth-inhibiting effects of DHT. This research using human hair follicle organs, in vitro dermal papilla cells, and in vivo animal models showed that ADSC-Exos not only encouraged healthy hair growth but also counteracted the inhibitory effects of DHT on hair growth. Additionally, we discovered that ADSC-Exos increased Ser9 phosphorylated glycogen synthase kinase-3β levels and facilitated nuclear translocation of β-catenin, which may have been blocked by the specific Wnt/β-catenin signaling pathway inhibitor dickkopf-related protein 1. Our findings suggested that ADSC-Exos are essential for hair regeneration, which is anticipated to open up new therapeutic possibilities for clinical alopecia, particularly for the treatment of AGA.

MiR-26b-3p Promotes Intestinal Motility Disorder by Targeting FZD10 to Inhibit GSK3β/β-Catenin Signaling and Induce Enteric Glial Cell Apoptosis.

Enteric glial cells (EGCs) are the major component of the enteric nervous system and affect the pathophysiological process of intestinal motility dysfunction. MicroRNAs (miRNAs) play an important role in regulating gastrointestinal homeostasis. However, the mechanism of miRNA-mediated regulation of EGCs in intestinal dysmotility remains unclear. In this study, we investigated the effect of EGC apoptosis on intestinal dysmotility, and the effect of miR-26b-3p on EGC proliferation and apoptosis in vivo and in vitro. A loperamide hydrochloride (Lop)-induced constipated mouse model and an in vitro culture system of rat EGCs were established. The transcriptome was used to predict the differentially expressed gene miR-26b-3p and the target gene Frizzled 10 (FZD10), and their targeting binding relationship was verified by luciferase. EGCs were transfected with miR-26b-3p mimic or antagomir, and the FZD10 expression was down-regulated by siRNA. Immunofluorescence and flow cytometry were used to detect EGC apoptosis. MiR-26b-3p and FZD10 expressions were examined using quantitative real-time PCR (qRT-PCR). The CCK-8 assay was used to detect EGC proliferation. The protein levels were detected by Western blotting and enzyme-linked immunosorbent assay (ELISA). The results showed that miR-26b-3p was up-regulated in the Lop group, whereas FZD10 was down-regulated, and EGC apoptosis was increased in the colon of intestinal dysmotility mice. FZD10 down-regulation and miR-26b-3p mimic significantly increased glycogen synthase kinase-3β phosphorylation (p-GSK3β) levels, decreased β-catenin expression, and promoted EGC apoptosis. MiR-26b-3p antagomir alleviated intestinal dysmotility, promoted EGC increased activity of EGCs, and reduced EGC apoptosis in vivo. In conclusion, this study indicated that miR-26b-3p promotes intestinal motility disorders by targeting FZD10 to block GSK3β/β-catenin signaling and induces apoptosis in EGCs. Our results provide a new research target for the treatment and intervention of intestinal dysmotility.

Pregnancy-induced physiological hypertrophic preconditioning attenuates pathological myocardial hypertrophy by activation of FoxO3a.

Previous studies show a woman's pregnancy is correlated with post-reproductive longevity, and nulliparity is associated with higher risk of incident heart failure, suggesting pregnancy likely exerts a cardioprotection. We previously reported a cardioprotective phenomenon termed myocardial hypertrophic preconditioning, but it is unknown whether pregnancy-induced physiological hypertrophic preconditioning (PHP) can also protect the heart against subsequent pathological hypertrophic stress. We aimed to clarify the phenomenon of PHP and its mechanisms. The pluripara mice whose pregnancy-induced physiological hypertrophy regressed and the nulliparous mice underwent angiotensin II (Ang II) infusion or transverse aortic constriction (TAC). Echocardiography, invasive left ventricular hemodynamic measurement and histological analysis were used to evaluate cardiac remodeling and function. Silencing or overexpression of Foxo3 by adeno-associated virus was used to investigate the role of FoxO3a involved in the antihypertrophic effect. Compared with nulliparous mice, pathological cardiac hypertrophy induced by Ang II infusion, or TAC was significantly attenuated and heart failure induced by TAC was markedly improved in mice with PHP. Activation of FoxO3a was significantly enhanced in the hearts of postpartum mice. FoxO3a inhibited myocardial hypertrophy by suppressing signaling pathway of phosphorylated glycogen synthase kinase-3β (p-GSK3β)/β-catenin/Cyclin D1. Silencing or overexpression of Foxo3 attenuated or enhanced the anti-hypertrophic effect of PHP in mice with pathological stimulation. Our findings demonstrate that PHP confers resistance to subsequent hypertrophic stress and slows progression to heart failure through activation of FoxO3a/GSK3β pathway.

Frizzled-9 triggers actin polymerization and activates mechano-transducer YAP to rescue simulated microgravity-induced osteoblast dysfunction.

Long-term spaceflight can result in bone loss and osteoblast dysfunction. Frizzled-9 (Fzd9) is a Wnt receptor of the frizzled family that is vital for osteoblast differentiation and bone formation. In the present study, we elucidated whether Fzd9 plays a role in osteoblast dysfunction induced by simulated microgravity (SMG). After 1-7 days of SMG, osteogenic markers such as alkaline phosphatase (ALP), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2) were decreased, accompanied by a decrease in Fzd9 expression. Furthermore, Fzd9 expression decreased in the rat femur after 3 weeks of hindlimb unloading. In contrast, Fzd9 overexpression counteracted the decrease in ALP, OPN, and RUNX2 induced by SMG in osteoblasts. Moreover, SMG regulated phosphorylated glycogen synthase kinase-3β (pGSK3β) and β-catenin expression or sublocalization. However, Fzd9 overexpression did not affect pGSK3β and β-catenin expression or sublocalization induced by SMG. In addition, Fzd9 overexpression regulated protein kinase B also known as Akt and extracellular signal-regulated kinase (ERK) phosphorylation and induced F-actin polymerization to form the actin cap, press the nuclei, and increase nuclear pore size, thereby promoting the nuclear translocation of Yes-associated protein (YAP). Our study findings provide mechanistic insights into the role of Fzd9 in triggering actin polymerization and activating YAP to rescue SMG-induced osteoblast dysfunction and suggest that Fzd9 is a potential target to restore osteoblast function in individuals with bone diseases and after spaceflight.

Novel synergistic treatment for depression: involvement of GSK3β-regulated AMPA receptors in the prefrontal cortex of mice.

Previous evidence has suggested a vital role of glycogen synthase kinase 3β-mediated α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors trafficking in depression. Considering the antidepressant effect of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors activation in the prefrontal cortex, we hypothesized that glycogen synthase kinase 3β-induced alterations in α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors function in the prefrontal cortex participate in depression. Herein, we confirmed that the levels of phosphorylated glycogen synthase kinase 3β and GluA1, the latter being a subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, were decreased in the prefrontal cortex of the chronic social defeat stress model mice presenting with depressive-like behaviors. We then found that a glycogen synthase kinase 3β (p.S9A) point mutation downregulated GluA1 and induced depressive-like behaviors in mice, whereas an agonist of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, PF-4778574 (2mg/kg) did not reversed the molecular changes. On the other hand, the antidepressant effect of PF-4778574 was dose dependent, and the single administration of PF-4778574 at a lower dose (0.5mg/kg) or of the glycogen synthase kinase 3β inhibitor SB216763 (5 and 10mg/kg) did not evoke an antidepressant effect. In contrast, co-treatment with PF-4778574 (0.5mg/kg) and SB216763 (10mg/kg) led to antidepressant effects similar to those of PF-4778574 (2mg/kg). Our results suggest that glycogen synthase kinase 3β-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors dysfunction in the prefrontal cortex is one of the key mechanisms of depression, and the combination of a lower dose of PF-4778574 with SB216763 shows potential as a novel synergistic treatment for depression.

Oat β-D-glucan ameliorates type II diabetes through TLR4/PI3K/AKT mediated metabolic axis

Diabetes is one of the major global public health problems. Our previous results found that oat β-D-glucan exhibited ameliorative effects on diabetic mice, but the underlying mechanism is unclear. The present study indicates that oat β-D-glucan increased glycogen content, decreased glycogen synthase (GS) phosphorylation and increased hepatic glycogen synthase kinase 3β (GSK3β) phosphorylation for glycogen synthesis via PI3K/AKT/GSK3-mediated GS activation. Moreover, oat β-D-glucan inhibited gluconeogenesis through the PI3K/AKT/Foxo1-mediated phosphoenolpyruvate carboxykinase (PEPCK) decrease. In addition, oat β-D-glucan enhanced glucose catabolism through elevated protein levels of COQ9, UQCRC2, COXIV and ATP5F complexes involved in oxidative phosphorylation, as well as that of TFAM, a key regulator of mitochondrial gene expression. Importantly, our results showed that oat β-D-glucan maintained hepatic glucose balance via TLR4-mediated intracellular signal. After TLR4 blocking with anti-TLR4 antibody, oat β-D-glucan had almost no effect on high glucose-induced HepG2 cells. These data revealed that oat β-D-glucan maintains glucose balance by regulating the TLR4/PI3K/AKT signal pathway.

Phosphoproteome Profiling of uEVs Reveals p-AQP2 and p-GSK3β as Potential Markers for Diabetic Nephropathy.

Diabetic nephropathy (DN) contributes to increased morbidity and mortality among patients with diabetes and presents a considerable global health challenge. However, reliable biomarkers of DN have not yet been established. Phosphorylated proteins are crucial for disease progression. However, their diagnostic potential remains unexplored. In this study, we used ultra-high-sensitivity quantitative phosphoproteomics to identify phosphoproteins in urinary extracellular vesicles (uEVs) as potential biomarkers of DN. We detected 233 phosphopeptides within the uEVs, with 47 phosphoproteins exhibiting significant alterations in patients with DN compared to those in patients with diabetes. From these phosphoproteins, we selected phosphorylated aquaporin-2 (p-AQP2[S256]) and phosphorylated glycogen synthase kinase-3β (p-GSK3β[Y216]) for validation, as they were significantly overrepresented in pathway analyses and previously implicated in DN pathogenesis. Both phosphoproteins were successfully confirmed through Phos-tag western blotting in uEVs and immunohistochemistry staining in kidney sections, suggesting that phosphoprotein alterations in uEVs reflect corresponding changes within the kidney and their potential as candidate biomarkers for DN. Our research proposes the utilization of phosphoproteins in uEVs as a liquid biopsy, presenting a highly feasible diagnostic tool for kidney disease.

A rat model of metabolic syndrome-related heart failure with preserved ejection fraction phenotype: pathological alterations and possible molecular mechanisms.

Heart failure with preserved ejection fraction (HFpEF) represents a syndrome involving multiple pathophysiologic disorders and clinical phenotypes. This complexity makes it challenging to develop a comprehensive preclinical model, which presents an obstacle to elucidating disease mechanisms and developing new drugs. Metabolic syndrome (MetS) is a major phenotype of HFpEF. Thus, we produced a rat model of the MetS-related HFpEF phenotype and explored the molecular mechanisms underpinning the observed pathological changes. A rat model of the MetS-related HFpEF phenotype was created by feeding spontaneously hypertensive rats a high-fat-salt-sugar diet and administering streptozotocin solution intraperitoneally. Subsequently, pathological changes in the rat heart and their possible molecular mechanisms were explored. The HFpEF rats demonstrated primary features of MetS, such as hypertension, hyperglycemia, hyperlipidemia, insulin resistance, and cardiac anomalies, such as left ventricular (LV) remodeling and diastolic impairment, and left atrial dilation. Additionally, inflammation, myocardial hypertrophy, and fibrosis were observed in LV myocardial tissue, which may be associated with diverse cellular and molecular signaling cascades. First, the inflammatory response might be related to the overexpression of inflammatory regulators (growth differentiation factor 15 (GDF-15), intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial cell adhesion molecule-1 (VCAM-1)). Secondly, phosphorylated glycogen synthase kinase 3β (GSK-3β) may stimulate cardiac hypertrophy, which was regulated by activated -RAC-alpha serine/threonine-protein kinase (AKT). Finally, the transforming growth factor-β1 (TGF-β1)/Smads pathway might regulate collagen production and fibroblast activation, promoting myocardial fibrosis. The HFpEF rat replicates the pathology and clinical presentation of human HFpEF with MetS and may be a reliable preclinical model that helps elucidate HFpEF pathogenesis and develop effective treatment strategies.