Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms. METHODS: Clinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation. RESULTS: The expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3ß (GSK3ß)/ß-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3ß/ß-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3ß activity through ERK-mediated phosphorylation, thereby affecting ß-catenin degradation. CONCLUSION: ACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3ß/ß-catenin signaling pathway.

Empagliflozin and pirfenidone confer renoprotection through suppression of glycogen synthase kinase-3ß and promotion of tubular regeneration in rats with induced metabolic syndrome.

Metabolic syndrome (MetS) is largely coupled with chronic kidney disease (CKD). Glycogen synthase kinase-3ß (GSK-3ß) pathway drives tubular injury in animal models of acute kidney injury; but its contribution in CKD is still elusive. This study investigated the effect empagliflozin and/or pirfenidone against MetS-induced kidney dysfunction, and to clarify additional underpinning mechanisms particularly the GSK-3ß signaling pathway. Adult male rats received 10%w/v fructose in drinking water for 20 weeks to develop MetS, then treated with either drug vehicle, empagliflozin (30 mg/kg/day) and/or pirfenidone (100 mg/kg/day) via oral gavage for subsequent 4 weeks, concurrently with the high dietary fructose. Age-matched rats receiving normal drinking water were used as controls. After 24 weeks, blood and kidneys were harvested for subsequent analyses. Rats with MetS showed signs of kidney dysfunction, structural changes and interstitial fibrosis. Activation of GSK-3ß, decreased cyclinD1 expression and enhanced apoptotic signaling were found in kidneys of MetS rats. There was abundant alpha-smooth muscle actin (α-SMA) expression along with up-regulation of TGF-ß1/Smad3 in kidneys of MetS rats. These derangements were almost alleviated by empagliflozin or pirfenidone, with evidence that the combined therapy was more effective than either individual drug. This study emphasizes a novel mechanism underpinning the beneficial effects of empagliflozin and pirfenidone on kidney dysfunction associated with MetS through targeting GSK-3ß signaling which can mediate the regenerative capacity, anti-apoptotic effects and anti-fibrotic properties of such drugs. These findings recommend the possibility of using empagliflozin and pirfenidone as promising therapies for management of CKD in patients with MetS.

Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

Targeting protein kinases that regulate signalling pathways in inflammation is an effective pharmacological approach to alleviate uncontrolled inflammatory diseases. In this context, the natural product indirubin and its 6-bromo-substituted analogue 6-bromoindirubin-3 -glycerol-oxime ether (6BIGOE; 1) were identified as potent inhibitors of glycogen synthase kinase-3ß (GSK-3ß). These inhibitors suppress the release of pro-inflammatory cytokines and prostaglandins (PG) from human monocytes. However, indirubin derivatives target several protein kinases such as cyclin-dependent kinases (CDKs) which has been a major concern for their application in inflammation therapy. Here, we report on a library of 13 5-bromo-substituted indirubin derivatives that have been designed to improve potency and target selectivity. Side-by-side comparison of reference compound 1 (6BIGOE) with 5-bromo derivatives revealed its isomer 2 (5BIGOE), as the most potent derivative able to supress pro-inflammatory cytokine and PG release in lipopolysaccharide-stimulated human monocytes. Analysis of protein kinase inhibition in intact monocytes, supported by our in silico findings, proposed higher selectivity of 1 for GSK-3ß inhibition with lesser potency against CDKs 8 and 9. In contrast, 2 supressed the activity of these CDKs with higher effectiveness than GSK-3ß, representing additional targets of indirubins within the inflammatory response. Encapsulation of 1 and 2 into polymer-based nanoparticles (NP) improved their pharmacological potential. In conclusion, the 5- and 6-brominated indirubins 1 and 2 as dual GSK-3ß and CDK8/9 inhibitors represent a novel concept for intervention with inflammatory disorders.

Impact of GSK-3ß and CK-1δ on Wnt signaling pathway in alzheimer disease: A dual target approach.

Alzheimer's disease (AD) is an enigmatic neurological illness that offers few treatment options. Recent exploration has highlighted the crucial connection of the Wnt signaling pathway in AD pathogenesis, shedding light on potential therapeutic targets. The present study focuses on the dual targeting of glycogen synthase kinase-3ß (GSK-3ß) and casein kinase-1δ (CK-1δ) within the framework of the Wnt signaling pathway as a possible technique for AD intervention. GSK-3ß and CK-1δ are multifunctional kinases known for their roles in tau hyperphosphorylation, amyloid processing, and synaptic dysfunction, all of which are major hallmarks of Alzheimer's disease. They are intricately linked to Wnt signaling, which plays a pivotal part in sustaining neuronal function and synaptic plasticity. Dysregulation of the Wnt pathway in AD contributes to cognitive decline and neurodegeneration. This review delves into the molecular mechanisms by which GSK-3ß and CK-1δ impact the Wnt signaling pathway, elucidating their roles in AD pathogenesis. We discuss the potential of small-molecule inhibitors along with their SAR studies along with the multi-targetd approach targeting GSK-3ß and CK-1δ to modulate Wnt signaling and mitigate AD-related pathology. In summary, the dual targeting of GSK-3ß and CK-1δ within the framework of the Wnt signaling pathway presents an innovative and promising avenue for future AD therapies, offering new hope for patients and caregivers in the quest to combat this challenging condition.

Osthole inhibits GSK-3ß/AMPK/mTOR pathway-controlled glycolysis and increases radiosensitivity of subcutaneous transplanted hepatocellular carcinoma in nude mice.

PURPOSE: Osthole possesses anti-tumor activities. However, whether osthole can have a radiosensitization effect on hepatic cancer remains unclear. Here, an HCC-LM3 cells-inoculated subcutaneous transplanted tumor was adopted to explore the effect of osthole. METHODS: The tumor-bearing mice were treated with 100 mg/kg osthole for 12 days, 4 Gy irradiation twice, or their combination. The tumor volume and weight, lactic acid content, glycolytic enzyme activities, and protein expression of glycogen synthase kinase 3ß (GSK-3ß), p­GSK-3ß, mammalian target of rapamycin (mTOR), p­mTOR, AMP-activated protein kinase (AMPK), p­AMPK, glucose transporter 1/3, and pyruvate kinase M2 were determined. The GSK-3ß-overexpressed HCC-LM3 or SK-Hep­1 cell models were also adopted to verify the effects of osthole on expression of these proteins. RESULTS: The tumor volume and weight, lactic acid content, and glycolytic enzyme activities in tumor tissues were lower in the osthole + radiation group than in the radiation group. Moreover, osthole could reverse the radiation-induced increments of p­GSK-3ß/GSK-3ß and p­mTOR/mTOR protein ratios and the expression of glucose transporter 1/3 and pyruvate kinase M2 proteins in tumor tissues, and increase the protein ratio of p­AMPK/AMPK. The effects of osthole on these glycolysis-related proteins were also observed in GSK-3ß-overexpressed HCC-LM3 or SK-Hep­1 cell models. CONCLUSION: Osthole has a radiosensitizing effect on subcutaneous transplanted hepatocellular carcinoma, and its mechanism may be related to inhibition of GSK-3ß/AMPK/mTOR pathway-controlled glycolysis.

Targeting ER stress/PKA/GSK-3ß/ß-catenin pathway as a potential novel strategy for hepatitis C virus-infected patients.

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). The HCC risk, while decreased compared with active HCV infection, persists in HCV-cured patients by direct-acting antiviral agents (DAA). We previously demonstrated that Wnt/ß-catenin signaling remained activated after DAA-mediated HCV eradication. Developing therapeutic strategies to both eradicate HCV and reverse Wnt/ß-catenin signaling is needed. METHODS: Cell-based HCV long term infection was established. Chronically HCV infected cells were treated with DAA, protein kinase A (PKA) inhibitor H89 and endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA). Western blotting analysis and fluorescence microscopy were performed to determine HCV levels and component levels involved in ER stress/PKA/glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin pathway. Meanwhile, the effects of H89 and TUDCA were determined on HCV infection. RESULTS: Both chronic HCV infection and replicon-induced Wnt/ß-catenin signaling remained activated after HCV and replicon eradication by DAA. HCV infection activated PKA activity and PKA/GSK-3ß-mediated Wnt/ß-catenin signaling. Inhibition of PKA with H89 both repressed HCV and replicon replication and reversed PKA/GSK-3ß-mediated Wnt/ß-catenin signaling in both chronic HCV infection and replicon. Both chronic HCV infection and replicon induced ER stress. Inhibition of ER stress with TUDCA both repressed HCV and replicon replication and reversed ER stress/PKA/GSK-3ß-dependent Wnt/ß-catenin signaling. Inhibition of either PKA or ER stress both inhibited extracellular HCV infection. CONCLUSION: Targeting ER stress/PKA/GSK-3ß-dependent Wnt/ß-catenin signaling with PKA inhibitor could be a novel therapeutic strategy for HCV-infected patients to overcomes the issue of remaining activated Wnt/ß-catenin signaling by DAA treatment. Video Abstract.

Development of [18F]Thiazolylacylaminopyridine-Based Glycogen synthase Kinase-3ß ligands for positron emission tomography imaging.

Glycogen synthase kinase-3ß (GSK-3ß) regulates numerous of CNS-specific signaling pathways, and is particularly implicated in various pathogenetic mechanisms of Alzheimer's disease (AD). A noninvasive method for detecting GSK-3ß in AD brains via positron emission tomography (PET) imaging could enhance the understanding of AD pathogenesis and aid in the development of AD therapeutic drugs. In this study, an array of fluorinated thiazolyl acylaminopyridines (FTAAP) targeting GSK-3ß were designed and synthesized. These compounds showed moderate to high affinities (IC50 = 6.0 - 426 nM) for GSK-3ß in vitro. A potential GSK-3ß tracer, [18F]8, was successfully radiolabeled. [18F]8 had unsatisfactory initial brain uptake despite its suitable lipophilicity, molecular size and good stability. Further structural refinement of the lead compound is needed to develop promising [18F]-labeled radiotracers for the detection of GSK-3ß in AD brains.

GSK3ß Inhibition Ameliorates Atherosclerotic Calcification.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to atherosclerotic calcification. In a previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induced ß-catenin and reduced mothers against DPP homolog 1 (SMAD1) in order to redirect osteoblast-like cells towards endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency and diabetic Ins2Akita/wt mice. Here, we report that GSK3ß inhibition or endothelial-specific deletion of GSK3ß reduces atherosclerotic calcification. We also find that alterations in ß-catenin and SMAD1 induced by GSK3ß inhibition in the aortas of Apoe-/- mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in atherosclerotic lesions through a similar mechanism to that in Mgp-/- mice.

GSK3ß Inhibition Reduced Vascular Calcification in Ins2Akita/+ Mice.

Endothelial-mesenchymal transition (EndMT) drives the endothelium to contribute to vascular calcification in diabetes mellitus. In our previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induces ß-catenin and reduces mothers against DPP homolog 1 (SMAD1) to direct osteoblast-like cells toward endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency. Here, we report that GSK3ß inhibition reduces vascular calcification in diabetic Ins2Akita/wt mice. Cell lineage tracing reveals that GSK3ß inhibition redirects endothelial cell (EC)-derived osteoblast-like cells back to endothelial lineage in the diabetic endothelium of Ins2Akita/wt mice. We also find that the alterations in ß-catenin and SMAD1 by GSK3ß inhibition in the aortic endothelium of diabetic Ins2Akita/wt mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in diabetic arteries through a similar mechanism to that in Mgp-/- mice.

Glycogen synthase kinase-3ß inhibition decreases inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

Bone is the preferential site of metastasis for breast cancer. Invasion of cancer cells induces the destruction of bone tissue and damnification of peripheral nerves and consequently induced central sensitization which contributes to severe pain. Herein, cancer induced bone pain (CIBP) rats exhibited destruction of tibia, mechanical allodynia and spinal inflammation. Inflammatory response mainly mediated by astrocyte and microglia in central nervous system. Our immunofluorescence analysis revealed activation of spinal astrocytes and microglia in CIBP rats. Transmission electron microscopy (TEM) observations of mitochondrial outer membrane disruption and cristae damage in spinal mitochondria of CIBP rats. Proteomics analysis identified abnormal expression of proteins related to mitochondrial organization and function. Intrathecally, injection of GSK-3ß activity inhibitor TDZD-8 significantly attenuated Drp1-mediated mitochondrial fission and recovered mitochondrial function. Inhibition of GSK-3ß activity also suppressed NLRP3 inflammasome cascade and consequently decreased mechanical pain sensitivity of CIBP rats. For cell research, TDZD-8 treatment significantly reversed TNF-α induced mitochondrial membrane potential (MMP) deficiency and high mitochondrial reactive oxygen species level. Taken together, GSK-3ß inhibition by TDZD-8 decreases spinal inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.