Promoted Akt Phosphorylation Research Articles

Interaction of Sp1 and Setd8 promotes vascular smooth muscle cells apoptosis by activating Mark4 in vascular calcification.

Vascular calcification (VC) is directly related to high mortality in chronic kidney disease (CKD), and cellular apoptosis of vascular smooth muscle cells (VSMCs) is a crucial process in the initiation of VC. Microtubule affinity-regulating kinase 4 (Mark4), known as a serine/threonine protein kinase, can induce cell apoptosis and autophagy by modulating Akt phosphorylation. However, the potential functions and molecular mechanisms of Mark4 in VSMCs apoptosis and calcification need to be further explored. Initially, our data indicated that the mRNA expression of Mark4 was prominently elevated in high phosphorus-stimulated human VSMCs compared with the other members in Marks. Consistently, Mark4 expression was found to be significantly increased in the calcified arteries of both CKD patients and rats. In vitro, silencing Mark4 suppressed apoptosis-specific marker expression by promoting Akt phosphorylation, finally attenuating VSMCs calcification induced by high phosphate. Mechanically, the transcription factor Sp1 was enriched in the Mark4 promoter region and modulated Mark4 transcription. Moreover, SET domain-containing protein 8 (Setd8) was proved to interact with Sp1 and jointly participated in the transcriptional regulation of Mark4. Finally, rescue experiments revealed that Setd8 contributed to VSMCs apoptosis and calcification by modulating Mark4 expression. In conclusion, these findings reveal that Mark4 is transcriptionally activated by Sp1, which is interacted with Setd8, to promote VSMCs calcification through Akt-mediated antiapoptotic effects, suggesting that Mark4 represents a potent and promising therapeutic target for VC in CKD.

Ortho-silicic Acid Prevents Glucocorticoid-Induced Femoral Head Necrosis by Promoting Akt Phosphorylation to Inhibit Endoplasmic Reticulum Stress-Mediated Apoptosis and Enhance Angiogenesis and Osteogenesis.

Glucocorticoid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent form of secondary osteonecrosis affecting the femoral head. Glucocorticoids can cause damage to both vascular endothelial cells and osteoblasts. Previous studies have demonstrated that silicon can improve the resistance of vascular endothelial cells to oxidative stress and positively impact bone health. However, the impact of silicon on SONFH has yet to be investigated. We examined the influence of ortho-silicic acid (OSA, Si(OH)4) on the apoptosis and proliferation of vascular endothelial cells after glucocorticoid induction. Additionally, we evaluated the expression of apoptosis-related genes such as cleaved-caspase-3, Bcl-2 and Bax. The impact of glucocorticoids and OSA on the function of vascular endothelial cells was evaluated through wound healing, transwell and angiogenesis assays. Osteogenic function was subsequently evaluated through alizarin red staining, alkaline phosphatase staining and expression levels of osteogenic genes like RUNX2 and ALP. Moreover, we investigated the potential role of OSA in vivo using the SONFH animal model. At concentrations below 100μM, OSA exhibits no toxicity on vascular endothelial cells and effectively reverses glucocorticoid-induced apoptosis in these cells. OSA increases the resilience of vascular endothelial cells against oxidative stress and enhances osteoblast differentiation. Our study revealed that glucocorticoids activate endoplasmic reticulum stress, a process that mediates the apoptosis of vascular endothelial cells. OSA ameliorated the endoplasmic reticulum stress associated with glucocorticoids through the increased expression of p-Akt levels. In vivo, OSA treatment effectively improved SONFH by enhancing vascular endothelial cell function and promoting osteogenic differentiation. OSA counteracted the adverse effects of glucocorticoids both in vitro and in vivo, demonstrating a beneficial therapeutic effect on SONFH.

Bifidobacterium breve-derived indole-3-lactic acid ameliorates colitis-associated tumorigenesis by directing the differentiation of immature colonic macrophages.

Aim: To elucidate dynamics and functions in colonic macrophage subsets, and their regulation by Bifidobacterium breve (B. breve) and its associated metabolites in the initiation of colitis-associated colorectal cancer (CAC). Methods: Azoxymethane (AOM) and dextran sodium sulfate (DSS) were used to create a CAC model. The tumor-suppressive effect of B. breve and variations of macrophage subsets were evaluated. Intestinal macrophages were ablated to determine their role in the protective effects of B. breve. Efficacious molecules produced by B. breve were identified by non-targeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The molecular mechanism was further verified in murine bone marrow-derived macrophages (BMDMs), macrophages derived from human peripheral blood mononuclear cells (hPBMCs), and demonstrated in CAC mice. Results: B. breve alleviated colitis symptoms, delayed colonic tumorigenesis, and promoted phenotypic differentiation of immature inflammatory macrophages into mature homeostatic macrophages. On the contrary, the ablation of intestinal macrophages largely annulled the protective effects of B. breve. Microbial analysis of colonic contents revealed the enrichment of probiotics and the depletion of potential pathogens following B. breve supplementation. Moreover, indole-3-lactic acid (ILA) was positively correlated with B. breve in CAC mice and highly enriched in the culture supernatant of B. breve. Also, the addition of ILA directly promoted AKT phosphorylation and restricted the pro-inflammatory response of murine BMDMs and macrophages derived from hPBMCs in vitro. The effects of ILA in murine BMDMs and macrophages derived from hPBMCs were abolished by the aryl hydrocarbon receptor (AhR) antagonist CH-223191 or the AKT inhibitor MK-2206. Furthermore, ILA could protect against tumorigenesis by regulating macrophage differentiation in CAC mice; the AhR antagonist largely abrogated the effects of B. breve and ILA in relieving colitis and tumorigenesis. Conclusion: B. breve-mediated tryptophan metabolism ameliorates the precancerous inflammatory intestinal milieu to inhibit tumorigenesis by directing the differentiation of immature colonic macrophages.

High-frequency repetitive transcranial magnetic stimulation promotes neural stem cell proliferation after ischemic stroke.

JOURNAL/nrgr/04.03/01300535-202408000-00031/figure1/v/2023-12-16T180322Z/r/image-tiff Proliferation of neural stem cells is crucial for promoting neuronal regeneration and repairing cerebral infarction damage. Transcranial magnetic stimulation (TMS) has recently emerged as a tool for inducing endogenous neural stem cell regeneration, but its underlying mechanisms remain unclear. In this study, we found that repetitive TMS effectively promotes the proliferation of oxygen-glucose deprived neural stem cells. Additionally, repetitive TMS reduced the volume of cerebral infarction in a rat model of ischemic stroke caused by middle cerebral artery occlusion, improved rat cognitive function, and promoted the proliferation of neural stem cells in the ischemic penumbra. RNA-sequencing found that repetitive TMS activated the Wnt signaling pathway in the ischemic penumbra of rats with cerebral ischemia. Furthermore, PCR analysis revealed that repetitive TMS promoted AKT phosphorylation, leading to an increase in mRNA levels of cell cycle-related proteins such as Cdk2 and Cdk4. This effect was also associated with activation of the glycogen synthase kinase 3β/β-catenin signaling pathway, which ultimately promotes the proliferation of neural stem cells. Subsequently, we validated the effect of repetitive TMS on AKT phosphorylation. We found that repetitive TMS promoted Ca2+ influx into neural stem cells by activating the P2 calcium channel/calmodulin pathway, thereby promoting AKT phosphorylation and activating the glycogen synthase kinase 3β/β-catenin pathway. These findings indicate that repetitive TMS can promote the proliferation of endogenous neural stem cells through a Ca2+ influx-dependent phosphorylated AKT/glycogen synthase kinase 3β/β-catenin signaling pathway. This study has produced pioneering results on the intrinsic mechanism of repetitive TMS to promote neural function recovery after ischemic stroke. These results provide a strong scientific foundation for the clinical application of repetitive TMS. Moreover, repetitive TMS treatment may not only be an efficient and potential approach to support neurogenesis for further therapeutic applications, but also provide an effective platform for the expansion of neural stem cells.

Cdh1 plays a protective role in nonalcoholic fatty liver disease by regulating PPAR/PGC-1α signaling pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) is a significant etiological factor in liver-related diseases, which can lead to severe consequences such as steatohepatitis, cirrhosis and death. Cdh1 is considered as a crucial protein involved in cell cycle regulation. The purpose of this study is to explore the biological role of Cdh1 in NAFLD. Materials and methodsNAFLD cell model was established, and L02 cells and AML12 cells were infected by shRNA lentivirus with Cdh1 knockdown in vitro, and the effect of Cdh1 deletion on cell lipid deposition was evaluated. The effects of Cdh1 deletion on Akt phosphorylation and PPAR/PGC-1α signaling pathway in L02 cells were examined. In addition, the NAFLD mouse model was constructed, and the conditional knockout mice of Cdh1 were selected to verify the results. ResultsIn vitro experiments showed that the Cdh1 deletion enhanced cell lipid deposition. In vivo experiments showed that conditional knockdown of Cdh1 aggravated fatty degeneration and damage of liver in mice. Cdh1 deletion promotes Akt phosphorylation and inhibits PPAR/PGC-1α signaling pathway in L02 cells. Conditional knockout of Cdh1 down-regulates PPAR/PGC-1α signaling pathway in NAFLD mouse model. ConclusionThe deletion of Cdh1 may promote Akt phosphorylation by up-regulating Skp2 and inhibit the PPAR/PGC-1α signaling pathway. Cdh1 serves a protective function in the occurrence and progression of NAFLD.

The effect of Par3 on the cellular junctions and biological functions of odontoblast-lineage cells.

Perfect intercellular junctions are key for odontoblast barrier function. However, whether Partitioning defective-3 (Par3) is expressed in odontoblasts and its potential effects on odontoblast junctions are unknown. Herein, we investigated the effect of Par3 on cellular junctions and the biological behavior of odontoblast-lineage cells (OLCs). Whole-transcriptome sequencing was used to analyze the effects of Par3 on OLCs and the underlying molecular mechanism. Par3 was detected under physiological and inflammatory conditions in OLCs. To investigate the regulatory effect of Par3 on junctions between mouse OLCs, the effects of Par3 downregulation on the proliferation, migration, cycle and apoptosis of OLCs were detected by 5-ethyl-2'-deoxyuridine (EdU) and Transwell assays and flow cytometry. Western blotting and alizarin red S and alkaline phosphatase (ALP) staining were used to observe the effect of Par3 downregulation on OLC mineralization. Whole-transcriptome sequencing was used to investigate the biological role of Par3 in OLCs and potential molecular mechanisms. Par3 was located along the odontoblast layer in the rat pulp tissue and in the cytoplasm of OLCs. Par3 expression was downregulated under inflammatory conditions. The OLC junctions were discontinuous, and total Zona occluden-1 (ZO-1) expression and expression of ZO-1 at the membrane in OLCs were reduced after Par3 silencing (P < 0.05). Expression of a junction-related protein (ZO-1) was downregulated after the downregulation of Par3 (P < 0.05), and ZO-1 moved from the cell membrane to the cytoplasm. OLC proliferation and migration were enhanced, but apoptosis and mineralization were inhibited in shPar3-transfected cells (P < 0.05). Sequencing identified 2996 differentially expressed genes (DEGs), which were mainly enriched in the response to stimuli and binding. Downregulation of Par3 could overactivate the PI3k-AKT pathway by promoting AKT phosphorylation (P < 0.05). Downregulation of Par3 may disrupt junctions between OLCs by affecting ZO-1 expression and distribution and promote OLC proliferation and migration but inhibit OLC mineralization. Par3 may interact with 14-3-3 proteins for PI3K-AKT pathway activation to affect OLC junctions and function.

The effects of the tumor suppressor gene PTEN on the proliferation and apoptosis of breast cancer cells via AKT phosphorylation.

The proliferation and apoptosis of cancer cells play important roles in breast carcinomas. However, to date, there have been few reports on the correlation between the expression of PTEN and AKT phosphorylation in breast cancer. This present study investigated the effects of the phosphatase and tensin homology deleted from chromosome 10 (PTEN) gene on the proliferation and apoptosis of breast cancer cells through protein kinase B (AKT) phosphorylation. Human breast cancer MDA-MB-231 cells were transfected with the pcDNA3.0 control vector or the pcDNA3.0-PTEN vector for 48 hours. The Cell Counting Kit 8 (CCK-8) was used to detect cell survival rates, double staining was performed to detect apoptosis, and Western blot (WB) analysis was conducted to detect protein expression. The effects of PTEN expression on the cell cycle and apoptosis of human breast cancer cell line MDA-MB-231, and on the levels of phosphorylated AKT protein were further analyzed. Moreover, the relationship between the PTEN gene and clinical features were also analyzed. The cell survival rate of cells transfected with pcDNA3.0-PTEN was significantly lower than that of cells transfected with the control pcDNA3.0 vector (55.65%±12.18% vs. 97.32%±12.45%, P=0.004). Compared with the pcDNA3.0 group, the apoptosis rate of the pcDNA3.0-PTEN group was significantly increased (20.65±2.18 vs. 2.32±0.45, P=0.001). The expression of PTEN protein in pcDNA3.0-PTEN group was higher than that in the pcDNA3.0 group, and the expression of the AKT and mTOR proteins was significantly lower than that in pcDNA3.0 group (P<0.05). The expression of PTEN in the lymph node metastasis positive group was significantly higher than that in the lymph node metastasis negative group (P<0.05). The expression of the AKT protein in breast cancer was higher than that in normal breast tissue, and the difference was statistically significant (P<0.01). Overexpression of the PTEN gene can promote AKT phosphorylation, increase the apoptotic index of breast cancer cells, and reduce the proliferative activity of breast cancer cells. This provided a new direction for the next treatment of breast cancer, but further clinical research is needed.

Effects and Mechanisms of Ban-Xia Xie-Xin Decoction on Type 2 Diabetes Mellitus: Network Pharmacology Analysis and Experimental Evidence.

Studies have indicated that Ban-Xia Xie-Xin Decoction (BXXXD) has therapeutic effects on type 2 diabetes mellitus (T2DM). However, due to the complexity of components and diversity of targets, the mechanisms are still not fully elucidated. In this research, we systematically analysed the targets of BXXXD through the method of network pharmacology and further validated them through experiments. The active components and therapeutic targets were identified, and these targets were analysed by the methods of gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) analysis. Then, based on these network pharmacology analyses, we validated the main targets through animal experiments. A total of 169 active components and 159 targets were identified. KEGG analysis showed that the mitogen-activated protein kinase (MAPK) signalling pathway, tumour necrosis factor (TNF) signalling pathway, the phosphatidylinositol 3' -kinase (PI3K), Akt signalling pathway, and other pathways were related to the treatment of T2DM by BXXXD. PPI network analysis showed that the key genes included signal transducers and activators of transcription 3 (STAT3), JUN, TNF, Recombinant V-Rel Reticuloendotheliosis Viral Oncogene Homolog A (RELA), Akt/PKB- 1 (Protein kinase B), TP53, mitogen-activated protein kinase-1 (MAPK-1), mitogen-activated protein kinase-3 (MAPK-3), interleukin- 6 (IL6), and mitogen-activated protein kinase-14 (MAPK- 14), respectively. Animal experiments showed that BXXXD could reduce blood glucose and improve insulin resistance, which may be related to the mechanisms of inhibiting TNF, interleukin-1 (IL-1), IL-6, and interleukin-17 (IL-17) and promoting Akt phosphorylation. Our research revealed the mechanisms of BXXXD in the treatment of diabetes, which laid a solid foundation for further studies on the molecular mechanisms of BXXXD in the treatment of T2DM.

Smad3-mediated lncRNA HSALR1 enhances the non-classic signalling pathway of TGF-β1 in human bronchial fibroblasts by binding to HSP90AB1.

Chronic obstructive pulmonary disease (COPD) is one of the diseases with high mortality and morbidity with complex pathogenesis. Airway remodeling is an unavoidable pathological characteristic. However, the molecular mechanisms of airway remodeling are incompletely defined. lncRNAs highly correlated with transforming growth factor beta 1(TGF-β1) expression were chosen, the lncRNA ENST00000440406 (named HSP90AB1 Assoicated LncRNA 1, HSALR1) was chosen for further functional experiments. Dual luciferase and ChIP assay were used to detect the upstream of HSALR1, transcriptome sequencing, Cck-8, Edu, cell proliferation, cell cycle assay, and WB detection of pathway levels confirmed the effect of HSALR1 on fibroblast proliferation and phosphorylation levels of related pathways. Mice was infected with adeno-associated virus (AAV) to express HSALR1 by intratracheal instillation under anesthesia and was exposure to cigarette smoke, then mouse lung function was performed and the pathological sections of lung tissues were analyzed. Herein, lncRNA HSALR1 was identified as highly correlated with the TGF-β1 and mainly expressed in human lung fibroblasts. HSALR1 was induced by Smad3 and promoted fibroblasts proliferation. Mechanistically, it could directly bind to HSP90AB1 protein, and acted as a scaffold to stabilize the binding between Akt and HSP90AB1 to promote Akt phosphorylation. In vivo, mice expressed HSALR1 by AAV was exposure to cigarette smoke (CS) for COPD modeling. We found that lung function was worse and airway remodeling was more pronounced in HSLAR1 mice compare to wild type (WT) mice. Our results suggest that lncRNA HSALR1 binds to HSP90AB1 and Akt complex component, and enhances activity of the TGF-β1 smad3-independent pathway. This finding described here suggest that lncRNA can participate in COPD development, and HSLAR1 is a promising molecular target of COPD therapy.

Exploration of Artemisinin Against IgA Nephropathy via AKT/Nrf2 Pathway by Bioinformatics and Experimental Validation.

Artemisinin (ART) is a safe and effective antimalarial drug. In recent years, antimalarial drugs have demonstrated a good therapeutic efficacy in IgA nephropathy, suggesting that this may become a new treatment option. We aimed to evaluate the effect and mechanism of artemisinin in IgA nephropathy. In this study, CMap database was used to predict the artemisinin therapeutic effect for IgA nephropathy. A network pharmacology approach was applied to explore the unknown mechanism of artemisinin in IgA nephropathy. We used molecular docking to predict the binding affinity of artemisinin with the targets. A mouse model of IgA nephropathy was established to investigate the therapeutic effect of artemisinin on IgA nephropathy. In vitro, the cell counting Kit-8 assay was used to evaluate the cytotoxicity of artemisinin. Flow cytometry and PCR assays were used to detect the effects of artemisinin on oxidative stress and fibrosis in lipopolysaccharide (LPS)-stimulated mesangial cells. Western blot and immunofluorescence were used to detect the expression of pathway proteins. CMap analysis showed artemisinin may reverse the expression levels of differentially expressed genes in IgA nephropathy. Eighty-seven potential targets of artemisinin in the treatment of IgA nephropathy were screened. Among them, 15 hub targets were identified. Enrichment analysis and GSEA analysis indicated that response to reactive oxygen species is the core biological process. AKT1 and EGFR had the highest docking affinity with artemisinin. In vivo, artemisinin could improve renal injury and fibrosis in mice. In vitro, artemisinin attenuated LPS-induced oxidative stress and fibrosis promoted AKT phosphorylation and Nrf2 nuclear translocation. Artemisinin reduced the level of fibrosis and oxidative stress with IgA nephropathy through the AKT/Nrf2 pathway, which provided an alternative treatment for IgAN.

MiR-21 alleviates renal tubular epithelial cells injury induced by ischemia by targeting TLR4

Renal ischemia is the initial stage of kidney damage, leading to mitochondrial metabolism disorders and cell necrosis. In this study, we aimed to investigate the biological functions and potential mechanisms of miR-21 in protecting renal tubular epithelial cells from oxidative stress and apoptosis following oxygen glucose deprivation (OGD). Following an OGD injury, miR-21 levels increased in HK-2 renal tubular epithelial cells. Overexpression of miR-21 decreased the protein expressions of cleaved caspase-3, BAX, P53, cell apoptosis and increased Bcl-2 expression in HK-2 cells with OGD injury. In vivo studies found that miR-21 agomir reduced renal tissue apoptosis, while miR-21 antagomir increased it. In addition, overexpression of miR-21 reduced levels of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in HK-2 cells with OGD injury. However, miR-21 inhibition exhibited the opposite effect. A dual-luciferase reporter assay demonstrated that miR-21 directly regulates Toll-like receptor 4 (TLR4) by targeting the 3′-UTR of TLR4 mRNA. Overexpression of miR-21 led to decreased TLR4 protein expression, and TLR4 knockdown was shown to greatly increase AKT activity in HK-2 cells by in vitro kinase assay. Additionally, TLR4 knockdown promoted AKT phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression, while TLR4 overexpression inhibited these processes. Furthermore, AKT activation abolished the effect of TLR4 on HIF-1α, while AKT inhibition decreased the expression of TLR4 on HIF-1α in TLR4 knockdown HK-2 cells. Further study revealed that HIF-1α inhibition abolished the protective effect of miR-21 overexpression on ROS, LDH levels and cell apoptosis in HK-2 cells after OGD injury, which is indicated by increased levels of ROS and LDH, as well as increased cell apoptosis after HIF-1α inhibition in miR-21-treated HK-2 cells. In conclusion, miR-21 defends OGD-induced HK-2 cell injury via the TLR4/AKT/HIF-1α axis.

Insights into the role of PHLPP2/Akt/GSK3β/Fyn kinase/Nrf2 trajectory in the reno-protective effect of rosuvastatin against colistin-induced acute kidney injury in rats.

Oxidative stress-mediated colistin's nephrotoxicity is associated with the diminished activity of nuclear factor erythroid 2-related factor 2 (Nrf2) that is primarily correlated with cellular PH domain and leucine-rich repeat protein phosphatase (PHLPP2) levels. This study investigated the possible modulation of PHLPP2/protein kinase B (Akt) trajectory as a critical regulator of Nrf2 stability by rosuvastatin (RST) to guard against colistin-induced oxidative renal damage in rats. Colistin (300,000 IU/kg/day; i.p.) was injected for 6 consecutive days, and rats were treated simultaneously with RST orally at 10 or 20 mg/kg. RST enhanced renal nuclear Nrf2 translocation as revealed by immunohistochemical staining to boost the renal antioxidants, superoxide dismutase (SOD) and reduced glutathione (GSH) along with a marked reduction in caspase-3. Accordingly, rats treated with RST showed significant restoration of normal renal function and histological features. On the molecular level, RST effectively decreased the mRNA expression of PHLPP2 to promote Akt phosphorylation. Consequently, it deactivated GSK-3β and reduced the gene expression of Fyn kinase in renal tissues. RST could attenuate colistin-induced oxidative acute kidney injury via its suppressive effect on PHLPP2 to endorse Nrf2 activity through modulating Akt/GSK3 β/Fyn kinase trajectory.

KIF14 mediates cabazitaxel-docetaxel cross-resistance in advanced prostate cancer by promoting AKT phosphorylation

Docetaxel is a first-line chemotherapy drug for castration-resistant prostate cancer (CRPC); yet, some CRPC patients develop docetaxel drug resistance. Cabazitaxel is approved in the post-docetaxel treatment setting. However, recent studies suggested cross-resistance between the development of drug resistance and current treatments. In this study, we used docetaxel-resistant cell lines DU145/DTX50 and PC-3/DTX30 to measure the responses to cabazitaxel. Our findings demonstrated that docetaxel resistance could lead to cross-resistance to cabazitaxel. After docetaxel-resistant cells were treated with cabazitaxel, transcriptome analysis was performed, and the results were analyzed in combination with survival analysis and correlation analysis with Gleason score to screen the cross-resistance genes. The continuously increased expression of kinesin family member 14 (KIF14) was identified as the main cause of cross-resistance to cabazitaxel in docetaxel-resistant cells. Silencing the expression of KIF14 could restore the sensitivity of resistant PCa cells to docetaxel and cabazitaxel, attenuate proliferation and promote apoptosis of the resistant PCa cells. Notably, the depressed expression of KIF14 inhibited the phosphorylation of Akt located downstream. In summary, KIF14 mediates the cross-resistance between docetaxel and cabazitaxel, and targeting KIF14 could be an effective measurement for reversing docetaxel or cabazitaxel chemotherapy failure or enhancing the anti-tumor effects of docetaxel or cabazitaxel.

Empagliflozin-Pretreated Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Attenuated Heart Injury.

Small extracellular vesicles derived from mesenchymal stem cells (MSCs) play important roles in cardiac protection. Studies have shown that the cardiovascular protection of sodium-glucose cotransporter 2 inhibitor (SGLT2i) is independent of its hypoglycemic effect. This study is aimed at investigating whether small extracellular vesicles derived from MSCs pretreated with empagliflozin (EMPA) has a stronger cardioprotective function after myocardial infarction (MI) and to explore the underlying mechanisms. We evaluated the effects of EMPA on MSCs and the effects of EMPA-pretreated MSCs-derived small extracellular vesicles (EMPA-sEV) on myocardial apoptosis, angiogenesis, and cardiac function after MI in vitro and in vivo. The small extracellular vesicles of control MSCs (MSC-sEV) and EMPA-pretreated MSCs were extracted, respectively. Small extracellular vesicles were cocultured with apoptotic H9c2 cells induced by H2O2 or injected into the infarcted area of the Sprague-Dawley (SD) rat myocardial infarction model. EMPA increased the cell viability, migration ability, and inhibited apoptosis and senescence of MSCs. In vitro, EMPA-sEV inhibited apoptosis of H9c2 cells compared with the control group (MSC-sEV). In the SD rat model of MI, EMPA-sEV inhibited myocardial apoptosis and promoted angiogenesis in the infarct marginal areas compared with the MSC-sEV. Meanwhile, EMPA-sEV reduced infarct size and improved cardiac function. Through small extracellular vesicles (miRNA) sequencing, we found several differentially expressed miRNAs, among which miR-214-3p was significantly elevated in EMPA-sEV. Coculture of miR-214-3p high expression MSC-derived small extracellular vesicles with H9c2 cells produced similar protective effects. In addition, miR-214-3p was found to promote AKT phosphorylation in H9c2 cells. Our data suggest that EMPA-sEV significantly improve cardiac repair after MI by inhibiting myocardial apoptosis. miR-214-3p at least partially mediated the myocardial protection of EMPA-sEV through the AKT signaling pathway.

KAT7 promotes radioresistance through upregulating PI3K/AKT signaling in breast cancer.

Chromatin-modifying enzymes are commonly altered in cancers, but the molecular mechanism by which they regulate cancers remains poorly understood. Herein, we demonstrated that Lysine acetyltransferase 7 (KAT7) was upregulated in breast cancer. KAT7 expression negatively correlated with the survival of breast cancer patients, and KAT7 silencing suppressed breast cancer radioresistance in vitro. Mechanistically, KAT7 activated Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) transcription, leading to enhanced PI3K/AKT signaling and radioresistance. Overexpression of AKT or PIK3CA restored radioresistance suppression induced by KAT7 inhibition. Moreover, overexpression of KAT7, but not KAT7 acetyltransferase activity-deficient mutants promoted AKT phosphorylation at the Ser473 site, PIK3CA expression and radioresistance suppression due to KAT7 inhibition. In conclusion, KAT7 has huge prospects for clinical application as a new target for predicting radioresistance in breast cancer patients.

Apolipoprotein A-IV restrains fat accumulation in skeletal and myocardial muscles by inhibiting lipogenesis and activating PI3K–AKT signalling

Background One of the pathological characteristics of obesity is fat accumulation of skeletal muscles (SKM) and the myocardium, involving mechanisms of insulin resistance and abnormal lipid metabolism. Apolipoprotein A-IV (ApoA-IV) is an essential gene in both glucose and lipid metabolisms. Materials and methods Using high-fat diet (HFD) induced obese apoA-IV-knockout mice and subsequent introduction of exogenous recombinant-ApoA-IV protein and adeno-associated virus (AAV)-transformed apoA-IV, we examined lipid metabolism indicators of SKM and the myocardium, which include triglyceride (TG) content, RT-PCR for lipogenic indicators and western blotting for AKT phosphorylation. Similarly, we used high-glucose-fed or palmitate (Pal)-induced C2C12 cells co-cultured with ApoA-IV protein to evaluate glucose uptake, the phosphoinositide 3-kinase (PI3K)–AKT pathway, and lipid metabolisms. Results In stable obese animal models, we find ApoA-IV-knockout mice show elevated TG content, enhanced expression of lipogenic enzymes and diminished phosphorylated AKT in SKM and the myocardium, but both stable hepatic expression of AAV-apoA-IV and brief ApoA-IV protein administration suppress lipogenesis and promote AKT phosphorylation. In a myoblast cell line C2C12, ApoA-IV protein suppresses Pal-induced lipid accumulation and lipogenesis but enhances AKT activation and glucose uptake, and the effect is abolished by a PI3K inhibitor. Conclusion We find that ApoA-IV reduces fat accumulation by suppressing lipogenesis and improves glucose uptake in SKM and the myocardium by regulating the PI3K–AKT pathway.

The NRF2 antagonist ML385 inhibits PI3K-mTOR signaling and growth of lung squamous cell carcinoma cells.

Lung squamous cell carcinoma (LUSC) currently has limited therapeutic options because of the relatively few validated targets and the lack of clinical drugs for some of these targets. Although NRF2/NFE2L2 pathway activation commonly occurs in LUSC, NRF2 has predominantly been studied in other cancer models. Here, we investigated the function of NRF2 in LUSC, including in organoid models, and we explored the activity of a small molecule NRF2 inhibitor ML385, which has not previously been investigated in LUSC. We first explored the role of NRF2 signaling in LUSC cancer cell line and organoid proliferation through NRF2 knockdown or ML385 treatment, both in vivo and in vitro. Next, we performed Western blot and immunofluorescence assays to determine the effect of NRF2 inhibition on PI3K-mTOR signaling. Finally, we used cell viability and clonogenic assays to explore whether ML385 could sensitize LUSC cancer cells to PI3K inhibitors. We find that downregulation of NRF2 signaling inhibited proliferation of LUSC cancer cell lines and organoids, both in vivo and in vitro. We also demonstrate that inhibition of NRF2 reduces PI3K-mTOR signaling, with two potential mechanisms being involved. Although NRF2 promotes AKT phosphorylation, it also acts downstream of AKT to increase RagD protein expression and recruitment of mTOR to lysosomes after amino acid stimulation. We also find that ML385 potentiates LUSC growth inhibition by a pan-PI3K inhibitor, which correlates with stronger inhibition of PI3K-mTOR signaling. Our data provide additional support for NRF2 promoting LUSC growth through PI3K-mTOR activation and support development of NRF2 inhibitors for the treatment of LUSC.

The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction

Abstract Background Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized. Purpose This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI. Methods Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis. Results We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI. Conclusion ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Simple SummaryA remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

RNA binding motif protein 3 (RBM3) promotes protein kinase B (AKT) activation to enhance glucose metabolism and reduce apoptosis in skeletal muscle of mice under acute cold exposure.

The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. RBM3, an exceptional cold shock protein, is rapidly upregulated in response to hypothermia to resist the adverse effects of cold stress. However, the mechanism of the protective effect and the rapid upregulation of RBM3 remains unclear. O-GlcNAcylation, an atypical O-glycosylation, is precisely regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the signal transduction of multiple cellular stress responses as a "stress and nutrition receptor." Therefore, our study aimed to explore the mechanism of RBM3 regulating glucose metabolism and promoting survival in skeletal muscle under acute cold exposure. Meanwhile, our study verifies whether O-GlcNAcylation mediated by OGT rapidly upregulates RBM3. The blood and skeletal muscle of mice were collected at the end of cold exposure treatment for 0, 2, and 4h. Changes in levels of RBM3, AKT, glycolysis apoptosis, and OGT were measured. The results show that acute cold exposure upregulated RBM3, OGT, and AKT phosphorylation and increased energy consumption, which enhanced glycolysis and prevent apoptosis. In the 32°C mild hypothermia model in vitro, overexpression of RBM3 enhanced AKT phosphorylation. Meanwhile, inactivation of AKT by wortmannin resulted in increased apoptosis and decreased glucose metabolism in skeletal muscle under acute cold exposure. In addition, OGT-mediated O-GlcNAcylation of p65 was confirmed in mouse myoblast cell line (C2C12) cells at mild hypothermia. O-GlcNAcylation level affected p65 activity and nuclear translocation. Compared with wild type (WT) mice, RBM3 and p65 phosphorylation were decreased in specific skeletal muscle Ogt (KO) mice, whereas AKT phosphorylation, glycolysis, and apoptosis were increased. Taken together, O-GlcNAcylation of p65 upregulates RBM3 to promote AKT phosphorylation, enhance glucose metabolism, and reduce apoptosis in skeletal muscle of mice under acute cold exposure.