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This study intended to explore the hypoglycemic and cardioprotective effects of 8-week aerobic interval training combined with liraglutide and elucidate the underlying mechanisms.Male Wistar rats were randomly divided into 5 groups - normal control group (CON), diabetic cardiomyopathy group (DCM), high-dose liraglutide group (DH), low-dose liraglutide group (DL), and aerobic interval training combined with liraglutide group (DLE). High-fat diet and streptozotocin (STZ) were used to induce the DCM model, and both the liraglutide administration group and combination therapy group allocated to 8 weeks of either liraglutide or liraglutide and exercise intervention. Cardiac functions were analyzed by electrocardiography. Blood biochemical parameters were measured to judge glycemic control conditions. Hematoxylin and eosin (HE) staining and Sirus red staining was used to identify cardiac morphology and collagen accumulation, respectively. Advanced glycation end products (AGEs) were determined by enzymatic methods. The mRNA expression of myocardial remodeling genes (BNP, GSK3β, α-MHC, β-MHC and PPARα) and the protein expression of GLP-1, GLP-1R were analyzed.DCM rats developed hyperglycemia, impaired cardiac function with accumulation of AGEs and collagen (P 0.05). The development of hyperglycemia and cardiac dysfunction was significantly attenuated with all interventions, as reduced cardiac fibrosis and improved cardiac function (P 0.05). Cardiac remodeling genes were normalized after all interventions, these positive modifications were due to increased GLP-1 and GLP-1R expression in DCM heart (P 0.05). Liraglutide combined with AIT significantly increased the diameters of cardiomyocytes, increased the α-MHC expressionx, reduced PPARαexpression and reduced the fluctuation of blood glucose level, which showed the safety and effective of medicine combined with exercise.Liraglutide combined with AIT intervention normalized blood glucose alleviates myocardial fibrosis and improves cardiac contractile function in DCM rats, supporting the efficacy and safety of the combination therapy.

Alzheimer's disease (AD) is the most common form of neurodegenerative dementia, characterized by cognitive deficits and memory dysfunction, which is clinically incurable so far. Novel small molecular compound 2JY-OBZ4 is one of structural analogue of Huperzine A (Hup-A), an anti-AD drug in China. In our previous work, 2JY-OBZ4 exhibited potent effects on tau hyperphosphorylation, Aβ production and acetylcholinesterase (AChE) activity. However, 2JY-OBZ4's anti-AD effects and the underlying molecular mechanisms remain unclear. We here reported that 2JY-OBZ4 resisted tau hyperphosphorylation at Thr181 and Ser396 sites in HEK293-hTau cells transfected with GSK-3β, decreased tau phosphorylation via upregulating the activity of PP2A in HEK293-hTau cells and reduced Aβ production through regulating protein levels of APP cleavage enzymes in N2a-hAPP cells. Meanwhile, we found that 2JY-OBZ4 had no adverse effects on cell viability of mice primary neuron even at high concentration, and ameliorated synaptic loss induced by human oligomeric Aβ42. 2JY-OBZ4 had moderate AChE inhibitory activity with the half maximal inhibitory concentration (IC50) to be 39.48 μg/ml

Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3β signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, with the incidence in men being about twice as compared to women. Gender differences may provide clues for finding key targets that mediate the death of dopaminergic (DA) neurons in PD. Luteinizing hormone (LH), analog of human chorionic gonadotropin (hCG), and their receptor, luteinizing hormone/choriogonadotropin receptor (LHCGR), are associated with the pathogenesis of PD. Movement-related symptoms are partially improved by hCG in PD patients. However, the relationship between hCG and PD, as well as its roles in mediating DA neuronal death, has not been elucidated. In this study, we investigated the potential of hCG as a treatment during PD progression. After establishment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models, we found that hCG restored the decrease of LHCGR activity caused by down-regulation of LH in the substantia nigra. Furthermore, the reduction of LHCGR activity led to DA neuronal death through knocking down the LHCGR in DA neurons by AAV-mTH-shRNA. Treatment with hCG alleviated the DA neuronal death induced by MPTP. Finally, hCG exerted neuroprotective effects by inhibiting the activation of glycogen synthase kinase 3 beta (GSK3β) in our MPTP-induced PD mouse and MPP

Transmembrane protein 97 (TMEM97) is a conserved integral membrane protein highly expressed in various human cancers, including colorectal cancer (CRC), and it exhibits pro-tumor roles in breast cancer, gastric cancer, and glioma. However, whether TMEM97 participates in CRC progression is not fully understood. The expression of mRNA and protein was evaluated by real-time qPCR, western blotting, immunofluorescent, and immunohistochemical staining. TMEM97 functions in cell proliferation, apoptosis, migration, and invasion were assessed by CCK-8, flow cytometry, and transwell assays. The roles of TMEM97 in CRC cells in vivo was investigated using a subcutaneous xenograft model. The transcriptional regulation of TMEM97 was explored by luciferase reporter and ChIP assays. The silencing of TMEM97 inhibited migration and invasion of CRC cells in vitro and led to suppressed growth and enhanced apoptosis in CRC cells and xenografts, whereas overexpression of TMEM97 displayed opposite effects. Mechanistically, TMEM97 knockdown caused a reduction of the proliferating marker PCNA and an increase of pro-apoptotic proteins (cleaved caspase 8/3/7 and cleaved PARP) in CRC cells. TMEM97 also positively regulated the β-catenin signaling pathway in CRC cells and xenografts by modulating the phosphorylated-GSK-3β and active (non-phospho) β-catenin levels. Interestingly, YY1, a well-recognized oncogenic transcription factor, was identified to bind to the TMEM97 promoter and enhance its transcriptional activity, and silencing of TMEM97 abolished YY1-mediated pro-tumor effects on CRC cells. Our results suggest that TMEM97 is transcriptionally activated by YY1 and promotes CRC progression via the GSK-3β/β-catenin signaling pathway, providing that TMEM97 might be a novel therapeutic target for preventing CRC development.

Bladder cancer (BC) is a common urological malignancy that still lacks an effective treatment. Doxorubicin (Dox) has been widely used in the treatment of various cancers, including BC. However, chemoresistance often hampers the clinical application of Dox, therefore, it is necessary to develop effective strategies to improve its efficacy. By using high-throughput screening, we identified OSU-T315, an integrin-linked kinase (ILK) inhibitor, that can augment the cytotoxicity of Dox against BC cells. We found that OSU-T315 and Dox synergistically induce apoptosis of BC cells via mitochondrial pathway in a caspase-dependent. Mechanically, it was found that OSU-T315 and Dox synergistically induced activation of Bax which is critical for the induction of apoptosis. Moreover, it was also found that the downregulation of BCL-2 and MCL-1 is essential for the activation of BAX induced by OSU-T315 and Dox. OSU-T315 was found to downregulate MCL-1 via the GSK-3β/FBXW7 axis in BC cells. Our findings suggest that combined treatment with OSU-T315 and Dox may be a promising strategy to treat BC.

Silibinin is a flavonoid extracted from the medicinal plant Silybum marianum (milk thistle), traditionally used to treat liver disease. Recent studies have shown that the antioxidative stress and anti-inflammatory effects of milk thistle are used in the treatment of neurological diseases. Silibinin has antioxidative stress and antiapoptotic effects and reduces cognitive impairment in models of Alzheimer’s disease (AD). However, the underlying mechanism of silibinin related to improvement of cognition remains poorly understood. In this study, we used the model of lateral ventricle injection of formaldehyde to examine the related mechanism of silibinin in improving cognitive impairment disorders. Oral administration of silibinin for three weeks significantly attenuated the cognitive deficits of formaldehyde-induced mice in a Y -maze test and Morris water maze test. Y -maze results show that silibinin increases the rate of spontaneous response alternation in FA-induced mice. Silibinin increases the target quadrant spending time and decreases escape latency in the Morris water maze test. We examined the effect of silibinin on the NRF2 signaling pathway, and silibinin promoted the nuclear transfer of NRF2 and increased the expression of HO-1 but did not significantly increase the protein expression of NRF2 in the hippocampus. Well, silibinin reduces the content of DHE and decreases the levels of apoptosis of mature neuron cells. We investigated the effect of silibinin on the content of formaldehyde degrading enzymes; biochemical analyses revealed that silibinin increased GSH and ALDH2 in formaldehyde-induced mice. In addition, as one of the pathological changes of AD, TAU protein is also hyperphosphorylated in FA model mice. Silibinin inhibits the expression of GSK-3β in model mice, thereby reducing the phosphorylation of TAU proteins ser396 and ser404 mediated by GSK3β. Based on our findings, we verified that the mechanism of silibinin improving cognitive impairment may be antioxidative stress, and silibinin is one of the potentially promising drugs to prevent formaldehyde-induced cognitive impairment.

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-3β activity inhibitor TDZD-8 significantly attenuated Drp1-mediated mitochondrial fission and recovered mitochondrial function. Inhibition of GSK-3β 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-3β inhibition by TDZD-8 decreases spinal inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

The mechanism of idiopathic oligohydramnios is still uncertain, and there is no effective and targeted treatment for it. Placental aquaporins (AQPs) were associated with idiopathic oligohydramnios. This study aimed to investigate the effect of tanshinone IIA on amniotic fluid volume (AFV) and its underlying molecular mechanisms related to placental AQPs (AQP1, AQP3, AQP8, AQP9). Results showed that compared with the women with normal AFV, placental AQP1, AQP3, AQP8, and AQP9 protein expressions were decreased in women with idiopathic oligohydramnios. Immunohistochemistry revealed localization of AQP1, AQP3, AQP8, and AQP9 mainly in trophoblast cells within labyrinth zone of mouse placenta. Also, AQP1 was located in fetal vascular endothelial cells. Pregnant mice were administered with tanshinone IIA (10 mg/kg or 50 mg/kg, n = 8, respectively) or vehicle (n = 8) from 9.5 to 18.5 gestational day (GD). Tanshinone IIA markedly increased the AFV in pregnant mice, without the effects on embryo numbers per litter, atrophic embryo rate, fetal weight, and placental weight, as well as increased the expressions of AQPs and inhibited the activity of GSK-3β in mice placenta. In JEG-3 cells, tanshinone IIA downregulated AQP1, AQP3, AQP8, AQP9 expressions and inhibited the activity of GSK-3β. Activating GSK-3β with MK-2206 eliminated these alterations. Thus, tanshinone IIA could increase AFV in pregnant mice, possibly through downregulating placental AQP1, AQP3, AQP8, and AQP9 expression via inhibiting the activity of GSK-3β. Tanshinone IIA may be optional for the treatment of idiopathic oligohydramnios.

In the adult mammalian brain, neural stem cells (NSCs) are the precursor cells of neurons that contribute to nervous system development, regeneration, and repair. MicroRNAs (miRNAs) are small non-coding RNAs that regulate cell fate determination and differentiation by negatively regulating gene expression. Here, we identified a post-transcriptional mechanism, centred around miR-130a-3p that regulated NSC differentiation. Importantly, overexpressing miR-130a-3p promoted NSC differentiation into neurons, whereas inhibiting miR-130a-3p function reduced the number of neurons. Then, the quantitative PCR, Western blot and dual-luciferase reporter assays showed that miR-130a-3p negatively regulated acyl-CoA synthetase long-chain family member 4 (Acsl4) expression. Additionally, inhibition of Acsl4 promoted NSC differentiation into neurons, whereas silencing miR-130a-3p partially suppressed the neuronal differentiation induced by inhibiting Acsl4. Furthermore, overexpressing miR-130a-3p or inhibiting Acsl4 increased the levels of p-AKT, p-GSK-3β and PI3K. In conclusion, our results suggested that miR-130a-3p targeted Acsl4 to promote neuronal differentiation of NSCs via regulating the Akt/PI3K pathway. These findings may help to develop strategies for stem cell-mediated treatment for central nervous system diseases.

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax ). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3. This novel system mimics human Bax regulation by GSK3 and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of “Contrato-Programa” UIDB/04050/2020- PE 20-23 and FCT ANR/BEX-BCM/0175/2012. Joana P. Guedes acknowledges the PhD fel lowship SFRH/BD/132070/2017 funded by FCT. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral programme in Applied and Environ mental Microbiology (DP_AEM), info:eu-repo/semantics/publishedVersion

Background and Aims: Liver regeneration is a complex process regulated by a variety of cells, cytokines and biological pathways. Aurora kinase A (AURKA) is a serine/threonine kinase that plays a role in centrosome maturation and spindle formation during the cell division cycle. The purpose of this study was to further explore the mechanism of AURKA on liver regeneration and to identify new possible targets for liver regeneration.Methods: The effect and mechanism of AURKA on liver regeneration were studied using a 70% hepatectomy model. Human liver organoids were used as an in vitro model to investigate the effect of AURKA on hepatocyte proliferation.Results: AURKA inhibition significantly reduced the level of β-catenin protein by reducing the phosphorylation level of glycogen synthase kinase-3β (GSK-3β), leading to the inhibition of liver regeneration. Further studies showed that AURKA co-localized and interacted with GSK-3β in the cytoplasm of hepatocytes. When phosphorylation of GSK-3β was enhanced, the total GSK-3β level remained unchanged, while AURKA was not affected, and β-catenin protein levels were increased. In addition, AURKA inhibition affected the formation and proliferation of human liver organoids. Furthermore, AURKA inhibition led to the polarization of M1 macrophages and the release of interleukin-6 and Tumour necrosis factor α, which also led to reduced liver regeneration and increased liver injury.Conclusions: These results provide more details on the mechanism of liver regeneration and suggest that AURKA is an important regulator of this mechanism.

Estrogen-induced premature closing of the growth plate in the long bones is a major cause of short stature after premature puberty. Recent studies have found that chondrocytes can directly trans-differentiate into osteoblasts in the process of endochondral bone formation, which indicates that cartilage formation and osteogenesis may be a continuous biological process. However, whether estrogen promotes the direct trans-differentiation of chondrocytes into osteoblasts remains largely unknown. Chondrocytes were treated with different concentrations of 17β-estradiol, and Alizarin Red staining and alkaline phosphatase activity assay were used to detected osteogenesis. Specific short hairpin RNA and tamoxifen were used to block the estrogen receptor (ER) pathway and osteogenic marker genes and downstream gene expression were detected using real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry staining. The findings showed that 17β-estradiol promoted the chondrocyte osteogenesis in vitro, even at high concentrations. In addition, blocking of the ERα/β pathway inhibited the trans-differentiation of chondrocytes into osteogenic cells. Furthermore, we found that dentin matrix protein 1 (DMP1), which is a direct downstream molecular of ER, was involved in 17β-estradiol/ER pathway-regulated osteogenesis. As well, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signal pathway also participates in ERα/β/DMP1-regulated chondrocyte osteogenesis. The GSK-3β/β-catenin signal pathway was involved in ERα/β/DMP1-regulated chondrocyte osteogenesis. These findings suggest that ER/DMP1/GSK-3β/β-catenin plays a vital role in estrogen regulation of chondrocyte osteogenesis and provide a therapeutic target for short stature caused by epiphyseal fusion.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and identification of novel targets is necessary for its diagnosis and treatment. This study aimed to investigate the biological function and clinical significance of tweety homolog 3 (TTYH3) in HCC. TTYH3 overexpression promoted cell proliferation, migration, and invasion and inhibited HCCM3 and Hep3B cell apoptosis. TTYH3 promoted tumor formation and metastasis

Neuroinflammation contributes to the pathogenesis of several neurodegenerative disorders. Glycogen synthase kinase-3β (GSK-3β) regulates the release of proinflammatory cytokines and promotes inflammatory responses in immune cells. Microglia are the resident mononuclear immune cells of the central nervous system. Here, we investigated the anti-neuroinflammatory effects of (2

Imbalanced osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is considered the core pathological characteristic of steroid-associated osteonecrosis of the femoral head (SONFH). N6-Methyladenosine (m6A) is the most common type of RNA modification in eukaryotic cells and participates in various physiological and pathological processes. However, the relationship between m6A modification and SONFH has not been reported. In the present study, we aimed to explore the roles of m6A modifications and methyltransferase METTL14 in SONFH. Our results showed that the m6A levels were down-regulated in femoral head tissues and BMSCs from SONFH patients, and this effect was attributed to the reduction of METTL14. Furthermore, METTL14 overexpression in BMSCs from SONFH patients enhanced cell proliferation and osteogenic differentiation. We further identified PTPN6 as the downstream target of METTL14 by mRNA sequencing. Mechanistically, METTL14 regulated PTPN6 expression by increasing PTPN6 mRNA stability in an m6A-dependent manner. Moreover, PTPN6 knockdown abrogated the beneficial effects of METTL14 overexpression on BMSCs. Additionally, we found that METTL14 activated the Wnt signaling pathway, and this effect was caused by the interaction of PTPN6 and GSK-3β. In conclusion, we elucidated the functional roles of METTL14 and m6A methylation in SONFH BMSCs and identified a novel RNA regulatory mechanism, providing a potential therapeutic target for SONFH.

Glucocorticoids (GCs) are widely prescribed for their anti-inflammatory and immunosuppressive properties as a treatment for a variety of diseases. The use of GCs is associated with important side effects, including diabetogenic effects. However, the underlying mechanisms of GC-mediated diabetogenic effects in β-cells are not well understood. In this study we investigated the role of glycogen synthase kinase 3 (GSK3) in the mediation of β-cell death and dysfunction induced by GCs. Using genetic and pharmacological approaches we showed that GSK3 is involved in GC-induced β-cell death and impaired insulin secretion. Further, we unraveled the underlying mechanisms of GC-GSK3 crosstalk. We showed that GSK3 is marginally implicated in the nuclear localization of GC receptor (GR) upon ligand binding. Furthermore, we showed that GSK3 regulates the expression of GR at mRNA and protein levels. Finally, we dissected the proper contribution of each GSK3 isoform and showed that GSK3β isoform is sufficient to mediate the pro-apoptotic effects of GCs in β-cells. Collectively, in this work we identified GSK3 as a viable target to mitigate GC deleterious effects in pancreatic β-cells.

Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2+/+ and Cdk5rap2+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.

Lithium chloride (LiCl) is an important mood-stabilizing therapeutic agent for bipolar disorders, which has also been shown to inhibit cancer cell metastasis. Investigations of LiCl-induced signaling have focused mainly on extracellular signal regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3 (GSK-3). However, little is known about the differences in cellular activities resulting from specific signaling via each of these pathways. In this study, we investigated the difference in responses between the Wnt/β-catenin and ERK pathways by LiCl or epidermal growth factor (EGF) treatment of osteosarcoma cells. In particular, we analyzed the mechanisms responsible for differences in cell mobility and cell proliferation when pERK or β-catenin is activated. In osteosarcoma cells treated with LiCl or EGF, active β-catenin and p-ERK protein levels were significantly increased compared to those in the control group. However, in wound healing and transwell invasion assays, U2OS and SaOS2 cell migration was significantly reduced by LiCl treatment but increased by EGF treatment. In addition, the proliferation of U2OS cells was reduced by LiCl treatment but increased by EGF treatment. Using immunofluorescence microscopy, we observed nuclear accumulation of phosphorylated ERK (pERK) with EGF treatment, but pERK was restricted to the perinuclear area with LiCl treatment. These results were confirmed using immunoblot assays after subcellular fractionation. Together, these data suggest that LiCl interferes with the translocation of pERK from the cytoplasm to the nucleus.

Glycogen synthesis kinase-3β (GSK-3β) is a kinase shown to regulate esophageal cancer (EC) progression. However, the significance of GSK-3β in phosphorylation of small kinetochore-associated protein (SKAP) has not been fully characterized. GSK-3β/SKAP expression was analyzed in EC tissues by RT-qPCR. The association between GSK-3β expression and the overall survival was analyzed using the Kaplan–Meier method. Transwell and wound healing assays were performed to assess the effects of GSK-3β/SKAP knockdown on EC cell migration and invasion. By in vitro kinase assay, the SKAP T294 site was identified as a phosphorylated target of GSK-3β. Moreover, we established two cell lines expressing either T294D (phosphor-mimic) or T294A (phosphor-deficiency) SKAP to analyze the effect of SKAP phosphorylation on EC cell invasion, migration, and epithelial–mesenchymal transition (EMT) process. GSK-3β was overexpressed and positively correlated with SKAP levels in EC tissues. Increased GSK-3β expression was associated with EC poor prognosis. Both of GSK-3β knockdown and silencing SKAP decreased EC cell migration and invasion. GSK-3β phosphorylated SKAP protein at Thr294 site. Additionally, a T294D mutant SKAP enhanced cell migration, invasion, and EMT process. Conversely, a T294A mutant SKAP inhibited EC cell malignancy. Meanwhile, cell invasion and migration abilities were inhibited after silencing GSK-3β in EC109-WT, EC109-T294A and EC109-T294D cells. Phosphorylation of SKAP induced by GSK-3β promoted EC cell migration and invasion.

MicroRNA-1290 (miR-1290) has been reported to be involved in many diseases and play a key role during the development process. However, the role of miR-1290 in atherosclerosis (AS) is still unclear. The current study showed that the expressions of miR-1290 were high in serum of patients with hyperlipidemia. The functional role of miR-1290 were then investigated in human umbilical vein endothelial cells (HUVECs). Here, we found that miR-1290 expressions were notably enhanced in HUVECs mediated by IL-8. miR-1290 inhibitor repressed monocytic THP-1 cells adhesion to HUVECs by regulating ICAM-1 and VCAM-1, inhibited proliferation through regulating cyclinD1 and PCNA, and inhibited inflammatory response by regulating IL-1β. Mechanistically, we verified that miR-1290 mimic was able to directly target the 3′-UTR of GSK-3β mRNA using luciferase reporter assay. Knockdown of GSK-3β (si-GSK-3β) promoted HUVECs adhesion and the expression of IL-1β, and partially restore the depression effect of miR-1290 inhibitor on HUVECs adhesion and inflammation. In contrast, si-GSK-3β inhibited the proliferation of HUVECs and the expression of cyclinD1 and PCNA. In summary, our study revealed that miR-1290 promotes IL-8-mediated the adhesion of HUVECs by targeting GSK-3β. However, GSK-3β is not the target protein for miR-1290 to regulate the proliferation of HUVECs. Our findings may provide potential target in atherosclerosis treatment.

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here, we sought to identify the potential roles of glycogen synthase kinase-3β (GSK-3β), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3β knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension and contractile functions were comparable at 2 weeks post-MI, cKO mice displayed significantly preserved LV chamber and contractile function versus control mice at 4 weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at 4 and 6 weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage, as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3β profoundly suppresses the expression and secretion of growth factors, including basic-fibroblast growth factor, angiopoietin-2, erythropoietin, stem cell factor, platelet-derived growth factor-BB, granulocyte colony-stimulating factor, and vascular endothelial growth factor, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3β promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte-GSK-3β could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

Hyperglycemia-induced oxidative stress in podocytes exerts a major role in the pathological process of diabetic nephropathy. Tripartite motif-containing protein 32 (TRIM32) has been reported to be a key protein in the modulation of cellular apoptosis and oxidative stress under various pathological processes. However, whether TRIM32 participates in the regulation of high glucose (HG)-induced injury in podocytes has not been investigated. This work aimed to assess the possible role of TRIM32 in mediating HG-induced apoptosis, oxidative stress, and inflammatory response in podocytes in vitro. Our results showed a marked increase in TRIM32 expression in HG-exposed podocytes and the glomeruli of diabetic mice. Loss-of-function experiments showed that TRIM32 knockdown improves the viability of HG-stimulated podocytes and suppresses HG-induced apoptosis, oxidative stress, and inflammatory responses in podocytes. Further investigation revealed that TRIM32 inhibition enhances the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, which is associated with the modulation of the Akt/glycogen synthase kinase-3β (GSK-3β) axis in podocytes following HG exposure. However, Akt suppression abrogated the TRIM32 knockdown-mediated activation of Nrf2 in HG-exposed podocytes. Nrf2 knockdown also markedly abolished the protective effects induced by TRIM32 inhibition o in HG-exposed podocytes. In summary, this work demonstrated that TRIM32 inhibition protects podocytes from HG-induced injury by potentiating Nrf2 signaling through modulation of Akt/GSK-3β signaling. The findings reveal the potential role of TRIM32 in mediating podocyte injury during the progression of diabetic nephropathy.

Background: The most prevalent kind of dementia, Alzheimer’s disease (AD), is a neurodegenerative disease. Previous research has shown that glycogen synthase kinase-3β (GSK-3β) is involved in the etiology and progression of AD, including amyloid-β (Aβ), phosphorylated tau, and mitochondrial dysfunction. NPD1 has been shown to serve a neuroprotective function in AD, although the mechanism is unclear. Objective: The effects of NPD1 on Aβ expression levels, tau protein phosphorylation, apoptosis ratio, autophagy activity, and GSK-3β activity in N2a/APP695swe cells (AD cell model) were studied, as well as the mechanism behind such effects. Methods: N2a/APP695swe cells were treated with NPD1, SB216763, or wortmannin as an AD cell model. The associated proteins of hyperphosphorylated tau and autophagy, as well as the activation of GSK3β, were detected using western blot and RT-PCR. Flow cytometry was utilized to analyze apoptosis and ELISA was employed to observe Aβ42. Images of autophagy in cells are captured using transmission electron microscopy. Results: In N2a/APP695swe cells, NPD1 decreased Aβ42 and hyperphosphorylated tau while suppressing cell death. NPD1 also promoted autophagy while suppressing GSK-3β activation in N2a/APP695swe cells. The outcome of inhibiting GSK-3β is comparable to that of NPD1 therapy. However, after activating GSK-3β, the opposite experimental results were achieved. Conclusion: NPD1 might minimize cell apoptosis, downregulate Aβ expression, control tau hyperphosphorylation, and enhance autophagy activity in AD cell models to promote neuronal survival. NPD1’s neuroprotective effects may be mediated via decreasing GSK-3β.

Bromodomain-containing protein 7 (BRD7) has been shown to interact with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85, in the insulin signaling pathway. Here, we show that upregulation of hepatic BRD7 improves glucose homeostasis even in the absence of either p85 isoform, p85α or p85β. However, BRD7 leads to differential activation of downstream effector proteins in the insulin signaling pathway depending on which isoform of p85 is present. In the presence of only p85α, BRD7 overexpression increases phosphorylation of insulin receptor (IR) upon insulin stimulation, without increasing the recruitment of p85 to IR substrate. Overexpression of BRD7 also increases activation of Akt in response to insulin, but does not affect basal phosphorylation levels of Akt. Meanwhile, the phosphorylation of glycogen synthase kinase 3β (GSK3β) is increased by overexpression of BRD7. On the other hand, in the presence of only p85β, BRD7 overexpression does not affect phosphorylation levels of IR, and Akt phosphorylation is not affected by insulin stimulation following BRD7 upregulation. However, BRD7 overexpression leads to increased basal phosphorylation levels of Akt and GSK3β. These data demonstrate that BRD7’s action on glucose homeostasis does not require the presence of both p85 isoforms, and p85α and p85β have unique roles in insulin signaling in the liver.

Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS) lead to the formation of cytoplasmic aggregates in neurons. They are believed to play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. However, the molecular pathogenic mechanisms behind FUS-associated ALS remain poorly understood. Here, we report GSK-3β as a potential modulator of FUS-induced toxicity. We demonstrated that RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration and mitochondrial dysfunction. Furthermore, we found that cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, we found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors.

We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual‐transgenic APP/PS1 mice. Six‐ or 12‐month‐old APP/PS1 and wild‐type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to β‐amyloid peptide oligomers (AβOs), LiCl and/or XAV939 (inhibitor of Wnt/β‐catenin) or transfected with small interfering RNA against the β‐catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aβ was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor‐GSK3β (ser9), β‐catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two‐month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AβOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of β‐catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/β‐catenin signalling.

Renal cell carcinoma (RCC) is a lethal malignancy of the genitourinary system. Follistatin-like 3 (FSTL3), which mediates cell differentiation and growth, acts as a biomarker of tumors and participates in cancer development and progression. Presently, the FSTL3’s functions in RCC were investigated. Quantitative reverse transcription PCR (qRT-PCR), Western Blot, and enzyme linked immunosorbent assay (ELISA) were conducted to verify FSTL3 expression in RCC tissues and cell lines. BrdU assay and CCK8 experiment were made to monitor cell proliferation. Transwell was implemented to examine the invasion of the cells. Flow cytometry analyzed cell apoptosis, and Western Blot evaluated the protein levels of E-cadherin, Twist, and Slug. In the meantime, the protein profiles of the GSK-3β, β-catenin, and TGF-β signaling pathways were ascertained. Moreover, the Xenograft tumor model was constructed in nude mice for measuring tumor growth in vivo. The statistics showed that FSTL3 presented an overexpression in RCC, and patients with a lower expression of FSTL3 manifested a better prognosis. Down-regulated FSTL3 hampered the proliferation, invasion, EMT, and tumor growth of RCC cells and caused cell apoptosis. On the contrary, FSTL3 overexpression enhanced the malignant behaviors of RCC cells. Furthermore, FSTL3 knockdown bolstered GSK-3β, suppressed β-catenin, and reduced BMP1-SMAD pathway activation. Inhibited β-catenin substantially mitigated FSTL3-mediated promoting functions in RCC. In short, FSTL3 functions as an oncogene in RCC by modulating the GSK-3β/β-catenin signaling pathway.

Adiponectin is an adipocyte‐derived hormone, which is closely associated with the development of Alzheimer's disease (AD) and has potential preventive and therapeutic significance. In the present study, we explored the relationship between adiponectin and circadian rhythm disorder in AD, the effect of adiponectin on the abnormal expression of Bmal1 mRNA/protein induced by amyloid‐β protein 31‐35 (Aβ31‐35), and the underlying mechanism of action. We found that adiponectin‐knockout mice exhibited amyloid‐β deposition, circadian rhythm disorders and abnormal expression of Bmal1. Adiponectin ameliorated the abnormal expression of the Bmal1 mRNA/protein caused by Aβ31‐35 by inhibiting the activity of glycogen synthase kinase 3β (GSK3β). These results suggest that adiponectin deficiency could induce circadian rhythm disorders and abnormal expression of the Bmal1 mRNA/protein, whilst exogenous administration of adiponectin may improve Aβ31‐35‐induced abnormal expression of Bmal1 by inhibiting the activity of GSK3β, thus providing a novel idea for the treatment of AD.

SUMMARY OBJECTIVE: In this study, we aimed at investigating the role of isoleucyl-tRNA synthetase in the growth, migration, and angiogenesis of human umbilical vein endothelial cells and the underlying molecular mechanism. METHODS: To assess the role of isoleucyl-tRNA synthetase, we silenced isoleucyl-tRNA synthetase in human umbilical vein endothelial cells using lentiviral 2 specific short hairpin RNAs (short hairpin RNAs 1 and 2) and examined silencing efficiency using real time quantitative polymerase chain reaction and western blot analyses. Short hairpin RNAs 1-isoleucyl-tRNA synthetase had greater knockdown efficiency, it was used in the entire downstream analysis. Short hairpin RNAs 1- isoleucyl-tRNA synthetase silencing effects on cell proliferation, cell colony generation, cell migration, as well as angiogenesis were assessed using cell counting kit-8, colony development, cell migration, and angiogenesis tube formation assays, respectively. RESULTS: Compared to the control group, anti-isoleucyl-tRNA synthetase short hairpin RNAs significantly silenced isoleucyl-tRNA synthetase expression in human umbilical vein endothelial cells, and suppressed their proliferation, migration, and angiogenic capacity. To characterize the underlying mechanism, western blot analyses showed that isoleucyl-tRNA synthetase knockdown suppressed phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. CONCLUSIONS: We have shown, for the first time, the critical role of isoleucyl-tRNA synthetase in human umbilical vein endothelial cells. Our data show that isoleucyl-tRNA synthetase knockdown suppresses human umbilical vein endothelial cell proliferation, migration, and angiogenesis. We have also shown that isoleucyl-tRNA synthetase knockdown suppresses phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. Together, these data highlight isoleucyl-tRNA synthetase as a potential antitumor anti-angiogenic target.

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington’s disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies., Mitochondrial dysfunction is a common hallmark of neurological disorders. Here, the authors identify CHIR99021 as a potent enhancer of mitochondrial function, which improved mitochondrial phenotypes in Huntington’s disease models. CHIR99021 was shown to stabilize calpastatin, which suppressed calpain activation and Drp1-induced mitochondrial fragmentation.

Objective Endothelial progenitor cells (EPCs) contribute to the recovery of neurological function after ischemic stroke. Indirect revascularization has exhibited promising effects in the treatment of cerebral ischemia related to moyamoya disease and intracranial atherosclerotic disease. The role of EPCs in augmenting the revascularization effect is not clear. In this study, we investigated the therapeutic effects of indirect revascularization combined with EPC transplantation in rats with chronic cerebral ischemia. Methods Chronic cerebral ischemia was induced by bilateral internal carotid artery ligation (BICAL) in rats, and indirect revascularization by encephalo-myo-synangiosis (EMS) was performed 1 week later. During the EMS procedure, intramuscular injection of EPCs and the addition of stromal cell-derived factor 1 (SDF-1), and AMD3100, an SDF-1 inhibitor, were undertaken, respectively, to investigate their effects on indirect revascularization. Two weeks later, the cortical microcirculation, neuronal damage, and functional outcome were evaluated according to the microvasculature density and partial pressure of brain tissue oxygen (PbtO2), regional blood flow, expression of phosphorylated Tau (pTau), TUNEL staining and the rotarod performance test, respectively. Results The cortical microcirculation, according to PbtO2 and regional blood flow, was impaired 3 weeks after BICAL. These impairments were improved by the EMS procedure. The regional blood flow was further increased by the addition of SDF-1 and decreased by the addition of AMD3100. Intramuscular injection of EPCs further increased the regional blood flow as compared with the EMS group. The rotarod test results showed that the functional outcome was best in the EMS combined with EPC injection group. Western blot analysis showed that the EMS combined with EPC treatment group had significantly decreased expressions of phosphorylated Tau and phosphorylated glycogen synthase kinase 3 beta (Y216 of GSK-3β). pTau and TUNEL-positive cells were markedly increased at 3 weeks after BICAL induction. Furthermore, the groups treated with EMS combined with SDF-1 or EPCs exhibited marked decreases in the pTau expression and TUNEL-positive cells, whereas AMD3100 treatment increased TUNEL-positive cells. Conclusion The results of this study suggested that indirect revascularization ameliorated the cerebral ischemic changes. EPCs played a key role in augmenting the effect of indirect revascularization in the treatment of chronic cerebral ischemia.

DHX33 is a member of DEAD/H box protein family, and is involved in both RNA and DNA metabolism. It plays diverse roles in multiple cellular activities. DHX33 overexpression has been found to promote the development of many human cancers. However, the underlying mechanism to explain its high expression in cancer cells remains incompletely resolved. In this study, with both human cancer cell lines and normal fibroblasts, we found glycogen synthase kinase 3β (GSK-3β) regulates DHX33 protein stability. This is through its direct phosphorylation of DHX33 on T482, which triggers ubiquitination mediate protein degradation. We further identified one of the major ubiquitination sites of DHX33 to be on its N-terminal K94, a critical residue previously found to be important and highly conserved for ATP binding and helicase activity. Our study for the first time reveals an important upstream regulator, GSK-3β, as a critical kinase to phosphorylate DHX33 at the post-translational level leading to its degradation. Moreover, cancer cells have frequent GSK3β deactivation to disrupt this signaling cascade. Therefore, DHX33 is stabilized in many cancer cells as compared to normal cells. Our study unveils an important post-translational regulation of DHX33 in cells and further unveils a novel mechanism for DHX33 overexpression in cancer cells.

NR2F1-AS1 is a natural antisense transcript with prominent roles in the carcinogenesis. It acts as an oncogene in almost all types of cancers except for cervical and colorectal cancers. It can act as a molecular sponge for miR-17, miR-371a-3p, miR-363, miR-29a-3p, miR-493-5p, miR-190a, miR-140, miR-642a, miR-363, miR-493-5p, miR-483-3p, miR-485-5p, miR-146a-5p, miR-877-5p, miR-338-3 P and miR-423-5p to influence expression of several cancer-related genes. Thus, the sponging role of NR2F1-AS1 is the most appreciated route of its contribution in the carcinogenesis. In addition, NR2F1-AS1 affects activity of IGF-1/IGF-1R/ERK, PI3K/AKT/GSK-3β and Hedgehog pathways. The current narrative review aims at summarization of the results of studies that highlighted the role of NR2F1-AS1 in the carcinogenesis.

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFβ1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFβ1 leads NDRG1, downstream of GSK3β, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFβ and GSK3β inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFβ to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFβ and GSK3β., Instituto de Salud Carlos III European Commission PI15/00336 PI19/01533 CP14/00197 CP19/00029 PIE16/00045, Ministry of Science and Innovation, Spain (MICINN), Instituto de Salud Carlos III, Spanish Government RTI2018.101309B-C22, Chair "Doctors Galera-Requena in cancer stem cell research" CMC-CTS963, European Regional Development Fund (European Union), Ministerio de Universidades FPU19/04450, Junta de Andalucia RH-0139-2020, Sistema Nacional de Garantia Juvenil (Fondo Social Europeo) 8064, Junta de Andalucia, Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades DOC_01686, Fundacion Cientifica Asociacion Espanola Contra el Cancer, Junta Provincial de Jaen (AECC) PRDJA19001BLAY

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10−7 M, 10−8 M, and 10−9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10−7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

Mesenchymal stem cells (MSCs) have been shown to be involved in bone injury repair. Programmed cell death 4 (PDCD4) is not only a tumor suppressor gene but also plays roles in the regulation of MSC function. The aim of the study was to uncover PDCD4 potential regulatory roles and mechanisms in the osteogenic differentiation and bone defect repair of MSCs. shRNA technique was used to knock down PDCD4 expression in umbilical cord-derived mesenchymal stem cells (shPDCD4-UCMSCs). Their phenotype was characterized by flow cytometry and the differentiation potential was verified. We found that PDCD4 knockdown did not affect the surface molecule expression of UCMSCs, but significantly enhanced their osteogenic differentiation and osteogenesis-related molecule expression. Mechanistically, glycogen synthase kinase-3β (GSK-3β) phosphorylation and β-catenin expression were significantly increased in shPDCD4-UCMSCs during the osteogenic differentiation process. The β-catenin inhibitor PNU-74654 reversed shPDCD4-increased osteogenesis and osteogenesis-related molecule expression. The results of animal experiments showed that shPDCD4-UCMSCs markedly improved the defect healing in rabbits. Our findings suggest that PDCD4 acts as a negative regulator of MSC osteogenic differentiation through GSK-3β/β-catenin pathway. Targeting PDCD4 may be a way to improve MSC-mediated therapeutic effects on bone injury.

Tribbles homolog 2 (TRIB2) is implicated in tumorigenesis and drug resistance in various types of cancers. However, the role of TRIB2 in the regulation of tumorigenesis and drug resistance of cancer stem cells (CSCs) is still elusive. In the present study, we showed increased expression of TRIB2 in spheroid-forming and aldehyde dehydrogenase-positive CSC populations of A2780 epithelial ovarian cancer cells. Short hairpin RNA-mediated silencing of TRIB2 expression attenuates the spheroid-forming, migratory, tumorigenic, and drug-resistant properties of A2780 cells, whereas overexpression of TRIB2 increases the CSC-like characteristics. TRIB2 overexpression induced GSK3β inactivation by augmenting AKT-dependent phosphorylation of GSK3β at Ser9, followed by increasing β-catenin level via reducing the GSK3β-mediated phosphorylation of β-catenin. Treatment of TRIB2-ovexpressed A2780 cells with the phosphoinositide-3-kinase inhibitor LY294002 abrogated TRIB2-stimulated proliferation, migration, drug resistance of A2780 cells. These results suggest a critical role for TRIB2 in the regulation of CSC-like properties by increasing the stability of β-catenin protein via the AKT-GSK3β-dependent pathways.

International audience; Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3␤), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3␤-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3␤by unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80-150) and high (2600-3600) CTG-repeats. Apart from GSK3␤ increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3␤. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3␤ inhibitor treatment responses.

The mitochondrial DNA m.3243A > G mutation is well-known to cause a variety of clinical phenotypes, including diabetes, deafness, and osteoporosis. Here, we report isolation and expansion of urine-derived stem cells (USCs) from patients carrying the m.3243A > G mutation, which demonstrate bimodal heteroplasmy. USCs with high levels of m.3243A > G mutation displayed abnormal mitochondrial morphology and function, as well as elevated ATF5-dependent mitochondrial unfolded protein response (UPRmt), together with reduced Wnt/β-catenin signaling and osteogenic potentials. Knockdown of ATF5 in mutant USCs suppressed UPRmt, improved mitochondrial function, restored expression of GSK3B and WNT7B, and rescued osteogenic potentials. These results suggest that ATF5-dependent UPRmt could be a core disease mechanism underlying mitochondrial dysfunction and osteoporosis related to the m.3243A > G mutation, and therefore could be a novel putative therapeutic target for this genetic disorder.

Background Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis. Results We uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited. Conclusions We show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.

Recent studies have demonstrated that one‐carbon metabolism plays a significant role in cancer development. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme of one‐carbon metabolism, has been reported to be dysregulated in many cancers. However, the specific role and mechanism of MTHFD2 in lung adenocarcinoma (LUAD) still remains unclear. In this study, we evaluated the clinicopathological and prognostic values of MTHFD2 in LUAD patients. We conducted a series of functional experiments in vivo and in vitro to explore novel mechanism of MTHFD2 in LUAD. The results showed that MTHFD2 was significantly up‐regulated in LUAD tissues and predicted poor prognosis of LUAD patients. Knockdown of MTHFD2 dramatically inhibited cell proliferation and migration by blocking the cell cycle and inducing the epithelial‐mesenchymal transition (EMT). In addition, MTHFD2 knockdown suppressed LUAD growth and metastasis in cell‐derived xenografts. Mechanically, we found that MTHFD2 promoted LUAD cell growth and metastasis via AKT/GSK‐3β/β‐catenin signalling. Finally, we identified miR‐30a‐3p as a novel regulator of MTHFD2 in LUAD. Collectively, MTHFD2 plays an oncogenic role in LUAD progression and is a promising target for LUAD diagnosis and therapy.

Genes with cross-cancer aberrations are most likely to be functional genes or potential therapeutic targets. Here, we found a total of 137 genes were ectopically expressed in eight cancer types, of which Holliday junction recognition protein (HJURP) was significantly upregulated in prostate cancer (PCa). Moreover, patients with higher HJURP mRNA and protein levels had poorer outcomes, and the protein levels served as an independent prognosis factor for the overall survival of PCa patients. Functionally, ectopic HJURP expression promoted PCa cells proliferation in vitro and in vivo. Mechanistically, HJURP increased the ubiquitination of cyclin-dependent kinase inhibitor 1 (CDKN1A) via the GSK3β/JNK signaling pathway and decreased its stability. This study investigated the role of HJURP in PCa proliferation and may provide a novel prognostic and therapeutic target for PCa.

ETS1 − an evolutionarily conserved transcription factor involved in the regulation of a number of cellular processes − is overexpressed in several malignancies, including ovarian cancer. Most studies on ETS1 expression have been focused on the transcriptional and RNA levels, with post-translational control mechanisms remaining relatively unexplored in the pathogenesis of malignancies. Here, we show that ETS1 forms a complex with glycogen synthase kinase-3β (GSK3β). Specifically, GSK3β-mediated phosphorylation of ETS1 at threonine 265 and serine 269 promoted protein stability, induced the transcriptional activation of matrix metalloproteinase (MMP)-9, and increased cell migration. In vivo experiments revealed that a GSK3β inhibitor was able to suppress both endogenous ETS1 expression and induction of MMP-9 expression. Upon generation of a specific antibody against phosphorylated ETS1, we demonstrated that phospho-ETS1 immunohistochemical expression in ovarian cancer specimens was correlated with that of MMP-9. Notably, the cumulative overall survival of patients with low phospho-ETS1 histoscores was significantly longer than that of those showing higher scores. We conclude that the GSK3β/ETS1/MMP-9 axis may regulate the biological aggressiveness of ovarian cancer and can serve as a prognostic factor in patients with this malignancy.

To investigate the role of platelet-rich plasma (PRP) from different sources in alleviating oxidative stress and ameliorating melanogenesis in UVB-irradiated PIG1 cells, PIG1 cells were irradiated with 80 mJ/cm2 UVB prior to 1% PRP application and the following experiments were taken: the viability of UVB-irradiated PIG1 cells, cellular malondialdehyde (MDA) and reactive oxygen species (ROS) content, and activities of antioxidant enzymes. Western blotting was utilized to detect the expression level of proteins associated with melanin synthesis, apoptosis, and DNA lesions. We found that PRP intervention promoted cell proliferation, reduced MDA and ROS content, increased the activities of series of antioxidant enzymes, and alleviated DNA damages in UVB-damaged PIG1 cells. It is important to note that PRP treatment inhibited UVB-induced melanogenesis via the PI3K/Akt/GSK3β signal pathway. Therefore, we suppose PRP treatment exerts a protective role through their antioxidation effect on UVB-damaged PIG1 cells and hinders melanogenesis induced by UVB irradiation.

Increasing proofs have declared that liver cancer stem cells (CSCs) are the main contributors to tumor initiation, metastasis, therapy resistance, and recurrence of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CSCs regulation remain largely unclear. Recently, PCNA-associated factor (PAF) was identified to play a key role in maintaining breast cancer cell stemness, but its role in liver cancer stem cells has not been declared yet. Herein, we found that both mRNA and protein expression levels of PAF were significantly higher in HCC tissues and cell lines than normal controls. CSC-enriched hepatoma spheres displayed an increase in PAF expression compared to monolayer-cultured cells. Both loss-of-function and gain-of-function experiments revealed that PAF enhanced sphere formation and the percentage of CD133(+) or EpCAM(+) cells in HCCLM3 and Huh7 cells. In the xenograft HCC tumor model, tumor initiation rates and tumor growth were suppressed by knockdown of PAF. Mechanistically, PAF can amplify the self-renewal of liver CSCs by activating β-catenin signaling. Taken together, our results demonstrate that PAF plays a crucial role in maintaining the hepatoma cell stemness by β-catenin signaling. Abbreviations: CSCs: cancer stem cells; HCC: hepatocellular carcinoma; PAF: pCNA-associated factor.

Although dysregulation and dysfunction of long noncoding RNAs (lncRNAs) have been implicated in malignant behavior of oral squamous cell carcinoma (OSCC), whether aberrant lncRNAs play a role in the carcinogenesis of oral leukoplakia (OL) as the best-known precursor of OSCC remains undetermined. Differentially expressed lncRNAs in the occurrence and progression of OL were studied by microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We found a novel key lncRNA n386251 that we named LOLA1 (lncRNA oral leukoplakia progressed associated 1) in the OL progression. The results of qRT-PCR revealed that LOLA1 aberrant expression was validated in tissue samples and cell lines from the normal oral mucosa, OL to OSCC. Fluorescent in situ hybridization showed that LOLA1 expression localized predominately at the cytoplasm of Leuk1 cells. Cell function assays showed that LOLA1 significantly influenced cell migration, invasion, and epithelial-mesenchymal transition (EMT) protein expression. Potential mechanism experiments revealed that AKT/GSK-3β signaling was involved in the regulatory mechanism of LOLA1 in OL progression. Remarkably, Kaplan-Meier analysis revealed that LOLA1 overexpression could predict malignant events of OL progression to OSCC. In conclusion, the current study for the first time profiled and validated the key lncRNAs related to OL progression. Importantly, we demonstrated that a novel lncRNA LOLA1 upregulation was associated with OL malignant progression, suggesting LOLA1 may be a predictive biomarker. Moreover, LOLA1 may promote migration, invasion, and EMT process in OL malignant progression via AKT/GSK-3β pathway.