Extracellular Regulated Protein Kinases Research Articles

Hyperglycemia induces PFKFB3 overexpression and promotes malignant phenotype of breast cancer through RAS/MAPK activation

BackgroundBreast cancer is the most common tumor in women worldwide. Diabetes mellitus is a global chronic metabolic disease with increasing incidence. Diabetes mellitus has been reported to positively regulate the development of many tumors. However, the specific mechanism of hyperglycemic environment regulating breast cancer remains unclear. PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3) is a key regulatory factor of the glycolysis process in diabetes mellitus, as well as a promoter of breast cancer. So, we want to explore the potential link between PFKFB3 and the poor prognosis of breast cancer patients with hyperglycemia in this study.MethodsCell culture was utilized to construct different-glucose breast cancer cell lines. Immunohistochemistry was adopted to analyze the protein level of PFKFB3 in benign breast tissues, invasive ductal carcinoma with diabetes and invasive ductal carcinoma without diabetes. The Kaplan–Meier plotter database and GEO database (GSE61304) was adopted to analyze the survival of breast cancer patients with different PFKFB3 expression. Western blot was adopted to analyze the protein level of PFKFB3, epithelial–mesenchymal transition (EMT)-related protein and extracellular regulated protein kinases (ERK) in breast cancer cells. Gene Set Cancer Analysis (GSCA) was utilized to investigate the potential downstream signaling pathways of PFKFB3. TargetScan and OncomiR were utilized to explore the potential mechanism of PFKFB3 overexpression by hyperglycemia. Transfections (including siRNAs and miRNA transfection premiers) was utilized to restrain or mimic the expression of the corresponding RNA. Cell functional assays (including cell counting, MTT, colony formation, wound-healing, and cell migration assays) were utilized to explore the proliferation and migration of breast cancer cells.ResultsIn this study, we demonstrated that the expression of PFKFB3 in breast cancer complicated with hyperglycemia was higher than that in breast cancer with euglycemia through cell experiment in vitro and histological experiment. PFKFB3 overexpression decreased the survival period of breast cancer patients and was correlated with a number of clinicopathological parameters of breast cancer complicated with diabetes. PFKFB3 promoted the proliferation and migration of breast cancer in a hyperglycemic environment and might be regulated by miR-26. In addition, PFKFB3 stimulated epithelial-mesenchymal transition of breast cancer in a hyperglycemic environment. In terms of downstream mechanism exploration, we predicted and verified the cancer-promoting effect of PFKFB3 in breast cancer complicated with hyperglycemia through RAS/MAPK pathway.ConclusionsIn conclusion, PFKFB3 could be overexpressed by hyperglycemia and might be a potential therapeutic target for breast cancer complicated with diabetes.

GPAT3 regulates the synthesis of lipid intermediate LPA and exacerbates Kupffer cell inflammation mediated by the ERK signaling pathway

In the process of inflammatory activation, macrophages exhibit lipid metabolism disorders and accumulate lipid droplets. Kupffer cells (KCs) are the resident hepatic macrophage with critical defense functions in the pathogenesis of several types of liver disease. How dysregulated lipid metabolism contributes to perturbed KCs functions remains elusive. Here we report that glycerol-3-phosphate acyltransferase 3 (GPAT3) plays a key role in KCs inflammation response. Our findings indicate that lipopolysaccharide (LPS)-mediated inflammatory activation markedly increased lipid droplets (LDs) accumulation in KCs. This increase could be attributed to significantly up-regulated GPAT3. The loss of GPAT3 function obviously reduced KCs inflammation reaction both in vivo and in vitro, and was accompanied by improved mitochondrial function and decreased production of lysophosphatidic acid (LPA), in turn inhibiting extracellular regulated protein kinases (ERK) signaling pathway. Overall, this study highlights the role of GPAT3 in inflammatory activation of KCs and could thus be a potential therapeutic target for the treatment of inflammation-related liver disease.

Banxia Xiexin decoction ameliorates dextran sulfate sodium (DSS)-induced ulcerative colitis via inhibiting serine-threonine protein kinase (Akt)/mitogen-activated protein kinase (MAPK) signaling pathway.

Banxia Xiexin decoction (BXD), a traditional Chinese medicine, was widely used in treating ulcerative colitis (UC). However, the active components of BXD and its mechanism in UC remain elusive. Therefore, we used network pharmacology in vivo experiments, molecular docking, and surface plasmon resonance strategy (SPR) to uncover BXD's potential mechanism. A UC rat model was established by orally administering 7% dextran sulfate sodium (DSS) in drinking water, BXD and palmatine were orally administered for 7days. Network pharmacology was used to investigate the main bioactive components and crucial targets of BXD in treating UC. Molecular docking was used to investigate interactions between components and crucial targets, verifying the results by SPR. By network pharmacology predicting, 20 active components and 44 candidate anti-UC targets of BXD were identified, and the crucial proteins were screeded from PPI network, including extracellular regulated protein kinases (ERK), AKT1, and tumor necrosis factor-α (TNF). In addition, some key active components (palmatine, sexangularetin, and skullcapflavone II) were screened out from the active components-targets network. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and in vivo experiments showed that protein-serine-threonine kinase (Akt)/MAPK pathway was involved in BXD treatment for UC; BXD and palmatine significantly ameliorated the severity of DSS-induced UC in rats. Our study might assist in further investigation of the active components in Chinese medicine.

Effect and mechanism of longkui yinxiao soup in treating psoriasis in mice.

Objective: Longkui Yinxiao Soup is a traditional Chinese medicine formula used to treat psoriasis for decades. Although Longkui Yinxiao Soup showed promising efficacy in clinical practice, the regulatory mechanisms of Longkui Yinxiao Soup remain elusive. This study aimed to explore the underlying mechanisms of Longkui Yinxiao Soup in a psoriasis-like mouse model. Methods: Longkui Yinxiao Soup was quality controlled by determining the contents of imperatorin and rhoifolin using high-performance liquid chromatography. The imiquimod-induced psoriasis-like mouse model was used to study the therapeutic effect and mechanism of Longkui Yinxiao Soup. The histopathological skin changes were observed by hematoxylin and eosin staining; the infiltration of proliferating proteins, proliferating cell nuclear antigen and Ki67, in skin tissues were observed by immunohistochemical analysis; and the inflammatory factors such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-23, and IL-17 in serum were detected using enzyme-linked immunosorbent assay. RNA sequencing and bioinformatic analysis were used to predict the mechanism of LYS against psoriasis. mRNA expressions of p38, extracellular regulated protein kinases (ERK), mitogen-activated protein kinase 3 (MEK3), mitogen-activated protein kinase 6 (MEK6), RAP1 GTPase activating protein (Rap1gap), and Rap1 were determined using real-time quantitative polymerase chain reaction. The expression levels of proteins related to Rap1-mitogen-activated protein kinase signaling pathways were measured by Western blotting. Results: A quality-control method for Longkui Yinxiao Soup was successfully established using imperatorin and rhoifolin as content determination indexes. Longkui Yinxiao Soup significantly ameliorated the psoriatic symptoms in mice. The serum levels of inflammatory cytokines such as IL-6, TNF-α, IL-23, and IL-17 were decreased, and the expression levels of antigen identified by monoclonal antibody Ki67 (Ki67) and PCNA in skin tissues were downregulated. Moreover, the inhibition of Rap1-MAPK signaling pathways by Longkui Yinxiao Soup was detected. Conclusion: This study confirmed the antipsoriatic activity of Longkui Yinxiao Soup in psoriasis-like mice. This might be due to the inhibition of inflammatory factor secretion, keratinocyte proliferation, and the Rap1-MAPK signal pathway.

Natural product Eriocalyxin B suppressed triple negative breast cancer metastasis both in vitro and in vivo

Breast cancer is the most commonly diagnosed cancer among women, and its metastasis to distant organs accounts for the majority of death. Eriocalyxin B (Eri B), an ent-kaurane diterpenoid isolating from Isodon eriocalyx var. laxiflora, has previously been reported to have anti-tumor and anti-angiogenic effects in breast cancer. Here, we investigated the effect of Eri B on cell migration and adhesion in triple negative breast cancer (TNBC) cells, as well as aldehyde dehydrogenases 1 family member A1 (ALDH1A1) expression, colony- and sphere-formation in cancer stem cell (CSC) enriched MDA-MB-231 cells. The in vivo anti-metastatic activities of Eri B were determined in 3 different breast tumor-bearing mouse models. Our results indicated that Eri B inhibited TNBC cell migration and adhesion to extracellular matrix proteins, and also reduced ALDH1A1 expression and colony formation in CSC-enriched MDA-MB-231 cells. The metastasis-related pathways, such as epidermal growth factor receptor/ mitogen-activated protein kinase kinases 1/2/ extracellular regulated protein kinase signaling altered by Eri B was firstly shown in MDA-MB-231 cells. The potent anti-metastatic efficacies of Eri B were demonstrated in breast xenograft-bearing mice and syngeneic breast tumor-bearing mice. Gut microbiome analysis results revealed the change in the diversity and composition of microbiome after Eri B treatment, and the potential pathways that are involved in the anti-cancer efficacy of Eri B. In conclusion, Eri B was shown to inhibit breast cancer metastasis in both in vitro and in vivo models. Our findings further support the development of Eri B as an anti­metastatic agent for breast cancer.

Heterostructures with Built-in Electric Fields for Long-lasting Chemodynamic Therapy.

Sustained signal activation by hydroxyl radicals (⋅OH) has great significance, especially for tumor treatment, but remains challenging. Here, a built-in electric field (BIEF)-driven strategy was proposed for sustainable generation of ⋅OH, thereby achieving long-lasting chemodynamic therapy (LCDT). As a proof of concept, a novel Janus-like Fe@Fe3 O4 -Cu2 O heterogeneous catalyst was designed and synthesized, in which the BIEF induced the transfer of electrons in the Fe core to the surface, reducing ≡Cu2+ to ≡Cu+ , thus achieving continuous Fenton-like reactions and ⋅OH release for over 18 h, which is approximately 12 times longer than that of Fe3 O4 -Cu2 O and 72 times longer than that of Cu2 O nanoparticles. In vitro and in vivo antitumor results indicated that sustained ⋅OH levels led to persistent extracellular regulated protein kinases (ERK) signal activation and irreparable oxidative damage to tumor cells, which promoted irreversible tumor apoptosis. Importantly, this strategy provides ideas for developing long-acting nanoplatforms for various applications.

Heterostructures with Built‐in Electric Fields for Long‐lasting Chemodynamic Therapy

AbstractSustained signal activation by hydroxyl radicals (⋅OH) has great significance, especially for tumor treatment, but remains challenging. Here, a built‐in electric field (BIEF)‐driven strategy was proposed for sustainable generation of ⋅OH, thereby achieving long‐lasting chemodynamic therapy (LCDT). As a proof of concept, a novel Janus‐like Fe@Fe3O4−Cu2O heterogeneous catalyst was designed and synthesized, in which the BIEF induced the transfer of electrons in the Fe core to the surface, reducing ≡Cu2+ to ≡Cu+, thus achieving continuous Fenton‐like reactions and ⋅OH release for over 18 h, which is approximately 12 times longer than that of Fe3O4−Cu2O and 72 times longer than that of Cu2O nanoparticles. In vitro and in vivo antitumor results indicated that sustained ⋅OH levels led to persistent extracellular regulated protein kinases (ERK) signal activation and irreparable oxidative damage to tumor cells, which promoted irreversible tumor apoptosis. Importantly, this strategy provides ideas for developing long‐acting nanoplatforms for various applications.

EGR1 is crucial for the chlorogenic acid-provided promotion on liver regeneration and repair after APAP-induced liver injury.

Improper use of acetaminophen (APAP) will induce acute liver failure. This study is designed to investigate whether early growth response-1 (EGR1) participated in the promotion on liver repair and regeneration after APAP-induced hepatotoxicity provided by natural compound chlorogenic acid (CGA). APAP induced the nuclear accumulation of EGR1 in hepatocytes regulated by extracellular-regulated protein kinase (ERK)1/2. In Egr1 knockout (KO) mice, the liver damage caused by APAP (300 mg/kg) was more severe than in wild-type (WT) mice. Results of chromatin immunoprecipitation and sequencing (ChIP-Seq) manifested that EGR1 could bind to the promoter region in Becn1, Ccnd1, and Sqstm1 (p62) or the catalytic/modify subunit of glutamate-cysteine ligase (Gclc/Gclm). Autophagy formation and APAP-cysteine adduct (APAP-CYS) clearance were decreased in Egr1 KO mice administered with APAP. The EGR1 deletion reduced hepatic cyclin D1 expression at 6, 12, or 18 h post APAP administration. Meanwhile, the EGR1 deletion also decreased hepatic p62, Gclc and Gclm expression, GCL enzymatic activity, and glutathione (GSH) content and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) activation and thus aggravated oxidative liver injury induced by APAP. CGA increased EGR1 nuclear accumulation; enhanced hepatic Ccnd1, p62, Gclc, and Gclm expression; and accelerated the liver regeneration and repair in APAP-intoxicated mice. In conclusion, EGR1 deficiency aggravated liver injury and obviously delayed liver regeneration post APAP-induced hepatotoxicity through inhibiting autophagy, enhancing liver oxidative injury, and retarding cell cycle progression, but CGA promoted the liver regeneration and repair in APAP-intoxicated mice via inducing EGR1 transcriptional activation.

Effects of IL-11/IL-11 Receptor Alpha on Proliferation and Steroidogenesis in Ovarian Granulosa Cells of Dairy Cows.

Granulosa cells (GCs) are essential for follicular growth, oocyte maturation, and steroidogenesis in the ovaries. Interleukin (IL)-11 is known to play a crucial role in the decidualization of the uterus, however, the expression of the IL-11 system (IL-11, IL-11Rα, and gp130) in the bovine ovary and its exact role in GCs have not been extensively studied. In this study, we identified the IL-11 signaling receptor complex in the bovine ovary and investigated the regulatory effects and underlying mechanism of IL-11Rα on the proliferation and steroidogenesis of GCs. We observed that the IL-11 complex was highly expressed in the GCs of large follicles. IL-11Rα knockdown significantly inhibited GC proliferation by inducing cell cycle arrest at the G1 phase, along with a significant downregulation of proliferating cell nuclear antigen (PCNA) and Cyclin D1 (CCND1) protein, and induced GC apoptosis by significantly upregulating the ratio of BCL-2-associated X protein (BAX) and B-cell lymphoma-2 (BCL-2). In addition, IL-11Rα knockdown attenuated the Janus kinase (JAK) 1-signal transducer and activator of transcription 3 (STAT3) signaling, which is related to cell proliferation and apoptosis. Furthermore, the enzyme-linked immunosorbent assay (ELISA) indicated that IL-11Rα silencing decreased the basal and forskolin (FSK)-stimulated secretions of estradiol and progesterone in GC culture medium concomitantly with a remarkable decrease in cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and steroidogenic acute regulatory protein (StAR). We subsequently determined that this reduction in steroidogenesis was in parallel with the decrease in phosphorylations of protein kinase A (PKA) substrates, cAMP-response element binding protein (CREB), extracellular regulated protein kinase (ERK) 1/2, and p38 mitogen-activated protein kinase (MAPK). Taken together, these data indicate that the effects of IL-11/IL-11Rα on the proliferation and steroidogenesis in bovine GCs is mediated by the JAK1-STAT3, PKA-CREB, p38MAPK, and ERK1/2 signaling pathways. Our findings provide important insights into the local action of the IL-11 system in regulating ovarian function.

Protein kinase D1 overexpression potentiates epidermal growth factor signaling pathway in MCF-7 cells.

Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified. By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells. Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting.

Mechanism of vascular endothelial cell-derived exosomes modified with vascular endothelial growth factor in steroid-induced femoral head necrosis

Steroid-induced avascular necrosis of the femoral head (SANFH) is an intractable orthopedic disease. This study investigated the regulatory effect and molecular mechanism of vascular endothelial cell (VEC)-derived exosomes (Exos) modified with vascular endothelial growth factor (VEGF) in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in SANFH. VECs were cultured in vitro and transfected with adenovirus Adv-VEGF plasmids. Exos were extracted and identified. In vitro/vivo SANFH models were established and treated with VEGF-modified VEC-Exos (VEGF-VEC-Exos). The internalization of Exos by BMSCs, proliferation and osteogenic and adipogenic differentiation of BMSCs were determined by the uptake test, cell counting kit-8 (CCK-8) assay, alizarin red staining, and oil red O staining. Meanwhile, the mRNA level of VEGF, the appearance of the femoral head, and histological analysis were assessed by reverse transcription quantitative polymerase chain reaction and hematoxylin-eosin staining. Moreover, the protein levels of VEGF, osteogenic markers, adipogenic markers, and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) pathway-related indicators were examined by Western blotting, along with evaluation of the VEGF levels in femur tissues by immunohistochemistry. Glucocorticoid (GC) induced adipogenic differentiation of BMSCs and inhibited osteogenic differentiation. VEGF-VEC-Exos accelerated the osteogenic differentiation of GC-induced BMSCs and inhibited adipogenic differentiation. VEGF-VEC-Exos activated the MAPK/ERK pathway in GC-induced BMSCs. VEGF-VEC-Exos promoted osteoblast differentiation and suppressed adipogenic differentiation of BMSCs by activating the MAPK/ERK pathway. VEGF-VEC-Exos accelerated bone formation and restrained adipogenesis in SANFH rats. VEGF-VEC-Exos carried VEGF into BMSCs and motivated the MAPK/ERK pathway, thereby promoting osteoblast differentiation of BMSCs in SANFH, inhibiting adipogenic differentiation, and alleviating SANFH.

In vitro and in vivo analyses on anti-NSCLC activity of apatinib: rediscovery of a new drug target V600E mutation

BackgroundApatinib (YN968D1) is the first small-molecule-targeting drug with anti-tumor activity created in China for the treatment of advanced gastric cancer (GC) and hepatocellular carcinoma (HCC). It showed significant variation in the efficacy for treating cancers, including advanced non-squamous non-small-cell lung cancer (NSCLC). Whether its efficacy could be optimized by subgrouping patients with certain genetic variation remains elusive.MethodsHere, we firstly used kinase screening to identify any possible target of apatinib against 138 kinases. The effects of apatinib on proliferation rates, cell cycle, cell apoptosis, and cell migration on cancer cell lines were analyzed; the in vitro potential pathways of apatinib on cancer cell lines were screened. The effect of apatinib on mouse cancer models in vivo was also analyzed.ResultsBased on HCC364 cells with BRAF V600E mutation, we have shown that apatinib could inhibit their growth, migration, cell cycle, and induce their apoptosis. Based on mice with transplanted HCC364 cells, we have also shown that apatinib could inhibit the tumor growth. Based on immunohistochemistry, we have demonstrated that apatinib could suppress the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase and extracellular regulated protein kinases. This may account at least part of the apatinib’s inhibitory effect on HCC364 cancer cells.ConclusionsBRAF V600E protein kinase is a target of apatinib by kinase screening. We have demonstrated that apatinib can effectively inhibit tumor cells with BRAF V600E mutation by in vitro and in vivo experiments. Our results have demonstrated that targeting BRAF V600E mutation, apatinib appears to be effective and safe for treating NSCLC and possibly other cancers with the same mutation.

Effects and Potential Mechanism of Zhuyu Pill Against Atherosclerosis: Network Pharmacology and Experimental Validation.

Atherosclerosis (AS) is an immunoinflammatory disease associated with dyslipidemia. Zhuyu Pill (ZYP) is a classic Chinese herbal compound that has been shown to exhibit anti-inflammatory and lipid-lowering effects on AS in our previous studies. However, the underlying mechanisms by which ZYP ameliorates atherosclerosis have not yet been fully investigated. In this study, network pharmacology and in vivo experiments were conducted to explore the underlying pharmacological mechanisms of ZYP on ameliorating AS. The active ingredients of ZYP were acquired from our previous study. The putative targets of ZYP relevant to AS were obtained from TCMSP, SwissTargetPrediction, STITCH, DisGeNET, and GeneCards databases. Protein-protein interactions (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted using the Cytoscape software. Furthermore, in vivo experiments were carried out for target validation inapolipoprotein E (ApoE) -/- mice. Animal experiments revealed that ZYP ameliorated AS mainly through lowering blood lipids, alleviating vascular inflammation, and decreasing the levels of vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), monocyte chemotactic protein-1 (MCP-1), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Additionally, the results of Real-Time quantitative PCR revealed that ZYP inhibited the gene expressions of mitogen-activated protein kinase (MAPK) p38, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK),and nuclear factor kappa-B (NF-κB) p65. The Immunohistochemistry and Western blot assays showed the inhibitory effect of ZYP on the proteins level of p38, p-p38, p65, and p-p65. This study has provided valuable evidence on the pharmacological mechanisms of action of ZYP in ameliorating AS that will be useful for forming the rationale of future research studying the cardio-protection and anti-inflammation effects of ZYP.

Hypoxia-inducible factor 1α inhibitor induces cell death via suppression of BCR-ABL1 and Met expression in BCR-ABL1 tyrosine kinase inhibitor sensitive and resistant chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) has a markedly improved prognosis with the use of breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitors (BCR-ABL1 TKIs). However, approximately 40% of patients are resistant or intolerant to BCR-ABL1 TKIs. Hypoxia-inducible factor 1α (HIF-1α) is a hypoxia response factor that has been reported to be highly expressed in CML patients, making it a therapeutic target for BCR-ABL1 TKI-sensitive CML and BCR-ABL1 TKI-resistant CML. In this study, we examined whether HIF-1α inhibitors induce cell death in CML cells and BCR-ABL1 TKI-resistant CML cells. We found that echinomycin and PX-478 induced cell death in BCR-ABL1 TKIs sensitive and resistant CML cells at similar concentrations while the cell sensitivity was not affected with imatinib or dasatinib in BCR-ABL1 TKIs resistant CML cells. In addition, echinomycin and PX-478 inhibited the c-Jun N-terminal kinase (JNK), Akt, and extracellular-regulated protein kinase 1/2 (ERK1/2) activation via suppression of BCR-ABL1 and Met expression in BCR-ABL1 sensitive and resistant CML cells. Moreover, treatment with HIF-1α siRNA induced cell death by inhibiting BCR-ABL1 and Met expression and activation of JNK, Akt, and ERK1/2 in BCR-ABL1 TKIs sensitive and resistant CML cells. These results indicated that HIF-1α regulates BCR-ABL and Met expression and is involved in cell survival in CML cells, suggesting that HIF-1α inhibitors induce cell death in BCR-ABL1 TKIs sensitive and resistant CML cells and therefore HIF-1α inhibitors are potential candidates for CML treatment. [BMB Reports 2023; 56(2): 78-83].

Intrahippocampal injection of IL-1β upregulates Siah1-mediated degradation of synaptophysin by activation of the ERK signaling in male rat.

Interleukin-1β (IL-1β) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1β injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1β, IL-1β + PD98059, and IL-1β + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1β exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1β in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1β both in vivo and in vitro. In primary cultured neurons incubated with IL-1β, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1β treatment. The present results suggest that exogenous IL-1β can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1β induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway.

Aloperine inhibits RANKL-induced osteoclast differentiation via suppressing the MAPK signaling pathways

To figure out the molecular mechanism of aloperine (ALO) on receptor activator of nuclear factor (NF)-kappa-B (κb) ligand (RANKL)-caused osteoclast differentiation. The histopathological analysis and tartrate-resistant acid phosphatase (TRAP) staining assays were applied to check the extent of bone loss of the femur. Then, the protein expressions of nitric oxide synthase 2, glutathione reductase, nuclear erythroid 2-related factor 2, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1, and catalase were checked in RAW264.7, a macrophage cell line, by enzyme-linked-immunosorbent serologic assay and Western blot analysis. Further, the TRAP staining and quantitative polymerase chain reaction assay were applied to characterize the level of RAW264.7 osteoclast differentiation. Besides, Western blot assay was used to check the protein expressions of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38 in RAW264.7. Finally, ERK activation blocker U0126 was used to inhibit mitogen-activated protein kinase (MAPK) signaling pathway to determine the effect of MAPK signaling pathway on RAW264.7 osteoclast differentiation. In this study, the results demonstrated that ALO could inhibit lipopolysaccharide-induced bone loss in vivo in a dose-dependent manner, with significant inhibitory effects at high doses. Further research indicated that ALO could inhibit RANKL-induced oxidative stress and osteoclast differentiation of RAW264.7. Then, ALO could inactivate MAPK signaling pathway. Finally, the results showed that inhibition of MAPK signaling pathway could increase ALO inhibition of RANKL-caused osteoclast differentiation. ALO inhibits RANKL-caused osteoclast differentiation by suppressing the MAPK signaling pathways.

Tanshinone IIA Inhibits Triple-Negative Breast Cancer Cells MDA-MB-231 via G Protein-Coupled Estrogen Receptor- (GPER-) Dependent Signaling Pathway.

Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.

KAP1 modulates osteogenic differentiation via the ERK/Runx2 cascade in vascular smooth muscle cells.

Osteoblast phenotypic transition in vascular smooth muscle cells (VSMCs) has been unveiled as a common cause of vascular calcification (VC). Krüppel-Associated Box (KRAB)-Associated Protein 1(KAP1) is a transcriptional corepressor that modulates various intracellular pathological processes from gene expression to DNA repair to signal transduction. However, the function and mechanism of KAP1 on the osteoblastic differentiation of VSMCs have not been evaluated yet. We demonstrate that the expression of KAP1 in VSMCs is significantly enhanced in vivo and in vitro calcification models. Downregulating the expression of KAP1 suppresses the osteoblast phenotypic transition of VSMCs, which is indicated by a decrease in the expression of osteoblast marker collagenase type I (COL I) and an increase in the expression of VSMC marker α-smooth muscle actin (α-SMA). Conversely, exogenous overexpression of KAP1 could promote osteoblast phenotypic transition of VSMCs. Moreover, KAP1 upregulated the expression of RUNX family transcription factor 2 (Runx2), an inducer of osteoblast that positively regulates many osteoblast-related genes, such as COL I. Evaluation of the potential mechanism demonstrated that KAP1 promoted osteoblast phenotypic transition of VSMCs by activating the extracellular regulated protein kinases (ERK) signaling pathway, which could activate Runx2. In support of this finding, KAP1-induced cell osteoblast phenotypic transition is abolished by treatment with PD0325901, a specific ERK inhibitor. The present study suggested that KAP1 participated in the osteoblast differentiation of VSMCs via the ERK/Runx2 cascade and served as a potential diagnostics and therapeutics target for vascular calcification.

A-Kinase Anchor Protein 95 Is Involved in ERK1/2-Elk-1 Signal Transduction in Colon Cancer.

To assess A-kinase anchor protein 95 (AKAP95), B-Raf, extracellular regulated protein kinases 1/2 (ERK1/2), and Elk-1 expression in colon cancer tissue, and characterize AKAP95 associations with B-Raf, ERK1/2, Elk-1, and colon cancer clinicopathological indices. The immunohistochemistry streptavidin-perosidase (SP) method was used to determine protein expression levels in 64 colon cancer and 32 para-carcinoma tissue specimens. (1) Positive AKAP95 expression rates in colon cancer tissue were higher when compared with para-carcinoma tissue (92.19% vs. 59.38%, P < 0.05). Similar findings were determined for B-Raf (76.56% vs. 25%, P < 0.05), ERK1/2 (90.63% vs. 31.25%, P < 0.05), and Elk-1 levels (92.19% vs. 40.63%, P < 0.05). (2) No significant associations were identified between AKAP95, B-Raf, ERK1/2, and Elk-1 protein expression and degree of differentiation, histological type, and lymph node metastasis in colon cancer samples (P > 0.05); however, in The Cancer Genome Atlas and Gene Expression Omnibus datasets, AKAP95 was closely related to immune infiltration, and highly expressed AKAP95 was negatively associated with overall survival and relapse free survival rates in colon cancer patients. (3) Correlations were observed between AKAP95 and ERK1/2, AKAP95 and Elk-1, B-Raf and ERK1/2, B-Raf and Elk-1, and ERK1/2 and Elk-1 (all P < 0.05), but no correlation was observed between AKAP95 and B-Raf (P > 0.05). AKAP95 may affect immune infiltration levels in colon cancer by participating in ERK1/2-Elk-1 signal transduction.

Isosteviol plays a protective role on hepatic ischemia and reperfusion injury in mice through MAPK/NF-κB signaling pathway.

Hepatic ischemia and reperfusion (I/R) injury is of common occurrence during liver surgery and transplantation, isosteviol (ISV) is an acid hydrolysate of stevioside, the major component of Stevia rebaudiana. Stevioside and its metabolites have been shown to have varieties of pharmacological activities, However, the effect of ISV on hepatic I/R injury has not determined. The purpose of this paper is to study the effect of ISV on mice with hepatic I/R injury and further investigate its underlying mechanism. The blood vessels supplying the left/middle lobe of the liver in mice were clamped to cause liver ischemia for 1h, and then removed the clamp to conduct reperfusion for 6 h. ISV or saline was injected intraperitoneally after reperfusion. The expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 in serum and tissues were evaluated by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The infiltration of neutrophils and macrophages into the liver tissue was determined by flow cytometry and myeloperoxidase. Liver hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and Annexin V probe were used to determine liver injury and hepatocyte apoptosis. western blots (WB) was used to investigate the activation of nuclear factor kappa-B (NF-κB) and c-JunNH2 terminal kinase (JNK), p38 and extracellular regulated protein kinase (ERK), while the expression of apoptosis-related proteins B-cell lymphoma-2 (BCL-2), BCL2-associated X protein (BAX), caspase-3 was detected. ISV reduced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels to alleviate liver injury. ISV significantly reduced the release of inflammatory cytokines and the accumulation of liver neutrophils and macrophages. Meanwhile, ISV can promote the expression of anti-apoptosis-related protein BCL-2 and inhibit the expression of pro-apoptotic protein BAX and the activation of the protease caspase-3, and reduce the occurrence of hepatocyte apoptosis. Finally, ISV can reduce the phosphorylation level and activation of NF-κB, JNK, p38 and ERK. ISV inhibits the occurrence of inflammation and hepatocyte apoptosis through mitogen-activated protein kinase (MAPK)/NF-κB signaling pathway to relieve liver injury.