Phosphoinositide 3-kinase Signaling Pathway Research Articles

Embryonic stem cell microenvironment enhances proliferation of human retinal pigment epithelium cells by activating the PI3K signaling pathway

BackgroundRetinal pigment epithelium (RPE) replacement has been proposed as an efficacious treatment for age-related macular degeneration (AMD), which is the primary cause of vision loss in the elderly worldwide. The embryonic stem cell (ESC) microenvironment has been demonstrated to enable mature cells to gain a powerful proliferative ability and even enhance the stem/progenitor phenotype via activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As the PI3K signaling pathway plays a pivotal role in proliferation and homeostasis of RPE, we hypothesize that the stemness and proliferative capability of RPE can be enhanced by the ESC microenvironment via activation of the PI3K signaling pathway.MethodsTo investigate whether the ESC microenvironment improves the stem cell phenotype and proliferation properties of human RPE (hRPE) cells by regulating the PI3K signaling pathway, primary hRPE cells were cocultured with either ESCs or human corneal epithelial cells (CECs) for 72 h, after which their proliferation, apoptosis, cell cycle progression, and colony formation were assayed to evaluate changes in their biological characteristics. Gene expression was detected by real-time PCR and protein levels were determined by western blotting or immunofluorescence. LY294002, an antagonist of the PI3K signaling pathway, was used to further confirm the mechanism involved.ResultsIn comparison to hRPE cells cultured alone, hRPE cells cocultured with ESCs had an increased proliferative capacity, reduced apoptotic rate, and higher colony-forming efficiency. The expression of the stem cell-associated marker KLF4 and the differentiation marker CRALBP increased and decreased, respectively, in hRPE cells isolated from the ESC coculture. Furthermore, PI3K pathway-related genes were significantly upregulated in hRPE cells after exposure to ESCs. LY294002 reversed the pro-proliferative effect of ESCs on hRPE cells. In contrast, CECs did not share the ability of ESCs to influence the biological behavior and gene expression of hRPE cells.ConclusionsOur findings indicate that the ESC microenvironment enhances stemness and proliferation of hRPE cells, partially via activation of the PI3K signaling pathway. This study may have a significant impact and clinical implication on cell therapy in regenerative medicine, specifically for age-related macular degeneration.

Ginger Extract Increases GLUT-4 Expression Preferentially Through AMPK Than PI3K Signalling Pathways in C2C12 Muscle Cells.

PurposeThere are two signal transduction pathways related to glucose metabolism in C2C12 mouse myoblast cells; one through AMP-activated protein kinase (AMPK), and the other through phosphoinositide 3-kinase (PI3K). Ginger is reported to have hypoglycemic effects. The aim of this study was to determine the exact mechanism of action of ginger in those pathways.MethodsC2C12 cells were seeded to four separate experimental groups; Control: treated with 50 μg/mL DMSO in the absence of any inhibitor; Treatment 1: treated with 50 μg/mL ethyl acetate ginger extract without any inhibitor; Treatment 2: treated with 50 μg/mL extract in the presence of 20 μM AMPK inhibitor; Treatment 3: treated with 50 μg/mL extract in the presence of 25 μM PI3K inhibitor. The amount of GLUT-4 protein (an important glucose transporter) was determined in cytosolic and membrane fractions using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.ResultsGLUT-4 concentration was significantly higher in the membrane fraction of cells treated with ethyl acetate ginger extract in the absence of any inhibitor in comparison with cells treated with this extract in the presence of each of the inhibitors (P-value < 0.05). GLUT-4 quantity in the membrane fractions in all groups was more than cytosolic fractions. The amount of GLUT-4 in membrane fraction of treated cells in the presence of PI3K inhibitor was higher than in the cells treated with this extract in the presence of AMPK inhibitor (P-value < 0.05).ConclusionEthyl acetate ginger extract affects the amount of GLUT-4 protein in membrane and cytosolic fractions of C2C12 myoblast cells mostly through AMPK pathway but less via PI3K.

Spinal cord injury causes insulin resistance associated with PI3K signaling pathway in hypothalamus

Spinal cord injury (SCI) is an independent risk factor for type 2 diabetes, and may induce insulin resistance that leads to this disease. Studies have shown that greater phosphoinositide 3-kinase (PI3K) activation in the hypothalamus leads to activation of the anti-inflammatory pathway, and the anti-inflammatory reflex may protect against insulin resistance and type 2 diabetes. However, the importance of this phenomenon in type 2 diabetes pathogenesis after SCI remains elusive. In the present study, the expression of c-Fos in the hypothalamus of rats with SCI was elevated, and the hypothalamus injury was observer following SCI. Then we showed that SCI could induce increased levels of blood glucose and glucose tolerance in rats. Also, we found that SCI could damage the liver, adipocyte and pancreas, and led to lipid position in liver. Western blots were used to detect the level of PI3K and p-Akt in the hypothalamus, and the results showed a significant downregulation of PI3K and p-Akt after SCI. Furthermore, to verify the activity of the PI3K signaling pathway, immunofluorescence was used to examine the expression of neurons positive for p-S6 (a marker of PI3K activation) after SCI. The results showed that the expression of p-S6-positive neurons decreased after SCI. In addition, the effect of SCI on peripheral inflammation was also investigated. Following SCI, the serum levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 increased. Collectively, our results suggest abnormality in glucose metabolism after SCI, and demonstrate that SCI may impair activation of the PI3K signaling pathway in the hypothalamus. The reduced activity of the PI3K signaling pathway in the hypothalamus may lead to peripheral inflammation, which might be the mechanism underlying the development of insulin resistance and type 2 diabetes following SCI.

In vivo immunotoxicity of Gd2 O3 :Eu3+ nanoparticles and the associated molecular mechanism.

The in vivo toxicity of Gd2 O3 :Eu3+ nanoparticles (NPs) used as dual-modal nanoprobes for molecular imaging has not been studied, and the corresponding molecular mechanism of immunotoxicity remains unknown. In this study, we investigated the cytotoxicity, in vitro apoptosis, and in vivo immunotoxicity of Gd2 O3 :Eu3+ NPs. The NPs showed little immunotoxicity to BALB/c mice. We explored the possible role of the phosphoinositide 3-kinase (PI3K) signaling pathway and found that reactive oxygen species could act as secondary messengers in cellular signaling, inhibiting PI3K expression in the liver. The immune suppression caused by PI3K inhibition helped the mice adapt to stress. The immunotoxicities caused by Gd2 O3 :Eu3+ and gadodiamide, a commonly used contrast agent, were not significantly different, and the mice were able to tolerate the immunotoxicity caused Gd2 O3 :Eu3+ NPs in vitro and in vivo experiments. The results suggest that Gd2 O3 :Eu3+ NPs are sufficiently biocompatible to be used safely in preclinical applications and show promise as bio-imaging agents. Moreover, the in vivo molecular mechanism of immunotoxicity caused by the Gd2 O3 :Eu3+ NPs provides a platform for further research on the immunotoxicity of nano-sized biomaterials.

Bifenthrin impairs the functions of Leydig and Sertoli cells in mice via mitochondrion-endoplasmic reticulum dysregulation

Bifenthrin (BF) is a synthetic insecticide that is widely used in fields, resulting in an increase in its exposure to animals. However, reports on the toxic effects of BF on mammalian species and the underlying mechanism are still lacking. Here, we elucidated the mechanism underlying the toxic effects of BF on mouse reproduction using cell lines of immature mouse Leydig (TM3) and Sertoli (TM4) cells, which are constituent cells of testes. Our results show that BF suppressed the proliferation and viability of TM3 and TM4 cells. Additionally, treatment with BF induced cell cycle arrest, apoptotic cell death, and DNA fragmentation. Mitochondrial dysfunction and disruption of calcium homeostasis were observed in BF-treated TM3 and TM4 cells. Further, bifenthrin modulated unfolded protein response and mitochondrion-associated membrane and mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) signaling pathways. The expression of the mRNAs related to cell cycle progression, steroidogenesis, and spermatogenesis was downregulated by BF, suggestive of testicular toxicity. Taken together, these results demonstrate the intracellular mechanism of action of BF to involve antiproliferative and apoptotic effects and testicular dysfunction in mouse testis.

The function of microRNA-211 expression in post-fracture bone cell apoptosis involving the transforming growth factor-β/ phosphoinositide 3-kinase signaling pathway.

BackgroundThe underlying mechanism of micro (mi)RNA-211 in bone cell apoptosis after fracture remains unclear. This study aimed to determine the effect and function of miRNA-211 in bone cell apoptosis in fracture patients.MethodsSerum samples were collected from patients with fractures and healthy controls. Serum miR-211 expression was detected by quantitative PCR. MC3T3-E1 cells were transfected with a transforming growth factor (TGF)-β inhibitor and phosphoinositide 3-kinase (PI3K) inhibitor. The viability of MC3T3-E1 cells was detected by the MTT assay, and apoptosis was detected by flow cytometry. Caspase-3/9 activity and the protein expression of TGF-β, PI3K, and p-Akt were detected by western blot and immunoprecipitation.ResultsIn the fracture group, miRNA-211 expression was significantly up-regulated compared with controls. We used miRNA-211 mimics to up-regulate miRNA-211 expression, and observed inhibited cell viability and induced apoptosis and lactate dehydrogenase (LDH) activity. miRNA-211 up-regulation also suppressed the expression of TGF-β, PI3K, and p-Akt proteins. Conversely, miRNA-211 down-regulation increased cell viability and reduced apoptosis and LDH activity, as well as inducing the expression of TGF-β, PI3K, and p-Akt. Inhibiting TGF-β decreased the effect of anti-miRNA-211 on osteocyte apoptosis.ConclusionOur data indicate that miRNA-211 functions via the TGF-β/PI3K/Akt signaling pathway in patients with fractures.

468-P: PDK1 Is a Key Regulator of Podocyte Viability and Maintenance of Glomerular Function

In diabetic states, altered activity of insulin signaling pathway is likely to play key roles in insulin resistance and in kidney dysfunction. Since the 3-phosphoinositide-dependent protein kinase 1 (PDK1) serves as a major signaling molecule in the regulation of cellular responses in multiple organs by mediating the phosphoinositide 3-kinase signaling pathway, we tested the hypothesis that podocyte-specific deletion of PDK1 leads to podocyte injury and proteinuric kidney disease. PDK1 was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Mice with a podocyte-specific deletion of PDK1 (PDK1 KO) developed proteinuria, and died due to end-stage renal disease by 5 weeks after birth. Histologically, features characteristic of glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis, and protein casts, were observed in PDK1 KO at 3 weeks of age. We also examined podocyte distribution in glomeruli using the podocyte markers synaptopodin and WT-1, and found that their expressions were lost in some glomeruli of PDK1 KO at 3 weeks of age although their expression patterns at 1 weeks of age PDK1 KO were almost comparable with control mice. We next examined a possible role of forkhead transcription factor, forkhead box O1 (FoxO1) in linking PDK1 of podocytes to kidney function. For this, we generated podocyte-specific PDK1 and FoxO1 double knockout mice (DKO) by introducing Foxo1-flox gene into PDK1 KO mice. DKO exhibited a reduced level of proteinuria compared with age-matched PDK1 KO, and the maximal survival time of DKO had extended to 6.6 weeks after birth. Taken together, PDK1-FoxO1 pathway may be one of the critical signal transduction pathways that links podocyte injury and kidney dysfunction in diabetic states. Disclosure W. Iwata: None. H. Unoki-Kubota: None. H. Kato: None. M. Matsumoto: None. M. Nangaku: Consultant; Self; Akebia Therapeutics, Astellas Pharma Inc., Bayer AG, Boehringer Ingelheim International GmbH, Daiichi Sankyo, GlaxoSmithKline plc., Japan Tobacco Inc., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Otsuka Pharmaceutical Co., Ltd., Scohia Pharma Inc. Research Support; Self; Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; AstraZeneca, Torii Pharmaceutical Co. Ltd. Y. Kaburagi: None.

Osteopontin enhances cisplatin resistance of human A549 lung cancer cells via stimulating the PI3K signaling pathway and upregulating ERCC1 expression.

BackgroundTo investigate the regulatory role of osteopontin (OPN) in cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC).MethodsOPN over-expression and interference vectors were constructed. Human NSCLC A549 cells were divided into normal control, shRNA-NC, shRNA-OPN, OPN, negative control (NC), control+DDP, shRNA-NC+DDP, shRNA-OPN+DDP, OPN+DDP, and NC+DDP groups. Cell proliferation was measured by MTT assay. Cell apoptosis was detected by flow cytometry. PI3K (phosphoinositide 3-kinase), p-ERK1/2 (phospho-extracellular signal-regulated kinases) and ERCC1 (excision repair cross-complementation group 1) mRNA and protein expression was determined by real-time PCR and Western blot.ResultsOPN overexpression and interference vectors were successfully constructed. When compared with control group, OPN group had significantly stimulated cell viability, reduced cell apoptosis, and increased expression of PI3K, p-ERK1/2 and ERCC1 (all P<0.05), whereas all other groups had lower cell viability, higher cell apoptosis, and inhibited protein expression. Moreover, OPN+DDP group had significantly higher proliferation, lower cell apoptosis, and enhanced PI3K, p-ERK1/2 and ERCC1 mRNA and protein expression compared with shRNA-OPN+DDP (all P<0.05).ConclusionsOPN can promote tumor cell growth and reduce cell apoptosis by activating PI3K pathway, which may induce the resistance of NSCLC to cisplatin. Moreover, the stimulation of ERCC1 expression may also increase the cisplatin resistance of NSCLCs.

Nrf2‐Activating Phytochemicals, Sulforaphane and Licochalcone A, Stimulate Cell Growth‐Regulating Kinases in HCT116 Human Colorectal Cancer Cells

Some phytochemicals have been reported to modulate phase II detoxifying/antioxidant enzyme expressions via both Keap1 (Kelch‐like ECH‐associated protein 1)‐dependent and independent regulations of a nuclear factor E2‐related factor 2/antioxidant response element (Nrf2/ARE) system. Based on multiple studies, various kinase signaling pathways are also responsible for regulation of Nrf2/ARE activity differently depending on cell types, microenvironmental factors, treatment doses, etc. Sulforaphane (SFN) and licochalcone A (LicA) are known as potent Nrf2/ARE inducers that can modulate several kinases including phosphoinositide 3‐kinase (PI3K)/Protein kinase B (Akt) and p38 mitogen‐activated protein kinase (MAPK) in propagating cancer cells. The purpose of the present study was to investigate the molecular mechanism through which SFN and LicA can activate Nrf2/ARE system and upregulate heme oxygenase‐1 (HO‐1) expression in HCT116 human colorectal cancer cells. Results revealed that SFN and LicA induced ARE‐transcriptional activity and subsequently increased HO‐1 expression through PI3K and p38 MAPK signaling pathways in HCT116 cells. In addition, HCT116 cell proliferation was significantly promoted when Nrf2/ARE activity was increased by either SFN‐induced PI3K activation or LicA‐induced p38 MAPK activation. These observations indicate that SFN and LicA regulated the Nrf2/ARE/HO‐1 system via phosphorylation of PI3K and p38 MAPK, respectively, in proliferation of HCT116 colorectal cancer cells. In conclusion, the present study highlights the working mechanism of potent Nrf2/ARE activators that possibly perturb the activities of various kinases in the growing cancer cells.Support or Funding InformationThis work was supported by the National Research Foundation of Korea grant (Grant No. 2017R1A2B4005087) funded by the Ministry of Science and ICT, Republic of Korea.

Clitoria ternatea anthocyanin extract suppresses inflammation in carrageenaninduced rat paw edema via down-regulating genes of phosphoinositide 3-kinase signaling pathway

Inflammation is characterized by an increase in vascular permeability leading to edema formation. Phosphoinositide 3-kinase (PI3K) signaling pathway plays the main role to regulate several key events in the inflammatory response to damage and infection. Our previous in silico study showed that anthocyanin and ternatin flavonoids may offer antiinflammatory potential via molecular docking model. Here, we explored whether Clitoria ternatea anthocyanin extract (CTA) had protective activity against inflammation via regulating gene expression related to PI3Ks in rat edema. CTA was extracted in methanol and acetone using maceration and lyophilization. Edema model was done by injecting carrageenan into rat paw. Oral supplementation of CTA (100, 250, and 500 mg/kg BW) and ibuprofen standard (15 mg/kg BW) was given to rats daily for 7 days. After sample treatment, the right hind paw of the rat was injected with carrageenan to induce edema condition. The ability of CTA to suppress the inflammation in paw edema was measured by quantifying the increased percentage of rat paw volume at 1, 3, and 5 hrs after injection. Rat paw tissue was collected, and gene expression related to PI3Ks, i.e. Akt1, PKB, Ilk, Pdk2, Pik3Ca, and p53 was determined using quantitative PCR (qPCR). Carrageenan showed a time-dependent increase in volume of rat paw starting from injection until the next 5 hrs. It reached maximum volume increase of rat paw at 3 hrs after injection. CTA treatment had a dose-dependent pattern in reducing the edema volume in carrageenan-induced paw edema. At 500 mg/kg BW, CTA significantly suppressed the inflammation in paw edema compared to that of positive control, indicating by the low percentage of paw edema volume at 1, 3, and 5 hrs after carrageenan injection. At gene level, qPCR data showed that CTA significantly down-regulated the expression of genes involved in PI3Ks, such as Akt1, PKB, Ilk, Pdk2, Pik3Ca, and p53 in paw tissues. Thus, CTA may have a protective effect against edema via suppressing genes of PI3Ks.

Abstract B48: PI3Kδ-specific inhibitors may act as adjuvants in the immunotherapy of glioblastoma

Abstract Glioblastoma is the most devastating form of primary brain tumor. Despite surgery, radiotherapy, and concomitant temozolomide chemotherapy, the survival rate remains disappointing. Multiple factors including a diverse array of signaling pathways play significant roles in glioblastoma pathogenesis. Of note, the phosphoinositide 3-kinase (PI3K) signaling pathway is often aberrant in glioblastoma, leading to uncontrolled cell proliferation, migration, and invasion into nearby normal brain tissues. The PI3Kδ isoform, predominantly found in leukocytes, is expressed in a number of tumors including glioblastoma. We had previously shown that PI3Kδ is also expressed in glioma cells such as the U87-MG cell line. Using siRNA knockdown technique, we demonstrated that it is important in glioma cell migration and invasion. Interestingly, its expression has been reported to associate with glioma cell resistance to erlotinib. To further study the mechanistic role of PI3Kδ in glioma pathogenesis, we created two knockout cell strains of U87-MG by CRISPR-Cas9 technology. We performed global transcriptome and pathway analysis comparing the parent cell line U87-MG with these knockout cell strains. Our results showed that the mRNA expression of Pleckstrin 2 (PLEK2) is dramatically reduced in PI3Kδ knockout cells. This is confirmed by quantitative RT-PCR. Western blot analysis showed that PLEK2 protein level is reduced by more than 90% and the phosphorylation of Akt at 308, but not 473, is also reduced. PLEK2 contributes to lamellipodia formation and cell spreading via actin cytoskeleton rearrangement, ultimately affecting cell migration and invasion. Our recent findings further strengthen the important role of PI3Kδ in glioma cell motility. It has been shown recently that inhibition of PI3Kα/δ directly enhances effector CD8+ T-cell activity. Thus, use of PI3Kδ-specific inhibitors may reduce expression of PLEK2 thereby reduces glioma cell migration and at the same time promotes antitumor immunity. PI3Kδ inhibitors may also be used as adjuvants together with immune checkpoint inhibitors such as pembrolizumab to prolong the survival of glioblastoma patients. Citation Format: Zulfikar Azam, Shao Wei, Tony To. PI3Kδ-specific inhibitors may act as adjuvants in the immunotherapy of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B48.

Cardioprotective properties of the platelet P2Y12 receptor inhibitor prasugrel on cardiac ischemia/reperfusion injury.

The effects of prasugrel, a third-generation thienopyridine, on myocardial infarction, and ischemia-induced ventricular arrhythmias was evaluated in open-chest anesthetized rats. The role of protein kinase C and phosphoinositide 3-kinase pathways in these effects was also examined. The effect of P2Y12 receptor inhibition by prasugrel (3-10mg/kg, po) on infarct size after 30-min coronary artery occlusion and 120-min reperfusion or on arrhythmias after 7-min coronary occlusion and 7-min reperfusion was evaluated. In the control group, 31.25 ± 3.01% of the risk zone infarcted. At both prasugrel doses, infarct size was significantly smaller than that in the control group: 5.03 ± 0.81% for 3mg/kg (p < 0.0001), and 8.78 ± 2.04% for 10mg/kg (p < 0.0001). The protein kinase C antagonist chelerythrine abolished the anti-infarct effect of prasugrel at 24.77 ± 1.73% as did the phosphoinositide 3-kinase antagonist wortmannin abolished the anti-infarct effect of prasugrel at 27.45 ± 2.74%. Ten mg/kg prasugrel reduced the duration of VT (p = 0.0152 vs control), and wortmannin, but not chelerythrine, reversed the effect of prasugrel on arrhythmias (p = 0.0295). The selective P2Y12 inhibitor prasugrel provides effective protection against myocardial infarction and ischemia-induced ventricular arrhythmias in rats. As in ischemic postconditioning, protein kinase C and phosphoinositide 3-kinase signaling pathways play a role in this protection.

Response of metastatic mouse invasive lobular carcinoma to mTOR inhibition is partly mediated by the adaptive immune system

ABSTRACT Effective treatment of invasive lobular carcinoma (ILC) of the breast is hampered by late detection, invasive growth, distant metastasis, and poor response to chemotherapy. Phosphoinositide 3-kinase (PI3K) signaling, one of the major druggable oncogenic signaling networks, is frequently activated in ILC. We investigated treatment response and resistance to AZD8055, an inhibitor of mammalian target of rapamycin (mTOR), in the K14-cre;Cdh1Flox/Flox;Trp53Flox/Flox (KEP) mouse model of metastatic ILC. Inhibition of mTOR signaling blocked the growth of primary KEP tumors as well as the progression of metastatic disease. However, primary tumors and distant metastases eventually acquired resistance after long-term AZD8055 treatment, despite continued effective suppression of mTOR signaling in cancer cells. Interestingly, therapeutic responses were associated with increased expression of genes related to antigen presentation. Consistent with this observation, increased numbers of tumor-infiltrating major histocompatibility complex class II-positive (MHCII+) immune cells were observed in treatment-responsive KEP tumors. Acquisition of treatment resistance was associated with loss of MHCII+ cells and reduced expression of genes related to the adaptive immune system. The therapeutic efficacy of mTOR inhibition was reduced in Rag1−/- mice lacking mature T and B lymphocytes, compared to immunocompetent mice. Furthermore, therapy responsiveness could be partially rescued by transplanting AZD8055-resistant KEP tumors into treatment-naïve immunocompetent hosts. Collectively, these data indicate that the PI3K signaling pathway is an attractive therapeutic target in invasive lobular carcinoma, and that part of the therapeutic effect of mTOR inhibition is mediated by the adaptive immune system.

Chitinase 3-Like 1 Contributes to Food Allergy via M2 Macrophage Polarization.

PurposeFood allergy is a hypersensitive immune response to specific food proteins. Chitinase 3-like 1 (CHI3L1, also known as YKL-40 in humans or BRP-39 in mice) is associated with various chronic diseases, such as cancer, rheumatoid arthritis, and allergic disease. CHI3L1 is involved in allergen sensitization and type 2 helper T (Th2) inflammation, but the role of CHI3L1 in food allergy remains unclear. In this study, we sought to investigate the role of CHI3L1 in the development of food allergy.MethodsWe measured serum levels of CHI3L1 in food allergic patients. Food allergy was induced in wild-type (WT) and CHI3L1 null mutant (CHI3L1−/−) BALB/c mice with ovalbumin (OVA). We investigated Th2 immune responses, M2 macrophage polarization, and mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) signaling pathways, and also performed transcriptome analysis.ResultsSerum levels of CHI3L1 were significantly higher in children with food allergy compared with those in healthy controls. Furthermore, CHI3L1 expression levels were elevated in WT mice after OVA treatment. Food allergy symptoms, immunoglobulin E levels, Th2 cytokine production, and histological injury were attenuated in food allergy-induced CHI3L1−/− mice compared with those in food allergy-induced WT mice. CHI3L1 expression was increased in OVA-treated WT intestinal macrophages and caused M2 macrophage polarization. Furthermore, CHI3L1 was involved in the extracellular signal-regulated kinases (ERK) and AKT signaling pathways and was associated with immune response and lipid metabolism as determined through transcriptome analysis.ConclusionsCHI3L1 plays a pivotal role in Th2 inflammation and M2 macrophage polarization through MAPK/ERK and PI3K/AKT phosphorylation in food allergy.

Targeting Fibroblast Growth Factor Receptor (FGFR) and Phosphoinositide 3-kinase (PI3K) Signaling Pathways in Medulloblastoma Cell Lines.

Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated. MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays. Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3. Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines.

Combination of KRAS gene silencing and PI3K inhibition for ovarian cancer treatment

The phosphoinositide 3-kinase (PI3K) and RAS signaling pathways are frequently co-activated and altered during oncogenesis. Owing to their regulatory cross-talk, the early attempts of targeting only one pathway have mostly ended up promoting the development of drug resistance. Here, we propose using small interfering RNA (siRNA) therapeutics to directly target the undruggable KRAS (siKRAS) in combination with the pan-PI3K inhibitor GDC-0941 (GDC) to simultaneously block both PI3K and RAS signaling, thereby exerting synergistic anti-tumor effects on ovarian cancers with PTEN deficiency and KRASG12D mutation. For successful delivery of siKRAS, tGC/psi—nanoparticle formulation of polymerized siRNA and thiol-modified glycol chitosan nanoparticle—was used for KRAS specific inhibition in vitro and in vivo. GDC or siKRAS monotherapy each impede downstream signaling, leading to some delay in cell proliferation and migration. When combined, however, they engender much higher inhibition of PI3K signaling and stimulation of apoptosis in an ovarian allograft model compared to monotherapies. Our results show the feasibility of developing new combination strategies for the management of multiple oncogenic mutations activating PI3K and RAS signaling.

Complement C3a Not C4a Activates Osteoclasts By Regulating the PI3K/PDK1/SGK3 Pathway in Patients with Multiple Myeloma

Objective: Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). We found that the serum levels of C3/C4 in MM patients were significantly positive correlated with the severity of bone disease in our previously study. Thus, we conduct detailed studies to explore the effect and potential mechanism of C3a/C4a on osteoclasts in patients with MM. Methods: By adding C3a/C4a to culture system of osteoclasts induced from mononuclear cells in vitro, and the expression of related gene, number and function of osteoclasts were detected. RNA-Seq analysis was used to detect the differentially expressed genes on osteoclasts between the complement group and control group, and the possible signal pathways were analyzed. Quantitative real-time PCR (qRT-PCR), Western blot and pathway inhibitors were used for further validation. R esults: In vitro, the osteoclasts area per view induced by C3a 1μg/ml (52.794±13.027 %) and 10μg/ml (51.797±12.464 %) was significantly increased than control group (0μg/ml) (33.668±8.427 %) (P&lt;0.001; P&lt;0.001), the mRNA relative expression of osteoclasts related genes OSCAR/TRAP/RANKL/Cathepsin K induced by C3a 1μg/ml (median 5.041; 3.726; 1.638; 4.752) and 10μg/ml (median 5.140; 3.702; 2.250; 5.172) was significantly increased than control group (0μg/ml) (median 3.137; 2.004; 0.573; 2.257) (1μg/ml P=0.001; P=0.003; P&lt;0.001; P=0.008; 1μg/ml P&lt;0.001; P=0.019; P&lt;0.001; P=0.002), and the absorption area of osteoclast resorption pit per view induced by C3a 1μg/ml (51.464±11.983 %) and 10μg/ml (50.219±12.067 %) was also significantly increased than control group (0μg/ml) (33.845±8.331 %) (P&lt;0.001; P&lt;0.001) in NDMM patients. There was no difference among the osteoclasts area, relative expression of osteoclasts related genes and absorption area of osteoclast resorption pit between C4a (1μg/ml and 10μg/ml) group and control group (0μg/ml). RNA-Seq was performed on total RNA of osteoclasts induced by C3a in 1μg/ml group and 0μg/ml group of 4 patients with NDMM. There were 184 differentially expressed genes that were detected by RNA-Seq analysis. KEGG Pathway enrichment bubble chart shows C3a may through Phosphoinositide 3-kinase (PI3K) signaling pathways (including PI3K-Akt pathway and AKT-independent signaling pathway) promotes the proliferation of osteoclast. Upregulated differentially expressed genes in this pathway among at least 3 patients with sequencing were validated by qRT-PCR and Western Blot. It was found that the relative expression level of Phosphoinositide dependent kinase-1 (PDK1) / Serum and glucocorticoid inducible protein kinases (SGK3) genes (median 2.078; 4.428) in C3a group (1μg/ml) was significantly higher than control group (0μg/ml) (median 1.336; 1.714) (P&lt;0.001; P=0.001). The relative grayscale levels of PDK1/ P-SGK3 protein (1.785±0.323; 2.190±0.274) in C3a group (1μg/ml) was significantly stronger than control group (0μg/ml) (0.8653±0.588; 0.176±0.152) (P=0.034; P&lt;0.001). Under the action of C3a in patients with NDMM, osteoclasts area per view in SGK inhibitor (EMD638683) 1μM group (39.244±9.089 %) and 10μM group (39.299±9.587 %) significantly reduced than control group (0μM) (54.884±12.837 %) (P&lt;0.001; P&lt;0.001), the relative expression of osteoclast related genes OSCAR/RANKL/TRAP/Cathepsin K in EMD638683 1μM group (median 0.869; 1.097; 0.902; 1.328) and 10μM group (median 0.703; 1.391; 0.843; 1.418) significantly decreased than control group (0μM) (median 2.270; 3.024; 2.208; 3.237) (1μM P=0.015; P=0.002; P=0.003; P=0.015; 10μM P=0.012; P=0.006; P&lt;0.001; P=0.017), and the absorption area per view of osteoclast resorption pit in EMD638683 1μM (35.383±7.794 %) group and 10μM group (32.886±8.993 %) significantly reduced than control group (0μM) (49.358±11.856 %) (P &lt; 0.001; P &lt; 0.001). Conclusions:ComplementC3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with MM, thus leading to the occurrence of bone diseases. SGK inhibitor has a significant inhibitory effect on osteoclasts in patients with MM. This study provide important evidences for the search for new therapeutic targets and strategies for myeloma bone disease patients. Disclosures No relevant conflicts of interest to declare.

A PDGFRβ-PI3K signaling axis mediates periosteal cell activation during fracture healing.

Insufficient and delayed fracture healing remain significant public health problems with limited therapeutic options. Phosphoinositide 3-kinase (PI3K) signaling, a major pathway involved in regulation of fracture healing, promotes proliferation, migration, and differentiation of osteoprogenitors. We have recently reported that knock-in mice with a global increase in PI3K signaling (gCblYF) show enhanced femoral fracture healing characterized by an extraordinary periosteal response to injury. Interestingly, of all growth factor receptors involved in fracture healing, PI3K directly binds only to PDGFR. Given these findings, we hypothesized a PDGFR-PI3K interaction is necessary for mediating robust periosteal cell activation following fracture. In this study, we isolated primary periosteal cells from gCblYF mice to analyze cross-talk between the PDGFRβ and PI3K signaling pathways. We found PDGFRβ signaling contributes to robust Akt phosphorylation in periosteal cells in comparison with other growth factor signaling pathways. Additionally, we performed femoral fractures on gCblYF mice with a conditional removal of PDGFRβ in mesenchymal progenitors using inducible alpha smooth muscle actin (αSMA) CreERT2 mice. Our studies showed that depletion of PDGFRβ signaling within these progenitors in the early phase of fracture healing significantly abrogates PI3K-mediated periosteal activation and proliferation three days after fracture. Combined, these results suggest that PDGFRβ signaling through PI3K is necessary for robust periosteal activation in the earliest phases of fracture healing.

Electroacupuncture attenuates chronic fibromyalgia pain through the phosphorylated phosphoinositide 3-kinase signaling pathway in the mouse brain.

Objective(s):Fibromyalgia (FM) is a central nervous system disorder characterized by widespread mechanical hyperalgesia due to unknown mechanisms. Several inflammatory mediators, such as interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor, are increased in the serum of FM patients. Although medications including pregabalin, duloxetine, and milnacipran are used to treat FM, the results are unsatisfying. In the present study we assessed whether electroacupuncture (EA) can reduce chronic FM pain and then proposed an underlying mechanism for this effect. Materials and Methods:Chronic FM pain was induced in mice by dual acid saline injection lasting up to 4 weeks. Results:Chronic FM pain was treated by EA manipulation, but not in the sham operated group. Phosphorylated phosphatidylinositol 3-kinase (pPI3K), protein kinase B, mechanistic target of rapamycin, and nuclear factor kappa-light-chain-enhancer of activated B cells were unaltered in the mouse dorsal root ganglion (DRG) and spinal cord (SC) after inducing FM and administering EA treatment. The pPI3K-associated nociceptive signaling pathway was increased in the thalamus of FM mice, but reversed by EA. Similar results were observed in the mouse somatosensory cortex. Conclusion:These data suggest that EA has a significant effect on a signaling pathway in brain areas of FM mice. These findings suggest the value of EA for clinical practice.

Effective Treatment of Metastatic Melanoma by Combining MAPK and PI3K Signaling Pathway Inhibitors.

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases. Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies. In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance. Thus, combined treatment targeting both pathways is a promising strategy to overcome this. Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines. Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis. Finally, an in vivo treatment experiment was carried out on NOD/SCID mice. We show that combined therapy was more effective than monotherapy. Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo. This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.