Characterization,expression analysis and localization pattern of toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella

In this study, the toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up‐regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells.     

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll‐like receptor 4 and p38 MAPK activation

Free fatty acid receptor G protein‐coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)‐stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana‐1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll‐like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen‐activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c‐Jun N‐terminal kinase inhibitor SP600125. LPS‐induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS‐induced inhibition of GPR120 expression is a reaction enhancing the LPS‐induced pro‐inflammatory response of macrophages.     

Endothelial toll‐like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a

We previously reported that the canonical innate immune receptor toll‐like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic‐specific, lung‐ and cell‐targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4?/? mice. Lung Ec‐silencing of TLR4 in wild‐type mice induced emphysema, highlighting the specific and distinct role of Ec‐expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16^INK4a, a senescence‐associated gene. Lung Ec‐p16^INK4a‐silencing prevented TLR4?/? induced emphysema, revealing a new functional role for p16^INK4ain lungs. TLR4 suppressed endogenous p16^INK4a expression via HDAC2‐mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence‐related gene expression.     

The evolutionary characteristics and structural biology of Gallus toll‐like receptor 21

Toll‐like receptors (TLRs) are an important part of the innate immune system, acting as a first line of defense against many invading pathogens. The ligand known to bind Gallus toll‐like receptor 21 (gTLR21) is the unmethylated cytosine phosphate guanine dideoxy nucleotide motif; however, the evolutionary characteristics and structural biology of gTLR21 are poorly elaborated. Our results suggest that gTLR21 is phylogenetically and evolutionarily related to the TLR11 family and is perhaps a close ortholog of the Mus TLR13. Structural biology of homology modeling of the gTLR21 ectodomain structure suggests that it has no Z‐loop like that seen in Mus TLR9. The cytosolic toll‐IL‐1 receptor region of gTLR21 contains a central 4‐stranded parallel β‐sheet (βA‐βD) surrounded by 5 α‐helices (αA‐αE) on both sides, a highly conserved structure also seen in other TLRs. Molecular docking analysis reveals that the gTLR21 ectodomain has the potential to distinguish between different ligands. Homodimer analysis results also suggest that Phe842 and Pro844 of the BB loop and Cys876 of the αC helix in gTLR21 are conserved in other cytosolic toll‐IL‐1 receptor domains of other TLRs and may contribute to the docking of homodimers. Our study on the evolutionary characteristics and structural biology of gTLR21 reveals that the molecule may have a broader role to play in innate immune system; however, further experimental validation is required to confirm our findings.     

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta‐ defensin‐ 2 via toll‐ like receptor 4 signalling

Recently, we demonstrated that intratracheal transplantation of human umbilical cord blood‐ derived mesenchymal stem cells (MSCs) attenuates Escherichia (E) coli‐ induced acute lung injury primarily by down‐ modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth of E. coli in vitro was significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up‐ regulation of toll‐ like receptors (TLR)‐ 2 and TLR‐ 4, and β‐ defensin 2 (BD2) in MSCs compared with fibroblasts after E. coli exposure. The increased BD2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by siRNA‐ mediated knockdown of TLR‐ 4, but not TLR‐ 2, and restored by BD2 supplementation. The in vivo down‐ modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection against E. coli‐ induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR‐ 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR‐ 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects against E. coli, both in vitro and in vivo. Furthermore, TLR‐ 4 from the transplanted MSCs plays a seminal role in attenuating in vivo E. coli‐ induced pneumonia and the ensuing acute lung injury through both its anti‐ inflammatory and antibacterial effects.     

Fisetin induces apoptosis in breast cancer MDA‐MB‐453 cells through degradation of HER2/neu and via the PI3K/Akt pathway

Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in breast cancer. The major snag faced by the human population is the development of chemoresistance to HER2 inhibitors by advanced stage breast cancer cells. Moreover, recent researchers focussed on fisetin as an antiproliferative and chemotherapeutic agent. Therefore, this study was intended to analyze the effects of fisetin on HER2/neu‐overexpressing breast cancer cell lines. Our results depicted that fisetin induced apoptosis of these cells by various mechanisms, such as inactivation of the receptor, induction of proteasomal degradation, decreasing its half‐life, decreasing enolase phosphorylation, and alteration of phosphatidylinositol 3‐kinase/Akt signaling.     

FGFRL1 affects chemoresistance of small‐cell lung cancer by modulating the PI3K/Akt pathway via ENO1

Fibroblast growth factor receptor‐like 1 (FGFRL1), a member of the FGFR family, has been demonstrated to play important roles in various cancers. However, the role of FGFRL1 in small‐cell lung cancer (SCLC) remains unclear. Our study aimed to investigate the role of FGFRL1 in chemoresistance of SCLC and elucidate the possible molecular mechanism. We found that FGFRL1 levels are significantly up‐regulated in multidrug‐resistant SCLC cells (H69AR and H446DDP) compared with the sensitive parental cells (H69 and H446). In addition, clinical samples showed that FGFRL1 was overexpressed in SCLC tissues, and high FGFRL1 expression was associated with the clinical stage, chemotherapy response and survival time of SCLC patients. Knockdown of FGFRL1 in chemoresistant SCLC cells increased chemosensitivity by increasing cell apoptosis and cell cycle arrest, whereas overexpression of FGFRL1 in chemosensitive SCLC cells produced the opposite results. Mechanistic investigations showed that FGFRL1 interacts with ENO1, and FGFRL1 was found to regulate the expression of ENO1 and its downstream signalling pathway (the PI3K/Akt pathway) in SCLC cells. In brief, our study demonstrated that FGFRL1 modulates chemoresistance of SCLC by regulating the ENO1‐PI3K/Akt pathway. FGFRL1 may be a predictor and a potential therapeutic target for chemoresistance in SCLC.     

Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice

Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues.     

The Nogo‐B receptor promotes human hepatocellular carcinoma cell growth via the Akt signal pathway

Nogo‐B receptor (NgBR) is a type I receptor with a single transmembrane domain and specifically binds to ligand Nogo‐B. A previous study demonstrated that NgBR was highly expressed in human breast invasive ductal carcinoma and promoted epithelial‐mesenchymal transition in breast tumor cells. Our recent work found that NgBR expression was associated with a poor prognosis in human patients with hepatocellular carcinoma (HCC). Here, we elucidate that the increased expression of NgBR contributes toward the increased cell growth of human HCC cells both in vitro and in vivo. Cell viability and clonogenic survival analysis results demonstrated that knockdown of NgBR inhibits the cell growth in human HCC cells, which correlates with a reduction in the phosphorylation of Akt levels. Furthermore, overexpression of NgBR by the cotransfected pIRES‐NgBR plasmid together with NgBR siRNA in human HCC cells can rescue impaired phosphorylation of Akt levels in NgBR knockdown human HCC cells. In addition, cell viability analyses showed that NgBR overexpression can rescue the cell growth inhibition presented in human HCC NgBR knockdown cells. Taken together, our results suggest that NgBR potentially acts as an oncogene in HCC by increasing Akt activity. Thus, NgBR may represent a new potential diagnostic and therapeutic target for the treatment of HCC.     

Myofibroblasts protect myoblasts from intrinsic apoptosis associated with differentiation via β1 integrin‐PI3K/Akt pathway

Skeletal myoblasts withdrawing from cell cycle is a prerequisite for myodifferentiation, while upon proliferation/differentiation transformation, a large portion of myoblasts will undergo apoptosis. Skeletal fibroblasts, residing in muscle tissue both during and post myogenesis, have been proofed to play pivotal roles in muscle development, while their effect on myoblast apoptosis being coincident with differentiation has not been reported. Using a membrane insert co‐culture system, we studied it and found that the mitochondrial pathway played a crucial role in myoblast apoptosis during differentiation, and fibroblasts promoted not only cell cycle withdrawal but also myoblast survival in a paracrine fashion, which was coupled with upregulations of β1 integrin, phosphorylated Akt and anti‐apoptotic protein Bcl2. To determine the effect of β1 integrin in the process, we transfected myoblasts with siRNA specific for β1 integrin before co‐culture and found that β1 integrin knockdown abolished anti‐apoptotic ability of myoblasts and inhibited Akt activation and Bcl2 expression. Blockage of PI3K/Akt pathway with wortmannin also seriously impaired the protective effect of fibroblasts on myoblasts and fibroblast‐induced Bcl2 expression. The data demonstrated that fibroblasts protected myoblasts from intrinsic apoptosis associated with differentiation, and β1 integrin‐PI3K/Akt pathway activation was required for the process.     

A polysaccharide of the marine alga Capsosiphon fulvescens induces apoptosis in AGS gastric cancer cells via an IGF-IR-mediated PI3K/Akt pathway

Because seaweed extracts have recently been found to have antioxidant and anti-tumor activities, we analyzed a hot-water-soluble polysaccharide (PS) of the marine alga Capsosiphon fulvescens for its potential as a functional foodstuff by determining its effects on cell growth and DNA synthesis. MTS assays showed that the C. fulvescens PS (Cf-PS) significantly inhibited the proliferation of cultured human cancer cells in a dose-dependent manner. Cf-PS-treated AGS cells exhibited a marked increase in caspase-3 activation and a decrease in Bcl-2 expression. In addition, phosphorylation of insulin-like growth factor-I receptor (IGF-IR) was decreased in Cf-PS-treated AGS cells as compared to non-treated control cells, which is consistent with PI3-kinase (PI3K)/Akt activation. Cf-PS also decreased IGF-I-stimulated recruitment of p85 to IGF-IR and IRS-1. These results indicate that Cf-PS inhibits cell proliferation and induces apoptosis by inhibiting IGF-IR signaling and the PI3K/Akt pathway.     

Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

Dynorphins act as endogenous anticonvulsants via activation of kappa opioid receptor (KOR). However, the mechanism underlying the anticonvulsant role remains elusive. This study aims to investigate whether the potential protection of KOR activation by dynorphin against epilepsy was associated with the regulation of PI3K/Akt/Nrf2/HO-1 pathway. Here, a pilocarpine-induced rat model of epilepsy and Mg²⁺-free-induced epileptiform hippocampal neurons were established. Decreased prodynorphin (PDYN) expression, suppressed PI3K/Akt pathway, and activated Nrf2/HO-1 pathway were observed in rat epileptiform hippocampal tissues and in vitro neurons. Furthermore, dynorphin activation of KOR alleviated in vitro seizure-like neuron injury via activation of PI3K/Akt/Nrf2/HO-1 pathway. Further in vivo investigation revealed that PDYN overexpression by intra-hippocampus injection of PDYN-overexpressing lentiviruses decreased hippocampal neuronal apoptosis and serum levels of inflammatory cytokines and malondialdehyde (MDA) content, and increased serum superoxide dismutase (SOD) level, in pilocarpine-induced epileptic rats. The protection of PDYN in vivo was associated with the activation of PI3K/Akt/Nrf2/HO-1 pathway. In conclusion, dynorphin activation of KOR protects against epilepsy and seizure-induced brain injury, which is associated with activation of the PI3K/Akt/Nrf2/HO-1 pathway.