High-Fat Diet Induces Inflammation of Meibomian Gland.

Purpose: To determine if a high-fat diet (HFD) induces meibomian gland (MG) inflammation in mice. Methods: Male C57BL/6J mice were fed a standard diet (SD), HFD, or HFD supplemented with the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist rosiglitazone for various durations. Body weight, blood lipid levels, and eyelid changes were monitored at regular intervals. MG sections were subjected to hematoxylin and eosin staining, LipidTox staining, TUNEL assay, and immunostaining. Quantitative RT-PCR and western blot analyses were performed to detect relative gene expression and signaling pathway activation in MGs. Results: MG acinus accumulated more lipids in the mice fed the HFD. Periglandular CD45-positive and F4/80-positive cell infiltration were more evident in the HFD mice, and they were accompanied by upregulation of inflammation-related cytokines. PPAR-γ downregulation accompanied activation of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways in the HFD mice. There was increased acini cell apoptosis and mitochondria damage in mice fed the HFD. MG inflammation was ameliorated following a shift to the standard diet and rosiglitazone treatment in the mice fed the HFD. Conclusions: HFD-induced declines in PPAR-γ expression and MAPK and NF-κB signaling pathway activation resulted in MG inflammation and dysfunction in mice.

Contextual Fear Memory Maintenance Changes Expression of pMAPK, BDNF and IBA-1 in the Pre-limbic Cortex in a Layer-Specific Manner.

Post-traumatic stress disorder (PTSD) is a debilitating and chronic fear-based disorder. Pavlovian fear conditioning protocols have long been utilised to manipulate and study these fear-based disorders. Contextual fear conditioning (CFC) is a particular Pavlovian conditioning procedure that pairs fear with a particular context. Studies on the neural mechanisms underlying the development of contextual fear memories have identified the medial prefrontal cortex (mPFC), or more specifically, the pre-limbic cortex (PL) of the mPFC as essential for the expression of contextual fear. Despite this, little research has explored the role of the PL in contextual fear memory maintenance or examined the role of neuronal mitogen-activated protein kinase (pMAPK; ERK 1/2), brain-derived neurotrophic factor (BDNF), and IBA-1 in microglia in the PL as a function of Pavlovian fear conditioning. The current study was designed to evaluate how the maintenance of two different long-term contextual fear memories leads to changes in the number of immune-positive cells for two well-known markers of neural activity (phosphorylation of MAPK and BDNF) and microglia (IBA-1). Therefore, the current experiment is designed to assess the number of immune-positive pMAPK and BDNF cells, microglial number, and morphology in the PL following CFC. Specifically, 2 weeks following conditioning, pMAPK, BDNF, and microglia number and morphology were evaluated using well-validated antibodies and immunohistochemistry (n = 12 rats per group). A standard CFC protocol applied to rats led to increases in pMAPK, BDNF expression and microglia number as compared to control conditions. Rats in the unpaired fear conditioning (UFC) procedure, despite having equivalent levels of fear to context, did not have any change in pMAPK, BDNF expression and microglia number in the PL compared to the control conditions. These data suggest that alterations in the expression of pMAPK, BDNF, and microglia in the PL can occur for up to 2 weeks following CFC. Together the data suggest that MAPK, BDNF, and microglia within the PL of the mPFC may play a role in contextual fear memory maintenance.

MicroRNA-370 carried by M2 macrophage-derived exosomes alleviates asthma progression through inhibiting the FGF1/MAPK/STAT1 axis.

Emerging evidence has suggested the functions of exosomes in allergic diseases including asthma. By using a mouse model with asthma induced by ovalbumin (OVA), we explored the roles of M2 macrophage-derived exosomes (M2Φ-Exos) in asthma progression. M2Φ-Exos significantly alleviated OVA-induced fibrosis and inflammatory responses in mouse lung tissues, as well as inhibited abnormal proliferation, invasion, and fibrosis-related protein production in platelet derived growth factor (PDGF-BB) treated primary mouse airway smooth muscle cells (ASMCs). The OVA administration in mice or the PDGF-BB treatment in ASMCs reduced the expression of miR-370, which was detected in M2Φ-Exos by miRNA sequencing. However, treating the mice or ASMCs with M2Φ-Exos reversed the inhibitory effect of OVA or PDGF-BB on miR-370 expression. We identified that the target of miR-370 was fibroblast growth factor 1 (FGF1). Downregulation of miR-370 by Lv-miR-370 inhibitor or overexpression of FGF1 by Lv-FGF1 blocked the protective roles of M2Φ-Exos in asthma-like mouse and cell models. M2Φ-Exos were found to inactivate the MAPK signaling pathway, which was recovered by miR-370 inhibition or FGF1 overexpression. Collectively, we conclude that M2Φ-Exos carry miR-370 to alleviate asthma progression through downregulating FGF1 expression and the MAPK/STAT1 signaling pathway. Our study may offer a novel insight into asthma treatment.

PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway.

Invasion and metastasis are the predominant causes of lethal outcomes in patients with hepatocellular carcinoma (HCC). However, the molecular mechanism underlying the invasive or metastatic process are still insufficiently understood. Here, we first integrated several public databases and identified a novel protein kinase, PDZ-binding kinase (PBK) that was frequently upregulated and correlated with poor prognosis in patients with HCC. Gain- or loss-of-function analysis revealed that PBK promoted migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, PBK enhanced uPAR expression by activating its promoter activity. Chromatin immunoprecipitation (ChIP) assay showed that ETV4 directly bound to the core region of uPAR promoter while PBK could enhance the binding of ETV4 to uPAR promoter. In orthotopic mouse model, PBK knockdown markedly inhibited the lung metastasis of HCC cells, while this effect was significantly restored by uPAR overexpression. Finally, there was a positive correlation between PBK and uPAR, ETV4 and uPAR in HCC clinical samples. Collectively, these findings revealed that PBK acted as a crucial kinase by promoting invasion and migration via the ETV4-uPAR signaling pathway, and it therefore could be a promising diagnostic biomarker and therapeutic target for HCC metastasis.

Development of efficient protocol for rice transformation overexpressing MAP kinase and their effect on root phenotypic traits.

Exhaustive studies on mitogen-activated protein kinase (MAPK) have reported the importance in regulating a variety of responses during plant growth and development. In particular, the potential MAPK genes, MPK3 and MPK6, seem to regulate a plethora of responses, conferring tolerance to varied abiotic, biotic, and developmental stimuli. This makes both MPK3 and MPK6 potential targets for further studies. It would be an important concern to overexpress and knock out these pivotal proteins and then, in turn, to monitor the plant response which is expected to correlate action of a gene to a trait in cellular and organismal contexts. However, overexpression of MAPK genes has remained a puzzle in plants. In the present study, we report the generation of stable transgenic lines overexpressing OsMPK3 in indica and japonica cultivars and OsMPK6 in japonica cultivar under the control of an inducible promoter. We also establish the crucial steps and troubleshooting for each of the indicated rice transformation medium components. Later, we study the potential role of these MAPKs in high-throughput analysis of root system architectural (RSA) traits. It was observed that OsMPK6 overexpression lines had a more robust and spread out root architectural system while OsMPK3 overexpression lines had a typical bushy phenotype.

LincK contributes to breast tumorigenesis by promoting proliferation and epithelial-to-mesenchymal transition.

BACKGROUND: Increasing evidence has demonstrated that mesenchymal stem cells (MSCs) play a role in the construction of tumor microenvironments. Co-culture between tumor cells and MSCs provides an easy and useful platform for mimicking tumor microenvironments and identifying the important members involved in tumor progress. The long non-coding RNAs (lncRNAs) have been shown to regulate different tumorigenic processes. In this study, we aimed to examine functional lncRNA deregulations associated with breast cancer malignancy instigated by MSC-MCF-7 co-culture. METHODS: The microarrays were used to profile the expression changes of lncRNAs in MCF-7 cells during epithelial-mesenchymal transition (EMT) induced by co-culture with MSCs. We found that an intergenic lncRNA KB-1732A1.1 (termed LincK, partly overlapped with GASL1) was significantly elevated. To investigate the biological function of LincK, the expression of EMT markers, cell migration, invasion, proliferation, and colony formation were evaluated in vitro and xenograft assay in nude mice were performed in vivo. Furthermore, we detected LincK expression in clinical samples using RNAscope® technology and verified aberrant expression of LincK in breast cancer data sets from The Cancer Genome Atlas (TCGA) by bioinformatic analysis. The underlying mechanisms of LincK were investigated using mRNA microarray analyses, Western blot, RNA pull down, and RNA immunoprecipitation. RESULTS: LincK induced an EMT progress in breast cancer cells (BCC) MCF-7, MDA-MB-453, and MDA-MB-231. The depletion of LincK decreased the growth, migration, and invasion in BCC, whereas the overexpression of LincK exerted the opposite effects. Moreover, knockdown of LincK repressed tumorigenesis, and ectopic expression of LincK promoted tumor growth in MCF-7 xenograft model. LincK ablation in MDA-MB-231 cells dramatically impaired lung metastasis when incubated intravenously into nude mice. Further, LincK was frequently elevated in breast cancer compared with normal breast tissue in clinical samples. Mechanistically, LincK may share common miRNA response elements with PBK and ZEB1 and regulate the effects of miR-200 s. CONCLUSION: LincK plays a significant role in regulating EMT and tumor growth and could be a potential therapeutic target in breast cancer.

Brain-Derived Neurotrophic Factor Increases Synaptic Protein Levels via the MAPK/Erk Signaling Pathway and Nrf2/Trx Axis Following the Transplantation of Neural Stem Cells in a Rat Model of Traumatic Brain Injury.

Brain-derived neurotrophic factor (BDNF) plays an important role in promoting the growth, differentiation, survival and synaptic stability of neurons. Presently, the transplantation of neural stem cells (NSCs) is known to induce neural repair to some extent after injury or disease. In this study, to investigate whether NSCs genetically modified to encode the BDNF gene (BDNF/NSCs) would further enhance synaptogenesis, BDNF/NSCs or naive NSCs were directly engrafted into lesions in a rat model of traumatic brain injury (TBI). Immunohistochemistry, western blotting and RT-PCR were performed to detect synaptic proteins, BDNF-TrkB and its downstream signaling pathways, at 1, 2, 3 or 4 weeks after transplantation. Our results showed that BDNF significantly increased the expression levels of the TrkB receptor gene and the phosphorylation of the TrkB protein in the lesions. The expression levels of Ras, phosphorylated Erk1/2 and postsynaptic density protein-95 were elevated in the BDNF/NSCs-transplanted groups compared with those in the NSCs-transplanted groups throughout the experimental period. Moreover, the nuclear factor (erythroid-derived 2)-like 2/Thioredoxin (Nrf2/Trx) axis, which is a specific therapeutic target for the treatment of injury or cell death, was upregulated by BDNF overexpression. Therefore, we determined that the increased synaptic proteins level implicated in synaptogenesis might be associated with the activation of the MAPK/Erk1/2 signaling pathway and the upregulation of the antioxidant agent Trx modified by BDNF-TrkB following the BDNF/NSCs transplantation after TBI.

The MAPKKK and MAPKK gene families in banana: identification, phylogeny and expression during development, ripening and abiotic stress.

The mitogen-activated protein kinase (MAPK) cascade, which is a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant development and stress responses. However, less information is known regarding the MAPKKK and MAPKK gene families in the important fruit crop banana. In this study, 10 MAPKK and 77 MAPKKK genes were identified in the banana genome, and were classified into 4 and 3 subfamilies respectively based on phylogenetic analysis. Majority of MAPKKK and MAPKK genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis indicated that MAPKKK-MAPKK genes is involved in tissue development, fruit development and ripening, and response to abiotic stress of drought, cold and salt in two banana genotypes. Interaction networks and co-expression assays demonstrated that MAPK signaling cascade mediated network participates in multiple stress signaling, which was strongly activated in Fen Jiao (FJ). The findings of this study advance understanding of the intricately transcriptional control of MAPKKK-MAPKK genes and provide robust candidate genes for further genetic improvement of banana.

miR-216b promotes cell growth and enhances chemosensitivity of colorectal cancer by suppressing PDZ-binding kinase.

PDZ-binding kinase (PBK/TOPK) acts as oncogene in various cancers and correlates with drug response. However, few studies have examined the expression and roles of PBK in colonrectal cancer (CRC). In this study, we found a significant increase in the expression of PBK in CRC tissues and cell lines. While overexpression of PBK promoted cell growth and decreased the toxicity effect of oxaliplation (OXA), targeting PBK with short hairpin RNA (shRNA) or novel PBK inhibitor HI-TOPK-032 effectively suppressed tumor growth and potentiated chemosensitivity in vitro and in vivo. Furthermore, there was a significant inverse correlation between the expressions of miR-216b and PBK. Further found that miR-216b could down-regulate PBK levels by binding to the 3' untranslated region (3'UTR) of PBK. Notably, while miR-216b decreased cell proliferation and enhanced sensitivity of CRC cells to oxaliplation, re-expression of PBK dramatically reversed these events. Collectively, our data indicated that miR-216b may function as a tumor suppressor though regulating PBK expression, which provided promising targets and possible therapeutic strategies for CRC treatment.

Blockade of NF-κB and MAPK pathways by ulinastatin attenuates wear particle-stimulated osteoclast differentiation in vitro and in vivo.

Ulinastatin, a urinary trypsin inhibitor (UTI), is widely used to clinically treat lipopolysaccharide (LPS)-related inflammatory disorders recently. Adherent pathogen-associated molecular patterns (PAMPs), of which LPS is the best-studied and classical endotoxin produced by Gram-negative bacteria, act to increase the biological activity of osteopedic wear particles such as polymethyl-methacrylate (PMMA) and titanium particles in cell culture and animal models of implant loosening. The present study was designed to explore the inhibitory effect of UTI on osteoclastogenesis and inflammatory osteolysis in LPS/PMMA-mediated Raw264.7 cells and murine osteolysis models, and investigate the potential mechanism. The in vitro study was divided into the control group, LPS-induced group, PMMA-stimulated group and UTI-pretreated group. UTI (500 or 5000 units/ml) pretreatment was followed by PMMA (0.5 mg/ml) with adherent LPS. The levels of inflammatory mediators including tumour necrosis factor-α (TNF-α), matrixmetallo-proteinases-9 (MMP-9) and interleukin-6 (IL-6), receptor activation of nuclear factor NF-κB (RANK), and cathepsin K were examined and the amounts of phosphorylated I-κB, MEK, JNK and p38 were measured. In vivo study, murine osteolysis models were divided into the control group, PMMA-induced group and UTI-treated group. UTI (500 or 5000 units/kg per day) was injected intraperitoneally followed by PMMA suspension with adherent LPS (2×108 particles/25 µl) in the UTI-treated group. The thickness of interfacial membrane and the number of infiltrated inflammatory cells around the implants were assessed, and bone mineral density (BMD), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), relative bone volume over total volume (BV/TV) of distal femur around the implants were calculated. Our results showed that UTI pretreatment suppressed the secretion of proinflammatory cytokines including MMP-9, IL-6, TNF-α, RANK and cathepsin K through down-regulating the activity of nuclear factor kappa B (NF-κB) and MAPKs partly in LPS/PMMA-mediated Raw264.7 cells. Finally, UTI treatment decreased the inflammatory osteolysis reaction in PMMA-induced murine osteolysis models. In conclusion, these results confirm the anti-inflammatory potential of UTI in the prevention of particle disease.

Staurosporine enhances ATRA-induced granulocytic differentiation in human leukemia U937 cells via the MEK/ERK signaling pathway.

Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia (AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinase C (PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA­induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen­activated protein kinase kinase (MEK) and extracellular signal­regulated kinase (ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer­binding protein ß (C/EBPß) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine­enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA­induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

MASM, a Matrine Derivative, Offers Radioprotection by Modulating Lethal Total-Body Irradiation-Induced Multiple Signaling Pathways in Wistar Rats.

Matrine is an alkaloid extracted from Sophora flavescens Ait and has many biological activities, such as anti-inflammatory, antitumor, anti-fibrosis, and immunosuppressive properties. In our previous studies, the matrine derivative MASM was synthesized and exhibited potent inhibitory activity against liver fibrosis. In this study, we mainly investigated its protection against lethal total-body irradiation (TBI) in rats. Administration of MASM reduced the radiation sickness characteristics and increased the 30-day survival of rats before or after lethal TBI. Ultrastructural observation illustrated that pretreatment of rats with MASM significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed that pretreatment with MASM had a dramatic effect on gene expression changes caused by TBI. Pretreatment with MASM prevented differential expression of 53% (765 genes) of 1445 differentially expressed genes induced by TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 21 pathways, such as metabolic pathways, pathways in cancer, and mitogen-activated protein kinase (MAPK) pathways. Our data indicated that pretreatment of rats with MASM modulated these pathways induced by TBI, suggesting that the pretreatment with MASM might provide the protective effects on lethal TBI mainly or partially through the modulation of these pathways, such as multiple MAPK pathways. Therefore, MASM has the potential to be used as an effective therapeutic or radioprotective agent to minimize irradiation damages and in combination with radiotherapy to improve the efficacy of cancer therapy.

De Novo Sequencing and Transcriptome Analysis of Pleurotus eryngii subsp. tuoliensis (Bailinggu) Mycelia in Response to Cold Stimulation.

Cold stimulation of Bailinggu's mycelia is the main factor that triggers primordia initiation for successful production of fruiting bodies under commercial cultivation. Yet, the molecular-level mechanisms involved in mycelia response to cold stimulation are still unclear. Here, we performed comparative transcriptomic analysis using RNA-Seq technology to better understand the gene expression regulation during different temporal stages of cold stimulation in Bailinggu. A total of 21,558 Bailinggu mycelia unigenes were de novo assembled and annotated from four libraries (control at 25 °C, plus cold stimulation treatments at -3 °C for a duration of 1-2 days, 5-6 days, and 9-10 days). GO and KEGG pathway analysis indicated that functional groups of differentially expressed unigenes associated with cell wall and membrane stabilization, calcium signaling and mitogen-activated protein kinases (MAPK) pathways, and soluble sugars and protein biosynthesis and metabolism pathways play a vital role in Bailinggu's response to cold stimulation. Six hundred and seven potential EST-based SSRs loci were identified in these unigenes, and 100 EST-SSR primers were randomly selected for validation. The overall polymorphism rate was 92% by using 10 wild strains of Bailinggu. Therefore, these results can serve as a valuable resource for a better understanding of the molecular mechanisms associated with Bailinggu's response to cold stimulation.

Activation of c-Jun predicts a poor response to sorafenib in hepatocellular carcinoma: Preliminary Clinical Evidence.

We determined the mitogen-activated protein kinase (MAPK) gene expression profile of acquired resistance in sorafenib-sensitive hepatocellular carcinoma (HCC) cells and aimed to identify c-Jun as an important molecule mediating the efficacy of sorafenib. Differences in gene expression of the MAPK signaling between untreated and sorafenib-treated HCC cell lines were investigated using real-time polymerase chain reaction array. Western blot and real-time PCR further evaluated the expression of c-Jun. Pathological specimens from 50 patients with advanced HCC were collected to measure p-c-Jun expression. Sorafenib-resistant HCC cells demonstrated greater levels of basal c-Jun mRNA and protein compared with sorafenib-sensitive HCC cells. Sorafenib activated p-c-Jun in a dose- and time-dependent manner in PLC/PRF/5 and MHCC97H cell lines. Decreased expression levels of 6 genes after sorafenib treatment suggested a robust inhibitory impact of sorafenib on MAPK signaling in HCC cells. c-Jun and p-c-Jun expression levels were inversely correlated with the efficacy of sorafenib; a high expression level of p-c-Jun was associated with resistance to sorafenib and poor overall survival in patients with clinical HCC. p-c-Jun may act as a biomarker for predicting responses of sorafenib treatment, thus advocating targeting of JNK/c-Jun signaling as an optimal therapeutic strategy in a subset of HCC.

Oncogenic roles of TOPK and MELK, and effective growth suppression by small molecular inhibitors in kidney cancer cells.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) and maternal embryonic leucine zipper kinase (MELK) have been reported to play critical roles in cancer cell proliferation and maintenance of stemness. In this study, we investigated possible roles of TOPK and MELK in kidney cancer cells and found their growth promotive effect as well as some feedback mechanism between these two molecules. Interestingly, the blockade of either of these two kinases effectively caused downregulation of forkhead box protein M1 (FOXM1) activity which is known as an oncogenic transcriptional factor in various types of cancer cells. Small molecular compound inhibitors against TOPK (OTS514) and MELK (OTS167) effectively suppressed the kidney cancer cell growth, and the combination of these two compounds additively worked and showed the very strong growth suppressive effect on kidney cancer cells. Collectively, our results suggest that both TOPK and MELK are promising molecular targets for kidney cancer treatment and that dual blockade of OTS514 and OTS167 may bring additive anti-tumor effects with low risk of side effects.

Theaflavin-3, 3'-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways.

Theaflavin-3, 3'-digallate (TF3) is a black tea polyphenol produced from polymerization and oxidization of the green tea ployphenols epicatechin gallate and (-)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anticancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the present study, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cell-induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by downregulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/mTOR/p70S6K/4E-BP1 pathway and Akt/c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally the impaired cancer cells induced angiogenesis. Nevertheless, TF3 did not have any influence on the MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

Type 5 adenylyl cyclase disruption leads to enhanced exercise performance.

The most important physiological mechanism mediating enhanced exercise performance is increased sympathetic, beta adrenergic receptor (ß-AR), and adenylyl cyclase (AC) activity. This is the first report of decreased AC activity mediating increased exercise performance. We demonstrated that AC5 disruption, that is, knock out (KO) mice, a longevity model, increases exercise performance. Importantly for its relation to longevity, exercise was also improved in old AC5 KO. The mechanism resided in skeletal muscle rather than in the heart, as confirmed by cardiac- and skeletal muscle-specific AC5 KO's, where exercise performance was no longer improved by the cardiac-specific AC5 KO, but was by the skeletal muscle-specific AC5 KO, and there was no difference in cardiac output during exercise in AC5 KO vs. WT. Mitochondrial biogenesis was a major mechanism mediating the enhanced exercise. SIRT1, FoxO3a, MEK, and the anti-oxidant, MnSOD were upregulated in AC5 KO mice. The improved exercise in the AC5 KO was blocked with either a SIRT1 inhibitor, MEK inhibitor, or by mating the AC5 KO with MnSOD hetero KO mice, confirming the role of SIRT1, MEK, and oxidative stress mechanisms. The Caenorhabditis elegans worm AC5 ortholog, acy-3 by RNAi, also improved fitness, mitochondrial function, antioxidant defense, and lifespan, attesting to the evolutionary conservation of this pathway. Thus, decreasing sympathetic signaling through loss of AC5 is not only a mechanism to improve exercise performance, but is also a mechanism to improve healthful aging, as exercise also protects against diabetes, obesity, and cardiovascular disease, which all limit healthful aging.

PBK/TOPK mediates promyelocyte proliferation via Nrf2-regulated cell cycle progression and apoptosis.

Acute myeloid leukemia (AML) is a disorder involving hematopoietic stem cells, characterized by blockage of hematopoietic cell differentiation and an increase in clonal neoplastic proliferation. AML is associated with poor patient outcome. PBK/TOPK is a protein kinase derived from PDZ-binding kinase (PBK)/T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK). Previous studies have shown that PBK/TOPK is expressed in hematologic tumors. In the present study, we aimed to investigate the role of PBK/TOPK in promyelocyte proliferation and to clarify the molecular mechanism. PBK/TOPK knockdown (KD) significantly decreased cell proliferation and viability in the NB4 and HL-60 promyelocytes. PBK/TOPK KD resulted in G2/M cell cycle arrest that attributed to a decrease in cdc2 and cyclin B expression. In addition, PBK/TOPK KD caused apoptosis, as evidenced by activation of the mitochondrial apoptotic pathway and an increase in TUNEL-positive cells. PBK/TOPK KD induced mitochondrial dysfunction and ROS generation, and inhibition of mitochondrial dysfunction and ROS production suppressed PBK/TOPK KD-induced cell cycle arrest and apoptosis. Moreover, PBK/TOPK KD decreased Nrf2 expression and ARE-binding activity. Overexpression of Nrf2 inhibited the PBK/TOPK KD-induced decrease in cdc2 and cyclin B expression and cell cycle arrest, and blocked ROS production and apoptosis. Based on literature and our results, it was demonstrated that Nrf2 may be a crucial regulator that mediates PBK/TOPK-exerted promotion of cell proliferation. PBK/TOPK stabilizes Nrf2, strictly regulates the ROS level, promotes cell cycle progression and inhibits apoptosis, contributing to the proliferation of promyelocytes. Our results provide new insights into the molecular mechanism of PBK/TOPK-mediated promyelocyte proliferation and shed light on the pathogenesis of AML.

Flavonoids isolated from Citrus platymamma induce mitochondrial-dependent apoptosis in AGS cells by modulation of the PI3K/AKT and MAPK pathways.

Citrus platymamma hort. ex Tanaka (Rutaceae family) has been widely used in Korean folk medicine for its wide range of medicinal benefits including an anticancer effect. In the present study, we aimed to investigate the molecular mechanism of the anticancer effects of flavonoids isolated from Citrus platymamma (FCP) on AGS cells. FCP treatment significantly inhibited AGS cell growth in a dose­dependent manner. Furthermore, FCP significantly increased the percentage of cells in the sub-G1 phase (apoptotic cell population), and apoptosis was confirmed by Annexin V double staining. Chromatin condensation and apoptotic bodies were also noted in the FCP-treated AGS cells. Moreover, immunoblotting results showed that FCP treatment significantly decreased the expression of procaspase-3, -6, -8 and -9, and PARP and increased cleaved caspase-3, cleaved PARP and the Bax/Bcl-xL ratio in a dose-dependent manner. In addition, the phosphorylation of AKT was significantly decreased, whereas extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were significantly increased in the FCP-treated AGS cells. Taken together, the cell death of AGS cells in response to FCP was mitochondrial-dependent via modulation of the PI3K/AKT and MAPK pathways. These findings provide new insight for understanding the mechanism of the anticancer effects of FCP. Thus, FCP may be a potential chemotherapeutic agent for the treatment of gastric cancer.

Expression of phosphorylated Akt, mTOR and MAPK in type I endometrial carcinoma: clinical significance.

BACKGROUND/AIM: The Akt/mTOR and MAPK pathways are frequently activated in various tumor types but data on endometrial carcinoma are limited. The aim of the present study was to investigate the clinical significance of the expression of phosphorylated MAPK, Akt and mTOR (p-MAPK, p-Akt, p-mTOR) in type I endometrial carcinoma. MATERIALS AND METHODS: The study comprised of 103 formalin-fixed paraffin-embedded (FFPE) type I endometrial carcino ma cases, retrospectively retrieved and assessed by immuno histochemistry for p-MAPK, p-Akt and p-mTOR expression. The expression of these proteins was also studied in non-neoplastic endometrial tissue adjacent to the tumor. RESULTS: The expression patterns of these molecules differed between malignant and non-tumorous tissue specimens. The immuno reactivity for p-Akt was exclusively detected in the neoplastic tissues. Expression levels of p-MAPK were higher in tumors compared to non-neoplastic endometrium (p<0.001), while p-mTOR was found to be over-expressed in non-neo plastic endometrium compared to carcinomas (p=0.001). Expression of p-Akt was correlated with p-MAPK protein levels (p=0.022, r=0.229). On the other hand, no association was found with clinicopathological parameters and with disease-free (DFS) or overall survival (OS) of the patients. CONCLUSION: Our findings support the de-regulation of the PI3K/Akt/mTOR and MAPK signaling pathways in type I endometrial carcinomas suggesting involvement of these pivotal pathways in endometrial carcinogenesis.