Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways.

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein-coupled receptors that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1, and activity-regulated cytoskeleton-associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, whereas only intracellular mGlu5 activates protein phosphatase 2 and leads to fragile X mental retardation protein degradation and de novo protein synthesis followed by a protein synthesis-dependent increase in Arc and post-synaptic density protein 95. However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 internalization and chemically induced long-term depression albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

Orexin B protects dopaminergic neurons from 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity associated with reduced extracellular signal-regulated kinase phosphorylation.

BACKGROUND: The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is a major pathological hallmark of Parkinson's disease (PD). Orexin B (OXB) has been reported to promote the growth of DA neurons. However, the roles of OXB in the degeneration of DA neurons still remained not fully clear. METHODS: An in vivo PD model was constructed by administrating 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Pole test was performed to investigate the motor function of mice and the number of DA neurons was detected by immunofluorescence (IF). A PD cell model was established by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+). OXB was added to the culture medium 2 h after MPP + treatment. Microscopic analysis was carried out to investigate the function of OXB in the cell model of PD 24 h after MPP + challenge. RNA-Seq analysis of the PD cell model was performed to explore the possible mechanisms. Western blot was used to detect the phosphorylation levels of extracellular signal-regulated kinase (ERK). RESULTS: OXB significantly decreased the DA neurons death caused by MPTP, alleviated MPP+-induced neurotoxicity in SH-SY5Y cells, and robustly enhanced the weight and motor ability of PD mice. Besides, RNA-Seq analysis demonstrated that the mitogen-activated protein kinase (MAPK) pathway was involved in the pathology of PD. Furthermore, MPP + led to increased levels of phosphorylation of ERK (p-ERK), OXB treatment significantly decreased the levels of p-ERK in MPP+-treated SH-SY5Y cells. CONCLUSIONS: This study demonstrated that OXB exerts a neuroprotective role associated with reduced ERK phosphorylation in the PD model. This suggests that OXB may have therapeutic potential for treatment of PD.

Vildagliptin Treatment Ameliorates Renal Interstitial Fibrosis in a Murine Model of Unilateral Ureteral Obstruction.

Renal interstitial fibrosis in mice can be modeled using unilateral ureteral obstruction (UUO). Here, we investigated the anti-fibrotic effects of the dipeptidyl peptidase-4 inhibitor vildagliptin in this model. We found that vildagliptin given in the drinking water at 10.6 ± 1.5 mg/kg/d prevented fibrosis. Mechanistically, UUO was associated with extracellular signal-regulated kinase (ERK) phosphorylation and with the accumulation of the toxic lipid peroxidation product expression of 4-hydroxy-2-nonenal (4-HNE). Both were significantly inhibited by vildagliptin. Similarly, UUO caused reductions in heme oxygenase-1 (HO-1) mRNA in the kidney, whereas interleukin-6 (IL-6) and cyclooxygenase-1 (COX-1) mRNA were increased; these effects were also prevented by vildagliptin. Taking these data together, we propose that vildagliptin reduces renal interstitial fibrosis resulting from UUO by means of its effects on ERK phosphorylation and the amounts of 4-HNE, HO-1, IL-6 and COX-1 in the kidney.

Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4.

WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, ß-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, ß-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF-ERK1/2 signaling pathways.

A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF-ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.

Regulator of G protein signaling 2 inhibits Gαq-dependent uveal melanoma cell growth.

Activating mutations in Gαq/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gαq/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gαq-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation GαqQ209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gαq. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cß, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gαq, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with GαqQ209L as a partial effector antagonist.

AAV-glycine receptor α3 alleviates CFA-induced inflammatory pain by downregulating ERK phosphorylation and proinflammatory cytokine expression in SD rats.

BACKGROUND: Glycine receptors (GlyRs) play key roles in the processing of inflammatory pain. The use of adeno-associated virus (AAV) vectors for gene therapy in human clinical trials has shown promise, as AAV generally causes a very mild immune response and long-term gene transfer, and there have been no reports of disease. Therefore, we used AAV for GlyRα1/3 gene transfer in F11 neuron cells and into Sprague-Dawley (SD) rats to investigate the effects and roles of AAV-GlyRα1/3 on cell cytotoxicity and inflammatory response. METHODS: In vitro experiments were performed using plasmid adeno-associated virus (pAAV)-GlyRα1/3-transfected F11 neurons to investigate the effects of pAAV-GlyRα1/3 on cell cytotoxicity and the prostaglandin E2 (PGE2)-mediated inflammatory response. In vivo experiment, the association between GlyRα3 and inflammatory pain was analyzed in normal rats after AAV-GlyRα3 intrathecal injection and after complete Freund's adjuvant (CFA) intraplantar administration. Intrathecal AAV-GlyRα3 delivery into SD rats was evaluated in terms of its potential for alleviating CFA-induced inflammatory pain. RESULTS: The activation of mitogen-activated protein kinase (MAPK) inflammatory signaling and neuronal injury marker activating transcription factor 3 (ATF-3) were evaluated by western blotting and immunofluorescence; the level of cytokine expression was measured by ELISA. The results showed that pAAV/pAAV-GlyRα1/3 transfection into F11 cells did not significantly reduce cell viability or induce extracellular signal-regulated kinase (ERK) phosphorylation or ATF-3 activation. PGE2-induced ERK phosphorylation in F11 cells was repressed by the expression of pAAV-GlyRα3 and administration of an EP2 inhibitor, GlyRαs antagonist (strychnine), and a protein kinase C inhibitor. Additionally, intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not induce obvious histopathological injury but increased ATF-3 activation in dorsal root ganglion (DRGs). CONCLUSIONS: Antagonists of the prostaglandin EP2 receptor, PKC, and glycine receptor can inhibit PGE2-induced ERK phosphorylation. Intrathecal AAV-GlyRα3 administration to SD rats significantly decreased CFA-induced inflammatory pain and suppressed CFA-induced ERK phosphorylation, did not significantly induce gross histopathological injury but elicited ATF-3 activation. We suggest that PGE2-induced ERK phosphorylation can be modulated by GlyRα3, and AAV-GlyRα3 significantly downregulated CFA-induced cytokine activation.

Inhibition of histone deacetylase 6 destabilizes ERK phosphorylation and suppresses cancer proliferation via modulation of the tubulin acetylation-GRP78 interaction.

BACKGROUND: The leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown. METHODS: The clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT-PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells. RESULTS: HDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth. CONCLUSIONS: HDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Prevention of Renal Interstitial Fibrosis by Suplatast in a Mouse Model.

Suplatast is a T helper 2 (Th2) cytokine inhibitor. Here, we tested its therapeutic effects using a mouse model of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO). In this model, suplatast was found to prevent the induced fibrosis in the obstructed kidney when given in the drinking water at 100 mg/kg/d. Mechanistically, suplaplast inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) that was otherwise increased by UUO. Similarly, suplaplast reduced the increased accumulation of KIM-1, transforming growth factor ß (TGF-ß), type I collagen, interleukin-4 (IL-4), janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)3 mRNA seen in the kidneys of UUO-treated mice. Furthermore, STAT3 phosphorylation, which was stimulated by UUO, was also significantly decreased by suplatast. Collectively, these data show that suplatast reduces UUO-induced renal interstitial fibrosis via mechanisms including a reduction of phosphorylation of ERK and JAK/STAT pathway signaling.

Phosphorylation of Neurofilament Light Chain in the VLO Is Correlated with Morphine-Induced Behavioral Sensitization in Rats.

Neurofilament light chain (NF-L) plays critical roles in synapses that are relevant to neuropsychiatric diseases. Despite postmortem evidence that NF-L is decreased in opiate abusers, its role and underlying mechanisms remain largely unknown. We found that the microinjection of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) into the ventrolateral orbital cortex (VLO) attenuated chronic morphine-induced behavioral sensitization. The microinjection of TSA blocked the chronic morphine-induced decrease of NF-L. However, our chromatin immunoprecipitation (ChIP)-qPCR results indicated that this effect was not due to the acetylation of histone H3-Lysine 9 and 14 binding to the NF-L promotor. In line with the behavioral phenotype, the microinjection of TSA also blocked the chronic morphine-induced increase of p-ERK/p-CREB/p-NF-L. Finally, we compared chronic and acute morphine-induced behavioral sensitization. We found that although both chronic and acute morphine-induced behavioral sensitization were accompanied by an increase of p-CREB/p-NF-L, TSA exhibited opposing effects on behavioral phenotype and molecular changes at different addiction contexts. Thus, our findings revealed a novel role of NF-L in morphine-induced behavioral sensitization, and therefore provided some correlational evidence of the involvement of NF-L in opiate addiction.

RSU-1 interaction with prohibitin-2 links cell-extracellular matrix detachment to downregulation of ERK signaling.

Cell-extracellular matrix (ECM) detachment is known to decrease extracellular signal-regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell-ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell-ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell-ECM adhesion-induced ERK activation. Furthermore, cell-ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150-206) effectively suppressed the cell-ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell-ECM detachment and links it to decreased ERK signaling.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance.

Transforming growth factor-beta 2 (TGF-ß2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-ß2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-ß2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-ß2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-ß2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm's canal (MSC) cell monolayers was decreased by TGF-ß2 treatment. SAHA inhibited the effects of TGF-ß2 on the permeability of these cells. TGF-ß2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-ß2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-ß2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-ß signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-ß. These results imply that SAHA prevents TGF-ß2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-ß signaling in TM and MSC cells.

RSK-Mediated Non-canonical Activation of EphA2 by Tamoxifen.

The long-term administration of tamoxifen to estrogen receptor α (ERα)-positive breast cancer patients is an established treatment that reduces mortality and recurrence. However, resistance to tamoxifen and an increased risk of endometrial cancer may occur; therefore, the mechanisms by which tamoxifen causes these adverse effects warrant further study. Tamoxifen has been shown to activate mitogen-activated protein kinase (MAPK) in an ERα-independent manner; therefore, we investigated its effects on the MAPK-mediated non-canonical activation of EphA2, a critical event regulating cell migration. Tamoxifen at slightly higher concentrations induced the rapid phosphorylation of EphA2 at Ser-897 via the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK-ribosomal S6 kinases (RSK) pathway in HeLa cells. In addition, tamoxifen significantly enhanced the migration ability of ERα-negative MDA-MB-231 breast cancer cells in RSK- and EphA2-dependent manners. Phosphorylated EphA2 was internalized and re-localized to the plasma membrane, including lamellipodia, in an RSK-dependent manner. Collectively, the present results provide novel insights into the tumor-promoting activity of tamoxifen.

Non-canonical Regulation of EGFR by the Air Pollutant 9,10-Phenanthrenequinone.

9,10-Phenanthrenequinone (9,10-PQ), a polycyclic aromatic hydrocarbon that is present in air pollutants, such as diesel exhaust gas and PM2.5, causes the production of excess reactive oxygen species. 9,10-PQ was recently shown to induce the activation of epidermal growth factor receptor (EGFR) by inhibiting protein tyrosine phosphatase 1B. In the present study, we focused on the non-canonical regulation of EGFR, including negative feedback and internalization. In contrast to previous findings, 9,10-PQ inhibited the constitutive tyrosine phosphorylation of EGFR via the mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Thr-669 in EGFR-overexpressing A431 and MDA-MB-468 cells. In addition, 9,10-PQ induced the clathrin-mediated endocytosis of EGFR via the p38 phosphorylation of Ser-1015 in HeLa and A549 cells. These results revealed that 9,10-PQ strongly induced the non-canonical regulation of EGFR by activating mitogen-activated protein kinase (MAPK).

Metastasis and MAPK Pathways.

Cancer is a leading cause of death worldwide. In many cases, the treatment of the disease is limited due to the metastasis of cells to distant locations of the body through the blood and lymphatic drainage. Most of the anticancer therapeutic options focus mainly on the inhibition of tumor cell growth or the induction of cell death, and do not consider the molecular basis of metastasis. The aim of this work is to provide a comprehensive review focusing on cancer metastasis and the mitogen-activated protein kinase (MAPK) pathway (ERK/JNK/P38 signaling) as a crucial modulator of this process.

Blocking ERK-DAPK1 Axis Attenuates Glutamate Excitotoxicity in Epilepsy.

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.

[Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells].

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

PTPN2 regulates the activation of KRAS and plays a critical role in proliferation and survival of KRAS-driven cancer cells.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

A soluble derivative of PrPC activates cell-signaling and regulates cell physiology through LRP1 and the NMDA receptor.

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

Angiotensin II type 1 receptor variants alter endosomal receptor-ß-arrestin complex stability and MAPK activation.

The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and ß-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. ß-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between ß-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor-ß-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin-BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with ß-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in ß-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with ß-arrestin2. We also found that despite ß-arrestin2 overexpression, T282M's and C289W's residency with ß-arrestin2 in endosomes was greatly reduced, leading to decreased ß-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/ß-arrestin2 trafficking and suggest conformationally dependent ß-arrestin-mediated MAPK activation as well as endosomal receptor-ß-arrestin complex stabilization in the mitogenic response of AT1R.