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Background: Endometrial cancer (EC) represents a serious health concern among women globally. Excessive activation of the protooncogene c-Myc (c-Myc) is associated with the proliferation and stemness of EC cells. Phosphocholine (PC), which is synthesized by choline kinase alpha (CHKA) catalysis, is upregulated in EC tumor tissues. The present study aimed to investigate the effect of PC accumulation on EC cells and clarify the relationship between PC accumulation and c-Myc activity in EC., Methods: The c-Myc and CHKA expression in EC tumor tissues were examined using immunohistochemistry. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, western blotting, BrdU staining, and tumorsphere formation assay were used to assess the effect of PC accumulation on EC cells. The mechanism by which PC accumulation inhibits c-Myc was evaluated using RNA-sequencing. Patient-derived organoid (PDO) models were utilised to explore the preclinical efficacy of PC against EC cells., Results: PC accumulation suppressed EC cell proliferation and stemness by inhibiting the activation of the mammalian target of rapamycin (mTOR)-c-Myc signaling. PC accumulation promoted excessive reactive oxygen species production, which reduced the expression of GTPase HRAS. This, in turn, inhibited the mTOR-c-Myc axis and induced EC cell apoptosis. Finally, PC impeded proliferation and downregulated the expression of the mTOR-MYC signaling in EC PDO models., Conclusions: PC accumulation impairs the proliferation ability and stem cell characteristics of EC cells by inhibiting the activated mTOR-c-Myc axis, potentially offering a promising strategy to enhance the efficacy of EC clinical therapy through the promotion of PC accumulation in tumor cells., Competing Interests: Declarations. Conflict of interests: The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of this article. Figures were created with BioRender ( https://biorender.com/ , accessed on November 10th, 2024). Ethical approval and consent to participate: The acquisition and use of human specimens were in concordance with the International Ethical Guidelines for Research Involving Human Subjects, as stated in the Helsinki Declaration, and were approved by the Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (No.: 202019001). The EC tumor tissue samples were collected after obtaining signed informed consent from the participants. The use of endometrial cancer tissue microarray was approved by the Ethics Committee of Shanghai Outdo Biotech Company (No.: SHYJS-CP-1504005). The animals experiments were approved by the Guangzhou Women and Children’s Medical Center Animal Welfare and Ethics Committee (KTDW-2024-00480). Consent for publication: Not applicable., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Eclipta prostrata (Linn.) is a traditional medicinal Chinese herb that displays multiple biological activities, such as encompassing immunomodulatory, anti-inflammatory, anti-tumor, liver-protective, antioxidant, and lipid-lowering effects. Ecliptasaponin A (ESA), a pentacyclic triterpenoid saponin isolated from Eclipta prostrata (Linn.), has been demonstrated to exert superior anti-inflammatory activity against many inflammatory disorders., Aim of the Study: Inflammation plays a critical role in acute myocardial infarction (AMI). This study aims to explore the treatment effects of ESA in AMI, as well as the underlying mechanism., Methods: An AMI mouse model was established in mice via left anterior descending coronary artery (LAD) ligation. After surgery, ESA was injected at doses of 0.5, 1.25, and 2.5 mg/kg, respectively. Myocardial infarction size, cardiomyocyte apoptosis and cardiac echocardiography were studied. The potential mechanism of action of ESA was investigated by RNA-seq, Western blot, surface plasmon resonance (SPR), molecular docking, and immunofluorescence staining., Results: ESA treatment not only significantly reduced myocardial infarct size, decreased myocardial cell apoptosis, and inhibited inflammatory cell infiltration, but also facilitated to improve cardiac function. RNA-seq and Western blot analysis proved that ESA treatment-induced differential expression genes mainly enriched in HMGB1/TLR4/NF-κB pathway. Consistently, ESA treatment resulted into the down-regulation of IL-1β, IL-6, and TNF-α levels after AMI. Furthermore, SPR and molecular docking results showed that ESA could bind directly to HMGB1, thereby impeding the activation of the downstream TLR4/NF-κB pathway. The immunofluorescence staining and Western blot results at the cellular level also demonstrated that ESA inhibited the activation of the HMGB1/TLR4/NF-κB pathway in H9C2 cells., Conclusion: Our study was the first to demonstrate a cardiac protective role of ESA in AMI. Mechanism study indicated that the treatment effects of ESA are mainly attributed to its anti-inflammatory activity that was mediated by the HMGB1/TLR4/NF-κB pathway., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disorder, has garnered increasing attention globally owing to its associated health complications. However, the lack of available therapeutic medications and inadequate management of complications in metabolic dysfunction-associated steatohepatitis (MASH) present significant challenges. There are little studies evaluating the effectiveness of POM in treating MASLD. In this study, we synthesized polyoxometalates (POM) for potential treatment of MASLD., Methods: We induced liver disease in mice using two approaches: feeding a high-fat diet (HFD) to establish MASLD or feeding a methionine-choline deficient (MCD) diet to induce hepatic lipotoxicity and MASH. Various metabolic parameters were detected, and biochemical and histological evaluations were conducted on MASLD. Western blotting, qRT-PCR and immunofluorescence assays were used to elucidate the molecular mechanism of POM in the treatment of MASLD., Results: POM therapy resulted in significant improvements in weight gain, dyslipidemia, liver injury, and hepatic steatosis in mice fed a HFD. Notably, in a more severe dietary-induced MASH model with MCD diet, POM significantly attenuated hepatic lipid accumulation, inflammation, and fibrosis. POM treatment effectively attenuated palmitic acid and oleic acid-induced lipid accumulation in HepG2 and Huh7 cells by targeting the AMPK pathway to regulate lipid metabolism, which was confirmed by AMPK inhibitor. Additionally, the activation of AMPK signaling by POM suppressed the expression of lipid synthesis genes, including sterol regulatory element-binding protein 1c (SREBP1c) and SREBP2, while concurrently upregulating the expression of sirtuin 1 (SIRT1) to promote fatty acid oxidation., Conclusion: These findings suggest that POM is a promising therapeutic strategy with high efficacy in multiple MASLD models., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (© 2024 Wang et al.)

Purpose: Gentamicin is a broad-spectrum antibiotic commonly used in clinical practice. However, the drug causes side effects of ototoxicity, leading to disruption in balance functionality. This study investigated the effect of gastrodin, a prominent compound present in Gastrodia, and the underlying mechanism on the development of gentamicin-induced vestibular dysfunction., Methods: Wild-type C57BL/6 mice were randomly assigned to three groups: control, gentamicin, and gentamicin + gastrodin groups. The extent of gentamicin-induced vestibular impairment was assessed through a series of tests including the swimming test, contact righting reflex test, and air-righting reflex. Alterations in vestibular hair cells were monitored through immunofluorescence assay, and cellular apoptosis was observed using TUNEL staining. The mRNA and protein expression of Notch1, Jagged1, and Hes1 was quantified through qRT-PCR, immunofluorescence, and western blot analyses., Results: Gentamicin treatment led to pronounced deficits in vestibular function and otolith organ hair cells in mice. Nevertheless, pretreatment with gastrodin significantly alleviated these impairments. Additionally, the Notch signaling pathway was activated by gentamicin in the utricle, contributing to a notable increase in the expression levels of apoptosis-associated proteins. By contrast, gastrodin treatment effectively suppressed the Notch signaling pathway, thereby mitigating the occurrence of apoptosis., Conclusion: Collectively, these findings underscore the crucial role of gastrodin in safeguarding against gentamicin-induced vestibular dysfunction through the modulation of the Notch signaling pathway. This study suggests the potential of gastrodin as a promising therapeutic agent for preventing vestibular injuries., Competing Interests: Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

Licorice is a traditional and versatile herbal medicine and food. Glabridin (Gla) is a kind of isoflavone extracted from the licorice root, which has anti-obesity, anti-atherosclerotic, and antioxidative effects. Alcoholic liver disease (ALD) is a widespread liver disease induced by chronic alcohol consumption. However, studies demonstrating the effect of Gla on ALD are rare. The research explored the positive effect of Gla in C57BL/6J mice fed by the Lieber-DeCarli ethanol mice diet and HepG2 cells treated with ethanol. Gla alleviated ethanol-induced liver injury, including reducing liver vacuolation and lipid accumulation. The serum levels of inflammatory cytokines were decreased in the Gla-treated mice. The reactive oxygen species and apoptosis levels were attenuated and antioxidant enzyme activity levels were restored in ethanol-induced mice by Gla treatment. In vitro, Gla reduced ethanol-induced cytotoxicity, nuclear factor kappa B (NF-κB) nuclear translocation, and enhanced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation. Anisomycin (an agonist of p38 MAPK) eliminated the positive role of Gla on ethanol-caused oxidative stress and inflammation. On the whole, Gla can alleviate alcoholic liver damage via the p38 MAPK/Nrf2/NF-κB pathway and may be used as a novel health product or drug to potentially alleviate ALD.

Inhibition of RTK pathways in cancer triggers an adaptive response that promotes therapeutic resistance. Because the adaptive response is multifaceted, the optimal approach to blunting it remains undetermined. TNF upregulation is a biologically significant response to EGFR inhibition in NSCLC. Here, we compared a specific TNF inhibitor (etanercept) to thalidomide and prednisone, two drugs that block TNF and also other inflammatory pathways. Prednisone is significantly more effective in suppressing EGFR inhibition-induced inflammatory signals. Remarkably, prednisone induces a shutdown of bypass RTK signaling and inhibits key resistance signals such as STAT3, YAP and TNF-NF-κB. Combined with EGFR inhibition, prednisone is significantly superior to etanercept or thalidomide in durably suppressing tumor growth in multiple mouse models, indicating that a broad suppression of adaptive signals is more effective than blocking a single component. We identify prednisone as a drug that can effectively inhibit adaptive resistance with acceptable toxicity in NSCLC and other cancers., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients., Competing Interests: Competing Interests Statement The authors declare no competing interests.

Background: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells., Methods: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid., Results: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy., Conclusions: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aberrant epidermal growth factor receptor (EGFR) signaling is widespread in cancer, making the EGFR an important target for therapy. EGFR gene amplification and mutation are common in glioblastoma (GBM), but EGFR inhibition has not been effective in treating this tumor. Here we propose that primary resistance to EGFR inhibition in glioma cells results from a rapid compensatory response to EGFR inhibition that mediates cell survival. We show that in glioma cells expressing either EGFR wild type or the mutant EGFRvIII, EGFR inhibition triggers a rapid adaptive response driven by increased tumor necrosis factor (TNF) secretion, which leads to activation in turn of c-Jun N-terminal kinase (JNK), the Axl receptor tyrosine kinase and extracellular signal-regulated kinases (ERK). Inhibition of this adaptive axis at multiple nodes rendered glioma cells with primary resistance sensitive to EGFR inhibition. Our findings provide a possible explanation for the failures of anti-EGFR therapy in GBM and suggest a new approach to the treatment of EGFR-expressing GBM using a combination of EGFR and TNF inhibition.

Deregulated Sonic Hedgehog (SHH) pathway facilitates the initiation, progression, and metastasis of Non-small cell lung cancer (NSCLC), confers drug resistance and renders a therapeutic interference option to lung cancer patients with poor prognosis. In this study, we screened and evaluated the specificity of a Chinese herb Scutellariabarbata D. Don extraction (SBE) in repressing SHH signaling pathway to block NSCLC progression. Our study confirmed that aberrant activation of the SHH signal pathway conferred more proliferative and invasive phenotypes to human lung cancer cells. This study revealed that SBE specifically repressed SHH signaling pathway to interfere the SHH-mediated NSCLC progression and metastasis via arresting cell cycle progression. We also found that SBE significantly sensitized lung cancer cells to chemotherapeutic agent DDP via repressing SHH components in vitro and in vivo. Mechanistic investigations indicated that SBE transcriptionally and specifically downregulated SMO and consequently attenuated the activities of GLI1 and its downstream targets in SHH signaling pathway, which interacted with cell cycle checkpoint enzymes to arrest cell cycle progression and lead to cellular growth inhibition and migration blockade. Collectively, our results suggest SBE as a novel drug candidate for NSCLC which specifically and sensitively targets SHH signaling pathway.

Epidermal growth factor receptor (EGFR) plays an important role in various types of human cancers. Overexpression of EGFR leads to a constitutive tyrosine phosphorylation of multiple tyrosine residues in the EGFR. Recently, we have demonstrated that overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling depending on the presence or absence of ligand. EGFR constitutively activates transcription factor IRF3, which results in transcription of its target genes. Addition of EGF causes a loss of IRF3 transcriptional activity and activation of canonical signaling pathways such as ERK. The mechanistic basis of this bimodal signaling appears to be the association of a distinct set of signaling proteins with EGFR in the absence or presence of ligand. In this chapter, we describe a detailed protocol for analyses of constitutive EGFR signaling with a focus on IRF3 target genes.

Apelin-13 has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test Apelin-13's effects on ischemic neurovascular unit (NVU) injuries and investigate whether the effects were dependent on vascular endothelial growth factor (VEGF). We detected the expression of VEGF and its receptors (VEGFRs) induced by Apelin-13 injection at 1d, 3d, 7d and 14d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of Apelin-13 on NVU in both in vivo and in vitro experiments as well as whether the effects were VEGF dependent by using VEGF antibody. We also assessed the related signal transduction pathways via multiple inhibitors. We demonstrated Apelin-13 highly increased VEGF and VEGFR-2 expression, not VEGFR-1. Importantly, Apelin-13 led to neurological functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death and astrocyte activation, which was markedly blocked by VEGF antibody. In cell cultures, Apelin-13 protected neurons, astrocytes and endothelial cells against oxygen-glucose deprivation (OGD) injuries. Moreover, the effect of Apelin-13 to up-regulate VEGF was suppressed by extracellular signal-regulated kinase (ERK) inhibitor U0126 and phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002. Our data suggest protective effects of Apelin-13 on ischemic NVU injuries are highly associated with the increase of VEGF binding to VEGFR-2, possibly acting through activation of ERK and PI3K/Akt pathways., (Copyright © 2016. Published by Elsevier Ltd.)

Constitutive activation of the EGFR is common in cancer due to EGFR wild-type (EGFRwt) overexpression or the presence of mutant EGFR. Signaling by constitutively active NSCLC EGFR mutants or the EGFRvIII mutant in glioblastoma has been studied intensively and the downstream signals are known. Normally, the EGFRwt is activated when it is exposed to ligand, resulting in activation of canonical signals such as ERK and Akt. The EGFRwt also becomes tyrosine phosphorylated and constitutively activated without ligand when it is overexpressed, but downstream signals are unclear. Recent studies have identified a noncanonical form of signaling triggered by EGFRwt exclusively in the absence of ligand that does not involve ERK or Akt activation but, instead, results in activation of the transcription factor IRF3. The addition of ligand turns off IRF3-dependent transcription and activates ERK and Akt. Thus, the EGFR triggers distinct and mutually exclusive signaling networks, depending on the presence of ligand. Furthermore, noncanonical EGFRwt signaling may influence response to treatment in cancer. Also, there are reports of both synergistic and antagonistic interactions between ligand-dependent EGFRwt and EGFRvIII signaling. Here, we discuss ligand-independent EGFR signal transduction by oncogenic EGFR mutants and EGFRwt, and review the interplay between EGFRwt and EGFRvIII., (©2015 American Association for Cancer Research.)

We investigate the role of β-catenin signaling in the response of macrophage to lipopolysaccharide (LPS) using RAW264.7 cells. LPS rapidly stimulated cytosolic β-catenin accumulation. β-catenin-mediated transcription was showed to be required for LPS induced gene expression and cell migration. Mechanically, ERK activation-primed GSK3β inactivation by Akt was demonstrated to mediate the LPS induced β-catenin accumulation. Overall, our findings suggest that suppression of GSK3β by ERK stimulates β-catenin signaling therefore contributes to LPS induced cell migration in macrophage activation.

Co-expression of erythropoietin (Epo) and erythropoietin receptor (EpoR) has been found in various non-hematopoietic cancers including hereditary and sporadic renal cell carcinomas (RCC), but the Epo/EpoR autocrine and paracrine mechanisms in tumor progression have not yet been identified. In this study, we used RNA interference method to down-regulate EpoR to investigate the function of Epo/EpoR pathway in human RCC cells. Epo and EpoR co-expressed in primary renal cancer cells and 6 human RCC cell lines. EpoR signaling was constitutionally phosphorylated in primary renal cancer cells, 786-0 and Caki-1 cells, and recombinant human Epo (rhEpo) stimulation had no significant effects on further phosphorylation of EpoR pathway, proliferation, and invasiveness of the cells. Down-regulation of EpoR expression in 786-0 cells by lentivirus-introduced siRNA resulted in inhibition of growth and invasiveness in vitro and in vivo, and promotion of cell apoptosis. In addition, rhEpo stimulation slightly antagonized the anti-tumor effect of Sunitinib on 786-0 cells. Sunitinib could induce more apoptotic cells in 786-0 cells with knockdown EpoR expression. Our results suggested that Epo/EpoR pathway was involved in cell growth, invasion, survival, and sensitivity to the multi-kinases inhibitor Sunitinib in RCC cells.

Background: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms., Methodology/principal Findings: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2., Conclusions: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

Atrial natriuretic peptide (ANP) and TGF-β play counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. We have demonstrated that ANP-cyclic GMP (cGMP)-protein kinase G (PKG) signaling inhibits TGF-β signaling by blocking TGF-β-induced nuclear translocation of mothers against decapentaplegic homolog (Smad)3 in pulmonary artery smooth muscle cells (PASMC). The current study tested the novel hypothesis that activation of the ANP-cGMP-PKG pathway limits TGF-β-induced Smad3 nuclear translocation by enhancing Smad3 binding to cytosolic anchoring proteins in isolated pulmonary artery smooth muscle cells. Cells were pretreated with vehicle or cGMP and then exposed to TGF-β1 treatment. Cytosolic fractions were isolated and immunoprecipitated with a selective anti-Smad3 antibody. Differential proteomic analysis of the cytosolic Smad3-interacting proteins by two-dimensional differential in-gel electrophoresis and mass spectroscopy followed by coimmunoprecipitation and immunostaining demonstrated that Smad3 was bound to β2-tubulin in a TGF-β1/cGMP-dependent manner: binding of Smad3 to β2-tubulin was decreased by TGF-β1 and increased by cGMP treatment. A site-directed mutagenesis study demonstrated that mutating Smad3 at Thr388, but not Ser309, two potential sites of PKG-induced hyperphosphorylation, inhibited cGMP-induced Smad3 binding to β2-tubulin. Further, luciferase reporter analysis showed that muation of T388 in Smad3 abolished the inhibitory effect of cGMP on TGF-β1-induced plasminogen activator inhibitor-1 (PAI-1) transcription. In addition, disruption of β2-tubulin with the microtubule depolymerizers nocodazole and colchicine promoted Smad3 dissociation from β2-tubulin, increased both TGF-β1-induced Smad3 nuclear translocation and PAI-1 mRNA expression, and abolished the inhibitory effects of cGMP on these processes. In contrast, the microtubule stabilizers paclitaxel and epothilone B increased cytosolic Smad3 binding to β2-tubulin and enhanced the inhibitory effect of cGMP on Smad3 nuclear translocation and PAI-1 expression in response to TGF-β1. These provocative findings suggest that sequestering Smad3 by β2-tubulin in cytosol is a key mechanism by which ANP-cGMP-PKG signaling interferes with downstream signaling from TGF-β and thus protects against pulmonary arterial remodeling in response to hypoxia stress.

This study utilized a transgenic mouse model that expresses an inducible dominant-negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII) to define the structural and functional responses of the left ventricle (LV) to pressure-overload stress in the absence of an intact TGF-beta signaling cascade. DnTGFbetaRII and nontransgenic (NTG) control mice (male, 8-10 wk) were randomized to receive Zn(2+) (25 mM ZnSO(4) in drinking H(2)O to induce DnTGFbetaRII gene expression) or control tap H(2)O and then further randomized to undergo transverse aortic constriction (TAC) or sham surgery. At 7 days post-TAC, interstitial nonmyocyte proliferation (Ki67 staining) was greatly reduced in LV of DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. At 28 and 120 days post-TAC, collagen deposition (picrosirius-red staining) in LV was attenuated in DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. LV end systolic diameter and end systolic and end diastolic volumes were markedly increased, while ejection fraction and fractional shortening were significantly decreased in TAC-DnTGFbetaRII+Zn(2+) mice compared with the other groups at 120 days post-TAC. These data indicate that interruption of TGF-beta signaling attenuates pressure-overload-induced interstitial nonmyocyte proliferation and collagen deposition and promotes LV dilation and dysfunction in the pressure-overloaded heart, thus creating a novel model of dilated cardiomyopathy.

Ciliary neurotrophic factor (CNTF) forms a functional receptor complex containing the CNTF receptor, gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature and stoichiometry of the receptor-mediated interactions in this complex have not yet been fully resolved. We show here that signaling by CNTF, but not by LIF or oncostatin M (OSM), was abolished in cells overexpressing a LIFR mutant with the N-terminal cytokine binding domain deleted. Our results illustrate molecular differences between the CNTF active receptor complex and those of LIF and OSM and provide further support for the hexameric model of the CNTF receptor complex.

Eph receptors and their cognate ligands ephrins are important players in axon guidance and neural patterning during development of the nervous system. Much of our knowledge about the signal transduction pathways triggered by Eph receptors has been related to the modulation of actin cytoskeleton, which is fundamental in mediating the cellular responses in growth cone navigation, cell adhesion, and cell migration. In contrast, little was known about whether long term activation of Eph receptor would regulate gene expression. Here we report a novel signaling pathway of EphA4, which involves activation of the tyrosine kinase Jak2 and the transcriptional activator Stat3. Transfection of COS7 cells with EphA4, but not the kinase-dead mutant, induced tyrosine phosphorylation of Jak2, Stat1, and Stat3. Treatment of cultured C2C12 myotubes with ephrin-A1 also induced tyrosine phosphorylation of Stat3, which was abolished by the Jak2 inhibitor AG490. Moreover, Jak2 was co-immunoprecipitated with EphA4 in muscle, and both proteins were concentrated at the neuromuscular junction (NMJ) of adult muscle. By using microarray analysis, we have identified acetylcholinesterase, the critical enzyme that hydrolyzed the neurotransmitter acetylcholine at the NMJ, as a downstream target gene of the Jak/Stat pathway in muscle. More importantly, ephrin-A1 increased the expression of acetylcholinesterase protein in C2C12 myotubes, which was abolished by AG490. In contrast, ephrin-A1 reduced the expression of fibronectin mRNA in C2C12 myotubes independently of Jak2. Finally, the expression level of acetylcholinesterase in limb muscle of EphA4 null mice was significantly reduced compared with the wild-type control. Taken together, these results have identified Jak/Stat proteins as the novel downstream targets of EphA4 signaling. In addition, the present study provides the first demonstration of a potential function of Eph receptors and Jak/Stat proteins at the NMJ.

Objective: To investigate the transduction of Wnt/Frizzled signaling pathway, especially the function of T cell factor 4 (TCF(4)), in renal carcinoma., Methods: A renal carcinoma yeast two hybrid library and a human TCF(4) yeast two hybrid expression vector were constructed. Proteins interacting with the bait protein human TCF(4) were obtained from the renal carcinoma yeast two hybrid library by reverse yeast two hybrid system., Results: 67 positive clones interacting with the bait protein TCF(4) were obtained by reverse yeast two hybrid system, including 18 beta-catenin clones, 24 TCF(4) clones and 25 unknown clones., Conclusion: Wnt/Frizzled signaling pathway exists in renal carcinoma. TCF(4), its important signal factor, interacts with beta-catenin and forms homodimer or homocopolymer by itself, thus displaying its constitutive transcriptional activity.

Cyclic AMP was the first second messenger to be identified. After five decades of research, much is currently known about its biological functions and clinical implications. Several components of the cAMP signalling pathways, such as the G-protein coupled receptors and the phosphodiesterases, have become sensitive and specific drug targets for a host of clinical applications. Surprisingly, very little effort has been invested so far into the study of cAMP signalling in parasites, and its significance in host/parasite interaction. Our laboratory has embarked on a study of cAMP signalling in Trypanosoma brucei. A newly identified adenylyl cyclase, GRESAG4.4B, a member of a small family of closely related genes, is being used as a model molecule for investigating the mechanisms which control cyclase activity in the T. brucei cell. On the other hand, a number of genes for different families of cAMP-specific phosphodiesterases have been identified and characterised. One enzyme, TbPDE1, is coded for by a single-copy gene. Knock-outs of this gene display an almost normal phenotype in culture, indicating that TbPDE1 is not an essential enzyme under culture conditions. A second phosphodiesterase which is being studied in detail, TbPDE2A, is clearly different from TbPDE1, and it is coded for by a member of a small gene family containing about six similar, but non-identical genes. TbPDE2A, as TbPDE1, is specific for cAMP. In its N-terminal, it contains a GAF domain which may represent an allosteric cGMP-binding site. The other members of the TbPDE2 family all exhibit strongly conserved catalytic domains, but vary widely in their N-terminal regulatory domains. With regard to downstream signalling by the cAMP generated through the interplay of adenylyl cyclases and phosphodiesterases, we have recently identified a single-copy gene (TbRSU1) which codes for a putative regulatory subunit of the cAMP-regulated protein kinase A. This protein exhibits considerable similarity with its mammalian counterparts. Immunoprecipitation co-precipitates a protein kinase activity with the characteristics of protein kinase A.