Search

Background: Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor. Results: RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane. Conclusions: Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis. [ABSTRACT FROM AUTHOR]

Taurine (2-aminoethanesulfonic acid) could reduce serum and liver cholesterol concentrations via regulating expression and the activity of cholesterol 7α-hydroxylase (CYP7A1). To investigate the possible role of calcineurin in taurine upregulating CYP7A1 expression in HepG2 cells under high cholesterol conditions. High cellular cholesterol conditions were achieved using 0.2 mM cholesterol in HepG2 cells. Calcineurin was decreased by FK506 and was depleted in CnAβ-/- cells. HepG2 cells were cultured in a taurine-containing medium for 24 or 48 h. The levels of total intracellular cholesterol were determined by an enzymatic method, and the expression levels of calcineurin, CYP7A1, and key transcriptional regulatory molecules were detected by western blotting. High-cholesterol resulted in increased CYP7A1 and calcineurin expressions. Taurine exhibited cholesterol-lowering effects regardless of low/high cholesterol conditions or calcineurin status. Taurine induced the expression of CYP7A1, which was abolished by inhibiting or deleting calcineurin. Taurine suppressed MEK1/2, p-c-Jun, and SHP-1, key molecules in one inhibitory pathway of CYP7A1 transcription. In contrast, suppression of MEK1/2 but not p-c-Jun or SHP-1 was reversed after completely knocking down calcineurin. Calcineurin is required for taurine's upregulation of CYP7A1 expression through inhibiting MEK1/2, which was partly responsible for taurine's cholesterol-lowering effect. [ABSTRACT FROM AUTHOR]

Hearing loss is one of the most common types of disability; however, there is only one FDA-approved drug to prevent any type of hearing loss. Treatment with the highly effective chemotherapy agent, cisplatin, and exposure to high-decibel noises are two of the most common causes of hearing loss. The mitogen-activated protein kinase (MAPK) pathway, a phosphorylation cascade consisting of RAF, MEK1/2, and ERK1/2, has been implicated in both types of hearing loss. Pharmacologically inhibiting BRAF or ERK1/2 is protective against noise- and cisplatin-induced hearing loss in multiple mouse models. Trametinib, a MEK1/2 inhibitor, protects from cisplatin-induced outer hair cell death in mouse cochlear explants; however, to the best of our knowledge, inhibiting MEK1/2 has not yet been shown to be protective against hearing loss in vivo. In this study, we demonstrate that trametinib protects against cisplatin-induced hearing loss in a translationally relevant mouse model and does not interfere with cisplatin's tumor-killing efficacy in cancer cell lines. Higher doses of trametinib were toxic to mice when combined with cisplatin, but lower doses of the drug were protective against hearing loss without any known toxicity. Trametinib also protected mice from noise-induced hearing loss and synaptic damage. This study shows that MEK1/2 inhibition protects against both insults of hearing loss, as well as that targeting all three kinases in the MAPK pathway protects mice from cisplatin- and noise-induced hearing loss. [ABSTRACT FROM AUTHOR]

Abstract Background Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of digestive system tumor related death in the world. Unfortunately, effective chemopreventive agent is lack for patients with ESCC in clinical practice, which leads to the extremely high mortality rate. Methods A library of prescribed drugs was screened for finding critical anti-tumor properties in ESCC cells. The phosphoproteomics, kinase array, pulldown assay and drug affinity responsive target stabilization assay (DARTS) were applied to explore mechanisms and searched for synergistic targets. Established models of PDX in mice were used to determine the therapeutic effect of domperidone. Results After screening a library of prescribed drugs, we discovered that domperidone has anti-tumor properties. Domperidone, acting as a gastroprokinetic agent, has been widely used in clinic for gastrointestinal motility disorders. Despite limited research, there are indications that domperidone may have anti-tumor properties. In this study, we determined that domperidone significantly inhibited ESCC proliferation in vitro and in vivo. We employed phosphoproteomics to reveal p-ERK, and p-SMAD3 down-regulation upon domperidone treatment. Then, the results of kinase assay and pulldown assay further validated that domperidone directly combined with MEK1/2 and CDK4, leading to the inhibition of their kinase activity. Furthermore, our results revealed that MEK/ERK and CDK4/SMAD3 signal pathway were major pathways in domperidone against ESCC. Conclusion Collectively, these findings suggest that domperidone serves as an effective “multi-target” inhibitor of MEK1/2 and CDK4, offering potential benefits for the chemoprevention of ESCC.

Simple Summary: Pancreatic ductal adenocarcinoma continues to be one of the deadliest cancers worldwide. Preclinical studies involving animals rarely include sex as a major biological variable in testing the efficacy of new drugs. In an animal model of pancreatic cancer, we analyzed the impact of sex on the pathological features of the disease and on an experimental small molecule-based therapy tested in vivo for the first time. While the therapy shows potential, the obtained results are confounded by sex-specific effects. This study, therefore, highlights the importance of sex-inclusive research while simultaneously providing a basis for further studies of the therapy tested. Preclinical studies rarely test the efficacy of therapies in both sexes. The field of oncology is no exception in this regard. In a model of syngeneic, orthotopic, metastasized pancreatic ductal adenocarcinoma we evaluated the impact of sex on pathological features of this disease as well as on the efficacy and possible adverse side effects of a novel, small molecule-based therapy inhibiting KRAS:SOS1, MEK1/2 and PI3K signaling in male and female C57BL/6J mice. Male mice had less tumor infiltration of CD8-positive cells, developed bigger tumors, had more lung metastasis and a lower probability of survival compared to female mice. These more severe pathological features in male animals were accompanied by higher distress at the end of the experiment. The evaluated inhibitors BI-3406, trametinib and BKM120 showed synergistic effects in vitro. This combinatorial therapy reduced tumor weight more efficiently in male animals, although the drug concentrations were similar in the tumors of both sexes. These results underline the importance of sex-specific preclinical research and at the same time provide a solid basis for future studies with the tested compounds. [ABSTRACT FROM AUTHOR]

Background: Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of digestive system tumor related death in the world. Unfortunately, effective chemopreventive agent is lack for patients with ESCC in clinical practice, which leads to the extremely high mortality rate. Methods: A library of prescribed drugs was screened for finding critical anti-tumor properties in ESCC cells. The phosphoproteomics, kinase array, pulldown assay and drug affinity responsive target stabilization assay (DARTS) were applied to explore mechanisms and searched for synergistic targets. Established models of PDX in mice were used to determine the therapeutic effect of domperidone. Results: After screening a library of prescribed drugs, we discovered that domperidone has anti-tumor properties. Domperidone, acting as a gastroprokinetic agent, has been widely used in clinic for gastrointestinal motility disorders. Despite limited research, there are indications that domperidone may have anti-tumor properties. In this study, we determined that domperidone significantly inhibited ESCC proliferation in vitro and in vivo. We employed phosphoproteomics to reveal p-ERK, and p-SMAD3 down-regulation upon domperidone treatment. Then, the results of kinase assay and pulldown assay further validated that domperidone directly combined with MEK1/2 and CDK4, leading to the inhibition of their kinase activity. Furthermore, our results revealed that MEK/ERK and CDK4/SMAD3 signal pathway were major pathways in domperidone against ESCC. Conclusion: Collectively, these findings suggest that domperidone serves as an effective "multi-target" inhibitor of MEK1/2 and CDK4, offering potential benefits for the chemoprevention of ESCC. [ABSTRACT FROM AUTHOR]

Abstract Background Disrupted germline differentiation or compromised testis development can lead to subfertility or infertility and are strongly associated with testis cancer in humans. In mice, SRY and SOX9 induce expression of Fgf9, which promotes Sertoli cell differentiation and testis development. FGF9 is also thought to promote male germline differentiation but the mechanism is unknown. FGFs typically signal through mitogen-activated protein kinases (MAPKs) to phosphorylate ERK1/2 (pERK1/2). We explored whether FGF9 regulates male germline development through MAPK by inhibiting either FGF or MEK1/2 signalling in the foetal testis immediately after gonadal sex determination and testis cord formation, but prior to male germline commitment. Results pERK1/2 was detected in Sertoli cells and inhibition of MEK1/2 reduced Sertoli cell proliferation and organisation and resulted in some germ cells localised outside of the testis cords. While pERK1/2 was not detected in germ cells, inhibition of MEK1/2 after somatic sex determination profoundly disrupted germ cell mitotic arrest, dysregulated a broad range of male germline development genes and prevented the upregulation of key male germline markers, DPPA4 and DNMT3L. In contrast, while FGF inhibition reduced Sertoli cell proliferation, expression of male germline markers was unaffected and germ cells entered mitotic arrest normally. While male germline differentiation was not disrupted by FGF inhibition, a range of stem cell and cancer-associated genes were commonly altered after 24 h of FGF or MEK1/2 inhibition, including genes involved in the maintenance of germline stem cells, Nodal signalling, proliferation, and germline cancer. Conclusions Together, these data demonstrate a novel role for MEK1/2 signalling during testis development that is essential for male germline differentiation, but indicate a more limited role for FGF signalling. Our data indicate that additional ligands are likely to act through MEK1/2 to promote male germline differentiation and highlight a need for further mechanistic understanding of male germline development.

Background: Disrupted germline differentiation or compromised testis development can lead to subfertility or infertility and are strongly associated with testis cancer in humans. In mice, SRY and SOX9 induce expression of Fgf9, which promotes Sertoli cell differentiation and testis development. FGF9 is also thought to promote male germline differentiation but the mechanism is unknown. FGFs typically signal through mitogen-activated protein kinases (MAPKs) to phosphorylate ERK1/2 (pERK1/2). We explored whether FGF9 regulates male germline development through MAPK by inhibiting either FGF or MEK1/2 signalling in the foetal testis immediately after gonadal sex determination and testis cord formation, but prior to male germline commitment. Results: pERK1/2 was detected in Sertoli cells and inhibition of MEK1/2 reduced Sertoli cell proliferation and organisation and resulted in some germ cells localised outside of the testis cords. While pERK1/2 was not detected in germ cells, inhibition of MEK1/2 after somatic sex determination profoundly disrupted germ cell mitotic arrest, dysregulated a broad range of male germline development genes and prevented the upregulation of key male germline markers, DPPA4 and DNMT3L. In contrast, while FGF inhibition reduced Sertoli cell proliferation, expression of male germline markers was unaffected and germ cells entered mitotic arrest normally. While male germline differentiation was not disrupted by FGF inhibition, a range of stem cell and cancer-associated genes were commonly altered after 24 h of FGF or MEK1/2 inhibition, including genes involved in the maintenance of germline stem cells, Nodal signalling, proliferation, and germline cancer. Conclusions: Together, these data demonstrate a novel role for MEK1/2 signalling during testis development that is essential for male germline differentiation, but indicate a more limited role for FGF signalling. Our data indicate that additional ligands are likely to act through MEK1/2 to promote male germline differentiation and highlight a need for further mechanistic understanding of male germline development. [ABSTRACT FROM AUTHOR]

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2—extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1β and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1β expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1β, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress growth, induce cell cycle defects, and apoptosis, as well as to rescue the expression of the pro-apoptotic BH3-only proteins BIM and BMF. A RNAseq analysis of GSCs revealed that HDACi repressed the pro-survival BCL-2 family genes MCL1 and BCL-XL. We therefore replaced MEKi with BCL-2 family inhibitors and observed enhanced apoptosis. Conversely, a ligand for the cancer stem cell receptor CD44 led to reductions in BMF, BIM, and apoptosis. Our data strongly support further testing of HDACi in combination with MEKi or BCL-2 family inhibitors in glioma. [ABSTRACT FROM AUTHOR]

Abstract Homo sapiens chromosome 2 clone RP11-339H12 (AC010883.5) is a dysregulated long non-coding RNA (lncRNA) that has never been investigated in cervical cancer (CC). Thus, the potential function and molecular mechanism remain unclear. Our study explored the biological function of AC010883.5 to determine the underlying mechanisms in CC and provide potential therapeutic targets for improving the clinical treatment strategy. We used quantitative real-time polymerase chain reaction to measure mitochondrial RNA levels and western blot to measure the protein levels of target genes. Further, we used Cell Counting Kit-8 and 5‐Bromo-2'-deoxyuridine incorporation assays to evaluate cell proliferation in vitro. Cell apoptosis was analyzed by flow cytometry. Cell invasion was analyzed by wound healing and Transwell migration assays was ued to analyze cell migration. Finally, the biological function and mechanism of AC010883.5 in CC growth were evaluated by in vivo xenograft assay. AC010883.5 was enhanced in CC tissues and cell lines, and enhanced AC010883.5 expression accelerated CC cell proliferation, migration, and invasion and induced epithelial–mesenchymal transition in vitro and in vivo. AC010883.5 also activated the mitogen-activated protein kinase (MAPK) signaling pathway by promoting phosphorylation of extracellular signal-regulated kinase 1/2 (i.e., ERK1/2) and MAPK kinase 1/2 (i.e., MEK1/2). Blocking the MAPK signaling pathway could counteract the pro-proliferative, pro-migrative, and pro-invasive effects of AC010883.5 over-expression. We found that the lncRNA, AC010883.5, is an oncogenic molecule involved in CC tumor progression via dysregulation of the MAPK signaling pathway, implying that AC010883.5 could be a tumor progression and therapeutic response biomarker.

Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As D3R regulates the activity of the Akt/mTOR and MEK/ERK1/2 pathways, we assessed the effects of SB-277011-A, a D3R antagonist, on the activity of these kinases during the reinstatement of cocaine-induced conditioned place preference (CPP) induced by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated an extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Cocaine-seeking behavior reactivation was correlated with decreased p-Akt, p-mTOR, and p-ERK1/2 activation in both nuclei of restrained animals. While a D3R blockade prevented stress-induced CPP reinstatement and plasma corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR, and ERK1/2 activation depending on the stressor and the dose used. Our data support the involvement of corticosterone in the SB-277011-A effects in restrained animals. Additionally, the ratios p-mTOR/mTOR and/or p-ERK1/2 /ERK1/2 in the BLA during stress-induced relapse seem to be related to the locomotor activity of animals receiving 48 mg/kg of the antagonist. Hence, our study indicates the D3R antagonist's efficacy to prevent stress-induced relapses in drug use through distinct modulation of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing nuclei. [ABSTRACT FROM AUTHOR]

Homo sapiens chromosome 2 clone RP11-339H12 (AC010883.5) is a dysregulated long non-coding RNA (lncRNA) that has never been investigated in cervical cancer (CC). Thus, the potential function and molecular mechanism remain unclear. Our study explored the biological function of AC010883.5 to determine the underlying mechanisms in CC and provide potential therapeutic targets for improving the clinical treatment strategy. We used quantitative real-time polymerase chain reaction to measure mitochondrial RNA levels and western blot to measure the protein levels of target genes. Further, we used Cell Counting Kit-8 and 5‐Bromo-2'-deoxyuridine incorporation assays to evaluate cell proliferation in vitro. Cell apoptosis was analyzed by flow cytometry. Cell invasion was analyzed by wound healing and Transwell migration assays was ued to analyze cell migration. Finally, the biological function and mechanism of AC010883.5 in CC growth were evaluated by in vivo xenograft assay. AC010883.5 was enhanced in CC tissues and cell lines, and enhanced AC010883.5 expression accelerated CC cell proliferation, migration, and invasion and induced epithelial–mesenchymal transition in vitro and in vivo. AC010883.5 also activated the mitogen-activated protein kinase (MAPK) signaling pathway by promoting phosphorylation of extracellular signal-regulated kinase 1/2 (i.e., ERK1/2) and MAPK kinase 1/2 (i.e., MEK1/2). Blocking the MAPK signaling pathway could counteract the pro-proliferative, pro-migrative, and pro-invasive effects of AC010883.5 over-expression. We found that the lncRNA, AC010883.5, is an oncogenic molecule involved in CC tumor progression via dysregulation of the MAPK signaling pathway, implying that AC010883.5 could be a tumor progression and therapeutic response biomarker. [ABSTRACT FROM AUTHOR]

[Display omitted] Inflammasomes are cytosolic supramolecular complexes that play a key role in the innate immune response. Overactivation of NLR family pyrin domain containing 3 (NLRP3) inflammasome leads to multiple diseases. Post-translational modifications (PTMs) are essential modulators of inflammasomes especially in activation phase. Here we found that MEK1/2 kinase activity was indispensable in NLRP3 inflammasome activation both in vitro and in vivo. Inhibition of MEK1/2 resulted in reactive oxygen species (ROS) scavenging and ubiquitination of NLRP3, which further blocked NLRP3 inflammasome activation. These effects were independent of ERK1/2, which were classic downstream of MEK1/2. These investigations proposed a mechanism that MEK1/2 regulated inflammation via non-transcriptional regulation of NLRP3 inflammasome and might help better understanding the effects and side-effects of MEK inhibitors in clinical use. [ABSTRACT FROM AUTHOR]

Berberine (BBR), a natural isoquinoline alkaloid exhibiting insulin sensitizing activity, has been applicated in the treatment of diabetes. However, until now, the exact target of BBR has not been well investigated. Here, primary hepatocytes pre-treated with TNF-α were used to evaluate the role of BBR on hepatic insulin sensitivity. Western blot and immunoprecipitation were used to investigate the effect of BBR on the crosstalk between TNF-α pathway and insulin signaling pathway. Molecular docking was used to verify the interactions between BBR and its potential targets. BBR inhibits the MEKK1 and MEK1/2, and thus suppresses the activation of their downstream ERK1/2. It attenuates the ERK1/2-induced serine phosphorylation of IRS-1 and thus enhances IRS-1 tyrosine phosphorylation and Akt activation. By molecular docking, BBR is proved to efficiently bind MEK1/2. MEKK1 is also considered as BBR target for its similarity in primary structure with MEK1/2. In conclusion, BBR ameliorates TNF-α-induced hepatic insulin resistance by targeting MEKK1 and MEK1/2. [ABSTRACT FROM AUTHOR]

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin‐mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small‐molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC‐1‐151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP‐2, FRA‐1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical‐size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex‐specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin‐mediated actions on bone formation in vivo and in a sex‐specific manner. [ABSTRACT FROM AUTHOR]

Abstract Neuroblastoma is the most common extra-cranial solid tumor in children, representing approximately 8% of all malignant childhood tumors and 15% of pediatric cancer-related deaths. Recent sequencing and transcriptomics studies have demonstrated the RAS-MAPK pathway’s contribution to the development and progression of neuroblastoma. This review compiles up-to-date evidence of this pathway’s involvement in neuroblastoma. We discuss the RAS-MAPK pathway’s general functioning, the clinical implications of its deregulation in neuroblastoma, and current promising therapeutics targeting proteins involved in signaling.

Search for the effective approaches to treat acute inflammation caused by combination of allergens and infectious agents is an important task for public health worldwide. House dust mites Dermatophagoides pteronyssinus are the source of allergens of the Der p groups and of microbial compounds, in particular, lipopolysaccharides (LPS). LPS and Der p 2 induce secretion of pro-inflammatory cytokines via activation of kinases p38 MAPK, MEK1/2, and PI3K. Participation of these kinases in the regulation of cells response to combined exposure to LPS and Der p 2 has not been sufficiently studied. We studied the effects of kinases (p38 MAPK, MEK1/2, and PI3K) inhibition on secretion of cytokines (TNF, IL-8, and IL-6) by peripheral blood mononuclear cells (PBMC) of healthy volunteers in response to E. coli LPS and rDer p 2. Contribution of kinases to the regulation of cell response to different agents (rDer p 2 and/or LPS) was revealed. It was found that p38 MAPK plays a key role in the regulation of secretion TNF by PBMC in response to the combination of LPS and rDer p 2. MEK1/2-dependent signaling is the main pathway for the synthesis of TNF and IL-8 in response to LPS and rDer p 2. PI3K-dependent signaling negatively regulates TNF production during rDer p 2-induced cell activation, but is not involved in the response to the combination of LPS and rDer p 2. PI3K-dependent signaling in the regulation of PBMC cytokine synthesis is most pronounced in response to their activation by rDer p 2. Understanding the mechanisms of immune cell responses to combinations of inflammatory agents could facilitate the search for new intracellular targets for anti-inflammatory therapy. [ABSTRACT FROM AUTHOR]

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is frequently de-regulated in human cancer. Melanoma in particular exhibits a high incidence of activating BRAFV600E/K and NRASQ61L/K mutations and such cells are addicted to the activity of these mutant oncoproteins. As a result three different BRAF inhibitors (BRAFi) have now been approved for BRAFV600E/K- mutant melanoma and have transformed the treatment of this disease. Despite this, clinical responses are typically transient as tumour cells develop resistance. These resistance mechanisms frequently involve reinstatement of ERK1/2 signalling and BRAFi are now deployed in combination with one of three approved MEK1/2 inhibitors (MEKi) to provide more durable, but still transient, clinical responses. Furthermore, inhibitors to ERK1/2 (ERK1/2i) have also been developed to counteract ERK1/2 signalling. However, recent studies have suggested that BRAFi/MEKi and ERK1/2i resistance can arise through activation of a parallel signalling pathway leading to activation of ERK5, an unusual protein kinase that contains both a kinase domain and a transcriptional transactivation domain. Here we review the evidence supporting ERK5 as a mediator of BRAFi/MEKi and ERK1/2i resistance. We also review the challenges in targeting ERK5 signalling with small molecules, including paradoxical activation of the transcriptional transactivation domain, and discuss new therapeutic modalities that could be employed to target ERK5.

A previous study from our team found that hyperbaric oxygen (HBO) pretreatment attenuated decompression sickness (DCS) spinal cord injury by upregulating heat shock protein 32 (HSP32) via the ROS/p38 MAPK pathway. Meanwhile, a MEK1/2-negative regulatory pathway was also activated to inhibit HSP32 overexpression. The purpose of this study was to determine if normobaric oxygen (NBO) might effectively induce HSP32 while concurrently inhibiting MEK1/2 and to observe any protective effects on spinal cord injury in DCS rats. The expression of HSP32 in spinal cord tissue was measured at 6, 12, 18, and 24 h following NBO and MEK1/2 inhibitor U0126 pretreatment. The peak time of HSP32 was observed at 12 h after simulated air diving. Subsequently, signs of DCS, hindlimb motor function, and spinal cord and serum injury biomarkers were recorded. NBO-U0126 pretreatment significantly decreased the incidence of DCS, improved motor function, and attenuated oxidative stress, inflammatory response, and apoptosis in both the spinal cord and serum. These results suggest that pretreatment with NBO and U0126 combined can effectively alleviate DCS spinal cord injury in rats by upregulating HSP32. This may lead to a more convenient approach for DCS injury control, using non-pressurized NBO instead of HBO.

Neuroblastoma is the most common extra-cranial solid tumor in children, representing approximately 8% of all malignant childhood tumors and 15% of pediatric cancer-related deaths. Recent sequencing and transcriptomics studies have demonstrated the RAS-MAPK pathway's contribution to the development and progression of neuroblastoma. This review compiles up-to-date evidence of this pathway's involvement in neuroblastoma. We discuss the RAS-MAPK pathway's general functioning, the clinical implications of its deregulation in neuroblastoma, and current promising therapeutics targeting proteins involved in signaling. [ABSTRACT FROM AUTHOR]

A previous study from our team found that hyperbaric oxygen (HBO) pretreatment attenuated decompression sickness (DCS) spinal cord injury by upregulating heat shock protein 32 (HSP32) via the ROS/p38 MAPK pathway. Meanwhile, a MEK1/2-negative regulatory pathway was also activated to inhibit HSP32 overexpression. The purpose of this study was to determine if normobaric oxygen (NBO) might effectively induce HSP32 while concurrently inhibiting MEK1/2 and to observe any protective effects on spinal cord injury in DCS rats. The expression of HSP32 in spinal cord tissue was measured at 6, 12, 18, and 24 h following NBO and MEK1/2 inhibitor U0126 pretreatment. The peak time of HSP32 was observed at 12 h after simulated air diving. Subsequently, signs of DCS, hindlimb motor function, and spinal cord and serum injury biomarkers were recorded. NBO-U0126 pretreatment significantly decreased the incidence of DCS, improved motor function, and attenuated oxidative stress, inflammatory response, and apoptosis in both the spinal cord and serum. These results suggest that pretreatment with NBO and U0126 combined can effectively alleviate DCS spinal cord injury in rats by upregulating HSP32. This may lead to a more convenient approach for DCS injury control, using non-pressurized NBO instead of HBO. [ABSTRACT FROM AUTHOR]

Abstract Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome‐wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)‐seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1‐regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression‐free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer.

Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome‐wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)‐seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1‐regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression‐free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer. [ABSTRACT FROM AUTHOR]

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

Abstract Background The large-conductance, voltage-gated, calcium (Ca (2+))-activated potassium channel (BKCa) plays an important role in regulating Ca (2+) signaling and cell physiological function, and is aberrantly expressed in some types of cancers. The present study focuses on identifying the oncogenic potential and clinical significance of BKCa in endometrial adenocarcinoma, as well as exploring the mechanistic relevance by 17β -estradiol (E2) inducing aberrant activation of MEK1/2 and ERK1/2 via BKCa. Methods The expression of BKCa, ERK1/2 and p-ERK1/2 were examined by immunohistochemical staining in 263 cases, including 185 primary types I endometrial cancer tissues, 38 atypical endometrial hyperplasia tissues and 40 normal endometrium tissues. Cell growth, cycle, apoptosis rate, migration and invasion was separately tested in Ishikawa cells using siRNA-BKCa and/or E2 treatment, as well as the expression of these interested proteins by western blot analysis. Results We showed that expression of BKCa is significantly elevated in 185 types I endometrial adenocarcinoma tissues compared to those of the normal endometrium and atypical endometrial hyperplasia tissues. Furthermore, in vitro observations revealed that down-regulation of BKCa expression inhibited cell growth by both enhancing apoptosis and blocking G1/S transition, suppressed cell migration and invasion in Ishakiwa cells, and decreased the expression of p-MEK1/2 and p-ERK1/2. Additionally, RNAi-mediated knockdown of BKCa attenuated the increased cellular growth and invasion, as well as the elevated expression of p-MEK1/2 and p-ERK1/2 proteins, induced by E2 stimulation. More importantly, the aberrant expression of BKCa and p-ERK1/2 were closely related with poor prognostic factors in type I endometrial cancer, and up-regulated expression of p-ERK1/2 was significantly associated with shorter disease-free survival (DFS) and overall survival (OS) and was an independent prognostic factor in type I endometrial cancer patients. Conclusion Our results demonstrated that BKCa and the key downstream effectors p-ERK1/2 could be involved in important signaling pathways in initiation and development of endometrial adenocarcinoma and may provide a new therapeutic approach for women with endometrial cancer.

Summary: Background: This Phase Ib study explored combination dosing of the allosteric MEK1/2 inhibitor cobimetinib and the ATP-competitive pan-AKT inhibitor ipatasertib. Methods: Patients with advanced solid tumors were enrolled to two dose escalation arms, each using a 3 + 3 design in 28-day cycles. In Arm A, patients received concurrent cobimetinib and ipatasertib on days 1–21. In Arm B, cobimetinib was administered intermittently with ipatasertib for 21 days. Primary objectives evaluated dose-limiting toxicities (DLTs), maximum tolerated doses (MTD), and the recommended Phase II dose (RP2D). Secondary objectives included analysis of pharmacokinetic parameters, MAPK and PI3K pathway alterations, changes in tissue biomarkers, and preliminary anti-tumor efficacy. Expansion cohorts included patients with PTEN-deficient triple-negative breast cancer and endometrial cancer. Results: Among 66 patients who received ≥1 dose of study drug, all experienced an adverse event (AE). Although no DLTs were reported, 6 patients experienced Cycle 1 DLT-equivalent AEs. The most common treatment-related AEs were diarrhea, nausea, vomiting, dermatitis acneiform, and fatigue. Thirty-five (53%) patients experienced drug-related AEs of ≥ grade 3 severity. Cobimetinb/ipatasertib MTDs were 60/200 mg on Arm A and 150/300 mg on Arm B; the latter was chosen as the RP2D. No pharmacokinetic interactions were identified. Biomarker analyses indicated pathway blockade and increases in IFNγ and PD-L1 gene expression following the combination. Three patients with endometrial or ovarian cancer achieved partial response, all with PTEN-low disease and two with tumor also harboring KRAS mutation. Conclusion: There was limited tolerability and efficacy for this MEK and AKT inhibitor combination. Nonetheless, pharmacodynamic analyses indicated target engagement and suggest rationale for further exploration of cobimetinib or ipatasertib in combination with other anticancer agents. ClinicalTrials.gov identifier: NCT01562275. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is categorized as an autoimmune disease that leads to bone or joint deformity due to altered immune response. Studies have concluded the role of inflammation and oxidative stress in the progression of RA and agents inhibiting these processes showed benificial effect against the disease. Xuebijing (XBJ) injection is an intravenous patent preparation made from five-traditional Chinese medicines. Previous studies showed its excellent pharmacological activities, such as against sepsis, inflammation, and oxidative stress which has encouraged us to investigate the protective effect of XBJ against rheumatoid arthritis cell line (MH7A). For this purpose, the effect of XBJ was quantified on several parameters on the human synovial MH7A cell line activated with tumor necrosis factor-α (TNF-α). The results of the study showed that the level of tested interleukines (IL- 1β, IL- 6, IL- 8) and collagenases 1, and 13, and matrix metallo-proteinases 1, and 13 (MMP-1, and MMP-13) were found significantly reduced in XBJ treated group as compared to TNF-α treated MH7A cells. The XBJ treated group showed reduction in mRNA protein expression of COX-2 and iNOS in RT-qPCR assay. The rate of cellular apoptosis was found increased in XBJ treated group with reduction of cell viability of MH7A cells. The XBJ also showed attenuation of the expression of p-MEK/1/2 and p-p65 in MH7A cells in a western blot analysis. Our results demonstrated that XBJ significantly inhibits the inflammatory response, prevents cell viability, and induces apoptosis in human RA synovial cells by preventing the activation of the MEK/NF-κB pathway. [ABSTRACT FROM AUTHOR]

The planarian flatworm is an ideal model to study the regeneration due to its robust regenerative ability. A variety of cellular response activities have been reported to be involved in the regeneration process, including the mitogen-activated protein kinase (MAPK) signaling. However, the mechanism of MAPK pathway in regenerative responses is still unclear. In this study, we employed the planarian, Dugesia japonica , as the model to investigate the function of MAP-extracellular signal-regulated kinase (MEK), an important component of MAPK signaling pathway, in the regeneration process. We found that MEK was required for the missing tissue response after several amputation and subsequent regeneration. MEK not only affected the size of blastema in the early stage of regeneration by regulating stem cell proliferation, but also determined the planarian's regeneration through balancing cell proliferation and apoptosis. In addition, the activation of Wnt pathway partially rescued regenerative defects induced by inhibition of MEK. Taken together, our results highlight a crucial role of MEK signaling in the planarian regeneration. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDA) responds poorly to treatment. Efforts have been exerted to prolong the survival time of PDA, but the 5‐year survival rates remain disappointing. Understanding the molecular mechanisms of PDA development is significant. MEK/ERK pathway signaling has been proven to be important in PDA. lncRNA–mRNA networks have become a vital part of molecular mechanisms in the MEK/ERK pathway. Herein, weighted gene coexpression network analysis was used to investigate the coexpressed lncRNA–mRNA networks in the MEK/ERK pathway based on GSE45765. Differently expressed long noncoding RNA (lncRNA) and messenger RNA (mRNA) were found and 10 modules were identified based on coexpression profiles. Gene ontology and Kyoto Encyclopedia of Genes and Genomes were then performed to analyze the coexpressed lncRNA and mRNA in different modules. PDA cells and tissues were used to validate the analysis results. Finally, we found that NONHSAT185150.1 and B4GALT6 were negatively correlated with MEK1/2. By analyzing GSE45765, the genome‐wide profiles of lncRNA–mRNA network after MEK1/2 was established, which might aid the development of drug‐targeting MEK1/2 and the investigation of diagnostic markers. [ABSTRACT FROM AUTHOR]

Abstract This study was designed to test the therapeutic potential of a MEK1/2 inhibitor (MEKi) in an experimental model of Pseudomonas aeruginosa pneumonia. The study found that treatment with MEKi reduced alveolar neutrophilic inflammation and led to faster recovery of weight compared to carrier-treated mice, without impairing bacterial clearance. Alveolar macrophages isolated from MEKi-treated mice also had increased M2 gene and protein expression, supporting the concept that MEKi modulates in vivo macrophage inflammatory responses. In summary, this report demonstrates the potential of MEKi to promote the resolution of inflammation in vivo during a primary lung infection without impairing bacterial clearance.

Hearing loss is one of the most common types of disability; however, there is only one FDA-approved drug to prevent any type of hearing loss. Treatment with the highly effective chemotherapy agent, cisplatin, and exposure to high decibel noises are two of the most common causes of hearing loss. The mitogen activated protein kinase (MAPK) pathway, a phosphorylation cascade consisting of RAF, MEK1/2, and ERK1/2, has been implicated in both types of hearing loss. Pharmacologically inhibiting BRAF or ERK1/2 is protective from noise and cisplatin-induced hearing loss in multiple mouse models. Trametinib, a MEK1/2 inhibitor, protects from cisplatin induced outer hair cell death in mouse cochlear explants; however, to the best of our knowledge, inhibiting MEK1/2 has not yet been shown to be protective from hearing loss in vivo . In this study, we demonstrate that trametinib protects from cisplatin-induced hearing loss in a translationally relevant mouse model and does not interfere with cisplatin's tumor killing efficacy in cancer cell lines. Higher doses of trametinib were toxic to mice when combined with cisplatin but lower doses of the drug were protective from hearing loss without any known toxicity. Trametinib also protected mice from noise-induced hearing loss and synaptic damage. This study shows that MEK1/2 inhibition protects from both insults of hearing loss and that targeting all three kinases in the MAPK pathway protect from cisplatin and noise-induced hearing loss in mice., Competing Interests: Conflicts of Interest T.T. is an inventor on US patent no. 11,433,073 and US serial no 17/736,330 filed for the use of trametinib for hearing protection, and patent application 62/500,677;WO2018204226, for the use of dabrafenib for hearing protection, and is a cofounder of Ting Therapeutics LLC.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens., (© 2024 The Author(s).)

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system. [ABSTRACT FROM AUTHOR]

In the human neuroblastoma SH‐SY5Y cell line, the glutathione transferase Omega 1‐1 (GSTO1‐1) appears to modulate Akt and MEK1/2 kinase activation. We observed a glutathionylation modification was involved in the activation of Akt but not MEK1/2. With the specific GSTO1‐1 inhibitor ML175, we show the enzyme activity of GSTO1‐1 is important for modulation as the inhibited GSTO1‐1 allowed activation of both Akt and MEK1/2. The inhibition of GSTO1‐1 showed a similar extent of activation of Akt and MEK1/2 as treatment by the endotoxin lipopolysaccharide. The GSTO1‐1 also either directly interacts with Akt and MEK1/2 or interacts with a protein complexed with Akt and MEK1/2 as both kinases coimmunoprecipitated with GSTO1‐1. The results suggest that GSTO1‐1 enzyme activity inhibits the activation of these two kinases to maintain basal levels. The possible regulation by GSTO1‐1 is of interest as both kinases have hundreds of potential downstream targets that are known to have contributions to various cellular processes including survival, growth, proliferation, and metabolism. [ABSTRACT FROM AUTHOR]

Trametinib was endorsed by the FDA in 2013 as a single agent for adult melanoma patients. Trametinib inhibits cell growth and proliferation in multiple tumor xenografts by preventing RAF phosphorylation of MEK and thus restricting accumulation of activated MEK. In this study, the focus of investigation was the mechanism of the interaction between trametinib and MEK1/2 via computational simulation. To specify the best interaction site of inhibitor with MEK1/2 based on the interaction energy ranking, first we performed a docking and then we studied the interactions of the ATP-bound MEK with trametinib, with RAF and the complex of the ATP-bound MEK-trametinib with RAF via molecular dynamic simulations. The results showed that trametinib inactivates the enzyme by bonding to a group of amino acids including Lys97/101, SER218/216, Asp208/212, and Met143/147 in MEK1/2. By bonding to the essential amino acids, trametinib inhibits the activity of the enzyme. All in all, the acquired results can be of great use in designing new inhibitors. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.

Abstract MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases, which act as an integration point by transferring extracellular signals to the nucleus. To investigate the function of MEK in teleost fish, we cloned MEK1 and MEK2 cDNA sequences from the orange-spotted grouper (Epinephelus coioides). EcMEK1 and EcMEK2 shared 80% amino acid identity with each other. EcMEK1 had 89–99% amino acid identity with teleosts or mammals, whereas EcMEK2 shared 85–97% amino acid identity. The exon structures of the grouper MEK1/2 genes were conserved with zebrafish and human MEK1/2. Tissue distribution analysis showed that EcMEK1 and EcMEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs. Both EcMEK1 and EcMEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells. Overexpression of EcMEK1 or EcMEK2 activated Activator protein 1 dependent luciferase. The phosphorylation levels of EcMEK1/2 and EcERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment. The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization. Highlights • Overexpression of EcMEK1 or EcMEK2 can activate AP1. • Grouper pMEK1/2 and pERK1/2 was increased by the stimulation of PMA. • Grouper MEK-ERK axis was activated in C. irritans infected or immune skin. [ABSTRACT FROM AUTHOR]

Background: The large-conductance, voltage-gated, calcium (Ca (2+))-activated potassium channel (BKCa) plays an important role in regulating Ca (2+) signaling and cell physiological function, and is aberrantly expressed in some types of cancers. The present study focuses on identifying the oncogenic potential and clinical significance of BKCa in endometrial adenocarcinoma, as well as exploring the mechanistic relevance by 17β -estradiol (E2) inducing aberrant activation of MEK1/2 and ERK1/2 via BKCa.Methods: The expression of BKCa, ERK1/2 and p-ERK1/2 were examined by immunohistochemical staining in 263 cases, including 185 primary types I endometrial cancer tissues, 38 atypical endometrial hyperplasia tissues and 40 normal endometrium tissues. Cell growth, cycle, apoptosis rate, migration and invasion was separately tested in Ishikawa cells using siRNA-BKCa and/or E2 treatment, as well as the expression of these interested proteins by western blot analysis.Results: We showed that expression of BKCa is significantly elevated in 185 types I endometrial adenocarcinoma tissues compared to those of the normal endometrium and atypical endometrial hyperplasia tissues. Furthermore, in vitro observations revealed that down-regulation of BKCa expression inhibited cell growth by both enhancing apoptosis and blocking G1/S transition, suppressed cell migration and invasion in Ishakiwa cells, and decreased the expression of p-MEK1/2 and p-ERK1/2. Additionally, RNAi-mediated knockdown of BKCa attenuated the increased cellular growth and invasion, as well as the elevated expression of p-MEK1/2 and p-ERK1/2 proteins, induced by E2 stimulation. More importantly, the aberrant expression of BKCa and p-ERK1/2 were closely related with poor prognostic factors in type I endometrial cancer, and up-regulated expression of p-ERK1/2 was significantly associated with shorter disease-free survival (DFS) and overall survival (OS) and was an independent prognostic factor in type I endometrial cancer patients.Conclusion: Our results demonstrated that BKCa and the key downstream effectors p-ERK1/2 could be involved in important signaling pathways in initiation and development of endometrial adenocarcinoma and may provide a new therapeutic approach for women with endometrial cancer. [ABSTRACT FROM AUTHOR]

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRβ by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRβ binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation. [ABSTRACT FROM AUTHOR]

Inhibitors of PARP1 are approved therapeutic agents in ovarian carcinomas. We determined whether the novel clinically relevant CHK1 inhibitor SRA737 interacted with PARP1 inhibitors to kill carcinoma cells. In multiple mammary and ovarian cancer lines SRA737 synergized with the PARP1 inhibitors olaparib and niraparib to cause cell death. The [SRA737 + niraparib] drug combination activated an ATM-AMPK-ULK1-mTOR pathway which resulted in the formation of autophagosomes, temporally followed by autolysosome formation. Phosphorylation of ULK1 S317 was essential for kinase activation against ATG13. The drug combination elevated eIF2α phosphorylation which was causal at increasing Beclin1 and ATG5 expression, reducing MCL-1 and BCL-XL levels, and causing CD95 activation. Knock down of CD95, eIF2α, ATM, AMPKα, ULK1, Beclin1 or ATG5 reduced drug combination lethality. Blockade of either caspase 9 function or that of AIF each partially prevented cell death. Expression of activated mTOR or of c-FLIP-s or of BCL-XL reduced cell killing. In vivo, SRA737 and niraparib interacted in an additive fashion to suppress the growth of mammary tumors. Multiplex analyses revealed that drug combination treated tumors had reduced their plasma levels of sERBB1, sERBB2, sVEGFR1, sVEGFR2, sIL-6R, HGF, PDGFAB/BB and CXCL16 and enhanced the levels of CCL26, IL-8 and MIF. Surviving tumors had activated ERK1/2 and AKT. This finding argues that IL-8/ERK/AKT signaling may be an evolutionary survival response to [SRA737 + niraparib]. [ABSTRACT FROM AUTHOR]

Summary: The survival and proliferation of chronic lymphocytic leukaemia (CLL) cells is driven by multiple signalling pathways, including those mediated by the B cell, Toll‐like and chemokine receptors. Many of these pathways converge on the same signalling molecules, including those involved in the Raf‐1/MEK/Erk1/2‐MAPK pathway. We investigated the effects of the MEK1/2 (also termed MAP2K1/2) inhibitor, binimetinib, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment. Binimetinib blocked CLL cell survival induced by stroma‐conditioned media and phorbol myristylate (PMA). Binimetinib was also significantly more toxic towards CLL cells cultured in the presence of either anti‐IgM antibody or stroma‐derived factor‐1α (SDF‐1α) and reduced CLL cell cycle progression and proliferation. Furthermore, binimetinib significantly increased the sensitivity of CLL cells co‐cultured with CD40 ligand (CD40L)‐expressing fibroblasts to the BH3‐mimetics ABT‐737 and Venetoclax (ABT‐199) via a mechanism involving down‐regulation of Mcl‐1 (MCL1) activity and Bim (BCL2L11) and Bcl‐xL (BCL2L1) expression. Collectively, these data suggest that binimetinib may have both cytotoxic and cytostatic effects on CLL cells by blocking microenvironment‐derived signals known to drive survival and proliferation. The combination of binimetinib with a BH3 mimetic may be an effective treatment strategy for CLL, particularly against the proliferative fraction of the disease within the tumour microenvironment. [ABSTRACT FROM AUTHOR]

This study investigated the relationship between MEK1/2 and nitric oxide (NO) in jasmonic acid (JA)-regulated metabolism of ascorbate and glutathione in maize leaves. The results showed that JA increased the activities of APX, GR, MDHAR, DHAR, GalLDH, and γ-ECS; the contents of AsA and GSH; and the production of NO. Above increases except for γ-ECS activity and NO production were all suppressed by pre-treatments with MEK1/2 inhibitors PD98059 and U0126. Above increases were all suppressed by pre-treatments with nitric oxide synthase (NOS) inhibitor l-NAME and NO scavenger cPTIO. The results of western blot showed that JA enhanced the phosphorylation level of MEK1/2. Pre-treatments with l-NAME and cPTIO suppressed JA-induced phosphorylation level of MEK1/2. Our results suggested that JA-induced NO activated MEK1/2 by increasing the phosphorylation level, which, in turn, resulted in the upregulation of ascorbate and glutathione metabolism in maize leaves. [ABSTRACT FROM AUTHOR]

Abstract: The Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time‐dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, β1 integrin, GLUT4, and IRβ that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast‐inducing effects on human adipose‐derived MSCs. In vivo, one‐year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone‐enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast‐inducing, bone‐enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy. [ABSTRACT FROM AUTHOR]

Membrane-associated guanylate kinase with inverted orientation protein 1 (MAGI-1) is a cytoplasmic scaffold protein that interacts with various signaling molecules; it negatively controls the cell growth of various types of cells and positively controls cell-cell interaction. In T cells, MAGI-1 has been shown to inhibit Akt activity through its interaction with PTEN and MEK1. In this study we found that MAGI-1 expression is decreased in multiple (9 out of 15) human T-cell leukemia cell lines, including adult T-cell leukemia (ATL), T-cell acute lymphoblastic leukemia and chronic T-cell lymphocytic leukemia. The overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced cellular growth. While the overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced the Akt and MEK activities, the knockdown of MAGI-1 in a MAGI-1-high ATL cell line augmented the Akt and MEK activities. Collectively, the findings of the present study suggest that the decreased expression of MAGI-1 in human T cells contributes to the development of several types of T-cell leukemia, partly through the stimulation of the Akt and MEK pathways. [ABSTRACT FROM AUTHOR]