Extracellular Signal-regulated Kinase Research Articles

Inhibition of phosphodiesterase 10A mitigates neuronal injury by modulating apoptotic pathways in cold-induced traumatic brain injury

Brain injury develops from a complex series of pathophysiological phases, resulting in acute necrotic or delayed apoptotic cell death after traumatic brain injury (TBI). Inhibition of apoptotic cell death is critical for the treatment of acute neurodegenerative disorders, such as TBI. Here, we investigated the role of phosphodiesterase 10A (PDE10A) in the development of neuronal injury, particularly in apoptotic cell death. Using the PDE10A inhibitor TAK-063, we found that PDE10A inhibition is associated with decreased brain injury, brain swelling, and blood brain barrier disruption 48 h after cold-induced TBI. Furthermore, a particularly notable result was observed with 3 mg/kg TAK-063, which reduced disseminated neuronal injury. Protein abundance analysis revealed that PDE10A inhibition activates survival kinases AKT and ERK-1/-2, which were associated with the decreased activation of MMP-9 and PTEN. Additionally, iNOS and nNOS levels significantly reduced in the TAK-063 group, playing roles in inflammation and apoptosis. A planar surface immunoassay was performed for in-depth analyses of the apoptotic signaling pathways. We observed that inhibition of PDE10A resulted in the decreased expression of TNFRSF1A, TNFRSF10B, and TNFRSF6 receptors, particularly inducing apoptotic cell death. Moreover, these findings correlated with reduced levels of pro-apoptotic proteins, including PTEN, p27, Cytochrome-c, cleaved Caspase-3, Bad, and p53. Interestingly, TAK-063 treatment reduced levels of anti-apoptotic proteins or enzymes, including XIAP, Claspin, and HIF1α, without affecting Bcl-x, MCL-1, SMAC, HO-1, HO-2, HSP27, HSP60, and HSP70. The findings suggest that PDE10A regulates cellular signaling predominantly pro-apoptotic pathways, and inhibition of this protein is a promising approach for the treatment of acute brain injury.

Anti-nociceptive effect of STW 5-II in rodent models of stress and post-inflammatory visceral hypersensitivity

AimsVisceral hypersensitivity is a therapy-resistant hallmark of irritable bowel syndrome (IBS). Many IBS patients’ symptoms develop following an acute colitis, and most report that stress worsens symptoms. STW 5-II, a combination of six herbal extracts, is a clinically proven treatment for IBS, but the mechanism is uncertain. Here, we employ two well-characterized rodent models to test the hypothesis that STW 5-II attenuates chronic colonic hypersensitivity. Main methodsSeparate cohorts of male rats were used for each model of colonic hypersensitivity. The first model used repeated water avoidance stress (1hr/day for 10 days), while the second model used intracolonic trinitrobenzene sulfonic acid to induce a short-lived colitis followed by post-inflammatory visceral hypersensitivity. Both models used sham treatment controls. Colonic sensitivity was quantified as the number of abdominal contractions to graded pressures (20–60 mmHg) of isobaric colorectal distension (CRD). Phosphorylation of extracellular signal-regulated kinase (pERK) was assessed via immunohistochemistry in the brain, spinal cord, and dorsal root ganglion (DRG). STW 5-II (10 ml/kg, p.o.) or vehicle (p.o.) was administered for 7 days, prior to CRD and pERK expression. Key findingsRats exposed to either model developed significant colonic hypersensitivity. Both models enhanced CRD-evoked pERK in DRGs, spinal cord, and brain. STW 5-II decreased colonic hypersensitivity and reduced CRD-evoked brain, spinal, and DRG pERK. SignificanceBoth models induced colonic hypersensitivity and enhanced pERK expression. STW 5-II inhibited colonic hypersensitivity and decreased noxious neuronal activation in both models, which could explain its clinically proven efficacy in relieving visceral hypersensitivity-related symptoms in IBS.

Conditional Pten inactivation in pituitary results in sex-specific prolactinoma formation

Pituitary tumors, including prolactinomas, present significant clinical challenges that require a deeper understanding of their molecular roots for improved diagnostics and therapies. Here, we investigate the role of the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K) pathway in pituitary tumorigenesis using a mouse model. Conditional knockout of Pten in all pituitary cell lineages resulted in prolactinoma formation exclusively in female mice, demonstrating the critical role of PTEN in pituitary homeostasis. While Pten inactivation induced Akt activation in all pituitary cells, only prolactin-producing cells exhibited tumorigenic changes, suggesting specific cell-type effects. Histological and molecular analyses of prolactinomas revealed similarities with human pituitary tumors, such as decreased vascularization and cell adhesion proteins and increased accumulation of cell cycle proteins. Notably, prolactinomas displayed diminished levels of phosphorylated extracellular signal-regulated kinase (ERK), implicating downregulation of ERK in tumorigenesis. Finally, we analyzed PTEN/PI3K activation in a collection of human pituitary tumors. Overall, our study delineates the intricate interplay between the PTEN and ERK signaling pathways, providing insights into sex-specific mechanisms of pituitary tumorigenesis and potential therapeutic strategies for prolactinomas.

Molecular Mechanisms of Skatole-Induced Inflammatory Responses in Intestinal Epithelial Caco-2 Cells: Implications for Colorectal Cancer and Inflammatory Bowel Disease.

Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in intestinal epithelial cells significantly contribute to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Given our previous findings that TNF-α is upregulated in intestinal epithelial Caco-2 cells induced by skatole, a tryptophan-derived gut microbiota metabolite, the present study aimed to explore the relationship between skatole and IL-6, alongside TNF-α. Skatole elevated the promoter activity of IL-6 as well as TNF-α, and increased IL-6 mRNA expression and protein secretion. In addition to activating NF-κB, the NF-κB inhibitor BAY 11-7082 reduced skatole-induced cell survival and the mRNA expression of IL-6 and TNF-α. NF-κB activation was attenuated by the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 and the p38 inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. U126 and SB203580 also decreased the skatole-induced increase in IL-6 expression. When skatole-induced AhR activation was inhibited by CH223191, in addition to promoting NF-κB activation, IL-6 expression was enhanced in a manner similar to that previously reported for TNF-α. Taken together, these results suggest that skatole-elicited NF-κB activation induces IL-6 and TNF-α expression, although AhR activation partially suppresses this process. The ability of skatole to increase the expression of IL-6 and TNF-α may significantly affect the development and progression of these diseases. Moreover, the balance between NF-κB and AhR activation appears to govern the skatole-induced increases in IL-6 and TNF-α expression. Therefore, the present findings provide new insights into the mechanisms linking tryptophan-derived gut microbiota metabolites with colorectal disease.

Gintonin Stimulates Glucose Uptake in Myocytes: Involvement of Calcium and Extracellular Signal-Regulated Kinase Signaling.

Ginseng has anti-hyperglycemic effects. Gintonin, a glycolipoprotein derived from ginseng, also stimulates insulin release from pancreatic beta cells. However, the role of gintonin in glucose metabolism within skeletal muscle is unknown. Here, we showed the effect of gintonin on glucose uptake, glycogen content, glucose transporter (GLUT) 4 expression, and adenosine triphosphate (ATP) content in C2C12 myotubes. Gintonin (3-30 μg/mL) dose-dependently stimulated glucose uptake in myotubes. The expression of GLUT4 on the cell membrane was increased by gintonin treatment. Treatment with 1-3 μg/mL of gintonin increased glycogen content in myotubes, but the content was decreased at 30 μg/mL of gintonin. The ATP content in myotubes increased following treatment with 10-100 μg/mL gintonin. Gintonin transiently elevated intracellular calcium concentrations and increased the phosphorylation of extracellular signal-regulated kinase (ERK). Gintonin-induced transient calcium increases were inhibited by treatment with the lysophosphatidic acid receptor inhibitor Ki16425, the phospholipase C inhibitor U73122, and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Gintonin-stimulated glucose uptake was decreased by treatment with U73122, the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, and the ERK inhibitor PD98059. These results show that gintonin plays a role in glucose metabolism by increasing glucose uptake through transient calcium increases and ERK signaling pathways. Thus, gintonin may be beneficial for glucose metabolism control.

Evaluation of Echinacea purpurea Extracts as Immunostimulants: Impact on Macrophage Activation.

Echinacea purpurea has been traditionally used to strengthen the immune system. Therefore, herein, we investigated the potential of E. purpurea aqueous extracts (AEs) obtained from flowers (F), leaves (L), or roots (R) as an immune booster in human primary monocyte-derived macrophages (hMDMs). Additionally, to identify the main class of compounds (phenolic/carboxylic acids vs. alkylamides) responsible for the bioactivity, the three AEs were fractioned by semi-preparative high-performance liquid chromatography (HPLC). The AEs and the isolated phenolic/carboxylic acidic fractions were not cytotoxic for hMDMs for all tested concentrations, as confirmed by the metabolic activity and DNA content assays. Moreover, AE drastically induced the production of the interleukin (IL)-6 and tumor necrosis factor (TNF)-α, with a minimal effect on IL-1β and prostaglandin E2 (PGE2), supporting their potential for macrophage activation. Interestingly, in the presence of the phenolic/carboxylic acidic fractions, this efficacy considerably decreased, suggesting a complementary effect between compounds. AE also triggered the phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling pathways and upregulated the cyclooxygenase (COX)-2 expression in hMDMs. Overall, AE-F was demonstrated to be the most powerful immunostimulant extract that can be related to their higher number in identified bioactive compounds compared to AE-L and AE-R. These results highlight the efficiency of E. purpurea AE to enhance the function of a key cell type of the immune system and their potential as immunostimulant formulations for patients with a compromised immune system due to certain diseases (e.g., acquired immunodeficiencies) and treatments (e.g., chemotherapy).

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.

Hypoxia Activates FGF-23-ERK/MAPK Signaling Pathway in Ischemia-Reperfusion-Induced Acute Kidney Injury

Introduction: Both hypoxia and fibroblast growth factor-23 (FGF-23) are key factors in ischemia-reperfusion (I/R)-induced acute kidney injury (AKI). This study aimed to explore the relationship between hypoxia and FGF-23 in AKI. Methods: An I/R-AKI animal model was established using male BALB/c mice. HK-2 cells, a part of the human proximal tubular epithelial cell line, were subjected to hypoxia/reoxygenation (H/R). qPCR was used to measure FGF-23 and HIF1α, and ELISA was used to measure inflammatory and oxidative stress cytokines. Western blotting was used to measure the phosphorylation of extracellular signal-regulated kinase (ERK) level. Results: In I/R mice, the levels of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), malondialdehyde (MDA), and the phosphorylation of ERK (p-ERK) were increased, whereas the levels of interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione peroxidase (GPx), and klotho were decreased, compared to the sham-operated mice. Silencing the FGF-23 expression in I/R mice normalized the levels of IL-6, IL-10, TNF-α, MDA, SOD, GPx, and p-ERK. In HK-2 cells, hypoxia-reperfusion (H/R) elevated the levels of IL-6, TNF-α, MDA, and p-ERK, but reduced IL-10, SOD, GPx, and klotho levels. Hypoxia induced apoptosis in HK-2 cells, but silencing of FGF-23 expression blocked the effects of hypoxia on cell apoptosis, pro-inflammatory factor levels, oxidative stress response, and p-ERK levels. Conclusion: FGF-23 is a key molecule in AKI, and hypoxia plays a crucial role in AKI by inducing cell apoptosis; however, its role is regulated by FGF-23. FGF-23 affects oxidative stress and the inflammatory response of kidney tissues by activating the ERK/mitogen-activated protein kinase (MAPK) signaling pathway.

Low shear stress protects chondrocytes from IL-1β-induced apoptosis by activating ERK5/KLF4 signaling and negatively regulating miR-143-3p

ObjectiveThis study investigated the protective effects of low fluid shear stress (FSS ≤ 2 dyn/cm²) against interleukin-1β (IL-1β)-induced chondrocyte apoptosis and explored the underlying molecular mechanisms.MethodsChondrocytes were cultured under four conditions: control, IL-1β stimulation, low FSS, and combined low FSS + IL-1β stimulation. Apoptosis was assessed using Hoechst staining and flow cytometry. Western blotting determined the expression of caspase-3 (CASP3), caspase-8 (CASP8), and NF-κB p65. Quantitative real-time PCR measured miR-143-3p expression. The roles of miR-143-3p and the extracellular signal-regulated kinase 5 (ERK5)/Krüppel-like factor 4 (KLF4) signaling pathway were further investigated using miR-143-3p mimics and inhibitors, an ERK5 inhibitor, and a KLF4 overexpression vector.ResultsIL-1β induced significant chondrocyte apoptosis, which was markedly inhibited by low FSS. Mechanistically, low FSS suppressed miR-143-3p expression, thereby enhancing ERK5 signaling. This activated ERK5 subsequently upregulated KLF4 expression, further mitigating IL-1β-induced damage. Importantly, miR-143-3p overexpression under low FSS conditions exacerbated IL-1β-induced apoptosis, while miR-143-3p inhibition attenuated it. Consistent with this, ERK5 inhibition augmented IL-1β-induced apoptosis, whereas KLF4 overexpression suppressed it.ConclusionLow FSS protects chondrocytes from IL-1β-induced apoptosis by suppressing miR-143-3p and activating the ERK5/KLF4 signaling pathway. This study reveals a novel mechanism by which mechanical stimulation protects cartilage.

MC-LR disrupts dopamine synthesis in the substantia nigra of midbrain by enhancing the chaperone-mediated autophagy pathway through direct binding to ERK2

Microcystins are environmental toxins produced by freshwater cyanobacteria. Microcystin-LR (MC-LR) is one of the most abundant and harmful isomers. MC-LR poses a serious threat to human health. MC-LR could penetrate the blood-brain barrier of mice and accumulate in the substantia nigra (SN) of the midbrain, leading to a reduction in dopamine levels and Parkinson's disease (PD)-like motor dysfunction in mice. The reduction in dopamine levels is a key factor contributing to movement disorders in humans with PD. Dopamine is synthesized in the dopaminergic neurons of the SN by the actions of tyrosine hydroxylase (TH) and dihydroxyphenylalanine decarboxylase (DDC). In this study, we found that MC-LR could enter dopaminergic neurons in the SN and directly bound to extracellular signal-regulated kinase 2 (ERK2), enhancing ERK2 stability. ERK2 further enhanced the transcriptional activity of Heat Shock Protein Family A Member 8 (HSPA8) and promoted the expression of Heat shock cognate 71 kDa protein (HSC70), which in turn amplified the chaperone-mediated autophagy (CMA) pathway and accelerated the degradation of TH and DDC. This affected the dopamine synthesis process, resulting in a significant reduction in dopamine levels. The study is the first to reveal that ERK2 was a direct target of MC-LR, and further enhanced CMA affecting dopamine synthesis, which has important theoretical and practical significance for environmental safety management.

Resveratrol and ceftriaxone encapsulated in hybrid nanoparticles to prevent dopaminergic neurons from degeneration for Parkinson's disease treatment

The purpose of this study is to evaluate the influence of phospholipid-polymer nanoparticles (PNPs) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling of dopaminergic neurons in degenerated brain. Resveratrol (RES)- and ceftriaxone (CEF)-entrapped PNPs with surface leptin (Lep) and transferrin (Tf) were fabricated to rescue both 1-methyl-4-phenylpyridinium (MPP+)-insulted SH-SY5Y cells and Wistar rats. Based on PNPs, anti-apoptosis of RES and CEF, and targeting of Lep and Tf were investigated. Experimental results revealed that 20–30 % alginic acid (Alg) yielded the maximal particle size, physical stability and entrapment efficiency of CEF, and the minimal release percentage of CEF. Increasing Alg content in PNPs decreased the entrapment efficiency of RES, and facilitated the release of RES. Optimized PNP composition was about 40 % Alg, 15 % phosphatidylserine and 45 % poly-ε-caprolactone. Lep-Tf-PNPs ameliorated brain permeability of RES and CEF without jeopardizing the blood-brain barrier, and promoted the viability of MPP+-insulted SH-SY5Y cells. Immunofluorescence images and western blots of MPP+-insulted SH-SY5Y cells showed that Lep-Tf-RES-CEF-PNPs upregulated dopamine transporter, tyrosine hydroxylase, B-cell lymphoma 2 (Bcl-2), cyclic AMP response element-binding protein and ERK5 expressions, and downregulated Bcl-2-associated X protein (Bax), α-synuclein (α-syn), phosphorylated tau protein (p-tau), c-Jun N-terminal kinase and ERK1/2 expressions. Lep-Tf-RES-CEF-PNPs unveiled a strong capacity to recover Bcl-2, Bax, α-syn and p-tau levels from MPP+ injury in the substantia nigra of rats. Hence, Lep-Tf-RES-CEF-PNPs can retard α-syn fibril formation, prevent tau protein from phosphorylation, and moderate MAPK/ERK and phosphatidylinositol 3-kinase/protein kinase B, and are promising for brain- and neuron-targeted pharmacotherapy to manage Parkinson's disease.

ARL8B promotes hepatocellular carcinoma progression and inhibits antitumor activity of lenvatinib via MAPK/ERK signaling by interacting with RAB2A

Tumor recurrence and metastasis are important factors affecting postoperative survival in hepatocellular carcinoma (HCC) patients. ADP Ribosylation factor-like GTPase 8B (ARL8B) plays a crucial role in many biological processes, including lysosomal function, immune response, and cellular communication, all of which are related to the occurrence and development of tumors. However, its role in HCC remains unclear. Herein, we revealed that ARL8B is consistently elevated in HCC tissues compared to normal liver tissues, suggesting an unfavorable outcome in HCC patients. Increased ARL8B levels promoted the malignant phenotype of HCC in vitro and in vivo. Notably, ARL8B also induced epithelial-to-mesenchymal transition (EMT) in HCC cells. Mechanistically, the results of bioinformatics analysis combined with mass spectrometry revealed the potential downstream target molecule RAB2A of ARL8B. ARL8B directly interacted with RAB2A and increased the levels of GTP-bound RAB2A, thereby contributing to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway. Interestingly, knockout of ARL8B in Hep3B cells enhanced the antitumor activity of lenvatinib in vitro and in vivo. Furthermore, AAV-shARL8B enhanced the inhibition of HCC growth through lenvatinib, providing new insights into its mechanism of action in lenvatinib-insensitive patients. In conclusion, ARL8B promotes the malignant phenotype of HCC and EMT via RAB2A mediated activation of the MAPK/ERK signaling pathway and is expected to be a valuable prognostic indicator and therapeutic target for HCC patients.

Reduction of endocytosis and EGFR signaling is associated with the switch from isolated to clustered apoptosis during epithelial tissue remodeling in Drosophila.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Removal of cells is coordinated with the increase in number of newly dividing cells to maintain barrier function of the tissue. In Drosophila metamorphosis, larval epidermal cells (LECs) are replaced by adult precursor cells called histoblasts. Removal of LECs must counterbalance the exponentially increasing adult histoblasts. Previous work showed that the LEC removal accelerates as endocytic activity decreases throughout all LECs. Here, we show that the acceleration is accompanied by a mode switching from isolated single-cell apoptosis to clustered ones induced by the endocytic activity reduction. We identify the epidermal growth factor receptor (EGFR) pathway via extracellular-signal regulated kinase (ERK) activity as the main components downstream of endocytic activity in LECs. The reduced ERK activity, caused by the decrease in endocytic activity, is responsible for the apoptotic mode switching. Initially, ERK is transiently activated in normal LECs surrounding a single apoptotic LEC in a ligand-dependent manner, preventing clustered cell death. Following the reduction of endocytic activity, LEC apoptosis events do not provoke these transient ERK up-regulations, resulting in the acceleration of the cell elimination rate by frequent clustered apoptosis. These findings contrasted with the common perspective that clustered apoptosis is disadvantageous. Instead, switching to clustered apoptosis is required to accommodate the growth of neighboring tissues.

Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays.

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene.

Targeting rapid TKI-induced AXL upregulation overcomes adaptive ERK reactivation and exerts antileukemic effects in FLT3/ITD acute myeloid leukemia.

Acute myeloid leukemia (AML) patients with the FMS-related receptor tyrosine kinase 3 internal tandem duplication (FLT3/ITD) mutation have a poorer prognosis, and treatment with FLT3 tyrosine kinase inhibitors (TKIs) has been hindered by resistance mechanisms. One such mechanism is known as adaptive resistance, in which downstream signaling pathways are reactivated after initial inhibition. Past work has shown that FLT3/ITD cells undergo adaptive resistance through the reactivation of extracellular signal-regulated kinase (ERK) signaling within 24 h of sustained FLT3 inhibition. We investigated the mechanism(s) responsible for this ERK reactivation and hypothesized that targeting tyrosine-protein kinase receptor UFO (AXL), another receptor tyrosine kinase that has been implicated in cancer resistance, may overcome the adaptive ERK reactivation. Experiments revealed that AXL is upregulated and activated in FLT3/ITD cell lines mere hours after commencing TKI treatment. AXL inhibition combined with FLT3 inhibition to decrease the ERK signal rebound and to exert greater anti-leukemia effects than with either treatment alone. Finally, we observed that TKI-induced AXL upregulation occurs in patient samples, and combined inhibition of both AXL and FLT3 increased efficacy in our in vivo models. Taken together, these data suggest that AXL plays a role in adaptive resistance in FLT3/ITD AML and that combined AXL and FLT3 inhibition might improve FLT3/ITD AML patient outcomes.

Chrysene contribution to bronchial asthma: Activation of TRPA1 disrupts bronchial epithelial barrier via ERK pathway

BackgroundElevated polycyclic aromatic hydrocarbon (PAH) levels are associated with exacerbation of asthma. Chrysene is one of the most prevalent unsubstituted PAHs in the environment. Transient receptor potential ankyrin 1 (TRPA1) can be used as a chemoreceptor to detect inhaled stimuli and plays an important role in the occurrence and deterioration of asthma. Whether exposure to a high concentration of chrysene in the environment can activate TRPA1 and contribute to the development of asthma, potentially through the dysfunction of the bronchial epithelial barrier, remains unclear. MethodsA cell-based assay was performed to verify the downregulation of the expression of E-cadherin and tight junction (TJ) proteins by chrysene in bronchial epithelial cells to explore the role of chrysene-mediated TRPA1 activation in the regulation of TJ protein expression through the extracellular signal-regulated protein kinase (ERK) pathway. Animal tests were conducted to determine whether chrysene could enhance airway hyperresponsiveness (AHR) induced by house dust mites (HDMs) and disrupt barrier function, thereby contributing to asthma. ResultsThe cell-based assay revealed that chrysene could disrupt the function of the bronchial epithelial barrier and decrease the expression levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and claudin-5 through the ERK pathway. Chrysene induced airway epithelial barrier dysfunction primarily through TRPA1 instead of transient receptor potential vanilloid 1. TRPA1 knockdown was able to attenuate chrysene-induced downregulation of TJ protein expression and downregulate ERK activation (p-ERK). Compared with exposure to HDM alone, coexposure to chrysene and HDM resulted in an increased incidence of AHR, disruption of barrier function, and eosinophilic inflammatory responses in a mouse model of asthma. Coexposure to chrysene and HDM increased TRPA1 expression. The animal test verified that the TRPA1 inhibitor HC030031 could suppress chrysene and HDM-induced asthma in mice. ConclusionsOur findings showed that chrysene contributed to the breakdown of the function of the bronchial epithelial barrier through the TRPA1–ERK axis and therefore acted as an adjuvant to contribute to asthma.

TRIM21 induces selective autophagic degradation of c-Myc and sensitizes regorafenib therapy in colorectal cancer

Kirsten rat sarcoma virus (KRAS) mutation is associated with malignant tumor transformation and drug resistance. However, the development of clinically effective targeted therapies for KRAS-mutant cancer has proven to be a formidable challenge. Here, we report that tripartite motif-containing protein 21 (TRIM21) functions as a target of extracellular signal-regulated kinase 2 (ERK2) in KRAS-mutant colorectal cancer (CRC), contributing to regorafenib therapy resistance. Mechanistically, TRIM21 directly interacts with and ubiquitinates v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) at lysine 148 (K148) via K63-linkage, enabling c-Myc to be targeted to the autophagy machinery for degradation, ultimately resulting in the downregulation of enolase 2 expression and inhibition of glycolysis. However, mutant KRAS (KRAS/MT)-driven mitogen-activated protein kinase (MAPK) signaling leads to the phosphorylation of TRIM21 (p-TRIM21) at Threonine 396 (T396) by ERK2, disrupting the interaction between TRIM21 and c-Myc and thereby preventing c-Myc from targeting autophagy for degradation. This enhances glycolysis and contributes to regorafenib resistance. Clinically, high p-TRIM21 (T396) is associated with an unfavorable prognosis. Targeting TRIM21 to disrupt KRAS/MT-driven phosphorylation using the antidepressant vilazodone shows potential for enhancing the efficacy of regorafenib in treating KRAS-mutant CRC in preclinical models. These findings are instrumental for KRAS-mutant CRC treatment aiming at activating TRIM21-mediated selective autophagic degradation of c-Myc.

Acetylcholine receptor-β inhibition by interleukin-6 in skeletal muscles contributes to modulating neuromuscular junction during aging

BackgroundAging-related strength decline contributes to physiological deterioration and is a good predictor of poor prognosis. However, the mechanisms underlying neuromuscular junction disorders affecting contraction in aging are not well described. We hypothesized that the autocrine effect of interleukin (IL)-6 secreted by skeletal muscle inhibits acetylcholine receptor (AChR) expression, potentially causing aging-related strength decline. Therefore, we investigated IL-6 and AChR β-subunit (AChR-β) expression in the muscles and sera of aging C57BL/6J mice and verified the effect of IL-6 on AChR-β expression.MethodsAnimal experiments, in vitro studies, bioinformatics, gene manipulation, dual luciferase reporter gene assays, and chromatin immunoprecipitation experiments were used to explore the role of the transcription cofactor peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) and its interacting transcription factors in the IL-6-mediated regulation of AChR-β expression.ResultsIL-6 expression gradually increased during aging, inhibiting AChR-β expression, which was reversed by tocilizumab. Both tocilizumab and the PGC1α agonist reversed the inhibiting effect of IL-6 expression on AChR-β. Compared to inhibition of signal transducer and activator of transcription 3, extracellular signal-regulated kinases 1/2 (ERK1/2) inhibition suppressed the effects of IL-6 on AChR-β and PGC1α. In aging mouse muscles and myotubes, myocyte enhancer factor 2 C (MEF2C) was recruited by PGC1α, which directly binds to the AChR-β promoter to regulate its expression.ConclusionsThis study verifies AChR-β regulation by the IL-6/IL-6R-ERK1/2-PGC1α/MEF2C pathway. Hence, evaluating muscle secretion, myokines, and AChRs at an earlier stage to determine pathological progression is important. Moreover, developing intervention strategies for monitoring, maintaining, and improving muscle structure and function is necessary.

The general glycan profiling of Dendrobium officinale and their protective effects on MIN6 cells via ERK signaling pathway

Based on structural elucidation of natural and hydrolyzed glycans, the general glycans profiling of D. officinale were unequivocally established for the first time as follows: [Display omitted] The results indicated that the structure of D. officinale glycans with low degree of polymerization (DP ≤ 22) was linear α-D-1,4-glucan, whereas the structure of glycans with high degree of polymerization (DP > 24) was linear acetylated 1,4-glucomannan. The content of acetyl groups and mannose to glucose (M/G) ratio increased with the degree of polymerization of D. officinale glycans. In addition, this study showed that natural D. officinale glycans protected pancreatic β-cell damage induced by glucotoxicity through the extracellular signal–regulated kinase (ERK)1/2 pathway.

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy

BackgroundStress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson’s disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear.MethodsMitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.ResultsAcute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.ConclusionsPathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.