Mitogen-activated Kinase Research Articles

The protective effect of irisin against hemorrhagic injury is mediated by PI3K and p38 pathways in hemorrhage/resuscitation.

The objective of this study is to investigate whether PI3kinase (PI3K) and p38 mitogen-activated kinase contributes to the protection of irisin during hemorrhage/resuscitation. Experimental groups were divided by receiving the different treatments during resuscitation: I) Hemorrhage: Adult male CD-1 mice were subjected to hemorrhage at a mean arterial blood pressure of 35~45 mmHg for 60 min followed by 120 min of resuscitation (n=13); II) Hemorrhage + Irisin: receiving irisin (5µg/kg) (n=13); III) Hemorrhage + Irisin + PI3K inhibitor: receiving both Ly294002 (1mg/kg, i.v.) and irisin (n=6); IV) Hemorrhage + Irisin + p38 inhibitor: receiving SB202190 (1mg/kg, i.v.) and irisin (n=6). As compared to hemorrhage/resuscitation control, irisin improved the cardiac function and recovery of hemodynamics in association with the decreased systemic IL-1, IL-6, and TNF-α, which was completely abrogated by PI3K or p38 inhibitions. Furthermore, inhibition of PI3K or p38 abolished irisin-induced reduction of the infiltration of inflammatory cells and TUNEL-positive apoptosis in the cardiac and skeletal muscles. Irisin reduced TNF-α and IL6 expression in cardiac and skeletal muscle, which was abrogated by inhibition of PI3K or p38. Irisin-treated hemorrhage increases the phosphorylation of PI3K and p38 in both cardiac and skeletal muscle, which was mitigated by inhibition of PI3K or p38. Conclusion: PI3K and p38 play a critical role in modulating the protective effect of irisin during the hemorrhage/resuscitation. Significance Statement 1). This study has identified a critical pathway in regulation of trauma/hemorrhage by using a preclinical and reproducible model, in which Irisin, as a hormone factor, stimulates PI3K and p38 pathways to induce the protection against traumatic conditions. 2). The study holds promise to develop a new therapeutic strategy to target irisin and its pathway related to PI3K and p38 to treat trauma and its comorbidities to reduce mortality for clinical implication.

Regulatory effects of the p38 mitogen-activated protein kinase-myosin light chain kinase pathway on the intestinal epithelial mechanical barrier and the mechanism of modified Pulsatilla decoction in the treatment of ulcerative colitis.

To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo. We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules. In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue. The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

Clarithromycin Modulates Neutrophilic Inflammation Induced by Prevotella intermedia in Human Airway Epithelial Cells.

Objectives: In the present study, we aimed to clarify the mechanisms by which periodontal pathogens, particularly Prevotella intermedia, induce severe neutrophilic inflammation. In addition, we aimed to test the efficacy of macrolides, which has not been resolved in the neutrophilic inflammation induced by P. intermedia. Methods: NCl-H292 human airway epithelial cells were pre-incubated with clarithromycin for 2 h before incubation with P. intermedia supernatants. Then, C-X-C motif chemokine ligand 8 (CXCL8) transcription and interleukin (IL)-8 production were measured. To elucidate the signaling pathway, mitogen-activated protein kinase inhibitors were added to the cell culture, and the cells were subjected to Western blotting. Results:P. intermedia supernatants promoted CXCL8 transcription and IL-8 production, and the reactions were significantly suppressed by clarithromycin pretreatment. Only trametinib, the selective mitogen-activated extracellular signal-regulated kinase inhibitor, downregulated CXCL8 transcription and IL-8 production. Furthermore, Western blotting revealed that stimulation with P. intermedia supernatants specifically induces extracellular signal-regulated kinases (ERK) 1/2 phosphorylation, which is suppressed by clarithromycin pretreatment. Notably, the interference analysis revealed that ERK3 might be dispensable for IL-8 production under the stimulation of P. intermedia supernatants. Conclusions: Our results provide new insight into the mechanism underlying P. intermedia-induced production of IL-8 from human airway epithelial cells. Furthermore, macrolides might have therapeutic potential in regulating periodontal pathogen-induced neutrophilic inflammation in the lungs.

Trametinib Sensitivity is Defined by a Myeloid Differentiation Profile in Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML) is a common blood cancer marked by heterogeneity in disease and diverse genetic abnormalities. Additional therapies are needed as the 5-year survival remains below 30%. Trametinib is a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor that is widely used in solid tumors and also in tumors with activating RAS mutations. A subset of patients with AML carry activating RAS mutations; however, a small-scale clinical trial with trametinib showed little efficacy. Here, we sought to identify transcriptomic determinants of trametinib sensitivity in AML. We tested the activity of trametinib against a panel of tumor cells from patients with AML ex vivo and compared this with RNA sequencing (RNA-Seq) data from untreated blasts from the same patient samples. We then used a correlation analysis between gene expression and trametinib sensitivity to identify potential biomarkers predictive of drug response. We found that a subset of AML tumor cells were sensitive to trametinib ex vivo, only a fraction of which (3/10) carried RAS mutations. On the basis of our RNA-Seq analysis we found that markers of trametinib sensitivity are associated with a myeloid differentiation profile that includes high expression of CD14 and CLEC7A (Dectin-1), similar to the gene expression profile of monocytes. Further characterization confirmed that trametinib-sensitive samples display features of monocytic differentiation with high CD14 surface expression and were enriched for the M4 subtypes of the FAB classification. Our study identifies additional molecular markers that can be used with molecular features including RAS status to identify patients with AML that may benefit from trametinib treatment.

19Fluorine-MRI Based Longitudinal Immuno-Microenvironment-Monitoring for Pancreatic Cancer.

Pancreatic cancer has a poor prognosis. Targeting Kirsten Rat Sarcoma (KRAS) mutation and its related pathways may enhance immunotherapy efficacy. While in vivo monitoring of therapeutic response and immune cell migration remains challenging, Fluorine-19 MRI (19F MRI) may allow noninvasive longitudinal imaging of immune cells. Evaluating the potential of 19F MRI for monitoring changes in the tumor immune microenvironment, in response to combined SHP2/MEK inhibition. Pre-clinical animal study. Murine genetically engineered pancreatic cancer model (N = 20, both sexes). 9.4-T, two-dimensional multi-slice Rapid Acquisition with Relaxation Enhancement sequence. Intravenous injection of 19F-perfluorocarbon (PFC) nanoparticles. Upon tumor detection by conventional 1H MRI screening, 19F MRI was performed in mice 24 hours after PFC nanoparticle administration. Animals were randomly assigned to four treatment groups: allosteric Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor SHP099, the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor Trametinib, the combination of both, or a vehicle control (4 to 6 mice each group), administered every other day per oral gavage. 1H and 19F MRI was repeated 7 days and 14 days later. Pancreatic immune cell infiltrates were analyzed by flow cytometry and multiplex immunohistofluorescence (mIHF) upon sacrifice. Independent t-tests and one-way analysis of variance. 19F MRI revealed continuous decrease of PFC-signals in tumors from vehicle controls (100%, 80%, and 74% on days 0, 7, and 14, respectively), contrasting with stable or increasing signals under KRAS-pathway-directed treatment. MEK inhibition showed 100%, 152%, and 84% and dual SHP2/MEK1/2 inhibition demonstrated signals of 100%, 134%, and 100% on days 0, 7, 14, respectively. mIHF analyses indicated CD11b+ macrophages/monocytes as primary contributors to the observed 19F MRI signal differences. 19F MRI might provide non-invasive longitudinal estimates for abundance and spatial distribution of CD11b+ macrophages/monocytes in pancreatic cancer. 1 TECHNICAL EFFICACY: Stage 2.

SKN-1 activation during infection of Caenorhabditis elegans requires CDC-48 and endoplasmic reticulum proteostasis.

During challenge of Caenorhabditis elegans with human bacterial pathogens such as Pseudomonas aeruginosa and Enterococcus faecalis, the elicited host response can be damaging if not properly controlled. The activation of Nrf (nuclear factor erythroid-related factor)/CNC (Cap-n-collar) transcriptional regulators modulates the response by upregulating genes that neutralize damaging molecules and promote repair processes. Activation of the C. elegans Nrf ortholog, SKN-1, is tightly controlled by a myriad of regulatory mechanisms, but a central feature is an activating phosphorylation accomplished by the p38 mitogen-activated kinase (MAPK) cascade. In this work, loss of CDC-48, an AAA+ ATPase, was observed to severely compromise SKN-1 activation on pathogen and we sought to understand the mechanism. CDC-48 is part of the endoplasmic reticulum (ER)-associated degradation (ERAD) complex where it functions as a remodeling chaperone enabling the translocation of proteins from the ER to the cytoplasm for degradation by the proteosome. Interestingly, one of the proteins retrotranslocated by ERAD, a process necessary for its activation, is SKN-1A, the ER isoform of SKN-1. However, we discovered that SKN-1A is not activated by pathogen exposure in marked contrast to the cytoplasmic-associated isoform SKN-1C. Rather, loss of CDC-48 blocks the antioxidant response normally orchestrated by SKN-1C by strongly inducing the unfolded protein response (UPRER). The data are consistent with the model of these 2 pathways being mutually inhibitory and support the emerging paradigm in the field of coordinated cooperation between different stress responses.

The SCOOP-MIK2 immune pathway modulates Arabidopsis root growth and development by regulating PIN-FORMED abundance and auxin transport.

Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor-like kinase (RLK) family at the cell surface. Various signaling peptide-RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN-FORMED (PIN)-regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1-INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen-activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response.

SIRT4 Has an Anti-Cancer Role in Cervical Cancer by Inhibiting Glutamine Metabolism via the MEK/ERK/C-Myc Axis.

Glutamine metabolism is crucial in cell proliferation, aging, and apoptosis across various cancer types. Existing research indicates that Sirtuin 4 (SIRT4), primarily located in mitochondria, modulates this process. This study aimed to clarify the regulatory relationship between SIRT4 and glutamine metabolism in cervical cancer. SIRT4 mRNA levels and their clinical correlation to cervical cancer were analyzed using the UALCAN database. Immunohistochemistry (IHC) was performed to assess SIRT4 protein expression in tissue samples from cervical cancer patients. Transient transfection was employed to create Hela and Siha cell lines with overexpressed SIRT4, mitogen-activated extracellular signal-regulated kinase (MEK), and glutaminase 1 (GLS1). The impact on cellular functions was studied using MTT, soft agar, transwell, and western blotting assays. Glutamate and ATP levels were also measured to evaluate metabolic changes. Low levels of SIRT4 mRNA in cervical cancer tissues correlated with tumor metastasis and poor survival rates. Overexpression of SIRT4 led to suppressed cell proliferation, colony growth, and motility, along with significant down-regulation of GLS expression, a key contributor to glutamine metabolism. Additionally, SIRT4 overexpression resulted in the inactivation of the MEK/ERK/c-myc signaling pathway, while overexpression of MEK reversed these effects. Notably, the inhibitory effects of SIRT4 on cell proliferation, colony formation, migration, and invasion in Hela and Siha cells were significantly attenuated following GLS1 overexpression. SIRT4 acts as an anti-cancer agent in cervical cancer by inhibiting glutamine metabolism through the MEK/ERK/c-myc signaling pathway, providing a novel sight for cervical cancer therapy.

Pharmacoinformational, basic and clinical studies of 7-hydroxymatairesinol in the context of searching for effective and safe therapeutic approaches to the treatment of mastopathy

7-hydroxymatairesinol (7(OH)MR) is the main lignan of spruce extracts, characterized by oncoprotective and anti-inflammatory properties. The results of chemoreactome modeling made it possible to formulate the molecular mechanisms of the pharmacological effects of 7(OH)MR for anti-inflammatory (inhibition of 5-lipoxygenase, matrix metalloproteinase MMP2, mitogen-activated kinase p38-alpha, leukotriene b4 receptor, prostacyclin receptor), oncoprotective (antioxidant effect due to inhibition heme oxygenase-2, inhibition of cyclin-dependent kinases 3 and 4, epidermal growth factor, mTOR protein). An assessment of the transcriptomic effects of 7(OH)MR in breast cancer cells showed significant dose-dependent effects on the transcription of 3468 of 12700 genes. The 7(OH)MR molecule, reducing the expression of proliferative genes (401 genes) and chronic inflammation (148 genes), while simultaneously increasing the transcription of oncoprotective immunity genes (100 genes), inhibits the proliferation of tumor cells. Experiments on solid Ehrlich carcinoma showed a significant oncoprotective effect of 7(OH)MR at doses of 60, 120 mg/day, even while taking estrogen (p = 0.007). A clinical study showed that 7(OH)MR (60 mg/day, 1 month) helps normalize estrogen metabolism and improve the clinical symptoms of mastopathy.

Epidermal growth factor receptor/mitogen-activated kinase inhibitor treatment induces a distinct inflammatory hair follicle response that includes collapse of immune privilege.

Inhibitors of epidermal growth factor receptor (EGFRi) or mitogen-activated kinase (MEKi) induce a folliculitis in 75-90% of patients, the pathobiology of which remains insufficiently understood. To characterize changes in the skin immune status and global transcriptional profile of patients treated with EGFRi; to investigate whether EGFRi affects the hair follicle's (HF) immune privilege (IP); and to identify early proinflammatory signals induced by EGFRi/MEKi in human scalp HFs ex vivo. Scalp biopsies were taken from patients exhibiting folliculitis treated long term with EGFRi ('chronic EGFRi' group, n = 9) vs. healthy scalp skin (n = 9) and patients prior to commencing EGFRi treatment and after 2 weeks of EGFRi therapy ('acute EGFRi' group, n = 5). Healthy organ-cultured scalp HFs were exposed to an EGFRi (erlotinib, n = 5) or a MEKi (cobimetinib, n = 5). Samples were assessed by quantitative immunohistomorphometry, RNA sequencing (RNAseq) and in situ hybridization. The 'chronic EGFRi' group showed CD8+ T-cell infiltration of the bulge alongside a partial collapse of the HF's IP, evidenced by upregulated major histocompatibility complex (MHC) class I, β2-microglobulin (B2 M) and MHC class II, and decreased transforming growth factor-β1 protein expression. Healthy HFs treated with EGFRi/MEKi ex vivo also showed partial HF IP collapse and increased transcription of human leucocyte antigen (HLA)-A, HLA-DR and B2 M transcripts. RNAseq analysis showed increased transcription of chemokines (CXCL1, CXCL13, CCL18, CCL3, CCL7) and interleukin (IL)-26 in biopsies from the 'chronic EGFRi' cohort, as well as increased IL-33 and decreased IL-37 expression in HF biopsies from the 'acute EGFRi' group and in organ-cultured HFs. The data show that EGFRi/MEKi compromise the physiological IP of human scalp HFs and suggest that future clinical management of EGFRi/MEKi-induced folliculitis requires HF IP protection and inhibition of IL-33.

A phase 1 study of the BET inhibitor ZEN003694 in combination with the MEK1/2 inhibitor binimetinib in solid tumors with RAS pathway alterations and triple-negative breast cancer (NCI number 10449).

TPS3182 Background: Bromodomain and extra-terminal (BET) proteins serve as epigenetic readers and regulate transcription by binding acetylated lysines in histones. BET inhibitors target this epigenetic machinery modifying the expression of oncogenes and have the potential to overcome drug resistance in different settings. Resistance to mitogen-activated extracellular kinase (MEK) inhibition appears to rely upon changes in gene expression, a process that can be potentially inhibited via BET blockade. Preclinical data have demonstrated synergistic efficacy of dual MEK and BET inhibition in MAPK-altered solid tumors. Triple negative breast cancer (TNBC) is a disease frequently found to have gene amplifications in BRAF, NF1 mutations, and upstream regulators of the MAPK pathway and preclinical work has shown efficacy with dual inhibition of MEK and BET. Methods: This phase 1, open-label, multicenter study was designed to evaluate the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the BET inhibitor ZEN003694 in combination with the MEK inhibitor binimetinib in solid tumors with RAS pathway alterations and TNBC. The study has two parts: dose escalation (part 1) which plans to recruit a maximum of 30 patients and dose expansion (part 2), which will comprise 12 patients. The primary objective of part 1 is to determine the MTD and recommended Phase 2 dose (RP2D), whereas the primary objective of dose expansion is to evaluate safety and toxicity. Secondary objectives include tolerability, pharmacokinetics (PK), and antitumor activity. Additional analyses will explore epigenetic changes such as ChIPseq to determine levels of H3K27ac and ATACseq to determine treatment-induced changes in open chromatin regions. ZEN003694 is given orally daily, and binimetinib is administered orally twice daily in 28-day cycles. Dose escalation is being conducted using a standard 3+3 cohort design to determine the MTD. Part 1 requires evaluable or measurable disease, whereas Part 2 requires measurable disease as per RECIST v1.1. Patients must have an ECOG performance status of ≤2. Included tumors are TNBC (estrogen receptor ≤1%, progesterone receptor ≤1%, human epidermal growth factor receptor 2 (HER2) 0–1+ or non-amplified) or any other solid tumor with actionable MAPK alterations (e.g. KRAS, NRAS, HRAS, or BRAF activating mutations, inactivating NF1 mutations, or BRAF fusions). Patients with any PI3K pathway activating genomic alterations are excluded. Prior therapy with BET, RAF, MEK, or ERK inhibitor is not permitted. This study is currently ongoing in dose escalation. Clinical trial information: NCI number 10449.

Cutaneous Reactions in Pediatric Patients Treated with MEK Inhibitors: A Retrospective Single-Center Study

Introduction: Mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors are in use for several indications for adults and children. Cutaneous toxicities are among the most common adverse effects. We aimed to describe the spectrum of cutaneous adverse events, its frequency, and severity in a cohort of pediatric patients. Methods: We reviewed all records of patients in our tertiary treatment center treated with MEK inhibitors between January 2016 and January 2023 for all indications. Results: Among 33 patients, 76% reported cutaneous adverse effects. The highest prevalence was in the group of patients treated with trametinib (90%), followed by the group treated with selumetinib (50%) and the group treated with a combination of trametinib and B-Raf proto-oncogene serine/threonine-protein kinase inhibitor (dabrafenib, 34%). Xerosis, dermatitis, paronychia, and hair heterochromia were most frequently reported. Severity was graded 1 or 2 for most adverse events, and 237 visits to the dermatology clinic related to these adverse events were recorded. Conclusions: Cutaneous adverse events are common in the pediatric population as in adults, but the clinical spectrum is different. Although considered mild, multiple dermatological consultations reflect the distress caused by these events. Dermatologists have a central role in the multidisciplinary care of pediatric patients receiving these agents.

Lignan 7-hydroxymatairesinol in the context of post-genomic pharmacology

Objective: to determine the molecular mechanisms of action of 7-hydroxymatairesinol (7(OH)MR), the main lignan of spruce extracts, characterized by oncoprotective and anti-inflammatory properties.Material and methods. The analysis of 7(OH)MR was carried out by chemoinformatic approach using the combinatorial theory of solvability and the topological theory of recognition. The postgenomic approach makes it possible to assess the effect of drugs on genome transcription (transcriptome) and on the proteome as a whole.Results. 7(OH)MP has anti-inflammatory (inhibition of 5-lipoxygenase, matrix metalloproteinase 2, mitogen-activated kinase p38-alpha, leukotriene В4 receptor, prostacyclin receptor), and oncoprotective (antioxidant effect due to inhibition of heme oxygenase-2, inhibition of cyclin-dependent kinases 3 and 4, epidermal growth factor, mTOR protein) pharmacological effects.Conclusion. By reducing the expression of proliferative genes and genes involved in chronic inflammation, the 7(OH)MP molecule inhibits the proliferation of tumor cells. Pharmacoinformatic modeling showed that the anti-inflammatory effects of 7(OH)MR may contribute to increased lifespan in animal models.

Lifileucel (Amtagvi): A cellular therapy for melanoma.

Lifileucel (Amtagvi – Iovance), a tumor-derived autologous T-cell immunotherapy, has received accelerated approval from the FDA for one-time treatment of adults with unresectable or metastatic melanoma previously treated with a programmed death receptor-1 (PD-1) inhibitor, and if BRAF V600 mutation-positive, a BRAF inhibitor with or without a mitogen-activated kinase (MEK) inhibitor.

Heavy metal stress and mitogen activated kinase transcription factors in plants: Exploring heavy metal-ROS influences on plant signalling pathways.

Due to their stationary nature, plants are exposed to a diverse range of biotic and abiotic stresses, of which heavy metal (HM) stress poses one of the most detrimental abiotic stresses, targeting diverse plant processes. HMs instigate the overproduction of reactive oxygen species (ROS), and to mitigate the adverse effects of ROS, plants induce multiple defence mechanisms. Besides the negative implications of overproduction of ROS, these molecules play a multitude of signalling roles in plants, acting as a central player in the complex signalling network of cells. One of the ROS-associated signalling mechanisms is the mitogen-activated protein kinase (MAPK) cascade, a signalling pathway which transduces extracellular stimuli into intracellular responses. Plant MAPKs have been implicated in signalling involved in stress response, phytohormone regulation, and cell cycle cues. However, the influence of various HMs on MAPK activation has not been well documented. In this review, we address and summarise several aspects related to various HM-induced ROS signalling. Additionally, we touch on how these signals activate the MAPK cascade and the downstream transcription factors that influence plant responses to HMs. Moreover, we propose a workflow that could characterise genes associated with MAPKs and their roles during plant HM stress responses.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Endothelial knockdown of the tumor suppressor, WWOX, increases inflammation in ventilator-induced lung injury.

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1β, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

Expression profile of mRNAs and miRNAs related to mitogen-activated kinases in HaCaT cell culture treated with lipopolysaccharide a and adalimumab

ABSTRACT Studies indicate that mitogen-activated protein kinases (MAPKs) exhibit activation and overexpression within psoriatic lesions. This study aimed to investigate alterations in the expression patterns of genes encoding MAPKs and microRNA (miRNA) molecules that potentially regulate their expression in human adult low-calcium high-temperature (HaCaT) keratinocytes when exposed to bacterial lipopolysaccharide A (LPS) and adalimumab. HaCaT cells underwent treatment with 1 µg/mL LPS for 8 hours, followed by treatment with 8 µg/mL adalimumab for 2, 8, or 24 hours. Untreated cells served as controls. The molecular analysis involved microarray, quantitative real-time polymerase chain reaction (RTqPCR), and enzyme-linked immunosorbent assay (ELISA) analyses. Changes in the expression profile of seven mRNAs: dual specificity phosphatase 1 (DUSP1), dual specificity phosphatase 3 (DUSP3), dual specificity phosphatase 4 (DUSP4), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase kinase kinase 2 (MAP3K2), mitogen-activated protein kinase kinase 2 (MAP2K2), and MAP kinase-activated protein kinase 2 (MAPKAPK2, also known as MK2) in cell culture exposed to LPS or LPS and the drug compared to the control. It was noted that miR-34a may potentially regulate the activity of DUSP1, DUSP3, and DUSP4, while miR-1275 is implicated in regulating MAPK9 expression. Additionally, miR-382 and miR-3188 are potential regulators of DUSP4 levels, and miR-200-5p is involved in regulating MAPKAPK2 and MAP3K2 levels. Thus, the analysis showed that these mRNA molecules and the proteins and miRNAs they encode appear to be useful molecular markers for monitoring the efficacy of adalimumab therapy.

Gene expression profile of mitogen-activated kinases and microRNAs controlling their expression in HaCaT cell culture treated with lipopolysaccharide A and cyclosporine A

ABSTRACT Studies indicate that mitogen-activated protein kinases (MAPKs) are activated and overexpressed in psoriatic lesions. The aim of the study was to assess changes in the expression pattern of genes encoding MAPKs and microRNA (miRNA) molecules potentially regulating their expression in human adult low-calcium high-temperature (HaCaT) keratinocytes exposed to bacterial lipopolysaccharide A (LPS) and cyclosporine A (CsA). HaCaT cells were treated with 1 µg/mL LPS for 8 h, followed by treatment with 100 ng/mL cyclosporine A for 2, 8, or 24 h. Untreated cells served as controls. The molecular analysis consists of microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay analyses. The statistical analysis of the obtained results was performed using Transcriptome Analysis Console and STATISTICA 13.5 PL with the statistical significance threshold of p < 0.05. Changes in the expression profile of six mRNAs: dual-specificity phosphatase 1 (DUSP1), dual-specificity phosphatase 4 (DUSP4), mitogen-activated protein kinase kinase 2 (MAP2K2), mitogen-activated protein kinase kinase 7 (MAP2K7), mitogen-activated protein kinase kinase kinase 2 (MAP3K2) and mitogen-activated protein kinase 9 (MAPK9) in cell culture exposed to LPS or LPS and the drug compared to the control. We observed that under the LPS and cyclosporine treatment, the expression o/ miR-34a, miR-1275, miR-3188, and miR-382 changed significantly (p < 0.05). We demonstrated a potential relationship between DUSP1 and miR-34a; DUSP4 and miR-34a, miR-382, and miR-3188; MAPK9 and miR-1275, MAP2K7 and mir-200-5p; MAP3K2 and mir-200-5p, which may be the subject of further research in the context of psoriasis.