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PurposeStressful training with insufficient recovery can impair muscle performance. Expression of mitogen-activated protein kinases (MAPK) has been reported at rest following overreaching and overtraining. The acute myocellular exercise response to stressful training with insufficient recovery has not been investigated. We investigated MAPK, androgen, and glucocorticoid receptor phosphorylation following a period of stressful training.MethodsSixteen resistance-trained men were matched on barbell squat 1 repetition maximum strength and randomized into a group that performed normal training or stressful training with insufficient recovery. The control group (CON) performed three speed-squat training sessions on non-consecutive days, while the stressful training group (NFOR) performed 15 training sessions over 7.5 days. Resting and post-exercise skeletal muscle biopsies were obtained prior to (T1) and after the training period (T2). Samples were analyzed for total and phosphorylated androgen receptor (AR), glucocorticoid receptor (GR), and MAPKs (ERK, JNK, and p38).ResultsTotal AR were down-regulated post-exercise at T2 in NFOR only. Phospho-AR at ser515 increased in both groups post-exercise at T1; however, ser515 only increased at T2 in NFOR. Phosphorylated ERK, JNK, and p38 increased post-exercise in CON and NFOR at T1 and T2. Post-exercise phospho-p38 was blunted in NFOR at T2 compared to T1. After the training intervention, resting phospho-p38 was higher in NFOR compared to T1. At T2, post-exercise phospho-GR at ser226 was lower compared to T1, and resting levels increased in NFOR.ConclusionSteroid receptors are phosphorylated after acute resistance exercise, and in addition to MAPKs, are differentially regulated after stressful training with insufficient recovery.

Crystallographic analysis depicting the interaction of the kinase inhibitor SCH772984 with ERK1/2 reveals a unique binding pocket distinct from off-targets such as haspin and is associated with slow binding kinetics and prolonged inhibitory activity. Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

This study focused on understanding the signaling mechanisms leading to GLUT-4 translocation and increased skeletal-muscle glucose uptake that follow creatine (Cr) supplementation in type 2 diabetes ( n  = 10). AMPK-α protein content presented a tendency to be higher ( p  = 0.06) after Cr supplementation (5 g/d for 12w). The changes in AMPK-α protein content significantly related ( p  < 0.001) to the changes in GLUT-4 translocation ( r  = 0.78) and Hb1Ac levels ( r  = −0.68), suggesting that AMPK signaling may be implicated in the effects of supplementation on glucose uptake in type 2 diabetes.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα 12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research. Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

Tumorigenesis driven by the oncogene BRAF V600E is shown both to depend on the BRAF substrates MEK1/2 associating with copper, and to be sensitive to copper-chelating drugs, suggesting merit in testing such drugs for the treatment of BRAF mutation-positive cancers. A role for copper in BRAF cancers A large proportion of melanomas and some other cancers harbour mutations in the BRAF gene, most of them at codon 600, causing constitutive activation of the MAPK (mitogen-activated protein kinase) pathway. Following the discovery that copper transport promotes MAPK signalling in Drosophila by binding to and activating the kinase MEK, Chris Counter and colleagues now show that oncogenic signalling by mutant BRAF requires copper binding to MEK, promoting activation of ERK1/2, the next kinases in the cascade. Interfering with copper availability by genetic means or with copper-chelating agents reduces BRAF-driven tumour growth in vivo in mouse models, and also of cancer cells that have become resistant to BRAF inhibitors. Thus copper chelators, already in the clinic for other indications, may prove useful for the treatment of BRAF-mutant tumours in combination with BRAF inhibitors, and potentially to prevent resistance. The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers 1 , 2 . BRAF V600E phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer 3 . Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma 4 , an effect that is increased when administered together with a BRAF V600E inhibitor 5 . We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu–MEK1 interaction 6 . Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF V600E -driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1–MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1 . Cu chelators used in the treatment of Wilson disease 7 decreased tumour growth of human or murine cells transformed by BRAF V600E or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF V600E mutation.

Transforming growth factor—β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal—regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase—1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Mitogen-activated protein kinase phosphatase-5 (MKP-5) is a regulator of extracellular signaling that is known to regulate lipid metabolism. In this study, we found that obesity caused by a high-fat diet (HFD) decreased the expression of MKP-5 in the pancreas and primary islet cells derived from mice. Then, we further investigated the role of MKP-5 in the protection of islet cells from lipotoxicity by modulating MKP-5 expression. As a critical inducer of lipotoxicity, palmitic acid (PA) was used to treat islet β-cells. We found that MKP-5 overexpression restored PA-mediated autophagy inhibition in Rin-m5f cells and protected these cells from PA-induced apoptosis and dysfunction. Consistently, a lack of MKP-5 aggravated the adverse effects of lipotoxicity. Islet cells from HFD-fed mice were infected using recombinant adenovirus expressing MKP-5 (Ad-MKP-5), and we found that Ad-MKP-5 was able to alleviate HFD-induced apoptotic protein activation and relieve the HFD-mediated inhibition of functional proteins. Notably, HFD-mediated impairments in autophagic flux were restored by Ad-MKP-5 transduction. Furthermore, the autophagy inhibitor 3-methyladenine (3-MA) was used to treat Rin-m5f cells, confirming that the MKP-5 overexpression suppressed apoptosis, dysfunction, inflammatory response, and oxidative stress induced by PA via improving autophagic signaling. Lastly, employing c-Jun amino-terminal kinas (JNK), P38, or extracellular-regulated kinase (ERK) inhibitors, we established that the JNK and P38 MAPK pathways were involved in the MKP-5-mediated apoptosis, dysfunction, and autophagic inhibition observed in islet β cells in response to lipotoxicity.

Cigarette smoking plays an important role in the progression of chronic kidney disease (CKD). Nicotine, one of the major components of cigarette smoking, has been demonstrated to increase proliferation of renal mesangial cells. In this study, we examined the effect of nicotine on podocyte injury. To determine the expression of nicotinic acetylcholine receptors (nAChR subunits) in podocytes, cDNAs and cell lysate of cultured human podocytes were used for the expression of nAChR mRNAs and proteins, respectively; and mouse renal cortical sections were subjected to immunofluorescant staining. We also studied the effect of nicotine on podocyte nephrin expression, reactive oxygen species (ROS) generation (via DCFDA loading followed by fluorometric analysis), proliferation, and apoptosis (morphologic assays). We evaluated the effect of nicotine on podocyte downstream signaling including phosphorylation of ERK1/2, JNK, and p38 and established causal relationships by using respective inhibitors. We used nAChR antagonists to confirm the role of nicotine on podocyte injury. Human podocytes displayed robust mRNA and protein expression of nAChR in vitro studies. In vivo studies, mice renal cortical sections revealed co-localization of nAChRs along with synaptopodin. In vitro studies, nephrin expression in podocyte was decreased by nicotine. Nicotine stimulated podocyte ROS generation; nonetheless, antioxidants such as N-acetyl cysteine (NAC) and TEMPOL (superoxide dismutase mimetic agent) inhibited this effect of nicotine. Nicotine did not modulate proliferation but promoted apoptosis in podocytes. Nicotine enhanced podocyte phosphorylation of ERK1/2, JNK, and p38, and their specific inhibitors attenuated nicotine-induced apoptosis. nAChR antagonists significantly suppressed the effects of nicotine on podocyte. Nicotine induces podocyte apoptosis through ROS generation and associated downstream MAPKs signaling. The present study provides insight into molecular mechanisms involved in smoking associated progression of chronic kidney disease.