Periostin regulates lysyl oxidase through ERK1/2 MAPK-dependent serum response factor in activated cardiac fibroblasts.

Collagen crosslinking, mediated by lysyl oxidase, is an adaptive mechanism of the cardiac repair process initiated by cardiac fibroblasts postmyocardial injury. However, excessive crosslinking leads to cardiac wall stiffening, which impairs the contractile properties of the left ventricle and leads to heart failure. In this study, we investigated the role of periostin, a matricellular protein, in the regulation of lysyl oxidase in cardiac fibroblasts in response to angiotensin II and TGFß1. Our results indicated that periostin silencing abolished the angiotensin II and TGFß1-mediated upregulation of lysyl oxidase. Furthermore, the attenuation of periostin expression resulted in a notable reduction in the activity of lysyl oxidase. Downstream of periostin, ERK1/2 MAPK signaling was found to be activated, which in turn transcriptionally upregulates the serum response factor to facilitate the enhanced expression of lysyl oxidase. The periostin-lysyl oxidase association was also positively correlated in an in vivo rat model of myocardial infarction. The expression of periostin and lysyl oxidase was upregulated in the collagen-rich fibrotic scar tissue of the left ventricle. Remarkably, echocardiography data showed a reduction in the left ventricular wall movement, ejection fraction, and fractional shortening, indicative of enhanced stiffening of the cardiac wall. These findings shed light on the mechanistic role of periostin in the collagen crosslinking initiated by activated cardiac fibroblasts. Our findings signify periostin as a possible therapeutic target to reduce excessive collagen crosslinking that contributes to the structural remodeling associated with heart failure.

Inhibition of Liver Cancer Cell Viability by Triazole through Up-regulation of p38 Phosphorylation and Targeting the Activation of p-ERK1/2 and Akt Protein Expression.

The present study was aimed to explore the effect of triazole on growth and viability of liver cancer cells. Cell growth was examined using the MTT test and expression of several proteins was assessed by western blotting assay. The Matrigel-coated Transwell assay was employed to examine the infiltration of cells. The data from MTT assay showed that MHCC97H and H4TG liver cancer cell viability was inhibited by triazole in a concentration-dependent manner. After treatment with 0.5, 1.0, 2.0, 4, 8, and 16 µM doses of triazole, the rate of H4TG cell viability was decreased to 96, 73, 58, 39, 29, and 28%, respectively. Treatment of MHCC97H cells with 0.5, 1.0, 2.0, 4, 8, and 16 µM doses of triazole resulted in a reduction in cell viability to 94, 70, 53, 35, 22, and 21%, respectively. Triazole treatment also led to a significant reduction in MHCC97H cell invasiveness compared to the control cells. In MHCC97H cells treated with triazole, there was a noticeable decrease in the levels of p-ERK1/2, and p-Akt protein expression. Treatment of MHCC97H cells with triazole resulted in a prominent increase in p-p38 level. In summary, triazole inhibits growth and viability of liver cancer cells through targeting the activation of p-ERK1/2 and Akt proteins. Therefore, triazole may be investigated further as a therapeutic agent for the treatment of liver cancer.

Inhibition of ERK1/2 or CRMP2 Disrupts Alcohol Memory Reconsolidation and Prevents Relapse in Rats.

Relapse to alcohol abuse, often caused by cue-induced alcohol craving, is a major challenge in alcohol addiction treatment. Therefore, disrupting the cue-alcohol memories can suppress relapse. Upon retrieval, memories transiently destabilize before they reconsolidate in a process that requires protein synthesis. Evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), governing the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase 1/2 (ERK1/2), in the reconsolidation process in a rat (Wistar) model of alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, while the PI3K-AKT pathway remained unaffected. Importantly, ERK1/2 inhibition after alcohol memory retrieval impaired alcohol-memory reconsolidation and led to long-lasting relapse suppression. Attenuation of relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2)-a translational product of mTORC1. Together, our findings indicate the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, with their inhibition as potential treatment targets for relapse prevention.

TGF-ß1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation.

Extracellular vesicles have emerged as important mediators of cell-to-cell communication in the pathophysiology of fibrotic diseases. One such disease is Peyronie's disease (PD), a fibrotic disorder of the penis caused by uncontrolled transformation of resident fibroblasts to alpha-smooth muscle actin positive myofibroblasts. These cells produce large amounts of extracellular matrix, leading to formation of a plaque in the penile tunica albuginea (TA), causing pain, penile curvature, and erectile dysfunction. We have used primary fibroblasts derived from the TA of PD patients to explore the role of transforming growth factor beta 1 (TGF-ß1), a key signalling factor in this process. TGF-ß1 treatment elicited a range of responses from the myofibroblasts: (i) they secreted extracellular vesicles (EVs) that were more numerous and differed in size and shape from those secreted by fibroblasts, (ii) these EVs prevented TGF-ß1-induced transformation of fibroblasts in a manner that was dependent on vesicle uptake and (iii) they prevented phosphorylation of Erk1/2, a critical component in modulating fibrogenic phenotypic responses, but did not affect TGF-ß1-induced Smad-signalling. We posit that this effect could be linked to enrichment of TSG-6 in myofibroblast-derived EVs. The ability of myofibroblast-derived vesicles to prevent further myofibroblast transformation may establish them as part of an anti-fibrotic negative feedback loop, with potential to be exploited for future therapeutic approaches.

Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway.

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network pharmacology. Employing network pharmacology and molecular docking techniques, we predicted the active components and targets of JWSJS and constructed a meticulous "drug-component-target" network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were utilized to discern the therapeutic pathways and targets of JWSJS. Autodock Vina 1.2.0 was deployed for molecular docking verification, and a 100-ns molecular dynamics simulation was conducted to affirm the docking results, followed by in vivo animal verification. The findings revealed that JWSJS shared 227 intersecting targets with diabetic nephropathy, constructing a protein-protein interaction network topology. KEGG enrichment analysis denoted that JWSJS mitigates diabetic nephropathy by modulating lipids and atherosclerosis, the PI3K-Akt signaling pathway, apoptosis, and the HIF-1 signaling pathway, with mitogen-activated protein kinase 1 (MAPK1), MAPK3, epidermal growth factor receptor (EGFR), and serine/threonine-protein kinase 1 (AKT1) identified as collective targets of multiple pathways. Molecular docking asserted that the core components of JWSJS (quercetin, palmitoleic acid, and luteolin) could stabilize conformation with three pivotal targets (MAPK1, MAPK3, and EGFR) through hydrogen bonding. In vivo examinations indicated notable augmentation in body weight and reductions in glycated serum protein (GSP), low-density lipoprotein cholesterol (LDL-C), uridine triphosphate (UTP), and fasting blood glucose (FBG) levels due to JWSJS. Electron microscopy coupled with hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) staining highlighted the potential of each treatment group in alleviating kidney damage to diverse extents, exhibiting varied declines in p-EGFR, p-MAPK3/1, and BAX, and increments in BCL-2 expression in the kidney tissues of the treated rats. Conclusively, these insights suggest that the protective efficacy of JWSJS on diabetic nephropathy might be associated with suppressing the activation of the EGFR/MAPK3/1 signaling pathway and alleviating renal cell apoptosis.

Micropeptide AF127577.4-ORF hidden in a lncRNA diminishes glioblastoma cell proliferation via the modulation of ERK2/METTL3 interaction.

Micropeptides hidden in long non-coding RNAs (lncRNAs) have been uncovered to program various cell-biological changes associated with malignant transformation-glioblastoma (GBM) cascade. Here, we identified and characterized a novel hidden micropeptide implicated in GBM. We screened potential candidate lncRNAs by establishing a workflow involving ribosome-bound lncRNAs, publicly available MS/MS data, and prognosis-related lncRNAs. Micropeptide expression was detected by western blot (WB), immunofluorescence (IF), and immunohistochemistry (IHC). Cell proliferation rate was assessed by calcein/PI staining and EdU assay. Proteins interacted with the micropeptide were analyzed by proteomics after co-immunoprecipitation (Co-IP). We discovered that lncRNA AF127577.4 indeed encoded an endogenous micropeptide, named AF127577.4-ORF. AF127577.4-ORF was associated with GBM clinical grade. In vitro, AF127577.4-ORF could suppress GBM cell proliferation. Moreover, AF127577.4-ORF reduced m6A methylation level of GBM cells. Mechanistically, AF127577.4-ORF diminished ERK2 interaction with m6A reader methyltransferase like 3 (METTL3) and downregulated phosphorylated ERK (p-ERK) level. The ERK inhibitor reduced p-ERK level and downregulated METTL3 protein expression. AF127577.4-ORF weakened the stability of METTL3 protein by ERK. Also, AF127577.4-ORF suppressed GBM cell proliferation via METTL3. Our study identifies a novel micropeptide AF127577.4-ORF hidden in a lncRNA, with a potent anti-proliferating function in GBM by diminishing METTL3 protein stability by reducing the ERK2/METTL3 interaction. This micropeptide may be beneficial for development of therapeutic strategies against GBM.

Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

OBJECTIVE: This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. MATERIALS AND METHODS: Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. RESULTS: BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. CONCLUSION: This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

ML241 Antagonizes ERK 1/2 Activation and Inhibits Rotavirus Proliferation.

Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.

Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.

ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2.

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

Inhibition of HSP20 Ameliorates Steatotic Liver Disease by Stimulating ERK2-Dependent Autophagy.

HSP20 emerges as a novel regulator of autophagy in the heart. Nonetheless, the detailed function of HSP20 in the liver and its effect on autophagy remain unknown. Here, we observed that HSP20 expression is increased in liver tissues from mice and patients with metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease. Liver-specific downregulation of HSP20 mitigates hepatic steatosis and insulin resistance in obese mice, while upregulating HSP20 promotes lipid deposition and hepatocyte cell death. Mechanistically, liquid chromatography-tandem mass spectrometry revealed that HSP20 interacts with phosphorylated extracellular regulated protein kinase 2 (ERK2) and prevents its dephosphorylation by dual specificity phosphatase 6, leading to ERK2-mediated repression of autophagy and resulting in aggravated saturated fatty acid (SFA)-triggered hepatocyte death. Importantly, such adverse effects could be ameliorated by ERK inhibitor. Our data reveal a framework of how HSP20 increases susceptibility of SFA-induced liver injury through enhancing ERK2 phosphorylation, which represents a plausible therapeutic intervention to combat MASLD.

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity.

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Transient Receptor Potential Ankyrin 1-dependent Activation of Extracellular Signal-regulated Kinase 2 in the Cerebral Cortices Contributes to Cortical Spreading Depolarization.

Extracellular signal-regulated kinase (ERK) are serine/threonine-selective proteins and ERK1/2 can be phosphorylated in peripheral and central brain regions after cortical spreading depolarization (CSD) and calcitonin gene-related peptide; However, it remains unclear about whether and how ERK activity modulates CSD that correlates to migraine aura. Here, we determined the role of ERK in regulating CSD and explored the underlying mechanism involving transient receptor potential ankyrin 1 (TRPA1), a stress-sensing cation channel. CSD was recorded using intrinsic optical imaging in mouse brain slices, and electrophysiology in rats. Phosphorylated ERK (pERK1/2) and interleukin-1ß (IL-1ß) protein levels were detected using Western blot or enzyme-linked immunosorbent assay, respectively. IL-1ß mRNA level was detected using qPCR. The results showed that an ERK inhibitor, SCH77298, markedly prolonged CSD latency and reduced propagation rate in mouse brain slices. Corresponding to this, CSD induction increased levels of cytosolic pERK1/2 in ipsilateral cerebral cortices of rats, the elevation of which correlated to the level of IL-1ß mRNA. Mechanistic analysis showed that pre-treatment of an anti-TRPA1 antibody reduced the cytosolic pERK2 level but not pERK1 following CSD in cerebral cortices of rats and this level of pERK2 correlated with that of cerebral cortical IL-1ß protein. Furthermore, an ERK activator, AES16-2M, but not its scrambled control, reversed the prolonged CSD latency by a TRPA1 inhibitor, HC-030031, in mouse brain slices. These data revealed a crucial role of ERK activity in regulating CSD, and elevation of pERK and IL-1ß production induced by CSD is predominantly TRPA1 channel-dependent, thereby contributing to migraine pathogenesis.

N6-methyladenosine-modified CircPSMA7 enhances bladder cancer malignancy through the miR-128-3p/MAPK1 axis.

Several studies have indicated that circular RNAs (circRNAs) play vital roles in the progression of various diseases, including bladder cancer (BCa). However, the underlying mechanisms by which circRNAs drive BCa malignancy remain unclear. In this study, we identified a novel circRNA, circPSMA7 (circbaseID:has_circ_0003456), showing increased expression in BCa cell lines and tissues, by integrating the reported information with circRNA-seq and qRT-PCR. We revealed that circPSMA7 is associated with a higher tumor grade and stage in BCa. M6A modification was identified in circPSMA7, and IGF2BP3 recognized this modification and stabilized circPSMA7, subsequently increasing the circPSMA7 expression. In vitro and in vivo experiments showed that circPSMA7 promoted BCa proliferation and metastasis by regulating the cell cycle and EMT processes. CircPSMA7 acted as a sponge for miR-128-3p, which showed antitumor effects in BCa cell lines, increasing the expression of MAPK1. The tumor proliferation and metastasis suppression induced by silencing circPSMA7 could be partly reversed by miR-128-3p inhibition. Thus, the METTL3/IGF2BP3/circPSMA7/miR-128-3p/MAPK1 axis plays a critical role in BCa progression. Furthermore, circPSMA7 may be a potential diagnostic biomarker and novel therapeutic target for patients with BCa.

MEK1-ERK1/2 signaling regulates the cardiomyocyte non-sarcomeric actin cytoskeletal network.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic ß- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic ß-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Expression and promoter methylation of mitogen-activated protein kinase 1 in tumor and marginal cells of breast cancer.

AIM: In the present study, we sought to explore potential differences in the expression and promoter methylation of mitogen-activated protein kinase 1 (MAPK1) between tumor and marginal cells of breast cancer lesions. METHODS: A total of 50 randomly selected patients with breast cancer (BCa) undergoing needle biopsy were enrolled. Clinical specimens containing both tumor and marginal cells were collected and preserved. After DNA extraction using specific primers, MAPK1 mRNA and promoter methylation were measured with spectrophotometry at 260/280 nm absorption wavelengths. To deliver a comparative analysis, data from The Cancer Genome Atlas (TCGA) program regarding breast cancer (BRCA), were downloaded from Xena Functional Genomics Explorer and separately analyzed. The suitability of MAPK1 expression and promoter methylation as biomarkers for BCa was analyzed with receiver operating characteristic (ROC) curves. RESULTS: We found a positive correlation between tumor stage and MAPK1 expression (P-value: 0.029) in BCa. Likewise, MAPK1 expression was significantly associated with lymph node metastasis (P-value: 0.018). There was a significant difference in the expression of MAPK1 mRNA between tumor and marginal cells of BCa and BRCA (P-value < 0.001). However, we did not find any statistically significant difference in MAPK1 promoter methylation between tumor and marginal cells of both BCa and BRCA. With an area under the curve (AUC) of 0.71, the diagnostic accuracy of MAPK1 expression in BCa and BRCA was validated. However, MAPK1 promoter methylation was not found to be a suitable biomarker. CONCLUSION: Our findings suggest that while MAPK1 expression, might be a promising biomarker for evaluating oncogenic activity in patients suspected of BCa. We were not able to detect a prognostic/diagnostic role for MAPK1 promoter methylation.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.