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Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

Automaticity involves Ca2+ handling at the cell membrane and sarcoplasmic reticulum (SR). Abnormal or acquired automaticity is thought to initiate ventricular arrhythmias associated with myocardial ischemia. Ca2+ flux from mitochondria can influence automaticity, and lysosomes also release Ca2+. Therefore, we tested whether lysosomal Ca2+ flux could influence automaticity. We studied ventricular human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), hiPSC 3D engineered heart tissues, and ventricular cardiomyocytes isolated from infarcted mice. Preventing lysosomal Ca2+ cycling reduced automaticity in hiPSC-CMs. Consistent with a lysosomal role in automaticity, activating the transient receptor potential mucolipin channel (TRPML1) enhanced automaticity, and two channel antagonists reduced spontaneous activity. Activation or inhibition of lysosomal transcription factor EB (TFEB) increased or decreased total lysosomes and automaticity, respectively. In adult ischemic cardiomyocytes and hiPSC 3D engineered heart tissues, reducing lysosomal Ca2+ release also inhibited automaticity. Finally, TRPML1 was upregulated in cardiomyopathic patients with ventricular tachycardia (VT) compared to those without VT. In summary, lysosomal Ca2+ handling modulates abnormal automaticity, and reducing lysosomal Ca2+ release may be a novel strategy for preventing ventricular arrhythmias.

Advanced or metastatic breast cancer affects multiple organs and is a leading cause of cancer-related death. Cancer metastasis is associated with epithelial-mesenchymal metastasis (EMT). However, the specific signals that induce and regulate EMT in carcinoma cells remain unclear. PRR16/Largen is a cell size regulator that is independent of mTOR and Hippo signalling pathways. However, little is known about the role PRR16 plays in the EMT process. We found that the expression of PRR16 was increased in mesenchymal breast cancer cell lines. PRR16 overexpression induced EMT in MCF7 breast cancer cells and enhances migration and invasion. To determine how PRR16 induces EMT, the binding proteins for PRR16 were screened, revealing that PRR16 binds to Abl interactor 2 (ABI2). We then investigated whether ABI2 is involved in EMT. Gene silencing of ABI2 induces EMT, leading to enhanced migration and invasion. ABI2 is a gene that codes for a protein that interacts with ABL proto-oncogene 1 (ABL1) kinase. Therefore, we investigated whether the change in ABI2 expression affected the activation of ABL1 kinase. The knockdown of ABI2 and PRR16 overexpression increased the phosphorylation of Y412 in ABL1 kinase. Our results suggest that PRR16 may be involved in EMT by binding to ABI2 and interfering with its inhibition of ABL1 kinase. This indicates that ABL1 kinase inhibitors may be potential therapeutic agents for the treatment of PRR16-related breast cancer.

We have described an arrhythmic mechanism seen only in cardiomyopathy that involves increased mitochondrial CaWe tested the role of SERCA2a in arrhythmias during ischemic cardiomyopathy.Myocardial infarction (MI) was induced in wild-type (Wt) and SERCA2a heterozygous knockdown (SERCACompared with Wt MI mice, SERCA2a heterozygous knockdown (SERCASERCA2a knockdown was antiarrhythmic after MI without affecting overall systolic performance. Possible antiarrhythmic mechanisms included reduced SR free Ca

Cardiomyopathies are associated with arrhythmias and cardiac ion channel downregulation. This downregulation is arrhythmogenic. Paradoxically, antiarrhythmic therapies are based on ion channel-blocking drugs that further downregulate these channels and exhibit proarrhythmic risk. Recent studies have shown that inhibition of the protein kinase RNA-like ER kinase (PERK) arm of the unfolded protein response (UPR) prevents select cardiac ion channel downregulation and plays a protective role against arrhythmias. Prevention of ion channel downregulation represents as a novel therapeutic strategy to treat arrhythmias in myocardial infarction and heart failure.

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias during cardiomyopathy. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in ischemic cardiomyopathy. Methods: Ischemic HF was induced in wild type FVB/NJ mice for three weeks by ligated left anterior descending coronary artery. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to sham hearts, the protein level of p-Src increased significantly in ischemic HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in ischemic HF mouse cardiomyocytes. Protein expression of CaMKII, PyK2, and p-PyK2 did not change during ischemic HF. MI activated c-Src by S-nitrosylation of caveolin-1 with subsequent release of the kinase. c-Src activation was associated with APD prolongation and arrhythmia following myocardial infarction in mice. Human heart tissue showed that ischemic HF patients had significantly increased p-Src associated with increased QTc prolongation and arrhythmia. Conclusions: During ischemic HF, c-Src activation directly increased MCU current that contributed to QT prolongation and arrhythmic risk in ischemic cardiomyopathy.

Background: The voltage-gated cardiac sodium channel α subunit ( SCN5A ; encoding Na v 1.5) plays a key role in cardiac conduction. The sodium channel is downregulated in cardiomyopathy, contributing to arrhythmic risk. In part, this downregulation is because of abnormal mRNA splicing and reduced mRNA stability. We recently reported that microRNA (miRNA)-448 contributes to the SCN5A mRNA instability in ischemic conditions. Objective: Long intergenic non-protein coding RNA 667 (LINC00667) is expressed in heart and can bind miRNA-448. We investigated the interaction of these two non-coding RNAs (ncRNAs) in regulating SCN5A . Methods: The expressions of SCN5A , miR-448 and LINC00667 were determined by quantitative real time polymerase chain reaction. The effects of ncRNAs on sodium current were determined in induced pluripotent stem cells-derived cardiomyocytes. The interaction between two ncRNAs was confirmed by pull-down assay. Results: LINC00667 has two binding sites for miR-448. A pull-down assay using biotin-tagged miR-448 showed direct binding of LINC00667 to miR-448. This indicates that LINC00667 could act as a competitor through the sponge function to miR-448. The expression of LINC00667 was decreased in human ischemic cardiomyopathy tissues compared to healthy controls. DFX treatment, a hypoxia mimetic chemical, or hypoxic condition (2% O 2 ) resulted in reduced LINC00667 and reduced SCN5A mRNA in cardiomyocyte. Furthermore, LINC00667 inhibition by siRNA or antisense oligo showed reduction of SCN5A mRNA. This reduction is expected to enhance the effect of miR-448 and is expected to be inhibited by treatment with a DNA construct containing the miR-448 binding site of LINC00667 or "miR-448 sponge". Conclusion: SCN5A is downregulated by hypoxia. In addition to upregulation of miR-448, which destabilizes SCN5A mRNA by direct binding, LINC00667 inhibition of miR-448 was relieved by hypoxia increasing the effect of miR-448 on SCN5A . Therefore, LINC00667 upregulation may represent a method to decrease arrhythmic risk during cardiomyopathy by raising SCN5A levels.

Introduction: Hypomagnesemia (HypoMg) induces mitochondrial dysfunction accompanied by disturbed Mg homeostasis in mouse hearts. Transient receptor potential cation channel subfamily M 7 (TRPM7) is a Mg transporter with both channel and kinase function located in the plasma membrane. Its upregulation has been observed in HypoMg. We investigated the role of TRPM7 in mitochondrial dysfunction mediated by HypoMg. Methods: Wild type (WT) C57BL/6J mice and transgenic mice with a K1646R mutation in the TRPM7 kinase domain (TRPM7 K1646R , with no kinase function) were fed with a low-Mg diet (HypoMg mice, 15-30 mg/kg Mg) or a normal Mg diet (control mice, 600 mg/kg Mg) for 6 weeks. Human cardiac RL-14 cells were cultured in a normal (with 0.81 mM MgSO 4 ) or a low-Mg medium (customized medium with 0.04 mM MgSO 4 ) for 48 h. TRPM7 knockdown was achieved by siRNA in RL-14 cells. Results: WT HypoMg mice showed significantly decreased serum Mg, diastolic dysfunction, increased mitochondrial ROS in isolated cardiomyocytes, increased TRPM7 mRNA and protein level, decreased ATP production and decreased total mitochondrial Mg content in heart tissues. TRPM7 K1646R mice on the low-Mg diet showed normal diastolic function, normal mitochondrial ROS levels in cardiomyocytes, and recovered ATP production and mitochondrial Mg content in hearts. Furthermore, TRPM7 K1646R prevent HypoMg-induced seizures and death (64.8% death in WT HypoMg mice). Hypomagnesemia in RL-14 cells resulted in increased mitochondrial ROS (1.77±0.08-fold of control, P Conclusions: HypoMg induces elevation of TRPM7 protein expression, and TRPM7 is involved in mitochondrial dysfunction, diastolic dysfunction, and death associated with HypoMg.

ARG2 has been reported to inhibit autophagy in vascular endothelial cells and keratinocytes. However, studies of its mechanism of action, its role in skin fibroblasts, and the possibility of promoting autophagy and inhibiting cellular senescence through ARG2 inhibition are lacking. We induced cellular senescence in dermal fibroblasts by using H2O2. H2O2-induced fibroblast senescence was inhibited upon ARG2 knockdown and promoted upon ARG2 overexpression. The microRNA miR-1299 suppressed ARG2 expression, thereby inhibiting fibroblast senescence, and miR-1299 inhibitors promoted dermal fibroblast senescence by upregulating ARG2. Using yeast two-hybrid assay, we found that ARG2 binds to ARL1. ARL1 knockdown inhibited autophagy and ARL1 overexpression promoted it. Resolvin D1 (RvD1) suppressed ARG2 expression and cellular senescence. These data indicate that ARG2 stimulates dermal fibroblast cell senescence by inhibiting autophagy after interacting with ARL1. In addition, RvD1 appears to promote autophagy and inhibit dermal fibroblast senescence by inhibiting ARG2 expression. Taken together, the miR-1299/ARG2/ARL1 axis emerges as a novel mechanism of the ARG2-induced inhibition of autophagy. Furthermore, these results indicate that miR-1299 and pro-resolving lipids, including RvD1, are likely involved in inhibiting cellular senescence by inducing autophagy.

LW1497 suppresses the expression of the hypoxia-inducing factor (HIF)-1α inhibiting malate dehydrogenase. Although hypoxia and HIF-1α are known to be important in cancer, LW1497 has not been therapeutically applied to cancer yet. Thus, we investigated the effect of LW1497 on the epithelial-mesenchymal transition (EMT) of lung cancer cells. A549 and H1299 lung cancer cells were induced to undergo via TGF-β1 treatment, resulting in the downregulation of E-cadherin and upregulation of N-cadherin and Vimentin concurrently with increases in the migration and invasion capacities of the cells. These effects of TGF-β1 were suppressed upon co-treatment of the cells with LW1497. An RNA-seq analysis revealed that LW1497 induced differential expression of genes related to hypoxia, RNA splicing, angiogenesis, cell migration, and metastasis in the A549 lung cancer cell lines. We confirmed the differential expression of Slug, an EMT-related transcription factor. Results from Western blotting and RT-PCR confirmed that LW1497 inhibited the expression of EMT markers and Slug. After orthotopically transplanting A549 cancer cells into mice, LW1497 was administered to examine whether the lung cancer progression was inhibited. We observed that LW1497 reduced the area of cancer. In addition, the results from immunohistochemical analyses showed that LW1497 downregulated EMT markers and Slug. In conclusion, LW1497 suppresses cancer progression through the inhibition of EMT by downregulating Slug.

Background Dietary Mg intake is associated with a decreased risk of developing heart failure, whereas low circulating Mg level is associated with increased cardiovascular mortality. We investigated whether Mg deficiency alone could cause cardiomyopathy. Methods and Results C57BL/6J mice were fed with a low Mg (low‐Mg, 15–30 mg/kg Mg) or a normal Mg (nl‐Mg, 600 mg/kg Mg) diet for 6 weeks. To test reversibility, half of the low‐Mg mice were fed then with nl‐Mg diet for another 6 weeks. Low‐Mg diet significantly decreased mouse serum Mg (0.38±0.03 versus 1.14±0.03 mmol/L for nl‐Mg; P P =0.011). Cellular ATP was decreased significantly in low‐Mg hearts. The changes were accompanied by mitochondrial dysfunction with mitochondrial reactive oxygen species overproduction and membrane depolarization. cMyBPC (cardiac myosin‐binding protein C) was S ‐glutathionylated in low‐Mg mouse hearts. All these changes were normalized with Mg repletion. In vivo (2‐(2,2,6,6‐tetramethylpiperidin‐1‐oxyl‐4‐ylamino)‐2‐oxoethyl)triphenylphosphonium chloride treatment during low‐Mg diet improved cardiac relaxation, increased ATP levels, and reduced S ‐glutathionylated cMyBPC. Conclusions Mg deficiency caused a reversible diastolic cardiomyopathy associated with mitochondrial dysfunction and oxidative modification of cMyBPC. In deficiency states, Mg supplementation may represent a novel treatment for diastolic heart failure.

Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmic risk after myocardial infarct (MI). MI induced by coronary artery ligation resulted in mice exhibited reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves of the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK knockout (PERKKO) reduced electrical remodeling in response to MI with shortened QTc intervals, less VT episodes, and higher survival rates (P

Heart disease is the number one mortality disease in the world. In particular, cardiac fibrosis is considered as a major factor causing myocardial infarction and heart failure. In particular, oxidative stress is a major cause of heart fibrosis. In order to control such oxidative stress, the importance of nuclear factor erythropoietin 2 related factor 2 (NRF2) has recently been highlighted. In this review, we will discuss the activation of NRF2 by docosahexanoic acid (DHA), eicosapentaenoic acid (EPA), and the specialized pro-resolving lipid mediators (SPMs) derived from polyunsaturated lipids, including DHA and EPA. Additionally, we will discuss their effects on cardiac fibrosis via NRF2 activation.

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (NI-HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in cardiomyopathy. Methods: NI-HF was induced by hypertension in Wt C57BL6 mice by unilateral nephrectomy, deoxycorticosterone acetate (DOCA) treatment and substituting drinking water with 1% saline for six weeks. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to control hearts, the protein level of p-Src S17 increased significantly accompanied by significant enhancement of tyrosine but not serine/threonine phosphorylation of MCU in NI-HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. It suggested that c-Src activation was directly related to mitochondrial-mediated arrhythmic risk. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in NI-HF mouse cardiomyocytes. Proline-rich tyrosine kinase 2 (Pyk2) did not contribute more phosphorylated MCU during cardiomyopathy as the protein expression of PyK2, p-PyK2 Y402 and p-PyK2 Y579/580 did not change during NI-HF. Correlation using human heart tissue showed that NI-HF patients had significantly increased p-Src S17 associated with an increased incidence of QTc elongation and arrhythmia. Conclusions: During cardiomyopathy, c-Src activation directly tyrosine phosphorylated MCU, increased MCU current, and increased mitochondrial/SR contact, suggesting this kinase is responsible for the increase in mitochondria-mediated arrhythmic risk seen in NI-HF.

Background: Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels and transporters. When activated, the protein kinase-like ER kinase (PERK) arm of the unfolded protein response (UPR) reduces protein translation and abundance. We hypothesize that inhibition of PERK could prevent cardiac ion channel downregulation and reduce arrhythmic risk after myocardial infarct (MI). Methods: The MI mouse model was induced by a left anterior descending coronary artery ligation. Pharmacological inhibition of PERK was achieved with a specific inhibitor, GSK2606414. Genetic inhibition of PERK was achieved by cardiac-specific PERK knockout in C57BL/6J mice (PERKKO). Echocardiography, telemetry, and electrophysiological measurements were performed to monitor cardiac function and arrhythmias. Results: Three weeks after surgery, the wild type MI mice exhibited decreased ejection fraction (EF%), ventricular tachycardia (VT) and prolonged QTc intervals. The UPR effectors (phospho-PERK, phospho-IRE1, and ATF6N) were elevated significantly (1.7- to 5.9-fold) at protein levels, and all major cardiac ion channels showed decreased protein expression in MI hearts. MI cardiomyocytes showed decreased currents for all major channels (I Na , I CaL , I to , I K1 , and I Kur : 60±6%, 53±9%, 27±6%, 55±7%, and 40±7% of sham, respectively, P90 : 291±43 ms of MI vs. 100±12 ms of sham, Pmax : 95±4 V/s of MI vs. 132±6 ms of sham, PNa and I to , shortened APD, and increased dV/dt max . PERKKO mice exhibited reduced electrical remodeling in response to MI with shortened QTc intervals, less VT episodes, and higher survival rates. Conclusion: PERK is activated during MI and contributes to arrhythmic risk by downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmic risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmic mechanism and maintaining ion channel levels is antiarrhythmic.

Introduction: Nephrilin peptide is a designed inhibitor of Rictor complex (also known as mTORC2), an evolutionarily conserved assembly believed to modulate responses to cellular stress. We previously demonstrated the ability of nephrilin peptide to suppress neuroinflammation, loss of body mass, glycaemic control and kidney function in a rat scald model, as well as sepsis mortality in a mouse model. The present study explores the effect of nephrilin plus iron formulations on clinically relevant outcomes in the rat scald model. Methods: Animals were treated with nephrilin by subcutaneous bolus injection on post-burn days 1–7. Equimolar ferric iron in the formulation improved the positive systemic effects of nephrilin on kidney function, glycaemic control, oxidative stress, early hyperinflammation, late inflammasome activation, hyperangiogenesis and body mass, all variables previously shown to bear upon clinically relevant burn injury outcomes. The sparing effects of nephrilin-iron were demonstrated in both sexes. Discussion: Surprisingly, optimum daily treatment doses were in the range of 2–4 mg/kg, while 8 mg/kg was less effective, suggesting the possibility of marginal pro-oxidant effects from the ‘free’ iron fraction. Thus, although ferric iron in the nephrilin formulation is clearly helpful, care must be exercised to select an optimum treatment dose. Conclusion: Iron increases the efficacy of nephrilin peptide in burns., Lay Summary Nephrilin peptide is a novel treatment for burn trauma. It addresses the serious and often long-lasting systemic effects of burn trauma on organ function, metabolism and the immune system. Trauma is often associated with anaemia. The present study asks whether the efficacy of nephrilin can be improved by the addition of iron. In a rat scald model, there is a significant improvement in outcomes when iron is added.

Cardiac ischemia is associated with arrhythmias; however, effective therapies are currently limited. The cardiac voltage-gated sodium channel α subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a miR, miR-448. miR-448 expression is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3′-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF-1α and NF-κB were major transcriptional regulators for MIR448. Moreover, hypoxia relieved MIR448 transcriptional suppression by RE1 silencing transcription factor. Therefore, miR-448 inhibition reduced arrhythmic risk after myocardial infarction. Here, we show that ischemia drove miR-448 expression, reduced Nav1.5 current, and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy., Ischemic induction of miR-448 negatively regulates the cardiac sodium channel Nav1.5, and inhibiting miR-448 raises Nav1.5 and reduces arrhythmic risk after myocardial infarction in mice.

Psoriasis is an immune cell‒mediated inflammatory disease of the skin with a mixed T helper type 1/T helper type17 cytokine environment combined with an innate immune response engaging toll-like receptors. Inflammatory diseases are characterized by dysregulated immune cell responses and elevated levels of adenosine at disease sites. Adenosine, acting through the A2AR, regulates inflammation, immune response, T-cell homeostasis, and tissue repair. We have identified a unique means to enhance A2AR function using a positive allosteric modulator. We show that oral administration of the A2AR-positive allosteric modulator AEA061 reduced ear swelling, skin thickness, erythema, scale formation, and inflammatory cytokine expression in A2Ar+/+ but not in A2Ar–/– mice with imiquimod-induced psoriasis-like dermatitis. Similar clinical and mRNA improvements were observed with topical administration. AEA061 also reduced clinical scores and cytokine expression in a mouse model of IL-23‒induced psoriasis-like dermatitis. In addition, AEA061 attenuated imiquimod-induced expression of IFN-α in plasmacytoid dendritic cells in vivo and IL-23 and IL-36α in conventional dendritic cells. TCR-mediated IL-17 expression in γδT cells in vivo and IL-17 production by CD4+ T cells enriched for γδT cells in vitro were also inhibited. Thus, the enhancement of A2AR responsiveness to the endogenous agonist adenosine through positive allosteric modulation is sufficient to enhance intrinsic homeostatic mechanisms attenuating disease progression.

Background Downregulated sodium currents in heart failure (HF) have been linked to increased arrhythmic risk. Reduced expression of the messenger RNA (mRNA)–stabilizing protein HuR (also known as ELAVL1) may be responsible for the downregulation of sodium channel gene SCN5A mRNA. Objective The purpose of this article was to investigate whether HuR regulates SCN5A mRNA expression and whether manipulation of HuR benefits arrhythmia control in HF. Methods Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of SCN5A. Optical mapping of the intact heart was adopted to study the effects of HuR on the conduction velocity and action potential upstroke in mice with myocardial infarct and HF after injection of AAV9 viral particles carrying HuR. Results HuR was associated with SCN5A mRNA in cardiomyocytes, and expression of HuR was downregulated in failing hearts. The association of HuR and SCN5A mRNA protected SCN5A mRNA from decay. Injection of AAV9 viral particles carrying HuR increased SCN5A expression in mouse heart tissues after MI. Optical mapping of the intact heart demonstrated that overexpression of HuR improved action potential upstroke and conduction velocity in the infarct border zone, which reduced the risk of reentrant arrhythmia after MI. Conclusion Our data indicate that HuR is an important RNA-binding protein in maintaining SCN5A mRNA abundance in cardiomyocytes. Reduced expression of HuR may be at least partially responsible for the downregulation of SCN5A mRNA expression in ischemic HF. Overexpression of HuR may rescue decreased SCN5A expression and reduce arrhythmic risk in HF. Increasing mRNA stability to increase ion channel currents may correct a fundamental defect in HF and represent a new paradigm in antiarrhythmic therapy.

Background: The cardiac voltage-gated sodium channel ( SCN5A ; encoding Na v 1.5) plays a key role in cardiac conduction. The sodium channel is downregulated in cardiomyopathy, contributing to arrhythmic risk. In part, this downregulation is because of abnormal mRNA splicing and reduced mRNA stability. The reason for decreased mRNA stability is unclear, however. Objective: Here, we examined whether microRNA-448 (miR-448)can contribute to SCN5A mRNA instability. Methods: Datasets from the Gene Expression Omnibus database were utilized to screen miR-448 in human and mouse heart tissues. The relationship between SCN5A and miR-448 was predicted by three independent prediction tools. The expressions of SCN5A and miR-448 were determined by quantitative real time polymerase chain reaction. Protein levels of Na v 1.5 were determined by Western blot. The effect of miR-448 mimic on sodium current was determined in induced pluripotent stem cells-derived cardiomyocytes. Mouse myocardial infarction was created by left anterior descending coronary artery ligation. Results: The expression of miR-448 is increased in stressed heart after myocardial infarction. The binding sequence for miR-448 is well-conserved in 3’-UTR of SCN5A and miR-448 directly bound to a consensus cis element, suppressing SCN5A expression and sodium currents. miR-448 expression wass increased by hypoxia. Activated by hypoxia, HIF1α and NF-κB activated were major transcriptional regulator for MIR448 . Conclusion: These results indicated that miR-448 contributed to post-transcriptional modification of SCN5A . miR-448 is likely one cause of sodium channel downregulation during ischemia and may represent a target for antagomir therapy to reduce arrhythmic risk associated with cardiomyopathy.. Keywords: SCN5A, miR-448, Cardiomyocyte, Hypoxia, NF-κB, HIF1α

Lung cancer is the number 1 cause of cancer-related casualties in the world. Appropriate diagnostic markers and novel targets for lung cancer are needed. Chitooligosaccharide deacetylase homolog (YDJC) catalyzes the deacetylation of acetylated carbohydrates; however, the role of YDJC in lung cancer progression has yet to be studied. A549 lung cancer orthotopic mouse model was used for mice experiments. We found that YDJC overexpression contributes to lung cancer progression in an orthotopic mouse model. Long-term treatment (48 h) induces YDJC expression in sphingosylphosphorylcholine (SPC)-induced epithelial-mesenchymal transition (EMT). Gene silencing of YDJC (siYDJC) reduced N-cadherin expression and increased E-cadherin expression in SPC-induced EMT. Overexpression of YDJC reverses them but overexpression of the deacetylase deficient mutant YDJCD13A could not. Interestingly, overexpression of CDC16, a YDJC binding partner, suppressed EMT. ERK2 is activated in siCDC16-induced EMT. YDJC overexpression reduces expression of protein phosphatase 2A (PP2A), whereas CDC16 overexpression induces PP2A expression. YDJC overexpression induced ubiquitination of PP2A but YDJCD13A could not. CDC16 overexpression increased the ubiquitination of YDJC. These results suggest that YDJC contributes to the progression of lung cancer via enhancing EMT by inducing the ubiquitination of PP2A. Therefore, YDJC might be a new target for antitumor therapy against lung cancer.

Triple-negative breast cancer (TNBC) is associated with a high mortality rate, which is related to the insufficient number of appropriate biomarkers and targets. Therefore, there is an urgent need to discover appropriate biomarkers and targets for TNBC. SARNP (Hcc-1 and CIP29) is highly expressed in several cancers. It binds to UAP56, an RNA helicase component of the TREX complex in messenger RNA (mRNA) splicing and export. However, the role of SARNP in mRNA splicing and export and in the progression of breast cancer, especially of TNBC, remains unknown. Therefore, we examined the role of SARNP in mRNA splicing and export and progression of TNBC. We confirmed that SARNP binds to UAP56 and Aly and that SARNP overexpression enhances mRNA splicing, whereas its knockdown suppressed mRNA export. The SARNP overexpression induced the proliferation of MCF7 cells, whereas its knockdown induced E-cadherin expression and downregulated vimentin and N-cadherin expressions in SK-BR-3 and MDA-MB-231 cells. SARNP downregulates E-cadherin expression by interaction with pinin. Mice injected with MDA-MB-231shSARNP cells exhibited a significant reduction in tumor growth and lung metastasis compared with those injected with MDA-MB-231shCon cells in vivo. These findings suggested that SARNP is involved in mRNA splicing and export. SARNP maintains mesenchymal phenotype by escaping from inhibitory interaction with pinin leading to the downregulation of E-cadherin expression.

// Hyun Ji Kim 1 , Hyun Jung Byun 1 , Mi Kyung Park 1 , Eun Ji Kim 1 , Gyeoung Jin Kang 1 , Chang Hoon Lee 1 1 College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea Correspondence to: Chang Hoon Lee, email: uatheone@dongguk.edu Keywords: sphingosylphosphorylcholine, RhebL1, AKT1, keratin reorganization, migration Received: December 12, 2016 Accepted: February 06, 2017 Published: February 15, 2017 ABSTRACT Sphingosylphosphorylcholine induces keratin phosphorylation and reorganization, and increases viscoelasticity of metastatic cancer cells such as PANC-1 cells. However, the mechanism involved in sphingosylphosphorylcholine-induced keratin phosphorylation and reorganization is largely unknown. Sphingosylphosphorylcholine dose- and time-dependently induces the expression of RhebL1. The involvement of RhebL1 in sphingosylphosphorylcholine-induced events including keratin 8 (K8) phosphorylation, reorganization, migration and invasion was examined. Gene silencing of RhebL1 suppressed the sphingosylphosphorylcholine-induced events and overexpression of RhebL1 enhanced those events even without sphingosylphosphorylcholine treatment. We examined whether the G protein function of RhebL1 induces K8 phosphorylation using constitutively active RhebL1Q64L and dominant negative RhebL1D60K. G protein activity of RhebL1 is involved in sphingosylphosphorylcholine-induced K8 phosphorylation. We found that RhebL1 binds and activates AKT1. G protein activity of RhebL1 is involved in the binding and activation of AKT1. MK2206 (AKT inhibitor) and gene silencing of AKT1 inhibited the sphingosylphosphorylcholine-induced events, whereas overexpression of activated-AKT1 induced K8 phosphorylation, reorganization, migration and invasion even without sphingosylphosphorylcholine treatment. The collective results indicate that RhebL1 is involved in sphingosylphosphorylcholine-induced events in A549 lung cancer cells via binding to AKT1 leading to activation of it. These results suggest that suppression of RhebL1 or inhibition of RhebL1’s binding to AKT1 might be a novel way that prevents changes in the physical properties of metastatic cancer cells.

Leukotriene B4 (LTB4) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB4 is known to be correlated with cancer progression. LTB4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown. We examined whether LTB4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB4-induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB4-or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pCBLT2) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells. Taken together, these results suggest that BLT2 is involved in LTB4-induced vimentin expression through ERK2 in PANC-1 cells.

Sphingosylphosphorylcholine (SPC) participates in several cellular processes including metastasis. SPC induces keratin reorganization and regulates the viscoelasticity of metastatic cancer cells including PANC-1 cancer cells leading to enhanced migration and invasion. The role of SPC and the relevant mechanism in invasion of breast cell are as yet unknown. SPC dose-dependently induces invasion of breast cancer cells or breast immortalized cells. Reverse transcription polymerase chain reaction and Western blot analyses of MCF10A and ZR-75-1 cells indicated that SPC induces expression and secretion of matrix metalloproteinase-3 (MMP3). From online KMPLOT, relapse free survival is high in patients having low MMP3 expressed basal breast cancer (n=581, p=0.032). UK370106 (MMP3 inhibitor) or gene silencing of MMP3 markedly inhibited the SPC-induced invasion of MCF10A cells. An extracellular signal-regulated kinase (ERK) inhibitor, PD98059, significantly suppressed the secretion and the gelatinolytic activity of MMP3, and invasion in MCF10A cells. Over-expression of ERK1 and ERK2 promoted both the expression and secretion of MMP3. In contrast, gene silencing of ERK1 and ERK2 attenuated the secretion of MMP3 in MCF10A cells. The effects of SPC-induced MMP3 secretion on β-catenin and TCF/lymphoid enhancer factor (LEF) promoter activity were examined since MMP3 indirectly activates canonical Wnt signaling. SPC induced translocation of β-catenin to nucleus and increased TCF/LEF promoter activity. These events were suppressed by UK370106 or PD98059. Wnt inhibitor, FH535 inhibited SPC-induced MMP3 secretion and invasion. Taken together, these results suggest that SPC induces MMP3 expression and secretion via ERK leading to Wnt activation.

Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear. We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells. Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK.

Sphingosylphosphorylcholine (SPC) evokes perinuclear reorganization of keratin 8 (K8) filaments and regulates the viscoelasticity of metastatic cancer cells leading to enhanced migration. Few studies have addressed the compounds modulating the viscoelasticity of metastatic cancer cells. We studied the effects of sphingosine (SPH), sphingosine 1-phosphate (S1P), FTY720 and FTY720-phosphate (FTY720P) on SPC-induced K8 phosphorylation and reorganization using Western blot and confocal microscopy, and also evaluated the elasticity of PANC-1 cells by atomic force microscopy. FTY720, FTY720P, SPH, and S1P concentration-dependently inhibited SPC-evoked phosphorylation and reorganization of K8, and migration of PANC-1 cells. SPC triggered reduction and narrow distribution of elastic constant K and conversely, FTY720 blocked them. A common upstream regulator of JNK and ERK, protein phosphatase 2A (PP2A) expression was reduced by SPC, but was restored by FTY720 and FTY72P. Butyryl forskolin, a PP2A activator, suppressed SPC-induced K8 phosphorylation and okadaic acid, a PP2A inhibitor, induced K8 phosphorylation. Gene silencing of PP2A also led to K8 phosphorylation, reorganization and migration. We also investigated the involvement of GPR12, a high-affinity SPC receptor, in SPC-evoked keratin phosphorylation and reorganization. GPR12 siRNA suppressed the SPC-triggered phosphorylation and reorganization of K8. GPR12 overexpression stimulated keratin phosphorylation and reorganization even without SPC. FTY720 and FTY720P suppressed the GPR12-induced phosphorylation and reorganization of K8. The collective data indicates that FTY720 and FTY720P suppress SPC-induced phosphorylation and reorganization of K8 in PANC-1 cells by restoring the expression of PP2A via GPR12. These findings might be helpful in the development of compounds that modulate the viscoelasticity of metastatic cancer cells and various SPC actions.

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-β1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-β1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.

Background Although transcription is the initial process of gene expression, posttranscriptional gene expression regulation has also played a critical role for fine‐tuning gene expression in a fast, precise, and cost‐effective manner. Although the regulation of sodium channel α‐subunit ( SCN 5A ) mRNA expression has been studied at both transcriptional and pre‐ mRNA splicing levels, the molecular mechanisms governing SCN 5A mRNA expression are far from clear. Methods and Results Herein, we show that, as evidenced by ribonucleoprotein immunoprecipitation assay, RNA binding protein Hu antigen R/ELAV like RNA binding protein 1 (HuR/ ELAVL 1) and myocyte enhancer factor‐2C (MEF2C) transcription factor mRNA are associated. HuR positively regulated transcription factor MEF 2C mRNA expression by protecting its mRNA from degradation. As demonstrated by both chromatin immunoprecipitation–quantitative polymerase chain reaction assay and an electrophoretic mobility shift assay, MEF 2C enhanced SCN 5A transcription by binding to a putative MEF 2C binding site within SCN 5A promoter region. Overexpression of HuR increased the expression of SCN 5A mRNA , and this effect was attenuated by the presence of MEF 2C small interfering RNA in cardiomyocytes. Conclusions In conclusion, our results suggested that HuR participates in a combined network at the DNA and RNA levels that regulates SCN 5A mRNA expression. HuR upregulates MEF 2C mRNA expression by protecting MEF 2C mRNA from degradation, and consequently, the elevated MEF 2C enhances SCN 5A mRNA transcription.

High-mobility group box 1 (HMGB1) enhances inflammatory reactions by potentiating the activity of pro-inflammatory mediators and suppressing the phagocytosis of apoptotic neutrophils. However, the effects of HMGB1 on phagocytosis induced by pro-resolving mediators, such as resolvins, have not been studied up until this point. In this study, we investigated the effects and underlying mechanism of HMGB1 on resolvin D1-induced phagocytosis of MDA-MB-231 cells, which were selected as a model system based on their phagocytic capability and ease of transfecting them with a plasmid or siRNA in several cancer cell lines. Then we confirmed effects of HMGB1 in THP-1 cells.Resolvin D1 (RvD1) enhanced phagocytosis in MDA-MB-231 and THP-1 cells. HMGB1 suppressed RvD1-induced phagocytosis in MDA-MB.231 and THP-1 cells. HMGB1 dose-dependently induced the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the inactivating enzyme in pro-resolving lipid mediators such as RvE1 and RvD1. Involvement of 15-PGDH in-HMGB-1-induced suppression of phagocytosis was examined using siRNA of 15-PGDH or 15-PGDH inhibitor, TD23. Surprisingly, the silencing of 15-PGDH increased phagocytotic activity of MDA-MB-231 cells. TD23 also enhanced phagocytosis of MDA-MB-231 and THP-1 cells.In conclusion, the release of HMGB1 during the inflammatory phase induces 15-PGDH expression, which suppresses the phagocytotic activity of macrophages. These processes might be involved in the mechanism that blocks the resolution of inflammation, thereby allowing acute inflammation to progress to chronic inflammation.

Regulatory T cells (Tregs) have key roles in the immune response by suppressing the differentiation and proliferation of various immune cells. The beneficial effects of docosahexaenoic acid (DHA) have been described for many diseases; however, the mechanism by which it modulates the immune system is poorly understood. Therefore, the aim of this study was to examine whether DHA suppresses allergic reactions and upregulates the generation of CD4(+)Foxp3(+) T cells. We also examined the effects of transfusing interleukin-10/transforming growth factor-β (TGF-β)-modified macrophages (M2 macrophages) treated with DHA into a mouse model of atopic dermatitis. Here, we show that administration of DHA upregulates the generation of TGF-β-dependent CD4(+) forkhead box protein 3 (Foxp3(+)) Tregs. DHA induced T-cell hypo-responsiveness and downregulated cytokines associated with T helper (Th)-1, Th2, and Th17 cells. The differentiation of Foxp3(+) Tregs into CD4(+) T cells was directly mediated by DHA-M2 macrophages, which deactivated effector macrophages and inhibited CD4(+) T-cell proliferation. DHA showed therapeutic effects in mice with experimental atopic dermatitis. These results show that DHA enhances the function of M2 macrophages and that the generation of Tregs effectively protects mice against an inflammatory immune disorder. Thus, DHA may be a useful therapeutic strategy for treating chronic inflammatory diseases.

Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation.

Here, we investigated whether over-activation of AKT pathway is important in the resistance to 5-fluorouracil (5-FU) in SNU-C5/5-FU cells, 5-FU-resistant human colon cancer cells. When compared to wild type SNU-C5 cells (WT), SNU-C5/5-FU cells showed over-activation of PI3K/AKT pathway, like increased phosphorylation of AKT, mTOR, and GSK-3β, nuclear localization of β-catenin, and decreased E-cadherin. Moreover, E-cadherin level was down-regulated in recurrent colon cancer tissues compared to primary colon cancer tissues. Gene silencing of AKT1 or treatment of LY294002 (PI3 kinase inhibitor) increased E-cadherin, whereas decreased phospho-GSK-3β. LY294002 also reduced protein level of β-catenin with no influence on mRNA level. PTEN level was higher in SNU-C5/WT than SNU-C5/5-FU cells, whereas the loss of PETN in SNU-C5/WT cells induced characteristics of SNU-C5/5-FU cells. In SNU-C5/5-FU cells, NF-κB signaling was activated, along with the overexpression of COX-2 and stabilization of survivin. However, increased COX-2 contributed to the stabilization of survivin, which directly interacts with cytoplasmic procaspase-3, while the inhibition of AKT reduced this cascade. We finally confirmed that combination treatment with 5-FU and LY294002 or Vioxx could induce apoptosis in SNU-C5/5-FU cells. These data suggest that inhibition of AKT activation may overcome 5-FU-resistance in SNU-C5/5-FU cells. These findings provide evidence that over-activation of AKT is crucial for the acquisition of resistance to anticancer drugs and AKT pathway could be a therapeutic target for cancer treatment.