Pharmacological Inhibition Of JNK Research Articles

JNK inhibition enhances cell-cell adhesion impaired by desmoglein3 gene disruption in keratinocytes.

c-Jun NH2-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell-cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell-cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell-cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell-cell contacts and the strength of cell-cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell-cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell-cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell-cell adhesion by increasing the linear localization of DSG1 in cell-cell contacts in KO cells. Inhibition of JNK signaling may improve cell-cell adhesion in diseases in which DSG3 expression is impaired.

Corilagin exhibits differential anticancer effects through the modulation of STAT3/5 and MAPKs in human gastric cancer cells.

Corilagin (CLG) is a hydrolyzable tannin and possesses various pharmacological activities. Here, we investigated the impact of CLG as an anti-tumor agent against human gastric tumor cells. We observed that CLG could cause negative regulation of JAKs-Src-STAT3/5 signaling axis in SNU-1 cells, but did not affect these pathways in SNU-16 cells. Interestingly, CLG promoted the induction of mitogen-activated protein kinases (MAPKs) signaling pathways in only SNU-16 cells, but not in the SNU-1 cells. CLG exhibited apoptotic effects that caused an increased accumulation of the cells in sub-G1 phase and caspase-3 activation in both SNU-1 and SNU-16 cell lines. We also noticed that CLG and docetaxel co-treatment could exhibit significantly enhanced apoptotic effects against SNU-1 cells. Moreover, the combinations treatment of CLG and docetaxel markedly inhibited cell growth, phosphorylation of JAK-Src-STAT3 and induced substantial apoptosis. Additionally, pharmacological inhibition of JNK, p38, and ERK substantially blocked CLG-induced activation of MAPKs, cell viability, and apoptosis, thereby implicating the pivotal role of MAPKs in the observed anti-cancer effects of CLG. Taken together, our data suggest that CLG could effectively block constitutive STAT3/5 activation in SNU-1 cells but induce sustained MAPKs activation in SNU-16 cells.

Monosodium urate crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response.

Monosodium urate crystals (MSUc) induce inflammation invivo without prior priming, raising the possibility of an initial cell-autonomous phase. Here, using genome-wide transcriptomic analysis and biochemical assays, we demonstrate that MSUc alone induce a metabolic-inflammatory transcriptional program in non-primed human and murine macrophages that is markedly distinct to that induced by LPS. Genes uniquely upregulated in response to MSUc belong to lipid and amino acid metabolism, glycolysis, and SLC transporters. This upregulation leads to a metabolic rewiring in sera from individuals and mice with acute gouty arthritis. Mechanistically, the initiating inflammatory-metabolic changes in acute gout flares are regulated through a persistent expression and increased binding of JUN to the promoter of target genes through JNK signaling-but not P38-in a process that is different than after LPS stimulation and independent of inflammasome activation. Finally, pharmacological JNK inhibition limits MSUc-induced inflammation in animal models of acute gouty inflammation.

A short peptide derived from pigment epithelial-derived factor exhibits an angioinhibitory effect

BackgroundPigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect.MethodsA series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides.ResultsThe MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice.ConclusionsThis is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.

Abstract PO-093: JNK2 suppresses the growth and invasion of pancreatic cancer and is opposed by JNK1

Abstract Introduction: Pancreatic cancer cells are exposed to multiple stressors with both endogenous (proliferation, metabolism and nutrient deprivation) and exogenous (medical treatment) origins. The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 are important stress-activated kinases, responsible for the cellular response to these stressors. Both were demonstrated to exert both tumor-suppressing and tumor-promoting effects in human cancers Most previous studies rely on nonspecific pharmacological JNK inhibition. In this study, we planned to determine the isoform-specific roles in PDAC. Methods: Specific JNK1- and JNK2-knockdown (KD) clones were established in cultured human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 by using specific shRNA plasmids. Anchorage dependent and independent growth, as well as migration and invasion, were analyzed in JNK-1/2 KD clones and compared to pharmacological JNK inhibition using SP600125. Xenograft growth was assessed using an orthotopic mouse model. Mechanistically, we assessed changes in EMT markers both in vitro and in vivo. Results: JNKs were expressed in both cultured pancreatic cancer cell lines as well as in resected human pancreatic cancer samples. Specific downregulation of JNK2 resulted in an overall increase in cell proliferation, invasion and alterations in cytoskeleton structure, while JNK1-KD revealed opposite effects. Nonspecific JNK-inhibition using SP600125 inhibited cell growth of all cell lines except PANC-1. Conclusion: JNK1 and JNK2 play opposing roles in human pancreatic cancer. Specifically targeting JNK1 with the retained function of JNK2 may provide a potential, individualized approach for targeting pancreatic cancer progression. Citation Format: Jingwei Shi, Xiaodong Tian, Marko Kornmann, Benno Traub. JNK2 suppresses the growth and invasion of pancreatic cancer and is opposed by JNK1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-093.

KCa3.1 inhibition-induced activation of the JNK/c-Jun signaling pathway enhances IL-10 expression in peripherally-induced regulatory T cells

The KCa3.1 inhibition up-regulates IL-10 expression in regulatory T (Treg) cells in the recovery phase of inflammatory bowel disease (IBD) model mice; however, the underlying signaling pathway remains unclear. We investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its transcription factors (TFs) in in vitro-induced mouse splenic Treg cells. The pharmacological inhibition of JNK reversed KCa3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of KCa3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the KCa3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced Treg cells.

Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells.

Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown that cadmium (Cd) induces neurotoxicity partly by impairing autophagy. However, the underlying mechanism is not fully elucidated. In this study, we show that Cd induced expansion of autophagosomes with a concomitant abnormal expression of autophagy-related (Atg) proteins in PC12 cells and primary murine neurons. 3-MA, a classical inhibitor of autophagy, attenuated Cd-induced expansion of autophagosomes and apoptosis in the cells. Further investigation demonstrated that Cd activated JNK pathway contributing to autophagosome expansion-dependent neuronal apoptosis. This is supported by the findings that pharmacological inhibition of JNK with SP600125 or expression of dominant negative c-Jun markedly attenuated Cd-induced expansion of autophagosomes and abnormal expression of Atg proteins, as well as apoptosis in PC12 cells and/or primary neurons. Furthermore, we noticed that chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM profoundly blocked Cd-elicited activation of JNK pathway and consequential expansion of autophagosomes, abnormal expression of Atg proteins, and apoptosis in the neuronal cells. Similar events were also seen following prevention of [Ca2+]i elevation with EGTA or 2-APB, implying a Ca2+-dependent mechanism involved. Taken together, the results indicate that Cd impairs autophagy leading to apoptosis by Ca2+-dependent activation of JNK signaling pathway in neuronal cells. Our findings highlight that manipulation of intracellular Ca2+ level and/or JNK activity to ameliorate autophagy may be a promising intervention against Cd-induced neurotoxicity and neurodegeneration.

Monosodium Urate Crystals Regulate a Unique JNK-Dependent Macrophage Metabolic and Inflammatory Response

The response of macrophages to monosodium urate crystals (MSUc) is incompletely understood partly due to the use of a toll-like receptor (TLR)-induced priming step in cell lines. Yet, MSUc induce inflammation in vivo without prior priming, raising the possibility of an initial cell-autonomous phase that is TLR-independent. Here, using genome wide transcriptomic analysis and biochemical assays we demonstrate that MSUc alone induce a metabolic-inflammatory transcriptional program in non-primed human and murine macrophages that is markedly distinct to that induced by LPS. Genes uniquely upregulated in response to MSUc belonged to lipid and amino acid metabolism, glycolysis, and SLC transporters. This upregulation led to a metabolic rewiring in sera from individuals and mice with acute gouty arthritis. Mechanistically, the initiating inflammatory-metabolic changes in acute gout flares are regulated through a persistent expression and increased binding of JUN to the promoter of target genes through JNK signaling -but not P38- in a process that is different than after LPS stimulation and unexpectedly independent of inflammasome activation. Finally, pharmacological JNK inhibition limited MSUc-induced inflammation in animal models of acute gouty inflammation. MSUc alone induce a unique strong macrophage activation and distinct phenotype that forces a reconsideration of initiating mechanisms in acute gout flares.

Salvianolic acid A inhibits the growth of diffuse large B-cell lymphoma through MAPK pathways

Treatment options are limited in patients with diffuse large B-cell lymphoma (DLBCL). Salvianolic acid A (SAA) is a water-soluble phenolic acid extracted from Salvia miltiorrhiza (Danshen) with anti-tumor properties. The anti-leukemic activity of SAA found in our recent research prompted us to investigate the therapeutic effect and mechanism of action of SAA in DLBCL. In the work described here, we found that SAA inhibited the viability of DLBCL cells by inducing cellular apoptosis, which was accompanied by upregulation of Bax and cleavage of PARP. Pre-incubation of SAA increased the phosphorylation of JNK, while it decreased the phosphorylation of p38 and ERK in DLBCL cells. Importantly, pharmacologic JNK inhibition partially mitigated the anti-survival effect of SAA, and inhibition of p38 and ERK synergized with SAA. Furthermore, SAA suppressed DLBCL tumor growth in a xenograft mouse model in vivo. Therefore, our data suggest the therapeutic utility of SAA in the management of DLBCL.

Mitochondrial dysfunction triggers a catabolic response in chondrocytes via ROS-mediated activation of the JNK/AP1 pathway.

Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper.

Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

BackgroundCurrent pharmacological therapies and treatments targeting pancreatic neuroendocrine tumors (PNETs) have proven ineffective, far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, a combination of anti-inflammatory herbal extracts, that has proven to be effective in various in vitro and in vivo cancer platforms, shows promise. However, its effects on pancreatic cancer, in particular, remain largely unexplored.MethodsIn the current study, we investigated the effects of Zyflamend on the survival of beta-TC-6 pancreatic insulinoma cells (β-TC6) and conducted a detailed analysis of the underlying molecular mechanisms.ResultsHerein, we demonstrate that Zyflamend treatment decreased cell proliferation in a dose-dependent manner, concomitant with increased apoptotic cell death and cell cycle arrest at the G2/M phase. At the molecular level, treatment with Zyflamend led to the induction of ER stress, autophagy, and the activation of c-Jun N-terminal kinase (JNK) pathway. Notably, pharmacological inhibition of JNK abrogated the pro-apoptotic effects of Zyflamend. Furthermore, Zyflamend exacerbated the effects of streptozotocin and adriamycin-induced ER stress, autophagy, and apoptosis.ConclusionThe current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer via modulation of the JNK pathway.5rXAVcWGwP9WqSeTdiEBY7Video abstract

Abstract 3778: MBNL1 regulated alternative splicing of MAP2K7 promotes de-differentiation of cancer cells by activating JNK

Abstract Therapeutic targeting of cancer stem cells (CSCs) is key to relapse-free, lasting response to cancer treatment. Several strategies against CSCs are currently in various stages of clinical development. However, there is need for additional targets. Here, we demonstrate that MBNL1, an RNA binding protein, is a suppressor of cancer-stemness. RNAi mediated down-regulation of MBNL1 promotes tumor-initiation, self-renewal, migration and invasion by up-regulating CSC-specific splice-isoforms. Particularly, the up-regulation of MAP2K7Δexon2 splice-isoform activates JNK signaling and establishes a feedback loop that promotes cancer-stemness. Pharmacological inhibition of JNK reverses CSC phenotypes in MBNL1low/MAP2K7Δexon2high CSC-like cells and these cells also show enhanced susceptibility to JNK inhibition. Remarkably, MBNL1 is down-regulated and MAP2K7Δexon2 splice-isoform is up-regulated in several cancer types. Down-regulation of MBNL1 is correlated with poor survival in gastric, lung and breast adenocarcinoma. In triple negative breast cancer, which is a highly aggressive disease characterised by enrichment of CSC-like cells, low MBNL1 correlates with poor relapse-free and distant metastasis-free survival. Our work shows, low MBNL1 and consequently high MAP2K7Δexon2 expression, is a biomarker for cancer stemness, high JNK activity and enhanced susceptibility to JNK inhibition. Hence, we have identified a novel alternative splicing driven mechanism of cancer cell de-differentiation and have identified biomarkers that can prognosticate patients for JNKi as a strategy to target CSCs. Citation Format: Debleena Ray. MBNL1 regulated alternative splicing of MAP2K7 promotes de-differentiation of cancer cells by activating JNK [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3778.

Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis

Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.

Zyflamend Induces Apoptosis in Pancreatic Cancer cells via Modulation of the JNK Pathway

Current pharmacological therapies and treatments targeting pancreatic neuroendocrine tumors (PNETs) have proven ineffective, far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, a combination of anti‐inflammatory herbal extracts, that has proven to be effective in various in vitro and in vivo cancer platforms, shows promise. However, its effects on pancreatic cancer, in particular, remain largely unexplored. Herein, we demonstrate that Zyflamend treatment decreased cell proliferation in a dose‐dependent manner, concomitant with increased apoptotic cell death and cell cycle arrest at the G2/M phase. At the molecular level, treatment with Zyflamend led to the induction of ER stress, autophagy, and the activation of c‐Jun N‐terminal kinase (JNK) pathway. Notably, pharmacological inhibition of JNK abrogated the pro‐apoptotic effects of Zyflamend. Furthermore, Zyflamend exacerbated the effects of streptozotocin and adriamycin‐induced ER stress, autophagy, and apoptosis. These studies identify Zyflamend as a potential novel approach to treat pancreatic cancer via modulation of the JNK pathway.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R00DK100736) to A.B.

Pharmacological Inhibition of MAP2K7 Induces Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages

Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.

The ubiquitin ligase PHR promotes directional regrowth of spinal zebrafish axons

To reconnect with their synaptic targets, severed axons need to regrow robustly and directionally along the pre-lesional trajectory. While mechanisms directing axonal regrowth are poorly understood, several proteins direct developmental axon outgrowth, including the ubiquitin ligase PHR (Mycbp2). Invertebrate PHR also limits regrowth of injured axons, whereas its role in vertebrate axonal regrowth remains elusive. Here we took advantage of the high regrowth capacity of spinal zebrafish axons and observed robust and directional regrowth following laser transection of spinal Mauthner axons. We found that PHR directs regrowing axons along the pre-lesional trajectory and across the transection site. At the transection site, initial regrowth of wild-type axons was multidirectional. Over time, misdirected sprouts were corrected in a PHR-dependent manner. Ablation of cyfip2, known to promote F-actin-polymerization and pharmacological inhibition of JNK reduced misdirected regrowth of PHR-deficient axons, suggesting that PHR controls directional Mauthner axonal regrowth through cyfip2- and JNK-dependent pathways.

Stretch‐induced activation of Hippo signaling in lung microvascular endothelial cells: A novel mechanism of overventilation‐induced pulmonary fibrosis

IntroductionMechanical ventilation is a mainstay of current therapy for patients with acute respiratory distress syndrome (ARDS). Yet despite its benefits, mechanical ventilation can also cause ventilator‐induced lung injury (VILI) that may ultimately progress to pulmonary fibrosis. In this study, we aimed to elucidate cellular mechanisms by which mechanical ventilation triggers pulmonary fibrosis. Specifically, we focused on the mechanosensitive transcriptional co‐factor TAZ, a key player in the Hippo‐signaling pathway involved in mechanosensation, cell proliferation, and profibrotic responses. As such, TAZ translocates to the nucleus of fibrotic lungs but not in healthy tissue. Interestingly, TAZ distribution upon cyclic stretch has not been extensively characterized and the underlying mechanisms are not well defined.ObjectivesTo examine the effect of mechanical stretch on the nuclear translocation of TAZ in endothelial cells, and to identify underlying regulatory mechanisms.MethodsTo mimic the effects of mechanical overventilation, human pulmonary microvascular endothelial cells (HPMECs) were subjected to stretch regimens at either 5%, mimicking low tidal volume ventilation, or 18% mimicking high tidal volume ventilation at 0.25 Hz for 1,3,6, 12h on a Flexcell 5000 Tension System and compared to static control (0%).ResultsMechanical stretch induced expression and release of the profibrotic cytokine TGF‐β1 from HPMECs and concomitantly caused translocation of TAZ from the cytosol to the nucleus in a dose‐ and time‐dependent manner. Specifically, the nuclear to cytoplasmic ratio (N/C) of TAZ increased after 3h of stretch at 18% compared to the static control whereas stretching these cells at 5% increased TAZ nuclear localization after 6h. Stretch‐induced TAZ translocation was not prevented by pharmacological inhibition of JNK or Src kinases, known regulators of the Hippo kinase LATS2; yet TAZ translocation was blocked by inhibition of Aurora kinases with SNS 314 mesylate (10UM). Consistently, inhibition of Aurora kinases decreased LATS2 phosphorylation at Ser83, a known modulation site of LATS2 activity.ConclusionThese data suggest that mechanical stretch activates TAZ and triggers release of pro‐fibrotic cytokines, at least partly, in an Aurora kinase and LATS2‐dependent mechanism. Interventions targeted at the Hippo‐signaling pathway as well as its upstream regulation may provide potential strategies to counteract the detrimental effects of mechanical ventilation.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ZPR1-Dependent Neurodegeneration Is Mediated by the JNK Signaling Pathway.

The zinc finger protein ZPR1 deficiency causes neurodegeneration and results in a mild spinal muscular atrophy (SMA)-like disease in mice with reduced Zpr1 gene dosage. Mutation of the survival motor neuron 1 (SMN1) gene causes SMA. Spinal muscular atrophy is characterized by the degeneration of the spinal cord motor neurons caused by chronic low levels of SMN protein. ZPR1 interacts with SMN and is required for nuclear accumulation of SMN. Patients with SMA express reduced levels of ZPR1. Reduced Zpr1 gene dosage increases neurodegeneration and severity of SMA disease in mice. Mechanisms underlying ZPR1-dependent neurodegeneration are largely unknown. We report that neurodegeneration caused by ZPR1 deficiency is mediated by the c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPK). ZPR1-dependent neuron degeneration is mediated by central nervous system (CNS)-specific isoform JNK3. ZPR1 deficiency activates the MAPK signaling cascade, MLK3 → MKK7 → JNK3, which phosphorylates c-Jun and activates caspase-mediated neuron degeneration. Neurons from Jnk3-null mice show resistance to ZPR1-dependent neurodegeneration. Pharmacologic inhibition of JNK reduces degeneration of ZPR1-deficient neurons. These data show that ZPR1-dependent neurodegeneration is mediated by the JNK signaling pathway and suggest that ZPR1 downregulation in SMA may contribute to JNK-mediated neurodegeneration associated with SMA pathogenesis.

AtRA induces cholesterol accumulation through CYP7A1 repression in the liver

All‐trans retinoic acid (atRA) is one of the most frequently used retinoids to treat certain cancers or dermatological diseases. Common side effects of atRA include hypercholesterolemia, leading to discontinuation of atRA uses. Previous studies have shown that atRA downregulates CYP7A1 (the rate‐liming enzyme converting cholesterol to a bile acid intermediate), potentially responsible for hypercholesterolemia. However, the detailed molecular mechanisms in atRA‐induced hypercholesterolemia is unclear. atRA increased cholesterol levels in a dose dependent manner in HepaRG cells, and this was accompanied by dramatic decrease in CYP7A1 mRNA and protein levels. CYP7A1 overexpression by lentiviral system reversed atRA‐induced cholesterol accumulation in HepaRG cells, suggesting that the cholesterol accumulation is mediated by CYP7A1 repression. CYP7A1 promoter reporter assays in HepG2 cells revealed that the HNF4α binding site in the promoter (−149/−118) is essential for atRA‐mediated CYP7A1 repression. HNF4α depletion by siRNA abrogated CYP7A1 repression by atRA in HepaRG cells. Western blot analysis revealed that atRA activates MAPKs signaling pathways including JNK, ERK, and p38 MAPKs in HepaRG cells. Pharmacological inhibition of JNK and ERK pathways but not p38 pathway by using a specific inhibitor (i.e., SP600125, PD98059, and SB203580 for JNK, ERK, and p38, respectively) attenuated atRA‐mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP‐1 (c‐Jun/c‐Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression in HepG2 cells. In DNA pull‐down assay, AP‐1 exhibited sequence‐specific binding to the HNF4α binding site in CYP7A1 promoter following atRA treatment in HepaRG cells. Chromatin immunoprecipitation assay also showed that AP‐1 binding to the CYP7A1 promoter (−181/−34) was increased following atRA treatment, whereas HNF4α binding was decreased. Collectively, results from this study indicate that atRA‐activated JNK and ERK pathways and downstream target AP‐1 represses HNF4α transactivation of CYP7A1 promoter. These results suggest an important role of CYP7A1 in atRA‐induced cholesterol accumulation and provide a basis to establish future strategy in cholesterol disorder by atRA.Support or Funding InformationThis study was supported by R01 HD089455.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.