Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

INTRODUCTION: Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. RESULTS: Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. CONCLUSION: PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

MKK4 Knockdown Plays a Protective Role in Hemorrhagic Shock-Induced Liver Injury through the JNK Pathway.

Hemorrhagic shock (HS) triggers tissue hypoxia and organ failure during severe blood loss, and the liver is sensitive to HS. Mitogen-activated protein kinase kinase 4 (MKK4) activates the c-Jun NH2-terminal kinase (JNK) pathway, and its expression is upregulated in the serum of HS patients and mouse livers at 1 h post-HS. However, the function of MKK4 in HS-induced liver injury is unclear. The role of MKK4 was investigated in vivo using rat models of HS. Before HS, lentivirus harboring shRNA against MKK4 was injected into rats via the tail vein to knock down MKK4 expression. HS was induced by bloodletting via intubation of the femoral artery followed by resuscitation. The results showed that MKK4 knockdown reduced HS-induced apoptosis in the liver by decreasing Bax expression and the cleavage of caspase 3 and promoting Bcl-2 expression. Moreover, the generation of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) in the liver was promoted, while superoxide dismutase (SOD) activity was inhibited by HS. However, the effect of HS on oxidative stress was abrogated by MKK4 knockdown. Furthermore, MKK4 knockdown restored MMP and complex I and complex III activities and promoted ATP production, suggesting that HS-induced mitochondrial dysfunction in the liver was ameliorated by MKK4 knockdown. The inhibitory effect of MKK4 knockdown on the phosphorylation and activation of the JNK/c-Jun pathway was confirmed. Overall, MKK4 knockdown may suppress oxidative stress and subsequent apoptosis and improve mitochondrial function in the liver upon HS by inhibiting the JNK pathway. The MKK4/JNK axis was shown to be a therapeutic target for HS-induced liver injury in this study.

Eriocitrin, a dietary flavonoid suppressed cell proliferation, induced apoptosis through modulation of JAK2/STAT3 and JNK/p38 MAPKs signaling pathway in MCF-7 cells.

Eriocitrin, a lemons flavanone, exhibits several biological properties, antiproliferative, and proapoptotic effects. However, its molecular mechanical action is not entirely clarified. Oxidative stress causes abnormal stimulation of signal transducer and activator of transcription 3 (STAT3) and c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs) signaling has been strongly connected with the ruling of cell survival and apoptosis of cancer cells. Herein, we investigated an antiproliferative and proapoptotic effect that Eriocitrin modulates STAT3/MAPKs signaling activation in MCF-7 cells. We noticed that Eriocitrin strongly enhances reactive oxygen species (ROS) generation, alteration of mitochondrial outer membrane potential, and enhances apoptotic morphological changes. Furthermore, Eriocitrin suppressed STAT3 phosphorylation via inhibiting an upstream molecule of JAK2 and Src kinase activation, thereby blocking STAT3 nuclear translocation. Similarly, Eriocitrin causes oxidative stress-mediated JNK/p38 MAPK signaling activation. We confirmed that Eriocitrin induced ROS-mediated apoptosis inhibited by the antioxidant substance of N-acetylcysteine. Eriocitrin induced apoptosis via suppression of STAT3 signaling regulated proteins, activating proapoptotic factors Bax, caspase 7, 8, 9 and suppressing Bcl-2, Bcl-x expression in MCF-7 cells. Overall, these results evidenced that Eriocitrin can affect multiple signaling events associated with tumorigenesis. From this evidence, Eriocitrin, a novel chemotherapeutic agent, can be used to treat breast cancer.

Induction of ZIP8, a ZIP transporter, via NF-κB signaling by the activation of IκBα and JNK signaling in cultured vascular endothelial cells exposed to cadmium.

Cadmium is an environmental pollutant that adversely affects various organs in the human body and is a well-known risk factor for cardiovascular diseases. These disorders are caused by the dysfunction of the vascular endothelial cells that cover the luminal surface of blood vessels. The ZIP transporter ZIP8 is one of the primary importers of cadmium, and its expression appears to be important for the sensitivity of vascular endothelial cells to cadmium. In the present study, we investigated the influence of ZIP8 on cadmium-induced cytotoxicity in vascular endothelial cells, the induction of ZIP8 expression by cadmium, and its action mechanism in vascular endothelial cells. The study revealed that: (1) cadmium cytotoxicity in vascular endothelial cells was potentiated by the overexpression of ZIP8, and the intracellular accumulation of cadmium in the cells was increased; (2) cadmium highly induced the expression of ZIP8, but not other ZIPs; (3) lead and methylmercury moderately induced ZIP8 expression, but the other tested metals did not; (4) the induction of ZIP8 expression by cadmium was mediated by both NF-κB and JNK signaling, and the accumulation of NF-κB in the nucleus was regulated by JNK signaling. Particularly, it was found that cadmium activated NF-κB to transfer it into nuclei and activated JNK to stabilize NF-κB in nuclei, resulting in the induction of ZIP8 expression. This induction appears to be crucial for cadmium cytotoxicity in vascular endothelial cells.

Perturbation of Cellular Redox Homeostasis Dictates Divergent Effects of Polybutyl Cyanoacrylate (PBCA) Nanoparticles on Autophagy.

Nanoparticles (NPs) are used in our everyday life, including as drug delivery vehicles. However, the effects of NPs at the cellular level and their impacts on autophagy are poorly understood. Here, we demonstrate that the NP drug delivery vehicle poly(butyl cyanoacrylate) (PBCA) perturbs redox homeostasis in human epithelial cells, and that the degree of redox perturbation dictates divergent effects of PBCA on autophagy. Specifically, PBCA promoted functional autophagy at low concentrations, whereas it inhibited autophagy at high concentrations. Both effects were completely abolished by the antioxidant N-acetyl cysteine (NAC). High concentrations of PBCA inhibited MAP1LC3B/GABARAP lipidation and LC3 flux, and blocked bulk autophagic cargo flux induced by mTOR inhibition. These effects were mimicked by the redox regulator H2O2. In contrast, low concentrations of PBCA enhanced bulk autophagic cargo flux in a Vps34-, ULK1/2- and ATG13-dependent manner, yet interestingly, without an accompanying increase in LC3 lipidation or flux. PBCA activated MAP kinase signaling cascades in a redox-dependent manner, and interference with individual signaling components revealed that the autophagy-stimulating effect of PBCA required the action of the JNK and p38-MK2 pathways, whose activities converged on the pro-autophagic protein Beclin-1. Collectively, our results reveal that PBCA exerts a dual effect on autophagy depending on the severity of the NP insult and the resulting perturbation of redox homeostasis. Such a dual autophagy-modifying effect may be of general relevance for redox-perturbing NPs and have important implications in nanomedicine.

A MEKK1 - JNK mitogen activated kinase (MAPK) cascade module is active in Echinococcus multilocularis stem cells.

BACKGROUND: The metacestode larval stage of the fox-tapeworm Echinococcus multilocularis causes alveolar echinococcosis by tumour-like growth within the liver of the intermediate host. Metacestode growth and development is stimulated by host-derived cytokines such as insulin, fibroblast growth factor, and epidermal growth factor via activation of cognate receptor tyrosine kinases expressed by the parasite. Little is known, however, concerning signal transmission to the parasite nucleus and cross-reaction with other parasite signalling systems. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatic approaches, cloning, and yeast two-hybrid analyses we identified a novel mitogen-activated kinase (MAPK) cascade module that consists of E. multilocularis orthologs of the tyrosine kinase receptor interactor Growth factor receptor-bound 2, EmGrb2, the MAPK kinase kinase EmMEKK1, a novel MAPK kinase, EmMKK3, and a close homolog to c-Jun N-terminal kinase (JNK), EmMPK3. Whole mount in situ hybridization analyses indicated that EmMEKK1 and EmMPK3 are both expressed in E. multilocularis germinative (stem) cells but also in differentiated or differentiating cells. Treatment with the known JNK inhibitor SP600125 led to a significantly reduced formation of metacestode vesicles from stem cells and to a specific reduction of proliferating stem cells in mature metacestode vesicles. CONCLUSIONS/SIGNIFICANCE: We provide evidence for the expression of a MEKK1-JNK MAPK cascade module which, in mammals, is crucially involved in stress responses, cytoskeletal rearrangements, and apoptosis, in E. multilocularis stem cells. Inhibitor studies indicate an important role of JNK signalling in E. multilocularis stem cell survival and/or maintenance. Our data are relevant for molecular and cellular studies into crosstalk signalling mechanisms that govern Echinococcus stem cell function and introduce the JNK signalling cascade as a possible target of chemotherapeutics against echinococcosis.

Mir24-2-5p suppresses the osteogenic differentiation with Gnai3 inhibition presenting a direct target via inactivating JNK-p38 MAPK signaling axis.

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.

JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.

Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice.

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway.

The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated mRNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the mRNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.

Dichloroacetate enhances the anti-tumor effect of sorafenib via modulating the ROS-JNK-Mcl-1 pathway in liver cancer cells.

Liver cancer is one of the most common and high recurrence malignancies. Besides radiotherapy and surgery, chemotherapy also plays an essential role in the treatment of liver cancer. Sorafenib and sorafenib-based combination therapies have been proven efficacy against tumors. However, previous clinical studies have indicated that some patients with liver cancer are resistant to sorafenib treatment and the existing strategies are not satisfactory in the clinic. Therefore, it is urgent to investigate strategies to improve the effectiveness of sorafenib for liver cancer and to explore effective drug combinations. In the present study, we found that dichloroacetate (DCA) could significantly enhance the anti-tumor effect of sorafenib on liver cancer cells, including reduced viability and dramatically promoted apoptosis in liver cancer cells. Moreover, compared to sorafenib alone, the combination of DCA and sorafenib markedly increased the degradation of anti-apoptotic protein Mcl-1 by enhancing its phosphorylation. Overexpression of Mcl-1 could significantly attenuate the synergetic effect of DCA and sorafenib on apoptosis induction in liver cancer cells. Furthermore, we found that the ROS-JNK pathway was obviously activated in the DCA combined sorafenib group. The levels of ROS and p-JNK were dramatically up-regulated in the two drug combination groups. Antioxidant NAC could alleviate the synergetic effects of DCA and sorafenib on ROS generation, JNK activation, Mcl-1 degradation, and cell apoptosis. Moreover, DCA and sorafenib's effects on Mcl-1 degradation and apoptosis could also be inhibited by JNK inhibitor 'SP'600125. Finally, the synergetic effects of DCA and sorafenib on tumor growth suppression, Mcl-1 degradation and induction of apoptosis were also validated in liver cancer xenograft in vivo. These findings indicate that DCA enhances the anti-tumor effect of sorafenib via the ROS-JNK-Mcl-1 pathway in liver cancer cells. This study may provide new insights to improve the chemotherapeutic effect of sorafenib, which may be beneficial for further clinical application of sorafenib in liver cancer treatment.

Pathophysiological Roles of Stress-Activated Protein Kinases in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is one of the most symptomatic progressive fibrotic lung diseases, in which patients have an extremely poor prognosis. Therefore, understanding the precise molecular mechanisms underlying pulmonary fibrosis is necessary for the development of new therapeutic options. Stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) are ubiquitously expressed in various types of cells and activated in response to cellular environmental stresses, including inflammatory and apoptotic stimuli. Type II alveolar epithelial cells, fibroblasts, and macrophages are known to participate in the progression of pulmonary fibrosis. SAPKs can control fibrogenesis by regulating the cellular processes and molecular functions in various types of lung cells (including cells of the epithelium, interstitial connective tissue, blood vessels, and hematopoietic and lymphoid tissue), all aspects of which remain to be elucidated. We recently reported that the stepwise elevation of intrinsic p38 signaling in the lungs is correlated with a worsening severity of bleomycin-induced fibrosis, indicating an importance of this pathway in the progression of pulmonary fibrosis. In addition, a transcriptome analysis of RNA-sequencing data from this unique model demonstrated that several lines of mechanisms are involved in the pathogenesis of pulmonary fibrosis, which provides a basis for further studies. Here, we review the accumulating evidence for the spatial and temporal roles of SAPKs in pulmonary fibrosis.

Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth.

Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health; however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Here, using a Drosophila intestinal model, we find that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. Our study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. We propose that intestinal tumors "hijack" innate immune signaling to eliminate enterocytes in order to support their own growth.

Role of JNK and p53 in Implementation of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue.

We studied the participation of JNK and p53 in the realization of the growth potential of different types of progenitors of the subventricular zone of mouse brain and secretion of neurotrophins by glial cells. The stimulating role of these signaling molecules in mitotic activity and specialization of multipotent neural stem cells was shown. It was found that JNK and p53 do not participate in the regulation of committed neuronal progenitor cells (clonogenic PSA-NCAM+ cells). A dependence of neurotrophic growth factors in individual populations of neuroglia on activity of these protein kinase and transcription factor was revealed. The role of JNK and p53 in astrocytes consists in stimulation of their secretion, and in microglial cells, on the contrary, in its inhibition. The secretory neurotrophic function of oligodendrogliocytes is not associated with JNK and p53 activity.

Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway.

B-cell acute lymphoblastic leukemia (B-ALL) is the common type of blood cancer. Although the remission rate has increased, the current treatment options for B-ALL are usually related to adverse reactions and recurrence, so it is necessary to find other treatment options. G protein signaling modulator 1 (GPSM1) is one of several factors that affect the basic activity of the G protein signaling system, but its role in B-ALL has not yet been clarified. In this study, we analyzed the expression of GPSM1 in the Oncomine database and found that the GPSM1 levels were higher in B-ALL cells than in peripheral blood mononuclear cells (PBMCs). Analyses of the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high GPSM1 levels had shorter survival times than those with low levels. Additionally, gene set enrichment analysis (GSEA) suggested that GPSM1 was positively correlated with proliferation, G protein-coupled receptor (GPCR) ligand binding, Gαs signaling and calcium signaling pathways. In further experiments, GPSM1 was found to be highly expressed in Acute lymphoblastic leukemia (ALL) cell lines, and downregulation of GPSM1 inhibited proliferation and promoted cell cycle arrest and apoptosis in BALL-1 and Reh cells. Moreover, knockdown of GPSM1 suppressed ADCY6 and RAPGEF3 expression in BALL-1 and Reh cells. Furthermore, we reported that GPSM1 regulated JNK expression via ADCY6-RAPGEF3. The present study demonstrates that GPSM1 promotes tumor growth in BALL-1 and Reh cells by modulating ADCY6-RAPGEF3-JNK signaling.

Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Selenomethionine alleviates LPS-induced JNK/NLRP3 inflammasome-dependent necroptosis by modulating miR-15a and oxidative stress in chicken lungs.

Selenium (Se) was involved in many physiological processes in humans and animals. microRNAs (miRNAs) also played important roles in lung diseases. However, the regulatory mechanism of miRNA in chicken lungs and the mechanism of lipopolysaccharide (LPS)-induced pneumonia remained unclear. To further study these mechanisms, we established a supplement of selenomethionine (SeMet) and/or LPS-treated chicken model and a cell model of LPS and/or high and low expression of miR-15a in chicken hepatocellular carcinoma (LMH) cells. We detected the expression of some selenoproteins, p-c-Jun N-terminal kinase (JNK), nod-like receptor protein 3 (NLRP3), caspase1, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL), miR-15a, and oxidative stress kits. Additionally, we observed the morphology of lungs by H.E. staining in vitro. The results indicated that necroptosis occurred in LPS-treated chicken and LMH cells. Moreover, LPS stimulation inhibited miR-15a, and increased the expression of JNK, NLRP3, caspase1, RIPK1, RIPK3, and MLKL. We also found that LPS treatment not only increased the content of H2O2 and MDA in the lungs but also increased the activities of iNOS and CAT and the content of GSH decreased. Conclusion: SeMet could reduce the oxidative damage and activate NLRP3 inflammasome reaction by stimulating miR-15a/JNK, thus reduced the pulmonary necroptosis induced by LPS.

Mobile element insertions and associated structural variants in longitudinal breast cancer samples.

While mobile elements are largely inactive in healthy somatic tissues, increased activity has been found in cancer tissues, with significant variation among different cancer types. In addition to insertion events, mobile elements have also been found to mediate many structural variation events in the genome. Here, to better understand the timing and impact of mobile element insertions and associated structural variants in cancer, we examined their activity in longitudinal samples of four metastatic breast cancer patients. We identified 11 mobile element insertions or associated structural variants and found that the majority of these occurred early in tumor progression. Most of the variants impact intergenic regions; however, we identified a translocation interrupting MAP2K4 involving Alu elements and a deletion in YTHDF2 involving mobile elements that likely inactivate reported tumor suppressor genes. The high variant allele fraction of the translocation, the loss of the other copy of MAP2K4, the recurrent loss-of-function mutations found in this gene in other cancers, and the important function of MAP2K4 indicate that this translocation is potentially a driver mutation. Overall, using a unique longitudinal dataset, we find that most variants are likely passenger mutations in the four patients we examined, but some variants impact tumor progression.

miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK, p-c-Jun, NFATc3, ANP and by Sirt-1 upregulation.

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3'UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3'UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy.

Benomyl induced oxidative stress related DNA damage and apoptosis in H9c2 cardiomyoblast cells.

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-κB and JNK protein levels were determined. At 4 µM concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-α and Akt1 ≥ 48.75 and ≥ 97.90; respectively. PTEN, JNK and NF-κB expressions were upregulated. The dramatic changes in JNK and NF-κB expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides.