Inhibition Of JNK Pathway Research Articles

Naringenin complexed with hydroxypropyl-β-cyclodextrin improves the sciatic nerve regeneration through inhibition of p75NTR and JNK pathway

Peripheral nerve injuries are common conditions that often lead to dysfunctions. Although much knowledge exists on the several factors that mediate the complex biological process involved in peripheral nerve regeneration, there is a lack of effective treatments that ensure full functional recovery. Naringenin (NA) is the most abundant flavanone found in citrus fruits and it has promising neuroprotective, anti-inflammatory and antioxidant effects. This study aimed to enhance peripheral nerve regeneration using an inclusion complex containing NA and hydroxypropyl-β-cyclodextrin (HPβCD), named NA/HPβCD. A mouse sciatic nerve crush model was used to evaluate the effects of NA/HPβCD on nerve regeneration. Sensory and motor parameters, hyperalgesic behavior and the sciatic functional index (SFI), respectively, improved with NA treatment. Western blot analysis revealed that the levels of p75NTR ICD and p75NTR full length as well phospho-JNK/total JNK ratios were preserved by NA treatment. In addition, NA treatment was able to decrease levels of caspase 3. The concentrations of TNF-α and IL-1β were decreased in the lumbar spine, on the other hand there was an increase in IL-10. NA/HPβCD presented a better overall morphological profile but it was not able to increase the number of myelinated fibers. Thus, NA was able to enhance nerve regeneration, and NA/HPβCD decreased effective drug doses while maintaining the effect of the pure drug, demonstrating the advantage of using the complex over the pure compound.

Baicalin relieves inflammation stimulated by lipopolysaccharide via upregulating TUG1 in liver cells.

Hepatitis has become a major social, health, and economic problem worldwide. Herein, we tested the beneficial influence of baicalin, a flavonoid extracted from the roots of Scutellaria baicalensis, on human normal liver L-02 and THLE2 cell apoptosis and inflammatory reaction stimulated by lipopolysaccharide (LPS) and possible molecular mechanisms. L-02 and THLE2 cell viability and apoptosis after LPS and/or baicalin treatment were tested using CCK-8 assay and Annexin V-FITC/PI apoptosis kit, respectively. qRT-PCR was used to measure the MCP-1, IL-6, TNF-α, and lncRNA taurine upregulated gene 1 (TUG1) expressions in L-02 and THLE2 cells. sh-TUG1 was transfected to knockdown TUG1. SB203580 was used as inhibitor of p38MAPK pathway, while SP600125 was used as inhibitor of JNK pathway. We discovered that LPS stimulation caused L-02 and THLE2 cell apoptosis and inflammatory reaction. Baicalin relieved the L-02 and THLE2 cell apoptosis and inflammatory reaction stimulated by LPS. Moreover, LPS lowered the TUG1 expression in L-02 cells, while baicalin promoted the TUG1 expression in L-02 and L-02 and THLE2 cells, as well as inactivated p38MAPK and JNK pathways in LPS-stimulated L-02 cells. Besides, knockdown of TUG1 activated p38MAPK and JNK pathways and promoted inflammatory cytokine expression in L-02 cells. In conclusion, this study further affirmed the beneficial influences of baicalin on LPS-stimulated human normal liver cell apoptosis and inflammatory reaction. Baicalin relived liver cell inflammation stimulated by LPS might be via upregulating TUG1 and then inactivating p38MAPK and JNK pathways.

The Protective Effect of Aesculus hippocastanum (Venoplant®) Against Concanavalin A-Induced Liver Injury

Aim: The present study was performed to investigate the effect of Aesculus hippocastanum (AH; Venoplant®) on concanavalin A (ConA)-induced acute liver injury and explore the mechanism in mice. Methods: ConA (20 mg/kg) was administered via tail vein injection to induce hepatic damage. The groups of AH (Venoplant®) were given at 65.8, 131.6, and 263.2 mg/kg by oral gavages for 20 days. The serum levels of aspartate transaminase (AST), alanine aminotransferase (ALT), total protein (TP), and albumin (Alb) were determined by automatic biochemical analyzer, and the Alb/globulin (A/G) ratio was calculated. Tumor necrosis factor-α (TNF-α) and IFN-γ levels were assayed by enzyme-linked immunosorbent assay. The liver tissue was attained by hematoxylin and eosin, and the histopathological changes were calculated. The cell apoptosis was assayed by terminal dUTP nick-end labeling. The malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) content of liver tissue were assayed by related kits. The activity of caspase-3 was detected by spectrophotometry. The expressions of cytochrome c, Bax, Bcl-2, c-Jun N-terminal kinase (JNK), and p-JNK were detected by western blot. Results: The results showed that the levels of ALT, AST, IFN-γ, and TNF-α in AH (Venoplant®) groups were significantly lower than those in ConA-injured group, while the levels of TP, Alb, and A/G were significantly higher. The SOD and GSH levels were significantly increased, and the MDA level was decreased; liver histopathology was changed consistently with the serological indicators, AH (Venoplant®) treatment significantly reduced the pathological damage and cell apoptosis; while in AH (Venoplant®) group, the expressions of cytochrome c, caspase-3, Bax/Bcl-2 ratio, and p-JNK were significantly decreased. Conclusion: AH (Venoplant®) could significantly protect the ConA-induced acute liver injury in mice via inhibition of reactive oxygen species and JNK pathway.

Docosahexaenoic acid inhibits both NLRP3 inflammasome assembly and JNK-mediated mature IL-1\u03b2 secretion in 5-fluorouracil-treated MDSC: implication in cancer treatment

Limitation of 5-fluorouracil (5-FU) anticancer efficacy is due to IL-1β secretion by myeloid-derived suppressor cells (MDSC), according to a previous pre-clinical report. Release of mature IL-1β is a consequence of 5-FU-mediated NLRP3 activation and subsequent caspase-1 activity in MDSC. IL-1β sustains tumor growth recovery in 5-FU-treated mice. Docosahexaenoic acid (DHA) belongs to omega-3 fatty acid family and harbors both anticancer and anti-inflammatory properties, which could improve 5-FU chemotherapy. Here, we demonstrate that DHA inhibits 5-FU-induced IL-1β secretion and caspase-1 activity in a MDSC cell line (MSC-2). Accordingly, we showed that DHA-enriched diet reduces circulating IL-1β concentration and tumor recurrence in 5-FU-treated tumor-bearing mice. Treatment with 5-FU led to JNK activation through ROS production in MDSC. JNK inhibitor SP600125 as well as DHA-mediated JNK inactivation decreased IL-1β secretion. The repression of 5-FU-induced caspase-1 activity by DHA supplementation is partially due to β-arrestin-2-dependent inhibition of NLRP3 inflammasome activity but was independent of JNK pathway. Interestingly, we showed that DHA, through β-arrestin-2-mediated inhibition of JNK pathway, reduces V5-tagged mature IL-1β release induced by 5-FU, in MDSC stably overexpressing a V5-tagged mature IL-1β form. Finally, we found a negative correlation between DHA content in plasma and the induction of caspase-1 activity in HLA-DR− CD33+ CD15+ MDSC of patients treated with 5-FU-based chemotherapy, strongly suggesting that our data are clinical relevant. Together, these data provide new insights on the regulation of IL-1β secretion by DHA and on its potential benefit in 5-FU-based chemotherapy.

Silence of lncRNA MIAT protects ATDC5 cells against lipopolysaccharides challenge via up-regulating miR-132

The over-expanding role of lncRNA myocardial infarction associated transcript (MIAT) in various human diseases has been recently revealed. This study attempted to see the role of MIAT in a cell model of osteoarthritis (OA). ATDC5 cells were subjected to lipopolysaccharides (LPS) to mimic a cell model of OA. The effects of MIAT on the model were tested by performing CCK-8 assay, flow cytometry, qRT-PCR, western blot and ELISA. The downstream miRNA and signalling pathways were studied by utilizing qRT-PCR and western blot. Transfection of ATDC5 cells with the shRNA specific against MIAT significantly attenuated LPS-evoked apoptosis and cytokines release. At the meantime, the viability loss and the cleavage of caspases were ameliorated as well. MIAT overexpressed lead to the opposite result. Further, miR-132 was found to be negatively regulated by MIAT. The protective effects of MIAT silence were flattened when miR-132 expression was suppressed. Besides that the inhibitory effects of MIAT silence on LPS-evoked NF-κB and JNK activation were eliminated by miR-132 silence. This study illustrated that silence of MIAT protected ATDC5 cells against LPS challenge. The chondroprotective effects of MIAT silence may be via up-regulation of miR-132 and inhibition of NF-κB and JNK pathways.

The protective role of the MKP-5-JNK/P38 pathway in glucolipotoxicity-induced islet β-cell dysfunction and apoptosis

Hyperglycemia and hyperlipidemia (glycolipotoxicity)-triggered islet β-cell dysfunction is known to drive the progression of obesity-related type 2 diabetes, however the underlying mechanisms have not been clearly elucidated. The current study aimed to investigate the role of mitogen-activated protein kinase phosphatase 5 (MKP-5) in islet cells under glucolipotoxic conditions. Using gene overexpression and knockdown approaches, we demonstrated that MKP-5 could alleviate glucolipotoxicity-induced apoptosis via the endoplasmic reticulum (ER) stress and mitochondrial apoptosis pathways owing to the altered regulation of caspase family members and ER stress-related molecules in MIN6 and primary islet cells. Overexpression of MKP-5 reversed the glucose and palmitic acid (GP)-induced impairment of insulin secretion as well as the abnormal decreases in the expression of islet functional genes, thereby maintaining the normal insulin secretory functionality, whereas the absence of MKP-5 aggravated islet cell dysfunction. In parallel, the production of ROS and increased inflammation-associated genes in response to GP were also reduced upon MKP-5 overexpression. Further, inhibition of JNK or P38 MAPK pathways resisted to glucolipotoxicity observed in MKP-5 knockdown MIN6 cells. These findings indicate that MKP-5 is an important mediator for glucolipotoxicity-induced islet cell dysfunction and apoptosis, with JNK and P38 as the critical downstream pathways.

C‑Jun N‑terminal kinase/transforming growth factor‑β/Smad3 pathway: Is it associated with endoplasmic reticulum stress‑mediated renal interstitial fibrosis?

The present study investigated the role of the c‑Jun N‑terminal kinase (JNK)/transforming growth factor‑β (TGF‑β)/Smad3 pathway in endoplasmic reticulum stress (ERS)‑mediated renal interstitial fibrosis, which would be beneficial for chronic kidney disease (CKD) therapy. In human renal biopsy tissue, the expression levels of glucose‑regulated protein78(GRP78) and phosphorylated (p)‑JNK were examined by immunohistochemical analysis. In renal tubular HK‑2 cells, tunicamycin(TM) was used to induce ERS, and the cells were then treated with the chemical ERS inhibitor 4‑phenylbutyrate (4‑PBA) or the chemical JNK pathway inhibitor SP600125, respectively. Western blotting was then performed in the cells to determine the expression levels of GRP78 and p‑JNK proteins, as well as TGF‑β/Smad3 pathway‑associated proteins, including TGF‑β1, p‑Smad3, connective tissue growth factor and α‑smooth muscle actin. The results revealed that GRP78 and p‑JNK were evidently expressed in the renal tissues of patients with CKD, and these expression levels were significantly higher in renal tissues with severe interstitial fibrosis compared with glomerular minor lesion tissues (P<0.01 and P<0.05, respectively). Furthermore, ERS and JNK pathway inhibition decreased the expression levels of TGF‑β/Smad3 pathway signals in cells incubated with TM. ERS pathway inhibition also attenuated the expression levels of p‑JNK in HK‑2 cells. In conclusion, ERS was observed to serve an important role in the pathogenesis of CKD and may induce renal interstitial fibrosis via the JNK/TGF‑β/Smad3 pathway.

Peimine inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+ /CaMKII/JNK pathway.

Prostate cancer (PC) is one of the most common malignant tumors in man. Peimine (PM) is a bioactive substance isolated from Fritillaria. Previous studies have shown that PM could inhibit the occurrence of a variety of cancers. However, the roles of PM in PC and its related mechanism have not been elucidated. Calcium (Ca2+ ) is an important intracellular messenger involved in a variety of cell processes. In this study, we found that the appropriate doses of PM (2.5, 5, and 10 μM) significantly inhibited the growth of PC cells (DU-145, LNCap, and PC-3), but has no significant effect on normal prostate cells (RWPE-1). In addition, PM treatment inhibited the invasion and migration of PC-3 cells and blocked the epithelial-mesenchymal transition process. These effects were exhibited a dose-dependent manner. Furthermore, the current results also showed that PM treatment significantly increased the Ca2+ concentration, the increased Ca2+ promoted the phosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK), further inhibited the growth and invasion of PC-3 cells, and induced its apoptosis. Ca2+ chelator BAPTA-AM (1 μM) could counteract the increase of intracellular Ca2+ concentration. Similarly, JNK pathway inhibitor SP600125 (10 μM) also inhibited cell growth and invasion and induced apoptosis. In addition, experiments in nude mice showed that PM inhibited tumor formation through Ca2+ /CaMKII/JNK signaling pathway. In conclusion, our results show that PM inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca2+ /CaMKII/JNK pathway.

ZCCHC9 promotes proliferation and invasion of lung cancer through regulating the JNK pathway.

ZCCHC9 is a type of CCHC type zinc-finger containing protein which was found to be expressed in some tissues including brain and testicles in mice. Expression and function of ZCCHC9 in human tissues including cancer was largely unknown. In this study, we investigated the expression and function of ZCCHC9 in human non-small cell lung cancer (NSCLC) and the related molecular mechanism. Immunochemistrical standing showed that ZCCHC9 was mainly located in the nucleus in bronchial epithelial cells and epithelial cells of submucosal glands (58.3% [14/24]). But in NSCLC cells ZCCHC9 was mainly located in the cytoplasm and the positive rate was 54.5% (60/110). Ectopic cytoplasmic expression of ZCCHC9 in cancer tissues was significantly associated with advanced TNM stages (III+IV), lymph node metastasis, and poor clinical outcome (P < 0.05). Overexpression of cytoplasmic ZCCHC9 using transfection of ZCCHC9 cDNA in A549 and NCI-H1299 cells significantly upregulated the proliferation and invasion of these cancer cells in vitro (P < 0.05). Western blot study showed that overexpression of cytoplasmic ZCCHC9 significantly upregulated expression of p-JNK, Cyclin D1, and MMP7 (P < 0.05). Next we used the inhibitor of JNK pathway to inhibit the activity of the JNK pathway and the results showed that co-addition of SP600125 significantly abolished the function of ZCCHC9 to promote the proliferation and invasion of cancer cells. These results indicate that cytoplasmic ZCCHC9 could promote the proliferation and invasion of NSCLC through the JNK pathway and may be a promising cancer maker.

Casticin inhibits esophageal cancer cell proliferation and promotes apoptosis by regulating mitochondrial apoptotic and JNK signaling pathways.

Casticin, a flavonoid isolated from Vitex species, has been found to have anti-tumor property in multiple human cancers. The present study aimed to investigate the effect of casticin on the proliferation and apoptosis of esophageal cancer (EC) cells, and further illustrate the underlying mechanisms. In in vitro studies, human EC cell lines TE-1 and ECA-109 were treated with various concentrations of casticin (low-, middle-, and high-dose groups). The results showed that casticin dose-dependently inhibited the proliferation and clonogenicity of EC cells and induced cell cycle arrest in sub-G1 and G2 phases. Furthermore, casticin markedly enhanced EC cell apoptosis as detected by flow cytometry and Hoechst 33342 staining. The level of anti-apoptotic Bcl-2 protein was decreased, while the levels of pro-apoptotic Bax, cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP were conversely increased in casticin-treated TE-1 and ECA-109 cells. Moreover, casticin decreased the mitochondrial membrane potential and increased the release of mitochondrial cytochrome C into cytoplasm. In addition, the JNK signaling pathway was involved in casticin-medicated anti-proliferation and pro-apoptosis. Cells pretreated with SP600125, a JNK pathway inhibitor, partially abolished the effect of casticin. Finally, the anti-tumor property of casticin was confirmed in in vivo xenograft models. Overall, we provided both in vitro and in vivo evidences that casticin inhibited the proliferation and induced apoptosis of EC cells, and the anti-tumor action of casticin was mediated, in part, by the mitochondrial-dependent apoptosis and the activation of JNK signaling pathway.

Ac34 protein of AcMNPV promoted progeny virus production and induced the apoptosis in host Sf9 cells.

To analyze the function of Ac34 in Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and elucidate the JNK apoptotic signaling pathway activation in host Spodoptera frugiperda 9 (Sf9) cells induced by the recombinant virus AcMNPV-Ac34-EGFP. AcMNPV is an important species of baculoviruses. First, viral propagation assay indicated that overexpression of Ac34 protein promoted replication of AcMNPV. Quantitative RT-PCR analysis showed that Ac34 increased the transcriptional level of late genes 38k and vp39, which suggested that Ac34 promoted the production of progeny virus by upregulating transcription of late genes. Second, AcMNPV-Ac34-EGFP inhibited the proliferation of Sf9 cells. Moreover, Sf9 cells infected with AcMNPV-Ac34-EGFP resulted in abundant expression of SfP53 and its accumulation in the nucleus. c-Jun N-terminal kinase (JNK) activation requires MKK4 and MKK7 mediated phosphorylation at Thr183 and Tyr185. We found increased levels of p-JNK1/2 in Sf9 cells infected by AcMNPV-Ac34-EGFP, with concomitant induction of Sf9 cell death. Furthermore, treatment of infected Sf9 cells with SP600125 (an inhibitor of JNK pathway) downregulated p-JNK1/2 and influenced the expression of virus-induced apoptosis protein SfP53, as well as Cytochrome C and Bax. AcMNPV-Ac34-EGFP virus upregulated the progeny virus production and triggered apoptosis via activation of the JNK pathway in Sf9 cells. In this work, we unveiled an effective virus replication factor-Ac34 and more importantly, developed a recombinant virus that can be used as an improved version of biopesticide.

Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells

AimsThe aim of this study was to evaluate gamma-aminobutyric acid (GABA)- and piperine-induced erythropoietin (EPO) and EPO-receptor expression. Materials and methodsThe effect of GABA and piperine on cell viability was examined using kidney epithelial cells. Expression levels of EPO and EPO-R mRNA and protein were evaluated in response to GABA and piperine treatments. GABA- and piperine-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Additionally, EPO function was evaluated using conditioned media containing EPO. The GABA receptor type involved in this process was identified. Key findingsMessenger RNA and protein expression levels of EPO and EPO-R significantly increased in response to treatment with GABA, piperine, or the combination of both, compared with control. GABA plus piperine synergistically enhanced EPO and EPO-R expression through p38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, but not through the extracellular signal-regulated kinase (ERK) MAPK pathway. SB203580 and SP600125 (p38 and JNK pathway inhibitors, respectively) attenuated GABA plus piperine-induced EPO and EPO-R expression. Treatment of macrophages with EPO-containing conditioned media induced mRNA expression of interleukin (IL)-10 and nuclear factor (NF)-κB due to the interaction between EPO and EPO-R. Interestingly, GABA-induced EPO and EPO-R expression was mediated through GABAA, not GABAB, receptor activation. SignificanceThese findings demonstrate that GABA plus piperine-mediated p38 and JNK MAPK activation increases EPO and EPO-R expression, resulting in up-regulation of IL-10 and NF-κB.

The combination of cantharidin and antiangiogenic therapeutics presents additive antitumor effects against pancreatic cancer

Cantharidin, one of the active components of mylabris, is believed to have antitumor activity. Cantharidin selectively inhibits protein phosphatase 2A (PP2A), which can repress multiple oncogenic kinases (ERK, JNK, PKC, and NF-κB). Researches in vitro have shown that cantharidin suppresses cell viability and metastasis in pancreatic cancer cells. This study aims to investigate the effects of cantharidin on pancreatic cancer xenografts in vivo. Xenograft models were established using cells stably expressing luciferase. Xenograft growth was evaluated by living imaging. Gene expression was determined using a microarray, real-time PCR, a RayBiotech antibody array, and the Milliplex assay. Surprisingly, cantharidin significantly accelerated xenograft growth. Living imaging showed a rapid distribution of D-luciferin in cantharidin-treated xenografts, suggesting a rich blood supply. Immunohistochemistry confirmed increased angiogenesis. Microarray and antibody array identified upregulated proangiogenic and downregulated antiangiogenic factors. The Milliplex assay suggested elevated secretion of IL-6, IL-8, TNF-α, and VEGF. Inhibitors of ERK, JNK, PKC, and NF-κB pathway attenuated the cantharidin-induced changes to proangiogenic gene expression. PKC pathway-inhibiting tamoxifen or antiangiogenic therapeutics, including Ginsenoside Rg3, bevacizumab, Apatinib, and Endostar, antagonized the proangiogenic effect of cantharidin or its derivatives. These regimens presented remarkable additive antitumor effects in vivo. Although cantharidin presents antitumor effects in vitro and has been applied in clinical practice, we revealed an unfavorable proangiogenic side effect. We recommend that the clinical application of cantharidin should be performed on the premise of antivascularization therapy.

Proinflammatory macrophages promote degenerative phenotypes in rat nucleus pulpous cells partly through ERK and JNK signaling

Intervertebral disc (IVD) degeneration is the major contributor to low back pain, a highly prevalent musculoskeletal problem that represents the leading cause of disability. Proinflammatory M1 macrophages were identified in degenerated IVDs. However, their role in the pathogenesis of IVD degeneration and the underlying mechanism was largely unknown. In this study, we explored the combined effects of molecules secreted by M1 macrophages on nucleus pulposus cells, by treating rat nucleus pulposus cells (rNP) with the conditioned medium collected from M1-polarized RAW264.7 cells (MФCM). We found that MФCM caused molecular changes associated with IVD degeneration, including increased expression of key matrix catabolic genes (Adamts4, Adamts5, Mmp3, and Mmp13), reduced the expression of major matrix-associated anabolic genes ( Sox9, Acan, and Col2a1), and upregulated transcription of inflammation-related genes ( IL-1b, IL-6, Ccl2, and Ccl3), in rNP cells. Moreover, we found that MФCM activated both ERK and JNK pathways in these cells, and that inhibition of JNK pathway attenuated MФCM-induced expression of both catabolic and inflammatory genes, whereas ERK inhibition only suppressed induction of catabolic, but not inflammatory genes. Together, our data demonstrated that proinflammatory macrophages promoted the degenerative phenotypes in rNP cells in part through ERK and JNK signaling, and suggested that inhibition of these pathways may serve as a potential therapeutic approach for the treatment of IVD degeneration.

Pharmacological c-Jun NH2-Terminal Kinase (JNK) Pathway Inhibition Reduces Severity of Spinal Muscular Atrophy Disease in Mice.

Spinal muscular atrophy (SMA) is a severe neurodegenerative disorder that occurs in early childhood. The disease is caused by the deletion/mutation of the survival motor neuron 1 (SMN1) gene resulting in progressive skeletal muscle atrophy and paralysis, due to the degeneration of spinal motor neurons (MNs). Currently, the cellular and molecular mechanisms underlying MN death are only partly known, although recently it has been shown that the c-Jun NH2-terminal kinase (JNK)-signaling pathway might be involved in the SMA pathogenesis. After confirming the activation of JNK in our SMA mouse model (SMN2+/+; SMNΔ7+/+; Smn−/−), we tested a specific JNK-inhibitor peptide (D-JNKI1) on these mice, by chronic administration from postnatal day 1 to 10, and histologically analyzed the spinal cord and quadriceps muscle at age P12. We observed that D-JNKI1 administration delayed MN death and decreased inflammation in spinal cord. Moreover, the inhibition of JNK pathway improved the trophism of SMA muscular fibers and the size of the neuromuscular junctions (NMJs), leading to an ameliorated innervation of the muscles that resulted in improved motor performances and hind-limb muscular tone. Finally, D-JNKI1 treatment slightly, but significantly increased lifespan in SMA mice. Thus, our results identify JNK as a promising target to reduce MN cell death and progressive skeletal muscle atrophy, providing insight into the role of JNK-pathway for developing alternative pharmacological strategies for the treatment of SMA.

Activation of melanocortin receptor 4 with RO27-3225 attenuates neuroinflammation through AMPK/JNK/p38 MAPK pathway after intracerebral hemorrhage in mice

BackgroundNeuroinflammation plays an important role in the pathogenesis of intracerebral hemorrhage (ICH)-induced secondary brain injury. Activation of melanocortin receptor 4 (MC4R) has been shown to elicit anti-inflammatory effects in many diseases. The objective of this study was to explore the role of MC4R activation on neuroinflammation in a mouse ICH model and to investigate the contribution of adenosine monophosphate-activated protein kinase (AMPK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (p38 MAPK) pathway in MC4R-mediated protection.MethodsAdult male CD1 mice (n = 189) were subjected to intrastriatal injection of bacterial collagenase or sham surgery. The selective MC4R agonist RO27-3225 was administered by intraperitoneal injection at 1 h after collagenase injection. The specific MC4R antagonist HS024 and selective AMPK inhibitor dorsomorphin were administered prior to RO27-3225 treatment to elucidate potential mechanism. Short- and long-term neurobehavioral assessments, brain water content, immunofluorescence staining, and western blot were performed.ResultsThe expression of MC4R and p-AMPK increased after ICH with a peak at 24 h. MC4R was expressed by microglia, neurons, and astrocytes. Activation of MC4R with RO27-3225 improved the neurobehavioral functions, decreased brain edema, and suppressed microglia/macrophage activation and neutrophil infiltration after ICH. RO27-3225 administration increased the expression of MC4R and p-AMPK while decreasing p-JNK, p-p38 MAPK, TNF-α, and IL-1β expression, which was reversed with inhibition of MC4R and AMPK.ConclusionsOur study demonstrated that activation of MC4R with RO27-3225 attenuated neuroinflammation through AMPK-dependent inhibition of JNK and p38 MAPK signaling pathway, thereby reducing brain edema and improving neurobehavioral functions after experimental ICH in mice. Therefore, the activation of MC4R with RO27-3225 may be a potential therapeutic approach for ICH management.

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.

Endogenous c-Jun N-terminal kinase (JNK) activity marks the boundary between normal and malignant granulosa cells

Granulosa cell tumor of the ovary (GCT) is a very rare tumor, accounting for only 2% of all ovarian tumors. It originates from sex cords in the ovary and can be divided into adult (95%) and juvenile (5%) types based on histologic findings. To date, no clear etiologic process has been identified other than a missense point mutation in the FOXL2 gene. Our previous works showed that c-Jun N-terminal kinase (JNK) pathway plays critical role in cell cycle progression and mitosis of normal and immortalized granulosa cells and follicle growth in rodent ovaries. These findings led us to investigate the role of JNK pathway in the granulosa cell tumor of the ovary. We used two different GCT cell lines (COV434 and KGN) and fresh GCT samples of adult and juvenile types obtained from the patients during surgery. We have discovered that endogenous kinase activity of JNK is markedly enhanced in the GCT samples and cell lines, whereas it was almost undetectable in mitotic non-malignant human granulosa cells. The inhibition of JNK pathway in GCT cell lines with two different pharmacologic inhibitors (SP600125 and AS601245) or siRNA resulted in a dose-dependent reduction in in vitro cell growth, increased apoptosis and diminished estradiol and AMH productions. JNK inhibition was also associated with a decrease in the number of cells positive for mitosis marker phospho-histone H3Ser 10 in the asynchronous cells; and diminished EdU uptake during S phase and cell cycle arrest at G2/M-phase transition in the synchronized cells. Ex vivo treatment of patient-derived GCT samples with JNK inhibitors for 24 h significantly decreased their in vitro growth and estradiol and AMH productions. Furthermore, in human GCT xenograft model, in vivo tumor growth was significantly reduced and plasma AMH levels were significantly decreased in SCID mice after administration of JNK inhibitors and siRNA. These findings suggest that targeting JNK pathway may provide therapeutic benefit in the treatment of granulosa cell tumors for which currently no curative therapy exists beyond surgery.

Stress-activated MAPKs and CRM1 regulate the subcellular localization of Net1A to control cell motility and invasion.

The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion.

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells.

BackgroundThe aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf.MethodsMouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry.ResultsExposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells.ConclusionsEven without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s.