Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway.

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 ℃, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 ℃ detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 ℃, 23 ℃, 20 ℃, 17 ℃, and 14 ℃). The cell viability significantly declined from 14 to 20 ℃; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 ℃ and 14 ℃; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 ℃ and 14 ℃, and PIDD was up-regulated at 20 ℃, 17 ℃, and 14 ℃. DUSP3 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃, and PARP was down-regulated at 17 ℃ and 14 ℃. JNK2 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

MAPK9 is Correlated with a Poor Prognosis and Tumor Progression in Glioma.

BACKGROUND: Glioma has a high incidence in young and middle-aged adults and a poor prognosis. Because of late diagnosis and uncontrollable recurrence of the primary tumor after failure of existing treatments, glioma patients tend to have a poor prognosis. Recent advances in research have revealed that gliomas exhibit unique genetic features. Mitogen-activated protein kinase 9 (MAPK9) is significantly upregulated in mesenchymal glioma spheres and may be a new target for glioma diagnosis. This study aimed to investigate the potential diagnostic significance and predictive value of MAPK9 in glioma. METHODS: Paraffin-embedded tumor tissues and paracancerous tissues were collected from 150 glioma patients seen at the General Hospital of Northern Theater Command. Immunohistochemistry and western blot assays were used to detect the expression levels of MAPK9. Prognosis and survival analyses were performed using SPSS 26 software for univariate/multivariate analysis and log-rank analysis. Cellular models were used to assess the effect of MAPK9 overexpression and knockdown in vitro. RESULTS: MAPK9 expression was higher in glioma tissues than in paraneoplastic tissues. Prognostic and survival analyses revealed that the MAPK9 expression level is an independent prognostic factor in glioma patients. In addition, overexpression of MAPK9 significantly promoted the proliferation and migration of primary glioma cells, possibly via the Wnt/ß-catenin-regulated EMT pathway. CONCLUSIONS: MAPK9 is an independent prognostic factor in glioma and is involved in tumor progression.

Farnesoid X receptor agonist attenuates subchondral bone osteoclast fusion and osteochondral pathologies of osteoarthritis via suppressing JNK1/2/NFATc1 pathway.

Osteoarthritis (OA) is a prevalent degenerative disease of the joint, featured by articular cartilage destruction and subchondral bone marrow lesions. Articular cartilage and subchondral bone constitute an osteochondral unit that guarantees joint homeostasis. During OA initiation, activated osteoclasts in subchondral bone ultimately result in impaired capacities of the subchondral bone in response to mechanical stress, followed by the degradation of overlying articular cartilage. Thus, targeting osteoclasts could be a potential therapeutic option for treating OA. Here, we observed that farnesoid X receptor (FXR) expression and osteoclast fusion and activity in subchondral bone were concomitantly changed during early-stage OA in the OA mouse model established by anterior cruciate ligament transection (ACLT). Then, we explored the therapeutic effects of FXR agonist GW4064 on the osteochondral pathologies in ACLT mice. We showed that GW4064 obviously ameliorated subchondral bone deterioration, associated with reduction in tartrate-resistant acid phosphatase (TRAP) positive multinuclear osteoclast number, as well as articular cartilage degradation, which were blocked by the treatment with FXR antagonist Guggulsterone. Mechanistically, GW4064 impeded osteoclastogenesis through inhibiting subchondral bone osteoclast fusion via suppressing c-Jun N-terminal kinase (JNK) 1/2/nuclear factor of activated T-cells 1 (NFATc1) pathway. Taken together, our results present evidence for the protective effects of GW4064 against OA by blunting osteoclast-mediated aberrant subchondral bone loss and subsequent cartilage deterioration. Therefore, GW4064 demonstrates the potential as an alternative therapeutic option against OA for further drug development.

c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1.

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and -independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection.

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus, and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. IMPORTANCE Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus, which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.

c-Jun N-terminal kinase (JNK) signaling contributes to cystic burden in polycystic kidney disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD.

Phosphorylation and Stabilization of PIN1 by JNK Promote Intrahepatic Cholangiocarcinoma Growth.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of the c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and therefore the key downstream effectors of this pathway, remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signaling in ICC that could open up therapeutic opportunities. APPROACH AND RESULTS: Using loss-of-function and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumors, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 through all-trans retinoic acid, a Food and Drug Administration-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation through PIN1 inhibition.

Placental Insulin/IGF-1 Signaling, PGC-1α, and Inflammatory Pathways Are Associated With Metabolic Outcomes at 4-6 Years of Age: The ECHO Healthy Start Cohort.

An adverse intrauterine environment is associated with the future risk of obesity and type 2 diabetes. Changes in placental function may underpin the intrauterine origins of adult disease, but longitudinal studies linking placental function with childhood outcomes are rare. Here, we determined the abundance and phosphorylation of protein intermediates involved in insulin signaling, inflammation, cortisol metabolism, protein glycosylation, and mitochondrial biogenesis in placental villus samples from healthy mothers from the Healthy Start cohort. Using MANOVA, we tested the association between placental proteins and offspring adiposity (fat mass percentage) at birth (n = 109) and infancy (4-6 months, n = 104), and adiposity, skinfold thickness, triglycerides, and insulin in children (4-6 years, n = 66). Placental IGF-1 receptor protein was positively associated with serum triglycerides in children. GSK3ß phosphorylation at serine 9, a readout of insulin and growth factor signaling, and the ratio of phosphorylated to total JNK2 were both positively associated with midthigh skinfold thickness in children. Moreover, peroxisome proliferator-activated receptor γ coactivator (PGC)-1α abundance was positively associated with insulin in children. In conclusion, placental insulin/IGF-1 signaling, PGC-1α, and inflammation pathways were positively associated with metabolic outcomes in 4- to 6-year-old children, identifying a novel link between placental function and long-term metabolic outcomes.

Isoform-specific functions of c-Jun N-terminal kinase 1 and 2 in lung ischemia-reperfusion injury through the c-Jun/activator protein-1 pathway.

BACKGROUND: c-Jun N-terminal kinase 1 (JNK1) and JNK2 regulate distinct pathological processes in lung diseases. Here we discriminated the respective roles of these kinases in lung transplantation-induced ischemia-reperfusion injury (IRI). METHODS: Rat pulmonary microvascular endothelial cells were transfected with JNK1 small-interfering RNA (siRNA) and JNK2 siRNA and then subjected to in vitro IRI. For the isoform confirmed to aggravate IRI, the delivery of short-hairpin RNA (shRNA) plasmid was performed by intratracheal administration 48 hours before transplantation into donor rats. After a 3-hour reperfusion, the samples were collected. RESULTS: JNK1 siRNA decreased but JNK2 siRNA increased JNK phosphorylation and activity, phosphorylated and total c-Jun, and activator protein-1 activity. Although JNK1 siRNA decreased apoptosis and the levels of malondialdehyde, interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF-α), it increased the levels of superoxide dismutase, S-phase percentage, and cyclin D1; JNK2 siRNA had a converse effect. JNK1 siRNA decreased the level of lactate dehydrogenase and increased the levels of VE-cadherin, nitric oxide, phosphorylated nitric oxide synthase, and cell viability; JNK2 si RNA had a converse effect. Compared with the control group, the JNK1 shRNA group exhibited a higher lung oxygenation index and lower lung apoptosis index, injury score, wet weight:dry weight ratio, and levels of IL-1, IL-6, and TNF-α. CONCLUSIONS: JNK1 aggravated, but JNK2 alleviated, IRI through differential regulation of the JNK1 pathway in in vitro ischemia-reperfusion. JNK1 silence attenuated lung graft dysfunction by inhibiting inflammation and apoptosis. These findings provide a theoretical basis for devising therapeutic strategies against IRI after lung transplantation.

Causal roles of stress kinase JNK2 in DNA methylation and binge alcohol withdrawal-evoked behavioral deficits.

Excessive binge alcohol intake is a common drinking pattern in humans, especially during holidays. Cessation of the binge drinking often leads to aberrant withdrawal behaviors, as well as serious heart rhythm abnormalities (clinically diagnosed as Holiday Heart Syndrome (HHS)). In our HHS mouse model with well-characterized binge alcohol withdrawal (BAW)-induced heart phenotypes, BAW leads to anxiety-like behaviors and cognitive impairment. We have previously reported that stress-activated c-Jun NH(2)-terminal kinase (JNK) plays a causal role in BAW-induced heart phenotypes. In the HHS brain, we found that activation of JNK2 (but not JNK1 and JNK3) in the prefrontal cortex (PFC), but not hippocampus and amygdala, led to anxiety-like behaviors and impaired cognition. DNA methylation mediated by a crucial DNA methylation enzyme, DNA methyltransferase1 (DNMT1), is known to be critical in alcohol-associated behavioral deficits. In HHS mice, JNK2 in the PFC (but not hippocampus and amygdala) causally enhanced total genomic DNA methylation via increased DNMT1 expression, which was regulated by enhanced binding of JNK downstream transcriptional factor c-JUN to the DNMT1 promoter. JNK2-specific inhibition either by an inhibitor JNK2I or JNK2 knockout completely offset c-JUN-regulated DNMT1 upregulation and restored the level of DNA methylation in HHS PFC to the baseline levels seen in sham controls. Strikingly, either JNK2-specific inhibition or genetic JNK2 depletion or DNMT1 inhibition (by an inhibitor 5-Azacytidine) completely abolished BAW-evoked behavioral deficits. In conclusion, our studies revealed a novel mechanism by which JNK2 drives BAW-evoked behavioral deficits through a DNMT1-regulated DNA hypermethylation. JNK2 could be a novel therapeutic target for alcohol withdrawal treatment and/or prevention.

CircRNF20 aggravates the progression of non-small-cell lung carcinoma by activating MAPK9.

OBJECTIVE: To explore the clinical significance of circRNF20 in non-small-cell lung carcinoma (NSCLC), and its regulatory effects on NSCLC cell functions by activating MAPK9. PATIENTS AND METHODS: Relative levels of circRNF20 and MAPK9 in NSCLC tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between circRNF20, MAPK9 and pathological factors in NSCLC patients was analyzed. Prognostic potentials of circRNF20 and MAPK9 in NSCLC were assessed by Kaplan-Meier method. The interaction between circRNF20 and MAPK9 was tested by Dual-Luciferase reporter assay. Regulatory effects of circRNF20 and MAPK9 on proliferative abilities in H358 and SPC-A1 cells were examined by Cell Counting Kit-8 (CCK-8) and colony formation assay. RESULTS: CircRNF20 and MAPK9 were upregulated in NSCLC tissues than normal ones. They were correlated to T stage and poor prognosis in NSCLC patients, while their levels were unrelated to gender, age, and incidences of lymphatic and distant metastasis. Knockdown of circRNF20 attenuated proliferative abilities in H358 and SPC-A1 cells. On the contrary, the overexpression of MAPK9 yielded the opposite results. MAPK9 was the target gene binding circRNF20, which was able to reverse the regulatory effect of circRNF20 on NSCLC proliferation. CONCLUSIONS: CircRNF20 and MAPK9 are upregulated in NSCLC cases, which are closely linked to T stage in NSCLC patients. They are independent prognostic factors for NSCLC. By activating MAPK9, circRNF20 stimulates NSCLC proliferation.

Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway.

Kaposi's sarcoma (KS), caused by Kaposi's sarcoma-associated herpesvirus (KSHV), is a highly angioproliferative disseminated tumor of endothelial cells commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) mediates KSHV-induced cell motility (PLoS Pathog. 2019 Jan 30;15(1):e1007578). However, the role of vIRF1 in KSHV-induced cellular transformation and angiogenesis remains unknown. Here, we show that vIRF1 promotes angiogenesis by upregulating sperm associated antigen 9 (SPAG9) using two in vivo angiogenesis models including the chick chorioallantoic membrane assay (CAM) and the matrigel plug angiogenesis assay in mice. Mechanistically, vIRF1 interacts with transcription factor Lef1 to promote SPAG9 transcription. vIRF1-induced SPAG9 promotes the interaction of mitogen-activated protein kinase kinase 4 (MKK4) with JNK1/2 to increase their phosphorylation, resulting in enhanced VEGFA expression, angiogenesis, cell proliferation and migration. Finally, genetic deletion of ORF-K9 from KSHV genome abolishes KSHV-induced cellular transformation and impairs angiogenesis. Our results reveal that vIRF1 transcriptionally activates SPAG9 expression to promote angiogenesis and tumorigenesis via activating JNK/VEGFA signaling. These novel findings define the mechanism of KSHV induction of the SPAG9/JNK/VEGFA pathway and establish the scientific basis for targeting this pathway for treating KSHV-associated cancers.

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma.

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

The IL-33-induced p38-/JNK1/2-TNFα axis is antagonized by activation of ß-adrenergic-receptors in dendritic cells.

IL-33, an IL-1 cytokine superfamily member, induces the activation of the canonical NF-κB signaling, and of Mitogen Activated Protein Kinases (MAPKs). In dendritic cells (DCs) IL-33 induces the production of IL-6, IL-13 and TNFα. Thereby, the production of IL-6 depends on RelA whereas the production of IL-13 depends on the p38-MK2/3 signaling module. Here, we show that in addition to p65 and the p38-MK2/3 signaling module, JNK1/2 are essential for the IL-33-induced TNFα production. The central roles of JNK1/2 and p38 in DCs are underpinned by the fact that these two MAPK pathways are controlled by activated ß-adrenergic receptors resulting in a selective regulation of the IL-33-induced TNFα response in DCs.

Deletion of JNK Enhances Senescence in Joint Tissues and Increases the Severity of Age-Related Osteoarthritis in Mice.

OBJECTIVE: To determine the role of JNK signaling in the development of osteoarthritis (OA) induced by joint injury or aging in mice. METHODS: In the joint injury model, 12-week-old wild-type control, JNK1-/- , JNK2-/- , and JNK1fl/fl JNK2-/- aggecan-CreERT 2 double-knockout mice were subjected to destabilization of the medial meniscus (DMM) (n = 15 mice per group) or sham surgery (n = 9-10 mice per group), and OA was evaluated 8 weeks later. In the aging experiment, wild-type control, JNK1-/- , and JNK2-/- mice (n = 15 per group) were evaluated at 18 months of age. Mouse knee joints were evaluated by scoring articular cartilage structure, toluidine blue staining, osteophytes, and synovial hyperplasia, by histomorphometric analysis, and by immunostaining for the senescence marker p16INK 4a . Production of matrix metalloproteinase 13 (MMP-13) in cartilage explants in response to fibronectin fragments was measured by enzyme-linked immunosorbent assay. RESULTS: There were no differences after DMM surgery between the wild-type and the JNK-knockout mouse groups in articular cartilage structure, toluidine blue, or osteophyte scores or in MMP-13 production in explants. All 3 knockout mouse groups had increased subchondral bone thickness and area of cartilage necrosis compared to wild-type mice. Aged JNK-knockout mice had significantly worse articular cartilage structure scores compared to the aged wild-type control mice (mean ± SD 52 ± 24 in JNK1-/- mice and 60 ± 25 in JNK2-/- mice versus 32 ± 18 in controls; P = 0.02 and P = 0.004, respectively). JNK1-/- mice also had higher osteophyte scores. Deletion of JNK resulted in increased expression of p16INK 4a in the synovium and cartilage in older mice. CONCLUSION: JNK1 and JNK2 are not required for the development of OA in the mouse DMM model. Deletion of JNK1 or JNK2 is associated with more severe age-related OA and increased cell senescence, suggesting that JNK may act as a negative regulator of senescence in the joint.

Lipid-encapsulated siRNA for hepatocyte-directed treatment of advanced liver disease.

Lipid-based RNA nanocarriers have been recently accepted as a novel therapeutic option in humans, thus increasing the therapeutic options for patients. Tailored nanomedicines will enable to treat chronic liver disease (CLD) and end-stage liver cancer, disorders with high mortality and few treatment options. Here, we investigated the curative potential of gene therapy of a key molecule in CLD, the c-Jun N-terminal kinase-2 (Jnk2). Delivery to hepatocytes was achieved using a lipid-based clinically employable siRNA formulation that includes a cationic aminolipid to knockdown Jnk2 (named siJnk2). After assessing the therapeutic potential of siJnk2 treatment, non-invasive imaging demonstrated reduced apoptotic cell death and improved hepatocarcinogenesis was evidenced by improved liver parenchyma as well as ameliorated markers of hepatic damage, reduced fibrogenesis in 1-year-old mice. Strikingly, chronic siJnk2 treatment reduced premalignant nodules, indicative of tumor initiation. Furthermore, siJnk2 treatment led to a significant activation of the immune cell compartment. In conclusion, Jnk2 knockdown in hepatocytes ameliorated hepatitis, fibrogenesis, and initiation of hepatocellular carcinoma (HCC), and hence might be a suitable therapeutic option, to define novel molecular targets for precision medicine in CLD.

Activation of JNK signaling promotes all-trans-retinal-induced photoreceptor apoptosis in mice.

Disrupted clearance of all-trans-retinal (atRAL), a component of the visual (retinoid) cycle in the retina, may cause photoreceptor atrophy in autosomal recessive Stargardt disease (STGD1) and dry age-related macular degeneration (AMD). However, the mechanisms underlying atRAL-induced photoreceptor loss remain elusive. Here, we report that atRAL activates c-Jun N-terminal kinase (JNK) signaling at least partially through reactive oxygen species production, which promoted mitochondria-mediated caspase- and DNA damage-dependent apoptosis in photoreceptor cells. Damage to mitochondria in atRAL-exposed photoreceptor cells resulted from JNK activation, leading to decreased expression of Bcl2 apoptosis regulator (Bcl2), increased Bcl2 antagonist/killer (Bak) levels, and cytochrome c (Cyt c) release into the cytosol. Cytosolic Cyt c specifically provoked caspase-9 and caspase-3 activation and thereby initiated apoptosis. Phosphorylation of JNK in atRAL-loaded photoreceptor cells induced the appearance of γH2AX, a sensitive marker for DNA damage, and was also associated with apoptosis onset. Suppression of JNK signaling protected photoreceptor cells against atRAL-induced apoptosis. Moreover, photoreceptor cells lacking Jnk1 and Jnk2 genes were more resistant to atRAL-associated cytotoxicity. The Abca4-/-Rdh8-/- mouse model displays defects in atRAL clearance that are characteristic of STGD1 and dry AMD. We found that JNK signaling was activated in the neural retina of light-exposed Abca4-/-Rdh8-/- mice. Of note, intraperitoneal administration of JNK-IN-8, which inhibits JNK signaling, effectively ameliorated photoreceptor degeneration and apoptosis in light-exposed Abca4-/-Rdh8-/- mice. We propose that pharmacological inhibition of JNK signaling may represent a therapeutic strategy for preventing photoreceptor loss in retinopathies arising from atRAL overload.

Both JNK1 and JNK2 Are Indispensable for Sensitized Extracellular Matrix Mineralization in IKKß-Deficient Osteoblasts.

Extracellular matrix mineralization is critical for osteogenesis, and its dysregulation could result in osteoporosis and vascular calcification. IKK/NF-κB activation inhibits differentiation of osteoblasts, and reduces extracellular matrix mineralization, however the underlying mechanisms are poorly understood. In this study, we used CRISPR/Cas9 system to permanently inactivate IKKß in preosteoblast cells and confirmed that such cells displayed dramatic increase in extracellular matrix mineralization associated with JNK phosphorylation. Such observation was also found in our study using IKKß-deficient primary murine osteoblasts. Interestingly, we found that in Ikbkb-/-Mapk8-/- or Ikbkb-/-Mapk9-/- double knockout cells, the enhanced mineralization caused by IKKß deficiency was completely abolished, and deletion of either Mapk8 or Mapk9 was sufficient to dampen c-Jun phosphorylation. In further experiments, we discovered that absence of JNK1 or JNK2 on IKKß-deficient background resulted in highly conserved transcriptomic alteration in response to osteogenic induction. Therefore, identification of the indispensable roles of JNK1 and JNK2 in activating c-Jun and promoting osteoblast differentiation on IKKß-deficient background provided novel insights into restoring homeostasis in extracellular matrix mineralization.

Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2.

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.

Overexpressed miR-200a promotes bladder cancer invasion through direct regulating Dicer/miR-16/JNK2/MMP-2 axis.

Invasive bladder cancer (BC) is one of the most lethal malignant urological tumors. Although miR-200a has been reported as an onco-miRNA that targets the PTEN gene in endometrioid carcinoma, its biological significance in BC invasion has been poorly explored. In the current study, we found that miR-200a was markedly overexpressed in both human BC tissues and BBN-induced muscle-invasive BC tissues. We further showed that miR-200a overexpression specifically promoted human BC cell invasion, but not migration, via transcriptional upregulation of matrix metalloproteinase (MMP)-2. Mechanistic studies indicated that the increased phosphorylation of c-Jun mediated the increasing levels of MMP-2 mRNA transcription. Further investigation revealed that Dicer was decreased in miR-200a overexpressed BC cells; this resulted in inhibition of miR-16 maturation and consequently led to increased JNK2 protein translation and c-Jun activation. Taken together, the studies here showed that miR-200a overexpression inhibited Dicer expression, in turn, resulted in inhibition of miR-16 maturation, leading to upregulation of JNK2 expression, c-Jun phosphorylation, MMP-2 transcription and, ultimately, BC invasion. Collectively, these results demonstrate that miR-200a is an onco-miRNA that is a positive regulator for BC invasion. This finding could be very useful in the ongoing development of new strategies to treat invasive BC patients.