Inhibition Of JNK Pathway Research Articles

Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

Quercetin (Que), a plant-derived flavonoid, possesses various biological functions. Moreover, Que exerts multiple beneficial actions in treatment of cardiovascular diseases and there are an inverse association between Que intakes and occurrence and development of various cardiovascular diseases. Some researchers have inferred that the mechanisms of Que to protect cardiomyocytes from ischemia/reperfusion (I/R) injury may be involved in modulation of intracellular signal pathways and regulation of proteins expression in vivo. The current study investigated whether Que has any protective effects on cardiomyocytes from hypoxia/reoxygenation (H/R) in vitro and its potential cardioprotective mechanisms. The cell viability of Que on H9c2 cardiomyoblast cells was assessed by MTT. Apoptosis was evaluated by both Hoechst33342 staining and Flow cytometric analysis (FACS). Furthermore, the effect of Que, SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) on mitogen-activated protein kinases (MAPKs) and the expression of apoptosis related proteins (Bcl-2, Bax and caspase-3) was determined by Western blotting. MTT assays showed that pretreatment with Que could increase the viability of H9c2 cardiomyocytes that suffered H/R. Both Hoechst33342 staining and FACS confirmed that Que could remarkably suppress the H/R-induced apoptotic cardiomyocytes. In addition, Que significantly alleviated H/R-induced the phosphorylation of JNK and p38, which further increased Bcl-2 expression and inhibited the activation of Bax and caspase-3 directly or indirectly. In summary, our results imply that Que can induce cardioprotection by inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways and modulate the expression of Bcl-2 and Bax proteins that provides a new experimental foundation for myocardial ischemia disease therapy.

Pharmacokinetic and tissue distribution studies of 1,9-pyrazoloanthrone, a c-Jun-N-terminal kinase inhibitor in Wistar rats by a simple and sensitive HPLC method

JNK pathway activates c-Jun(s) which are responsible for cell apoptosis; as a result, inhibitors of JNK pathway have the potential to prevent dopaminergic neurons from death and decrease the loss of dopamine in substantia nigra pars compacta (SNpc). Recent in-vitro studies show that 1,9-pyrazoloanthrone (1,9-P) a potent JNK-3 inhibitor prevents the apoptosis of dopaminergic cells of brain. In the present study we formulated liposomes to increase the bioavailability of 1,9-P in the brain and developed a simple, sensitive and selective high performance liquid chromatographic method and validated for the estimation of 1,9-P in Wistar rat plasma and tissue samples. Plasma and tissue samples were extracted by protein precipitation technique using acetonitrile (ACN) and rasagiline as the internal standards. Chromatography was performed on Hibar C18 column with mobile phase of ammonium acetate (10mM, pH 8.0 adjusted with ammonia) and ACN at a flow rate of 1mL/min. The lower limit of quantification of the developed method was found to be 2.0ng/mL and 4.0ng/g in plasma and tissue samples respectively. The liposomes of 1,9-P administered to animals at the dose equivalent to 15mg/kg orally demonstrated remarkable absorption into the systemic circulation with maximum concentration (∼7500ng/mL) within 2.0h. The order of the area under curve was found to be kidney>liver>brain>lungs>spleen>heart. The liposomes of 1,9-P were rapidly taken up into brain and showed a good brain concentration after 2.0h; sustenance up to 4.0h was achieved which is better than 1,9-P solution.

The Bone Marrow-Mediated Protection of Myeloproliferative Neoplastic Cells to Vorinostat and Ruxolitinib Relies on the Activation of JNK and PI3K Signalling Pathways.

The classical BCR-ABL-negative Myeloproliferative Neoplasms (MPN) are a group of heterogeneous haematological diseases characterized by constitutive JAK-STAT pathway activation. Targeted therapy with Ruxolitinib, a JAK1/2-specific inhibitor, achieves symptomatic improvement but does not eliminate the neoplastic clone. Similar effects are seen with histone deacetylase inhibitors (HDACi), albeit with poorer tolerance. Here, we show that bone marrow (BM) stromal cells (HS-5) protected MPN-derived cell lines (SET-2; HEL and UKE-1) and MPN patient-derived BM cells from the cytotoxic effects of Ruxolitinib and the HDACi Vorinostat. This protective effect was mediated, at least in part, by the secretion of soluble factors from the BM stroma. In addition, it correlated with the activation of signalling pathways important for cellular homeostasis, such as JAK-STAT, PI3K, JNK, MEK-ERK and NF-κB. Importantly, the pharmacological inhibition of JNK and PI3K pathways completely abrogated the BM protective effect on MPN cell lines and MPN patient samples. Our findings shed light on mechanisms of tumour survival and may indicate novel therapeutic approaches for the treatment of MPN.

Vitexin reduces neutrophil migration to inflammatory focus by down-regulating pro-inflammatory mediators via inhibition of p38, ERK1/2 and JNK pathway

BackgroundVitexin is a flavonoid found in plants of different genus such as Vitex spp. and Crataegus spp. Despite being an important molecule present in phytomedicines and nutraceuticals, the mechanisms supporting its use as anti-inflammatory remains unclear. PurposeTo investigate the cellular and molecular mechanisms involved in acute anti-inflammatory effect of vitexin with regard to neutrophil recruitment and macrophages activation. MethodsAnti-inflammatory properties of vitexin were evaluated in four models of neutrophil recruitment. The regulation of inflammatory mediators release was assessed in vivo and in vitro. Vitexin (5, 15 and 30 mg/kg p.o) effects on leukocytes migration to peritoneal cavity induced by zymosan (ZY), carrageenan (CG), n-formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) were evaluated in Swiss-Webster mice and the effects on the levels of TNF-α, IL-1β and IL-10 cytokines, and NO concentration were in the LPS-peritonitis. RAW 264.7 macrophages viability were determined by Alamar Blue assay as well as the capacity of vitexin in directly reducing the concentrations of TNF-α, IL-1β, IL-10, NO and PGE2. Additionally, vitexin effects upon the transcriptional factors p-p38, p-ERK1/2 and p-JNK were evaluated by western blotting in cells activated with LPS. ResultsVitexin was not cytotoxic (IC50 > 200 µg/ml) in RAW 264.7 and at all doses tested it effectively reduced leukocyte migration in vivo, particularly neutrophils in the peritoneal lavage, independently of the inflammatory stimulus used. It also reduced TNF-α, IL-1β and NO releases in the peritoneal cavity of LPS-challenged mice. Vitexin had low cytotoxicity and was able to reduce the releases of TNF-α, IL-1β, NO, PGE2 and increase in IL-10 release by LPS activated RAW 264.7 cells. Vitexin was also able to regulate transcriptional factors for pro-inflammatory mediators, reducing the expression of p-p38, p-ERK1/2 and p-JNK in LPS-elicited cells. ConclusionsVitexin presented no in vitro cytotoxicity. Inhibition of neutrophil migration and pro-inflammatory mediators release contributes to the anti-inflammatory activity of vitexin. These effects are associated with the inactivation of important signaling pathways such as p38, ERK1/2 and JNK, which act on transcription factors for eliciting induction of inflammatory response.

Genetic inhibition of JNK3 ameliorates spinal muscular atrophy.

Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH2-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA.

Abstract 2934: TRAIL-resistance in pancreatic cancer stem cells can be regulated through JNK pathway inhibition without impacting resident stem cell physiology

Abstract Cancer Stem Cells (CSC) are considered the drivers of drug-resistance and metastasis in patients with pancreatic ductal adenocarcinomas (PDAC). C-Jun N-terminal kinase (JNK) signaling has been shown to be of importance in cancer, glioblastoma CSC maintenance, and hepatocellular carcinoma cell growth. We aimed to sensitize resistant CSCs to the apoptotic effects of TNFa-related apoptosis-inducing ligand (TRAIL) by JNK inhibition (JNKi) through critical regulation of the TRAIL receptors in PDAC, without affecting the physiology of normal tissue-resident stem cells. CSC-enriched tumorsphere cultures of Panc1, MiaPaca2, and L3.6pl were treated with JNKi to evaluate sphere-forming ability as well as gene expression of CSC markers Oct3/4, Nanog, Sox2, and CD44 by qRT-PCR. The regulation of apoptosis decoy and death TRAIL receptors were analyzed on low dose JNKi and TRAIL pressure. In addition, a TRAIL-resistant cell line from parental L3.6pl cells was created to investigate the effect of our established regimen. To resemble the desmoplastic environment of PDAC, human adipose tissue-derived stem cells (ASCs) were exposed to hypoxic conditions. Finally, we tested the efficacy of the combination of JNKi and TRAIL in vivo by injecting L3.6pl or MiaPaCa2 pancreatic cancer cells orthotopically into athymic nu/nu mice. We found that JNKi, even at low concentrations, significantly reduced the number of spheres and decreased the expression of CSC markers to levels closer to those detected in parental cells. JNKi downregulates the TRAIL decoy receptor DcR1 while upregulating pro-apoptotic death receptors DR4/5 and thereby sensitizing cells with acquired TRAIL-resistance to apoptosis induction. The combination of JNKi and TRAIL significantly impacts on CSCs, but leaves ASCs - even under hypoxic stress conditions - unaffected. Treatment of orthotopic xenografts of L3.6pl, Panc1 or MiaPaCa2 with JNK (259.6 ± 72.58 mg) or TRAIL (260.6 ± 52.82 mg) alone for 4 weeks showed only modest effects compared to control (292.3 ± 66.88 mg), whereas the synergism of JNKi and TRAIL significantly reduced tumor weight (84.23 ± 25.44 mg; p < 0.0174) and occurrence of metastasis. The concept of selective treatment of pancreatic CSCs without impacting normal stem cell physiology warrants promising potential for further clinical evaluation. As an innovative approach, TRAIL-secreting ASC could increase the local concentration of TRAIL in the immediate tumor microenvironment while systemic JNKi treatment would sensitize PDAC for pro-apoptotic therapy. Citation Format: Alejandro Recio Boiles, Matthias Ilmer, Jody Vykoukal, Eckhard Alt. TRAIL-resistance in pancreatic cancer stem cells can be regulated through JNK pathway inhibition without impacting resident stem cell physiology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2015-2934

Abstract 3238: Taiwanin A targets against nonsteroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma

Abstract Taiwanin A (α,β-bis(piperonylidene)-γ-butyrolactone) is isolated from Taiwania cryptomerioides. Similar to paclitaxel, Taiwanin A also extracted from tree barks and have anti-tumor activity in breast, liver, and lung cancer cell lines. The objective of this study is to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of Non-steroidal anti-inflammatory drug-activated gene (NAG-1). NAG-1 has been reported to have antitumor and pro-apoptotic activity, suggesting potential target for cancer therapy. Silencing NAG-1 mRNA expression in A549 reduces the cytotoxicity caused by Taiwanin A. Furthermore, the c-Jun-N-terminal kinase/Ste20-related protein proline/alanine-rich kinase (JNK/SPAK) pathway played a key role in the action of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor, SP600125, resulted in the inhibitory effect of NAG-1 and recovery of Taiwanin A treated cells. Xenograft tumor model showed that Taiwanin A dose-dependently significantly decreases tumor mediated growth in nude mice by increasing NAG-1 expression accompanied tumor apoptosis. These data support the conclusion that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression via the JNK pathway both in vivo and in vitro. This result might be contributed to compound design for increasing cytotoxicity activity in the future. Citation Format: Hong-Meng Chuang, Horng-Jyh Harn. Taiwanin A targets against nonsteroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3238. doi:10.1158/1538-7445.AM2015-3238

Effect of Vascular Cell Adhesion Molecule -1 Overexpression on Adipogenic Differentiation of Murine Mesenchymal Stem Cells and Its Mechanism

To investigate the effect of vascular cell adhesion molecule-1 (VCAM-1) gene overexpression on adipogenic differentiation of mouse mesenchymal stem cells(MSC) and explore its molecular mechanism. VCAM-1 overexpression MSC (MIGR1-VCAM-1/MSC) and the empty plasmid transfection MSC (MIGR1/MSC) were induced to adipogenic differentiation, oil-red-O staining and real-time PCR were used to detect the adipogenic differentiation ability and the mRNA expression level of key transcription factors C/EBP α and PPAR γ. The activation of P38, ERK and JNK pathways were analyzed by Western blot. Furthermore, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the induced culture system and the alteration of the MSC adipogenic differentiation ability were evaluated. no matter in self or induced differentiation groups, the lipid droplets in MIGR1-VCAM-1/MSC became larger, the amount of adipocyte increased than that in MIGR1/MSC (P<0.01), the mRNA expression level of C/EBPα and PPARγ were upregulated, and JNK pathway were down-regulated while the P38 and ERK pathway were significantly up-regulated. The inhibition of JNK pathway of MIGR1-VCAM-1/MSC could lead to increased mRNA expression level of C/EBP α and PPAR γ, the amount of adipocytes increased (P<0.01), however, the inhibition of the P38 and ERK pathway of MIGR1-VCAM-1/MSC could lead to decreased mRNA expression level of C/EBP α and PPAR γ, and the lipid droplets and the number of adipocytes became smaller and less. The overexpression of VCAM-1 may promote MSC to differentiate into adipocytes through inhibiting JNK signaling pathway, activating P38 and ERK pathways.

Glycated serum albumin stimulates expression of endothelial cell specific molecule-1 in human umbilical vein endothelial cells: Implication in diabetes mediated endothelial dysfunction.

Pro-inflammatory conditions induced by products of protein glycation in diabetes substantially enhance the risk of endothelial dysfunction and related vascular complications. Endothelial cell specific molecule-1 (ESM-1) or endocan has been demonstrated as a potential biomarker in cancer and sepsis. Its role in diabetes-induced pathologies remains unknown. The expression of ESM-1 gene is under cytokine regulation, indicating its role in endothelium-dependent pathological disorders. In this study, we investigated the effect of advanced glycated human serum albumin (AGE-HSA) on the production of ESM-1. We show that AGE-HSA exerts a modulating role on the expression of ESM-1 in human umbilical vein endothelial cells. It up-regulates expression of ESM-1 protein in a dose-dependent manner which correlates with its messenger RNA (mRNA) transcription. RAGE and galectin-3, both AGE receptors, show antagonistic action on its expression. While gene silencing of RAGE has down-regulatory effect, that of galectin-3 has up-regulatory effect on AGE-induced expression of ESM-1. Inhibition of MAPKKK and JNK pathways did not alter the expression. In contrast, phosphatidylinositol 3 kinase (PI3K) inhibition significantly up-regulated ESM-1 expression. In conclusion, these results suggest that AGE-induced activation of human umbilical vein endothelial cells promotes formation of endocan which is an endothelial dysfunction marker and may be related to vascular disease in diabetes.

Osmostress-induced apoptosis in Xenopus oocytes: role of stress protein kinases, calpains and Smac/DIABLO.

Hyperosmotic shock induces cytochrome c release and capase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.

Protection by genistein on cortical neurons against oxidative stress injury via inhibition of NF-kappaB, JNK and ERK signaling pathway

Context: Genistein, one of the isoflavones derived from soybean seeds, has been reported to exert multiple bioactivities. However, the mechanism of its action on the central nervous system is not fully understood.Objective: To investigate the cytoprotection of genistein and its molecular mechanism against H2O2-induced cell death in primary rat cortical neurons.Materials and methods: Genistein (0.01, 0.1, and 1 μM) were added into the primary rat neurons 24 h before and co-cultured with 500 μM H2O2 for 1 h. Neuronal injury was assessed by MTT, lactate dehydrogenase (LDH) assay, and Hoechst33258 staining. Intracellular reactive oxygen species (ROS) generation induced by H2O2 was determined. Neuronal apoptosis was evaluated by Bcl-2/Bax ratio as well as by caspase-9 and caspase-3 activities. The protein levels and phosphorylation of NF-κB/p65, IκB, JNK, and ERK were detected by western blots.Results: Genistein pretreatment attenuated H2O2-mediated neuronal viability loss, nuclear condensation, and ROS generation in a concentration-dependent manner. Genistein exerted anti-apoptotic effects by reversing the apoptotic factors Bcl-2 and Bax ratio, along with the suppression of caspase-9 and caspase-3 activities. In addition, genistein down-regulated the expression of NF-κB/p65, and suppressed the phosphorylation of p65 and IκB. Genistein also inhibited H2O2-induced activation of the MAPK-signaling pathway including JNK and ERK.Discussion and conclusion: The results indicated that genistein effectively protects cortical neurons against oxidative stress at least partly via inactivation of NF-κB as well as MAPK-signaling pathways, and suggested the possibility of this antioxidant for the prevention and treatment of stroke.

Inhibition of metastasis of oral squamous cell carcinoma by anti-PLGF treatment.

Neovascularization plays a critical role in cancer metastasis. However, the molecular mechanism regulating the neovascularization in oral squamous cell carcinoma (OSCC) is poorly understood. Placental growth factor (PLGF) has been known to regulate pathological angiogenesis and has been recently shown to regulate matrix metalloproteinases (MMPs) for extracellular matrix degradation during neovascularization. Here we aimed to examine whether PLGF may regulate MMPs in the metastasis of OSCC. We found that PLGF and MMP9 levels strongly correlated in OSCC in the patients, both increased in the OSCC from the patients with metastasis of the primary OSCC. Thus, we used several human OSCC cell lines to examine the relationship between PLGF and MMP9. We found that overexpression of PLGF in OSCC cells increased expression of MMP9, while inhibition of PLGF in OSCC cells decreased expression of MMP9. However, adaptation of MMP9 levels in OSCC cells did not affect the levels of PLGF. These data suggest that PLGF may regulate MMP9 in OSCC cells, but not vice versa. Moreover, inhibition of ERK1/2, but not inhibition of PI3k or JNK pathways, substantially abolished the effect of PLGF on MMP9, suggesting that PLGF may increase expression of MMP9 via ERK/MAPK signaling pathway. Thus, our data demonstrate that PLGF-induced cancer neovascularization may be partially mediated through its effect on MMP9 activation in OSCC.

Preventive effects of bitter melon (Momordica charantia) against insulin resistance and diabetes are associated with the inhibition of NF-κB and JNK pathways in high-fat-fed OLETF rats

Bitter melon (BM; Momordica charantia) has been used as a treatment method for various diseases including cancer and diabetes. The objective of this study was to investigate whether BM has preventive effects against insulin resistance and diabetes and to identify the underlying mechanism by which BM ameliorates insulin resistance in obese and diabetic rats. The rats were separated into three groups as follows: (a) high-fat (HF) diet control, (b) HF diet and 1% BM and (c) HF diet and 3% BM. After 6 weeks of assigned treatments, body weight and food intake were not altered by BM administration. Bitter melon treatment significantly improved glucose tolerance and insulin sensitivity. The levels of proinflammatory cytokines were significantly down-regulated in liver, muscle and epididymal fats from BM-treated rats. The activation of nuclear factor-κB (NF-κB) in the liver and muscle was decreased by BM compared with HF controls. The 3% BM supplementation significantly increased the levels of phospho-insulin receptor substrate-1 (Tyr612) and phospho-Akt (Ser473). It also significantly decreased the levels of phospho-NF-κB (p65) (Ser536) and phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185) in liver, muscle and epididymal fats. The findings of this study indicate that BM exerted preventive effects against insulin resistance and diabetes through the modulation of NF-κB and JNK pathways. Therefore, BM may be useful in the prevention of insulin resistance and diabetes.

Taiwanin A targets non-steroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma

Taiwanin A (α,β-bis(piperonylidene)-γ-butyrolactone) is extracted from Taiwania cryptomerioides. Taiwanin A is extracted from tree bark and exhibits antitumor activity in breast, liver, and lung cancer cell lines. The objective of this study was to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 has been reported to exhibit antitumor and proapoptotic activities, suggesting potential use in cancer therapy. Inhibiting NAG-1 mRNA expression in A549 reduced the cytotoxicity caused by Taiwanin A. Furthermore, the c-Jun-N-terminal kinase/Ste20-related protein proline/alanine-rich kinase (JNK/SPAK) pathway played a key role in the influence of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor SP600125 resulted in an inhibitory effect on NAG-1 and recovery of Taiwanin-A-treated cells. A xenograft tumor model demonstrated that Taiwanin A dose-dependently significantly decreases tumor-mediated growth in nude mice by increasing the NAG-1 expression accompanying tumor apoptosis. These data supported the hypothesis that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression through the JNK pathway both in vivo and in vitro. This result can contribute to a compound design for increasing cytotoxicity activity in the future.

Secretoglobin 3A2 attenuates lipopolysaccharide-induced inflammation through inhibition of ERK and JNK pathways in bronchial epithelial cells.

Secretoglobin (SCGB) 3A2, previously known as uteroglobin-related protein 1, is a secreted protein highly expressed in the epithelial cells of the airways. It has been demonstrated that SCGB3A2 is involved in allergic airway inflammation such as bronchial asthma. However, the role of SCGB3A2 in lipopolysaccharide (LPS)-induced airway inflammation has yet to be reported. The goal of this study was therefore to clarify the role of SCGB3A2 in LPS-induced airway inflammation. We stimulated BEAS-2B, human bronchial epithelial cells, with LPS and analyzed messenger RNA (mRNA) expression of tumor necrosis factor (TNF)-α and CXCL8 with or without pre-incubation of SCGB3A2. The mRNA expression of TNF-α and CXCL8 was clearly upregulated 3 h after LPS stimulation, and pre-incubation of SCGB3A2 significantly inhibited the upregulation of the mRNA expression. The pre-incubation of SCGB3A2 also inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase in BEAS-2B cells. Furthermore, PD98059, a specific inhibitor for ERK, as well as SP600125, a specific inhibitor for JNK, inhibited LPS-induced mRNA upregulation of inflammatory mediators. These results demonstrate the novel biological activity of SCGB3A2, which is that it attenuates LPS-induced inflammation in bronchial epithelial cells through inhibition of ERK and JNK activation.

BMPRIA mediated signaling is essential for temporomandibular joint development in mice.

The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ), also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC)-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa) inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development.

Ultraviolet A-induced cathepsin K expression is mediated via MAPK/AP-1 pathway in human dermal fibroblasts.

BackgroundCathepsin K (CatK), a cysteine protease with the potent elastolytic activity, plays a predominant role in intracellular elastin degradation in human dermal fibroblasts (HDFs), and contributes to solar elastosis. In previous studies, CatK expression was downregulated in photoaged skin and fibroblasts, but upregulated in acute UVA-irradiated skin and fibroblasts. The underlying mechanisms regulating UVA-induced CatK expression remain elusive.ObjectiveThis study investigates mechanisms involved in the regulation of UVA-induced CatK expression in HDFs.MethodsPrimary HDFs were exposed to UVA. Cell proliferation was analyzed using a colorimetric assay of relative cell number. Quantitative real-time RT-PCR and Western blot were performed to detect CatK expression in HDFs on three consecutive days after 10 J/cm2 UVA irradiation, or cells treated with increasing UVA doses. UVA-activated MAPK/AP-1 pathway was examined by Western blot. Effects of inhibition of MAPK pathway and knockdown of Jun and Fos on UVA-induced CatK expression were also measured by real-time RT-PCR and Western blot.ResultsUVA significantly increased CatK mRNA and protein expression in a dose-dependent manner. UVA-induced CatK expression occurred along with UVA-activated phosphorylation of JNK, p38 and Jun, UVA-increased expression of Fos. Inactivation of JNK and p38MAPK pathways both remarkably decreased UVA-induced CatK expression, which was suppressed more by inhibition of JNK pathway. Furthermore, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatK expression.ConclusionUVA is capable of increasing CatK expression in HDFs, most likely by activation of MAPK pathway and of AP-1, which has been shown to be the case for matrix metalloproteinases. As current strategies for selecting anti-photoaging agents focus on their ability to decrease MMPs' expression through inhibiting UV- activated MAPK pathway, future strategies should also consider their effect on CatK expression.

Palmitate induces SHIP2 expression via the ceramide-mediated activation of NF-κB, and JNK in skeletal muscle cells

AimsElevated plasma free fatty acids impair the insulin signaling by induction of the expression of protein phosphatases. However, the effect of palmitate on SH2-containing inositol 5′-phosphatase 2 (SHIP2) expression has not been investigated. Here we investigated the effects of palmitate on SHIP2 expression and elucidated the underlying mechanisms in skeletal muscle cells. Main methodsSHIP2 mRNA and protein levels were measured in C2C12 myotubes exposed to palmitate. Specific inhibitors were used to identify the signaling pathways involved in SHIP2 expression. Key findingsThe results showed that 0.5mM palmitate significantly upregulates the mRNA and protein levels of SHIP2 in C2C12 cells. To address the role of palmitate intracellular metabolites in SHIP2 expression, the myotubes were treated with palmitate in the presence of ceramide and diacylglycerol synthesis inhibitors. The results demonstrated that only ceramide synthesis inhibition could prevent palmitate-induced SHIP2 expression in these cells. In addition, the incubation of muscle cells with different concentrations of C2-ceramide dose-dependently enhanced SHIP2 expression. Furthermore, the inhibition of both JNK and NF-κB pathways could prevent ceramide-induced SHIP2 expression in myotubes. SignificanceThese findings suggest that palmitate contributes to SHIP2 overexpression in skeletal muscle via the mechanisms involving the activation of ceramide-JNK and NF-κB pathways.

SDC2 and SDC4 Working Together to Keep the Homeostasis of IVD? A Preliminary Study

Introduction Our previous work have proved that Syndecan 4 (SDC4) plays an important role in TNFa and IL1b induced disc degeneration by promoting extracellular matrix degradation, Syndecan 2 (SDC2) and SDC4 both are members of SDC family, however, expression and role of SDC2 in intervertebral disc is still unclear. The role of this study is to investigate the expression pattern and regulation of SDC2 and its and role in intervertebral disc degeneration. Materials and Methods Nucleus pulposus (NP) cells and annulus fibrosis (AF) cells were cultured separately. mRNA were extracted and expression of SDC2 in NP and AF cells were examined by q-PCR method. Expression of SDC2 was examined by q-PCR in 4, 8, 24hs after NP cells were treated by TNFa (20mg/L), IL1-b(10mg/L) and TGF-b (20mg) retrospectively. NP cells were pretreated with p38, JNK and NFkB signaling pathway inhibitors before treatment with TNFa, then expression of SDC2 was examined by qPCR. Results SDC2 was expressed in both NP and AF cells. NP cells have a higher expression level than AF cells of SDC2 in mRNA level. TNFa and IL1b could inhibit SDC2 expression in NP cells robustly. What's more, SDC2 expression was induced after TGFb treatment in NP cells. Inhibition of SDC2 expression by TNFa could be reduced by p38 and NFkB pathway inhibitor. JNK inhibitor has no role in Inhibition of SDC2 expression by TNFa. Conclusion Inflammatory cytokines could inhibit SDC2 expression and induce SDC4 expression in NP cells, p38 and NFkB pathway participant in this process. The different regulation pattern of SDC2 and SDC4 might play important role in keeping intervertebral disc homeostasis. Disclosure of Interest None declared

Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways

The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways.