Mitogen-activated Protein Kinase Superfamily Research Articles

Radiobrominated probe targeting activated p38α in inflammatory diseases.

p38α, a member of the mitogen-activated protein kinase superfamily, is activated by external stimuli, followed by nuclear translocation for the regulation of inflammatory responses at the transcriptional and translational levels in inflammatory diseases. Thus, activated p38α would be an appropriate target molecule for in vivo noninvasive imaging and targeted radionuclide therapy. For this purpose, we designed a radiobrominated compound, 6-(4-[77Br]bromo-2-fluorophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)-pyrido[2,3-d]pyrimidin-7(8H)-one ([77Br]4-BR), based on a potent p38α selective inhibitor, R1487, for use with single-photon emission computed tomography. We synthesized [77Br]4-BR and evaluated its effectiveness as an activated p38α imaging probe compared with our previous radioiodinated probe (6-(2-fluoro-4-[125I]iodophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)-pyrido[2,3-d]pyrimidin-7(8H)-one ([125I]4-IR)) in a mouse inflammatory model. We designed [77Br]4-BR by replacing the radioiodine of [125I]4-IR or the fluorine of R1487 with radiobromine at the 4-position of the phenoxy ring. We synthesized 4-BR via a four-step process. The inhibitory potency of 4-BR was measured using an ADP-Glo™ kinase assay system. Radiosynthesis of [77Br]4-BR was performed via an organotin-radiobromine exchange reaction using the corresponding tributyltin precursor. Radioactivity biodistribution was evaluated in normal ddY mice and turpentine oil-induced inflammation model mice for 120min after intravenous administration of [77Br]4-BR. The temporal changes in radioactivity in blood fractions were compared between [77Br]4-BR and [125I]4-IR. 4-BR was synthesized at a total yield of 9.1% and showed a p38α inhibitory potency similar to that of 4-IR. [77Br]4-BR was successfully obtained from a tributyltin precursor with high radiochemical yield (89.9%), purity (95.9%), and molar activity (2.0 TBq/µmol). [77Br]4-BR showed accumulation of high radioactivity in the inflamed tissue (3.4% ± 0.9% ID/g, peaking at 15min), rapid delivery throughout the body, and rapid blood clearance with approximately half of the blood radioactivity existing as an intact form at 60min. Although the maximum radioactivity accumulation in inflamed tissue after [77Br]4-BR administration was approximately half that of [125I]4-IR because of its faster blood clearance and lower free fraction in the input function, the inflamed tissue-to-blood ratio was comparable between [77Br]4-BR and [125I]4-IR. [77Br]4-BR would be a promising imaging agent for detecting activated p38α in inflammatory diseases.

Biphasic Regulation of Mitogen-Activated Protein Kinase Phosphatase 3 in Hypoxic Colon Cancer Cells.

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

Development of radioiodinated pyrimidinopyridone derivatives as targeted imaging probes of activated p38α for single photon emission computed tomography.

p38α, a member of the mitogen-activated protein kinase superfamily, is ubiquitously expressed in a variety of mammalian cells. Activated p38α induces inflammatory responses to external stimuli, suggesting that non-invasive detection of activated p38α would be valuable for diagnosing inflammatory diseases. For this purpose, we designed radiolabeled compounds [123I]2-IR and [123I]4-IR based on a potent p38α selective inhibitor R1487 for use with single photon emission computed tomography (SPECT). In this study, we used 125I instead of 123I due to its more usable radiochemical properties, synthesized [125I]2-IR and [125I]4-IR, and evaluated their effectiveness as activated p38α imaging probes. [123I]2-IR and [123I]4-IR were designed by introduction of a 123I atom at the 2- or 4-ositions of the phenoxy ring, preserving the pyrimidinopyridone structure of R1487. We synthesized 2-IR and 4-IR via a 7-step process. The inhibitory potencies of 2-IR, 4-IR, and p38α inhibitors were measured using an ADP-Glo™ kinase assay system. Radioiodination of 2-IR and 4-IR was performed via an organotin-radioiodine exchange reaction using the corresponding tributyltin precursors. Biodistributions were evaluated by determining radioactivity in tissues of interest after intravenous administration of [125I]2-IR and [125I]4-IR in normal ddY mice and turpentine oil-induced inflammation model mice. In vivo inhibition study was also performed in inflammation model mice after intravenous administration of [125I]4-IR with pretreatment of p38α inhibitors. We synthesized 2-IR and 4-IR at total yields of 17.5% and 19.2%, respectively. 4-IR had higher p38α inhibitory potency than 2-IR; both compounds were significantly less potent than R1487. [125I]2-IR and [125I]4-IR were successfully obtained from tributyltin precursors with high radiochemical yield (> 65%), purity (> 97%), and molar activity (~ 81GBq/µmol). [125I]4-IR showed high radioactivity accumulation in the inflamed tissue (7.0 ± 1.2%D/g), rapid delivery throughout the body, and rapid blood clearance, resulting in a high inflammation-to-blood ratio (6.2 ± 0.4) and a high inflammation-to-muscle ratio (5.2 ± 1.3) at 30min, while [125I]2-IR showed low radioactivity accumulation in inflamed tissue over the experimental period. Further, radioactivity accumulation in inflamed tissue after [125I]4-IR administration was significantly decreased by pretreatment with selective inhibitors. [123I]4-IR would be a promising imaging agent for detection of activated p38α.

Targeting DUSP7 signaling alleviates hepatic steatosis, inflammation and oxidative stress in high fat diet (HFD)-fed mice via suppression of TAK1

The non-alcoholic fatty liver disease (NAFLD), as a critical liver disease, is still lack of effective treatments because the molecular mechanism revealing the NAFLD pathogenesis remains unclear. Dual specific phosphatase 6 (DUSP7) shows effects on inflammatory response and is a negative feedback mechanism of the mitogen-activated protein kinase (MAPK) superfamily, which are critical factors in regulating NAFLD progression. However, the effects of DUSP7 on hepatic steatosis are still not fully understood. Here, we found that DUSP7 functioned as a negative regulator of NAFLD and in various metabolic disorders. DUSP7 expression was markedly reduced in liver samples from patients with simple hepatic steatosis or non-alcoholic steatohepatitis (NASH), as well as in liver tissues from high fat diet (HFD)-challenged mice or genetically obese (ob/ob) mice. DUSP7 knockout markedly accelerated insulin resistance, glucose intolerance, liver dysfunction, fibrosis and hepatic steatosis in HFD-fed mice. In addition, inflammatory response was significantly exacerbated in HFD-challenged mice with DUSP7 deletion, which was associated with the elevated activation of nuclear factor-κB (NF-κB) and MAPKs signaling pathways. Moreover, oxidative stress was detected in liver of HFD-induced mice, and this phenomenon was aggravated in mice with DUSP7 knockout. Importantly, we demonstrated that DUSP7 physically interacted with transforming growth factor β (TGF-β)-activated kinase (TAK1). DUSP7 deletion considerably promoted the activation of TAK1 in mice after HFD feeding, contributing to the lipid deposition, inflammatory response and reactive oxygen species (ROS) production. Taken together, DUSP7 might function as a protective factor against NAFLD development and metabolic disorder through alleviating dyslipidemia, inflammation and oxidative stress by directly interacting with TAK1 in hepatocytes, which was involved in the suppression of fibrosis. Thus, we may provide an effective strategy for the treatment of hepatic steatosis via targeting DUSP7.

The Role of JNk Signaling Pathway in Obesity-Driven Insulin Resistance.

Obesity is not only closely related to insulin resistance but is one of the main factors leading to the formation of Type 2 Diabetes (T2D) too. The c-Jun N-terminal kinase (JNK) family is a member of the mitogen-activated protein kinase (MAPK) superfamily. JNK is also one of the most investigated signal transducers in obesity and insulin resistance. JNK-centric JNK signaling pathway can be activated by growth factors, cytokines, stress responses, and other factors. Many researches have identified that the activated phosphorylation JNK negatively regulates insulin signaling pathway in insulin resistance which can be simultaneously regulated by multiple signaling pathways related to the JNK signaling pathway. In this review, we provide an overview of the composition of the JNK signaling pathway, its regulation of insulin signaling pathway, and the relationship between the JNK signaling pathway and other pathways in insulin resistance.

JNK signaling pathway regulates the development of ovaries and synthesis of vitellogenin (Vg) in the swimming crab Portunus trituberculatus.

The development of Portunus trituberculatus egg cells is directly related to the nutritional status of the fertilized egg, which affects the key production stages of offspring hatching. Vitellogenin plays a key role in the nutrient supply required for the development of the egg cells. The c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) superfamily and plays an important role in cell proliferation, transformation, differentiation, and apoptosis. At present, there are no reports on the involvement of the JNK signaling pathway in the reproductive regulation of P. trituberculatus. In this study, rapid amplification of complementary DNA ends amplification technology was used to clone the full length of JNK complementary DNA, which has a length of 2094bp, including an open reading frame (ORF) of 1266bp encoding a 421-amino acid protein. The protein includes the S_TKC conserved domain with a TPY phosphorylation site, which is a typical feature of the JNK gene family. Observing tissue sections found the oocytes in the inhibitor group developed slowly, while the oocytes in the activated group showed accelerated development. Meanwhile, Portunus trituberculatus JNK and vitellogenin (Vg) genes exhibited the same trend in the hepatopancreas and ovaries, and the expression of the SP600125 group was downregulated (P < 0.05), while the anisomycin group was upregulated (P < 0.05). In addition, JNK enzyme activity and vitellin (Vn) content in the ovarian tissue showed that the JNK activity of the SP600125 group decreased, while activity increased in the anisomycin group. The accumulation of Vn content in the SP600125 group decreased, and that in the anisomycin group increased. In summary, after injection with inhibitor or activator, the JNK signaling pathway of P. trituberculatus was inhibited or activated, the accumulation of Vn in the ovary was reduced or increased, and ovarian development was inhibited or accelerated, respectively. These results indicated that the JNK signaling pathway is involved in the regulation of Vg synthesis and ovarian development in P. trituberculatus. The results of this study further add to the knowledge of the breeding biology of P. trituberculatus and provide a theoretical reference for the optimization of breeding techniques in aquaculture production systems.

The Expression of ERK1/2 in Female Yak (Bos grunniens) Reproductive Organs.

Simple SummaryExtracellular signal-regulated kinases1/2 (ERK1/2) plays a significant role in regulating the reproductive processes of mammals. The goal of our research is to investigate the expression and distribution of ERK1/2 in the main reproductive organs of the yak during different stages. Using immunohistochemistry, western blot, and relative quantitative real-time polymerase chain reaction techniques, we found that the expression of ERK1 and ERK2 proteins and their mRNA in the yak’s ovary, oviduct, and uterus varies with the stage of the reproductive cycle. The variation character of ERK1 and ERK 2 expression in the yak’s main reproductive organs during different stages implies that they play an important role in regulating the reproductive functions under different physiological statuses.The main reproductive organs undergo different histological appearances and physiological processes under different reproductive statuses. The variation of these organs depends on a delicate regulation of cell proliferation, differentiation, and apoptosis. Extracellular signal-regulated kinases1/2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) super family. They have important roles in regulating various biological processes of different cells, tissues, and organ types. Activated ERK1/2 generally promotes cell survival, but under certain conditions, ERK1/2 also have the function of inducing apoptosis. It is widely believed that ERK1/2 play a significant role in regulating the reproductive processes of mammals. The goal of our research is to investigate the expression and distribution of ERK1/2 in the yak’s main reproductive organs during different stages. In the present study, samples of the ovary, oviduct, and uterus of 15 adult female yak were collected and used in the experiment. The ERK1/2 proteins, localization, and quantitative expression of their mRNA were investigated using immunohistochemistry (IHC), western blot (WB) and relative quantitative real-time polymerase chain reaction (RT-PCR). The results indicated that ERK1/2 proteins and their mRNA were highly expressed in the ovary of the luteal phase and gestation period, in the oviduct of the luteal phase, and in the uterus of the luteal phase and gestation period. Immunohistochemical analysis revealed a strong distribution of ERK1/2 proteins in follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium, and luminal epithelium. These results demonstrated that the expression of ERK1 and ERK2 proteins and their mRNA in the yak’s ovary, oviduct, and uterus varies with the stage of the reproductive cycle. The variation character of ERK1 and ERK 2 expression in the yak’s main reproductive organs during different stages implies that they play an important role in regulating the reproductive function under different physiological statuses.

Extracellular signal-regulated kinases (ERK) 1 and 2 as a key molecule in pain research.

Pain can be divided into nociceptive, inflammatory, and neuropathic pain. It is important to understanding the molecular mechanism of pain signaling in the development of pain relief therapies. Twenty years ago, extracellular signal-regulated kinases (ERK) 1 and 2, which are members of the mitogen-activated protein kinase superfamily, were identified as molecules activated in neurons by the exposure of peripheral tissues to noxious stimuli. Further studies have revealed that peripheral nerve injury induces ERK activation in glial cells, sensory neurons, and second-order neurons, albeit at different time points. Moreover, inhibition of ERK suppresses pathological pain in animals with peripheral nerve injury. Therefore, ERK is currently recognized as an important molecule in pain signaling and a potential novel target for pain treatment. This review introduces recent advances in revealing the regulation of ERK in pain research.

Deletion of the PDZ-binding kinase (Pbk) gene does not affect male fertility in mice.

The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.

Identification of mapk gene family in Lateolabrax maculatus and their expression profiles in response to hypoxia and salinity challenges

Mitogen-activated protein kinases (MAPK) superfamily exerts crucial roles in the regulation of intracellular metabolism, gene expression as well as integral activities in diverse cellular processes. In our study, 14 mapk genes were identified in spotted sea bass (Lateolabrax maculatus) and these genes were divided into three subfamilies according to phylogenetic analysis, including 6 erk genes, 3 jnk genes and 5 p38-mapk genes. Syntenic and tertiary structure analysis confirmed their annotation and orthologies. The expression profiles of several stress-responsive mapk genes of spotted sea bass were examined in gill using quantitative real-time PCR after salinity (0‰, 12‰, 30‰, 45‰) and hypoxia challenges (DO = 1.0 ± 0.2 mg/L). After salinity challenge, significant up-regulations were observed in the expressions of mapk8 (jnk1), mapk9 (jnk2), mapk11 (p38β), mapk14a (p38a) and mapk14b (p38b). mapk8 and mapk9 were more sensitive to hypotonic challenge (0‰) than hyperosmotic (45‰) and isosmotic challenges (12‰), while the highest expression of mapk11, mapk14a and mapk14b were observed in hyperosmotic challenge (45‰). After hypoxia challenge, the expression levels of mapk1 (erk2), mapk3 (erk1) and mapk14a (p38a) in treatment group (DO = 1.0 ± 0.2 mg/L) were significantly up-regulated in comparison with control group (DO = 8.0 ± 0.5 mg/L) in time-dependent manner. These results indicated that these mapk genes in spotted sea bass may play important roles in response to salinity and hypoxia challenges.

The expression of ERK and JNK in patients with an endemic osteochondropathy, Kashin-Beck disease

Kashin–Beck disease (KBD) is a chronic, endemic osteochondropathy. Its etiopathogenesis is still obscure until now. Epidemiological observation has shown that low selenium play a crucial role in the pathogenesis of KBD. Extracellular signal-regulated kinases (ERKs) and C-Jun N-terminal kinase (JNK), members of the mitogen-activated protein kinase (MAPK) superfamily, play an important role in cell proliferation and differentiation. Nuclear factor-ĸB (NF-ĸB), an important signaling mediator for inflammatory and immune responses, is involved in the regulation of osteoclastogenesis. In the present study, we investigated the expression of ERK and JNK signal molecular, as well as nuclear factor-ĸB in the pathogenesis of Kashin-Beck disease, evaluated the effect of selenium on ERK signal pathway. The expression levels of ERK and JNK signal pathway, as well as nuclear factor-ĸB were investigated for 218 patients and 209 controls by immunoblot analysis in whole blood. Evaluated the effect of selenium on ERK signal pathway by Na2SeO3 treatment. The protein levels of pRaf-1, pMek1/2 and pErk1/2 decreased significantly in KBD patients, p-JNK and NF-ĸB increased in KBD patients. Furthermore, Na2SeO3 treatment improved the reduction of proteins in ERK signal pathway. These findings indicated that ERK and JNK signaling pathways, as well as the expression level of NF-κB signaling molecular are important contributor to the pathogenesis of KBD. Selenium stimulates the phosphorylation of the ERK signaling pathway.

Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line.

p38 mitogen-activated protein kinase (MAPK) belongs to the MAPK superfamily, phosphorylating serine and/or threonine residues of the target proteins. The activation of p38 MAPK leads to cell growth, differentiation, inflammation, survival or apoptosis. In this study, we tested the effect of two highly specific and potent inhibitors of p38 MAPK (namely, SB203580 and SB202190) on human breast cancer cell line MDA-MB-231 to elucidate the controversial role of p38 MAPK on cell proliferation and/or cell migration/metastasis further. It was determined that the IC50 value of SB203580 was 85.1µM, while that of SB202190 was 46.6µM, suggesting that SB202190 is slightly more effective than SB203580. To verify the effect of each inhibitor on cell proliferation and cytotoxicity, the cells were treated with various doses of SB203580 and SB202190 and examined using iCELLigence system. No significant effect of 1 and 5µM of both inhibitors were seen on cell proliferation as compared to the DMSO-treated control cells for up to 96h. On the other hand, both SB203580 and SB202190 significantly prevented cell proliferation at a concentration of 50µM. SB202190 was again more effective than SB203580. Afterwards, we tested the effect of each inhibitor on cell migration using wound assay. Both SB203580 and SB202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50µM. However, interestingly it was observed that a low and noncytotoxic dose of 5µM of SB203580 and SB202190 also did cause significant cell migration inhibition at 48h of the treatment, corroborating the fact that p38 MAPK pathway has a critical role in cell migration/metastasis. Then, we tested whether each p38 MAPK inhibitor has any effect on cell adhesion during a treatment period of 3h using iCELLigence system. A concentration of only 50µM of SB202190 reduced cell adhesion for about 1.5h (p<0.001); after that period of time, cell adhesion in 50µM SB202190-treated cells returned to the level of the control cells. To determine the mechanism of growth and cell migration inhibitory effects of p38 MAPK inhibitors, the activation/inactivation of various proteins and enzymes was subsequently analyzed by PathScan® Intracellular Signaling Array kit. The ERK1/2 phosphorylation level was not modified by low concentrations (1 or 5µM) of SB202190 and SB203580; while a high concentration (50µM) of both inhibitors caused significant reductions in the ERK1/2 phosphorylation. In addition, it was determined that both p38 MAPK inhibitors caused significant increases on the Ser15 phosphorylation of mutant p53 in MDA-MB-231 under these experimental conditions; while SB202190 was more potent than SB203580.

Association of Topoisomerase II (TOP2A) and Dual-Specificity Phosphatase 6 (DUSP6) Single Nucleotide Polymorphisms with Radiation Treatment Response and Prognosis of Lung Cancer in Han Chinese.

BackgroundMutations of DNA topoisomerase II (TOP2A) are associated with chemotherapy resistance, whereas dual-specificity phosphatase 6 (DUSP6) negatively regulates members of the mitogen-activated protein (MAP) kinase superfamily to control cell proliferation. This study assessed TOP2A and DUSP6 single nucleotide polymorphisms (SNPs) in non-small cell lung cancer (NSCLC) patients for association with chemoradiotherapy responses and prognosis.Material/MethodsA total of 140 Chinese patients with histologically confirmed NSCLC were enrolled and subjected to genotyping of TOP2A rs471692 and DUSP6 rs2279574 using Taqman PCR. An independent sample t test was used to analyze differences in tumor regression after radiotherapy versus SNP risk factors. Kaplan-Meier curves analyzed overall survival, followed by the log-rank test and Cox proportional hazard models.ResultsThere were no significant associations of TOP2A rs471692 and DUSP6 rs2279574 polymorphisms or clinicopathological variables with response to chemoradiotherapy (p>0.05). Comparing overall survival of 87 patients with stage I–III NSCLC treated with radiotherapy or chemoradiotherapy to clinicopathological variables, the data showed that tumor regression, weight loss, clinical stage, and cigarette smoking were independent prognostic predictors (p=0.009, 0.043, 0.004, and 0.025, respectively). Tumor regression rate >0.34 was associated with patent survival versus tumor regression rate ≤0.34 (p=0.007).ConclusionsTOP2A rs471692 and DUSP6 rs2279574 SNPs were not associated with chemoradiotherapy response, whereas tumor regression, weight loss, clinical stage, and cigarette smoking were independent prognostic predictors for these Chinese patients with NSCLC.

C-Jun N-terminal kinase (JNK) is involved in immune defense against bacterial infection in Crassostrea hongkongensis

c-Jun N-terminal kinase (JNK) is a universal and essential subgroup of the mitogen-activated protein kinase (MAPK) superfamily, which is highly conserved from yeast to mammals and functions in a variety of physiological and pathological processes. In this study, we report the first oyster JNK gene homolog (ChJNK) and its biological functions in the Hong Kong oyster Crassostrea hongkongensis. The ChJNK protein consists of 383 amino acids and contains a conserved serine/threonine protein kinase (S_TKc) domain with a typical TPY motif. Phylogenetic analysis revealed that ChJNK shared a close evolutionary relationship with Crassostrea gigas JNK. Quantitative RT-PCR analyses revealed broad expression patterns of ChJNK mRNA in various adult tissues and different embryonic and larval stages of C. hongkongensis. When exposed to Vibrio alginolyticus or Staphylococcus haemolyticus, ChJNK mRNA expression levels were significantly up-regulated in the hemocytes and gills in a time-dependent manner. Additionally, subcellular localization studies that ChJNK is a cytoplasm-localized protein, and that its overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. In summary, this study provided the first experimental demonstration that oysters possess a functional JNK that participates in host defense against bacterial infection in C. hongkongensis.

3D-QSAR studies on disubstituted dibenzosuberone derivatives as p38α MAP kinase inhibitors using CoMFA and COMSIA

The p38 mitogen-activated protein kinases belong to the mitogen-activated protein kinase superfamily. They are strongly activated by different stress signals and inflammatory cytokines. p38α mitogen-activated protein kinase is broadly expressed in most cell types and plays an important role in the coordination of the cellular responses such as proliferation, differentiation & survival. The role of p38α in various cancers and cellular functions makes it an interesting kinase drug target. In the present study, a series of recently published disubstituted dibenzosuberones derivatives were taken as p38α inhibitors and three-dimensional quantitative structure-activity relationship study was performed. A reasonable Comparative Molecular Field Analysis (q2 = 0.689, NOC = 6, r2 = 0.942) and Comparative Molecular Similarity Indices Analysis (q2 = 0.645, NOC = 6, r2 = 0.921) models were developed. All the developed models were validated using external test set, bootstrapping, progressive sampling and rm2 metric calculations. The validation of models showed acceptable statistical values and proved to be predictive and robust. The contour map analysis revealed the important insight on favorable substituents to increase the potency of the compounds. Negative substitution near the oxygen atom of R1 position and hydrophobic & hydrogen bond acceptor groups near the phenyl ring of R1 position could enhance the activity. Likewise, positive hydrophobic substitution near the phenyl ring at R3 position favors the increase in the inhibitory activity. Our finding could be helpful to design a more potent derivative of p38α kinase inhibitors in future.Open image in new window

Insight into the selective inhibition of JNK family members through structure-based drug design

The c-Jun N-terminal kinase (JNK) family, which comprises JNK1, JNK2 and JNK3, belongs to the mitogen-activated protein kinase (MAPK) superfamily, whose members regulate myriad biological processes, including those implicated in tumorigenesis and neurodegenerative disorders.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

Effects of SPAK knockout on sensorimotor gating, novelty exploration, and brain area-dependent expressions of NKCC1 and KCC2 in a mouse model of schizophrenia.

SPAK (Sterile 20/SPS1-related proline/alanine-rich kinase) is a protein kinase belonging to the mitogen-activated protein kinase (MAPK) superfamily that has been found to be extensively distributed across the body. The SPAK downstream substrates NKCC1 and KCC2 in the central nervous system are important in the interpretation of developmental mental disorders. The present study aimed to clarify the role of SPAK-NKCC1/KCC2 using a rodent schizophrenia-like model. The mouse paradigm of isolation rearing (IR) was employed, as it simulates the sensorimotor gating abnormalities of schizophrenia. SPAK transgenic mice were used and were divided into four groups: social-wild type, social-SPAK(-/-), isolation-wild type, and isolation-SPAK(-/-). The prepulse inhibition (PPI) test and the novel object recognition test (NORT) were used to measure schizophrenia-associated dysfunctions in gating ability and the novelty recognition, respectively. Finally, the protein expressions of NKCC1/KCC2 in the prefrontal cortex and hippocampus were detected to determine correlations with the behavioral data. Our results demonstrated that SPAK-null mice had superior PPI and novelty recognition relative to wild type controls, with a concomitant increase in KCC2 in the prefrontal cortex. IR disrupted PPI and NORT performances with an associated increase in KCC2. Furthermore, rearing environment and gene manipulation had mutually interactive effects, as the IR-induced effects on PPI and NORT were reversed by SPAK knockout, and the increase in KCC2 and the decreased in the NKCC1/KCC2 ratio in the prefrontal cortex induced by SPAK knockout were reversed by IR. Our data supported the gene-environment hypothesis and demonstrated the potential value of SPAK manipulation in future schizophrenia studies.

DUSP10 gene polymorphism and risk of colorectal cancer in the Han Chinese population

Dual-specificity protein phosphatases (DUSP) negatively regulate members of the mitogen-activated protein kinase superfamily, which is associated with cellular proliferation and differentiation. A recent study suggests that DUSP10 is frequently upregulated in colorectal cancer (CRC). Our study aimed to assess whether DUSP10 contributes to the risk of CRC. We analyzed nine tag single nucleotide polymorphisms (tSNPs) of DUSP10 in a case-control study of Han Chinese by the χ²-test and the SHEsis software. We found that two tSNPs (rs908858, P=0.00004; rs11118838, P=0.02510) were significantly associated with CRC using the χ²-test. Using the SHEsis software, six tSNPs (rs12041033, rs17010629, rs12724393, rs12036163, rs11118838, rs12044821) were found in the same linkage disequilibrium block. Within this linkage disequilibrium block, haplotype 'CTCAAC' showed an increased risk of CRC by 42%. By global haplotype analysis, we found that the haplotype 'ACTCAACTA' may increase the risk of CRC by ∼53%; the haplotype 'GCCCACCCA' may decrease the risk by ∼46%. Our results, combined with previous studies, suggest that certain mutations in DUSP10 correlate with the incidence of CRC. Thus, the function of the DUSP10 gene product may contribute toward CRC in the Han Chinese population.

Identification and analysis of the jnk1 gene in polyploid hybrids of red crucian carp (Carassius auratus red var.) and common carp (Cyprinus carpio L.)

c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase superfamily. The allotetraploid crucian carp is a product of distant hybridization of female red crucian carp with male common carp. It is the first natural case of an allotetraploid with stable genetic characters, including fertility of both female and male animals. In this study, 2 jnk1 cDNAs (including jnk1a and jnk1b) have been cloned from the polyploid crucian carp system, consisting of the allotetraploid crucian carp, the triploid crucian carp, and their original parents (red crucian and common carp). We show that jnk1a and jnk1b represent 2 splice forms arising from the jnk1 gene. On the basis of the genetic structure of jnk1a gene in the polyploid crucian carp system, we demonstrated that the allotetraploid crucian carp is phylogenetically closer to its paternal parent (common carp) than to its maternal parent. We further show a similarity between the triploid crucian carp and its original female parent (red crucian carp). Comparisons of genetic structures indicated that the jnk1b genes of allotetraploid and triploid crucian carp are more similar to those of the original paternal parent rather than the original female parent (red crucian carp). RT-PCR analysis indicated that both the jnk1a and jnk1b genes are widely expressed in fish embryos and in the adult organs, displaying distinct features of embryonic-stage and organ specificity in the polyploid crucian carp system.