Dissection of MKK6 and p38 Signaling Using Light-Activated Protein Kinases.

Stress-activated signaling pathways orchestrate cellular behaviors and fates. Studying the precise role(s) of stress-activated protein kinases is challenging, because stress conditions induce adaptation and impose selection pressure. To meet this challenge, we have applied an optogenetic system with a single plasmid to express light-activated p38α or its upstream activator, MKK6, in conjunction with live-cell fluorescence microscopy. In starved cells, decaging of constitutively active p38α or MKK6 by brief exposure to UV light elicits rapid p38-mediated signaling, release of cytochrome c from mitochondria, and apoptosis with different kinetics. In parallel, light activation of p38α also suppresses autophagosome formation, similarly to stimulation with growth factors that activate PI3K/Akt/mTORC1 signaling. Active MKK6 negatively regulates serum-induced ERK activity, which is p38-independent as previously reported. Here, we reproduce that result with the one plasmid system and show that although decaging active p38α does not reduce basal ERK activity in our cells, it can block growth factor-stimulated ERK signaling in serum-starved cells. These results clarify the roles of MKK6 and p38α in dynamic signaling programs, which act in concert to actuate apoptotic death while suppressing cell survival mechanisms.

Genetic regulation of liver lipids in a mouse model of insulin resistance and hepatic steatosis.

To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high-fat/high-sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition.

Optical control of MAP kinase kinase 6 (MKK6) reveals that it has divergent roles in pro-apoptotic and anti-proliferative signaling.

The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.

MEK6 Overexpression Exacerbates Fat Accumulation and Inflammatory Cytokines in High-Fat Diet-Induced Obesity.

Obesity is a state of abnormal fat accumulation caused by an energy imbalance potentially caused by changes in multiple factors. MEK6 engages in cell growth, such as inflammation and apoptosis, as one of the MAPK signaling pathways. The MEK6 gene was found to be related to RMR, a gene associated with obesity. Because only a few studies have investigated the correlation between MEK6 and obesity or the relevant mechanisms, we conducted an experiment using a TgMEK6 model with MEK6 overexpression with non-Tg and chow diet as the control to determine changes in lipid metabolism in plasma, liver, and adipose tissue after a 15-week high-fat diet (HFD). MEK6 overexpression in the TgMEK6 model significantly increased body weight and plasma triglyceride and total cholesterol levels. p38 activity declined in the liver and adipose tissues and lowered lipolysis, oxidation, and thermogenesis levels, contributing to decreased energy consumption. In the liver, lipid formation and accumulation increased, and in adipose, adipogenesis and hypertrophy increased. The adiponectin/leptin ratio significantly declined in plasma and adipose tissue of the TgMEK6 group following MEK6 expression and the HFD, indicating the role of MEK6 expression in adipokine regulation. Plasma and bone-marrow-derived macrophages (BMDM) of the TgMEK6 group increased MEK6 expression-dependent secretion of pro-inflammatory cytokines but decreased levels of anti-inflammatory cytokines, further exacerbating the results exhibited by the diet-induced obesity group. In conclusion, this study demonstrated the synergistic effect of MEK6 with HFD in fat accumulation by significantly inhibiting the mechanisms of lipolysis in the adipose and M2 associated cytokines secretion in the BMDM.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-κB.

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Circ_016719 plays a critical role in neuron cell apoptosis induced by I/R via targeting miR-29c/Map2k6.

BACKGROUND: Stroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown. METHODS: Mice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B-I, LC3B-II, Beclin 1, and p62 expression. RESULTS: Significantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B-I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719. CONCLUSIONS: Our results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.

Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1.

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.

MKK6 Functions in Two Parallel MAP Kinase Cascades in Immune Signaling.

Arabidopsis (Arabidopsis thaliana) MAP KINASE (MPK) proteins can function in multiple MAP kinase cascades and physiological processes. For instance, MPK4 functions in regulating development as well as in plant defense by participating in two independent MAP kinase cascades: the MEKK1-MKK1/MKK2-MPK4 cascade promotes basal resistance against pathogens and is guarded by the NB-LRR protein SUMM2, whereas the ANPs-MKK6-MPK4 cascade plays an essential role in cytokinesis. Here, we report a novel role for MKK6 in regulating plant immune responses. We found that MKK6 functions similarly to MKK1/MKK2 and works together with MEKK1 and MPK4 to prevent autoactivation of SUMM2-mediated defense responses. Interestingly, loss of MKK6 or ANP2/ANP3 results in constitutive activation of plant defense responses. The autoimmune phenotypes of mkk6 and anp2 anp3 mutant plants can be largely suppressed by a constitutively active mpk4 mutant. Further analysis showed that the constitutive defense response in anp2 anp3 is dependent on the defense regulators PAD4 and EDS1, but not on SUMM2, suggesting that the ANP2/ANP3-MKK6-MPK4 cascade may be guarded by a TIR-NB-LRR protein. Our study shows that MKK6 has multiple functions in plant defense responses in addition to cytokinesis.

Mitogen-activated protein kinase kinase 6 is involved in the immune response to bacterial di-/tripeptide challenge in grass carp Ctenopharyngodon idella.

Mitogen-activated protein kinase kinase 6 (MKK6) is an essential component of the p38MAPK signaling pathway, which is involved in the modulation of inflammation, cell apoptosis and survival responses in mammals. However, the function of MKK6s in teleosts is still unclear. In this study, a fish MKK6 homolog (CiMKK6) was first identified from the grass carp (Ctenopharyngodon idella), a freshwater fish. CiMKK6 cDNA encodes a putative protein of 357 amino acids that contains conserved structural characteristics of the MKK6 family, including the S_TKc domain, SVAKT motif and DVD site. The deduced CiMKK6 protein exhibits high sequence homology with other reported fish MKK6s and shares the closest relationship with MKK6 from Danio rerio. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMKK6 mRNA was widely expressed in all tested tissues and stages of embryonic development. Additionally, the transcript levels of CiMKK6 in the intestine were significantly upregulated in response to bacterial muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) stimulation. Moreover, subcellular localization analysis indicated that CiMKK6 was distributed in both the cytoplasm and the nucleus of HEK293T cells. Finally, overexpression of CiMKK6 significantly enhanced the transcriptional activity of the AP-1 reporter gene in HEK293T cells. Overall, these findings may help better clarify the immune function of teleost MKK6s and provide new insight into the immune defense mechanisms of grass carp.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection.

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

Quantitative proteome analysis identifies MAP2K6 as potential regulator of LIFR-induced radioresistance in nasopharyngeal carcinoma cells.

Using Tandem Mass Tags (TMT) labeling and LC-MS/MS analysis of peptides from two cell lines (CNE2 and its radioresistant subclone CNE2-IR), we identified 754 proteins differentially expressed in CNE2-IR compared to CNE2. MAP2K6 was identified as a candidate radioresistance-related protein kinase. In vitro functional analysis revealed that over-expression of MAP2K6 significantly enhanced cell survival and colony formation following irradiation in NPC cells. Further, knockdown of MAP2K6 in radioresistant NPC cells led to decreased colony formation and increased apoptotic cells following irradiation. However, the effect of MAP2K6 in regulating the radioresistance in NPC cells did not seem to depend on p38/MAPK activity. Importantly, MAP2K6 might be required for leukemia inhibitory factor receptor (LIFR)-regulated radioresistance, as the expression levels of MAP2K6 affected LIFR/p70S6K signaling activation in NPC cells. Further, MAP2K6 kinase activity is required to activate LIFR/p70S6K signaling and to confer pro-survival effect on NPC cells. In conclusion, MAP2K6 might be an important regulator of LIFR-induced radioresistance in NPC.

Modulation of adenylate cyclase signaling in association with MKK3/6 stabilization under combination of SAC and berberine to reduce HepG2 cell survivability.

Cancer cells often have faulty apoptotic pathways resulting in sustenance of survivability, tumour metastasis and resistance to anticancer drugs. Alternate strategies are sought to improve therapeutic efficacy and therefore HepG2 cells were treated with S-allyl-cysteine (SAC) and berberine (BER) to analyze their mechanistic impact upon necroptosis along with its interacting relationship to apoptosis. In the present study we observed that SAC and BER exposure reduced NFκß nuclear translocation through adenylate cyclase-cAMP-protein kinaseA axis and eventually evaded c-FLIP inhibition. Effective RIP1 k63-polyubiquitination and persistent MKK3/MKK6 expression during drug treatment potentiated caspase8 activity via p53-DISC conformation. Resultant tBid associated lysosomal protease mediated AIF truncation induced DNA fragmentation and persuaded effector caspase mediated scramblase activation resulting induction of necroptosis in parallel to apoptotic events. SAC+BER effectively reduced Rb-phosphorylation resulting insignificant nuclear E2F presence led to ending of cell proliferation. Therefore necroptosis augmented the drug response and may be targeted alongside cell proliferation inhibition in formation of efficient therapeutics against liver cancer.

MAPK pathways regulation by DUSP1 in the development of osteosarcoma: Potential markers and therapeutic targets.

Osteosarcoma (OS) is the most frequent primary bone tumor that affect children and adolescents. This tumor is highly aggressive with high risk of metastasis and the implementation of new drugs has not been successful. The search for biomarkers or new therapeutic targets is urgently needed and can help in advances of OS treatment. MAPKs are major signaling transduction molecules that play an important role in regulating a variety of cellular responses. DUSP1 is a phosphatase that dephosphorylates the MAPKs. Both MAPKs and DUSPs have been implicated as major modulators of critical signaling pathways that are dysregulated in various diseases. In a previous study, we found an increase in MAPK7 gene expression contributed for worst overall survival and treatment response. We analyzed gene expression of MAPK pathways that participate in MAPK7 regulation, and DUSP1 gene using paired 28 pre/post-chemotherapy and 12 metastasis OS samples. To understand the DUSP1 role in the pathogenesis of OS, we assessed the function of DUSP1 in four OS cell lines through a series of cellular assays combined with gene silencing technique. Our findings showed increased MAP2K6, MAP4K3, and DUSP1 gene expression in post-chemotherapy OS samples presenting poor prognosis. We also found that the suppression of DUSP1 gene expression resulted in decreased proliferation, migration, and invasion in OS cells. These results suggest that members of MAPK family may be possible prognostic markers in OS and DUSP1 has a relevant role in the OS pathogenesis and can be an attractive therapeutic target in new strategies of OS treatment.

MAP2K6-FP Enhances the Sensitiveness of Paclitaxel for Ovarian Cancer via Inducing Autophagy.

BACKGROUND: Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Recent studies have revealed an association between autophagy and drug resistance. METHODS: We previously synthesized a MAPK kinase-recombinant fusion protein, MAP2K6-FP, that contains 3 domains: a protein transduction domain TAT, a human ovarian cancer HO8910 cell-specific binding peptide, and a potential antitumor effector domain MKK6(E). In this study, we investigated the effect of MAP2K6-FP on HO8910 cells treated with paclitaxel. RESULTS: The IC50 (concentration by which 50% cell growth was inhibited) was 20 µM for paclitaxel alone, 1.5 µg/mL for MAP2K6-FP alone, and 0.3 µg/mL for MAP2K6-FP and 15 µM for paclitaxel if combined, respectively. In addition, immunohistochemistry assay demonstrated that tumor tissues from ovarian cancer patients showed higher expression of LC-3, the autophagy-related protein, compared with normal ovarian tissues. MAP2K6-FP (0, 2.5, 5, 10, 20, and 40 µg/mL) dose-dependently increased the LC-3 expression in HO8910 cells. Immunofluorescence assay showed that paclitaxel alone increased the expression of LC-3 in HO8910 cells, which was further enhanced by the combination with MAP2K6-FP. Downregulation of LC-3 expression using LC-3 small interfering RNA inhibited the cytotoxicity effect of MAP2K6-FP. Furthermore, either MAP2K6-FP alone or in combination with paclitaxel increased the ratio of expressions of Beclin-1/Bcl-2, another autophagy-related markers, compared with paclitaxel alone. CONCLUSIONS: MAP2K6-FP enhanced the sensitiveness of paclitaxel for ovarian cancer via inducing autophagy.

Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

p38α MAPK is required for tooth morphogenesis and enamel secretion.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and ß4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

Genetic Analyses Reveal Functions for MAP2K3 and MAP2K6 in Mouse Testis Determination.

Testis determination in mammals is initiated by expression of SRY in somatic cells of the embryonic gonad. Genetic analyses in the mouse have revealed a requirement for mitogen-activated protein kinase (MAPK) signaling in testis determination: targeted loss of the kinases MAP3K4 and p38 MAPK causes complete XY embryonic gonadal sex reversal. These kinases occupy positions at the top and bottom level, respectively, in the canonical three-tier MAPK-signaling cascade: MAP3K, MAP2K, MAPK. To date, no role in sex determination has been attributed to a MAP2K, although such a function is predicted to exist. Here, we report roles for the kinases MAP2K3 and MAP2K6 in testis determination. C57BL/6J (B6) embryos lacking MAP2K3 exhibited no significant abnormalities of testis development, whilst those lacking MAP2K6 exhibited a minor delay in testis determination. Compound mutants lacking three out of four functional alleles at the two loci also exhibited delayed testis determination and transient ovotestis formation as a consequence, suggestive of partially redundant roles for these kinases in testis determination. Early lethality of double-knockout embryos precludes analysis of sexual development. To reveal their roles in testis determination more clearly, we generated Map2k mutant B6 embryos using a weaker Sry allele (Sry(AKR)). Loss of Map2k3 on this highly sensitized background exacerbates ovotestis development, whilst loss of Map2k6 results in complete XY gonadal sex reversal associated with reduction of Sry expression at 11.25 days postcoitum. Our data suggest that MAP2K6 functions in mouse testis determination, via positive effects on Sry, and also indicate a minor role for MAP2K3.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Genomic analysis of gum disease and hypertrichosis in foxes.

Since the 1940s, a proliferative gingival disease called hereditary hyperplastic gingivitis (HHG) has been described in the farmed silver fox, Vulpes vulpes (Dyrendahl and Henricson 1960). HHG displays an autosomal recessive transmission and has a pleiotropic relationship with superior fur quality in terms of length and thickness of guard hairs. An analogous human disease, hereditary gingival fibromatosis (HGF), is characterized by a predominantly autosomal dominant transmission and a complex etiology, occurring either as an isolated condition or as a part of a syndrome. Similar to HHG, the symptom most commonly associated with syndromic HGF is hypertrichosis. Here we explore potential mechanisms involved in HHG by comparison to known genetic information about hypertrichosis co-occurring with HGF, using an Affymetrix canine genome microarray platform, quantitative PCR, and candidate gene sequencing. We conclude that the mitogen-activated protein kinase pathway is involved in HHG, however despite involvement of the mitogen-activated protein kinase kinase 6 gene in congenital hypertrichosis with gingival fibromatosis in humans, this gene did not contain any fixed mutations in exons or exon-intron boundaries in HHG-affected foxes, suggesting that it is not causative of HHG in the farmed silver fox population. Differential up-regulation of MAP2K6 gene in HHG-affected foxes does implicate this gene in the HHG phenotype.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.