The role of transcription factor Ap1 in the activation of the Nrf2/ARE pathway through TET1 in diabetic nephropathy.

TET1 mediates demethylation in tumors, but its role in diabetic nephropathy (DN), a prevalent diabetic complication, is unclear. We attempted to probe the possible mechanism of TET1 in DN. A DN rat model was established and verified by marker detection and histopathological observation. The in vitro model was established on human mesangial cells (HMCs) induced by high glucose (HG), and verified by evaluation of fibrosis and inflammation. The differentially expressed mRNA was screened out by microarray analysis. The most differentially expressed mRNA (TET1) was reduced in DN rats and HG-HMCs. The upstream and downstream factors of TET1 were verified, and their roles in DN were analyzed by gain- and loss-function assays. TET1 was decreased in DN rats and HG-HMCs. High expression of TET1 decreased biochemical indexes and renal injury of DN rats and hampered the activity, fibrosis, and inflammation of HG-HMCs. Ap1 lowered TET1 expression, and enhanced inflammation in HG-HMCs, and accentuated renal injury in DN rats. TET1 overexpression inhibited the effect of Ap1 on DN. TET1 promoted the transcription of Nrf2. The Ap1/TET1 axis mediated the Nrf2/ARE pathway activity. Overall, TET1 overexpression weakened the inhibitory effect of Ap1 on the Nrf2/ARE pathway, thus alleviating inflammation and renal injury in DN.

Anti-inflammatory and antioxidant properties of chemical constituents of Broussonetia papyrifera.

Extensive phytochemical analysis of the CHCl3-soluble part of an ethanolic extract of branches and twigs of Broussonetia papyrifera led to the isolation of fourteen compounds, including a novel 5,11-dioxabenzo[b]fluoren-10-one derivative named broussofluorenone C (12). The isolated compounds 1-14 were characterized based on their NMR and HRMS data, and examined for their anti-inflammatory activities in LPS-stimulated THP-1 cells as well as for their cellular antioxidant effects. Compounds 7-10 and 12 showed inhibitory effects on NF-κB/AP-1 activation and compounds 7-9 were subsequently confirmed to suppress the secretion of both IL-1ß and TNF-α in LPS-stimulated THP-1 cells more significantly than the prednisone used as a positive control. In the CAA assay, compound 10 exhibited the greatest antioxidant effect, greater than that of the quercetin used as a positive control. The results show possible beneficial effects and utilization of B. papyrifera wood in the treatment of inflammatory diseases as well as oxidative stress.

Treating murine Kala-azar with a Mayan plant induces immunochemical changes.

Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P. andrieuxii effective to treat experimental Kala-azar in Balb/c mice.

Inflammatory intracellular pathways activated by electronegative LDL in monocytes.

AIMS: Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes. METHODS AND RESULTS: We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. CONCLUSIONS: LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes.

Coenzyme Q0 regulates NFκB/AP-1 activation and enhances Nrf2 stabilization in attenuation of LPS-induced inflammation and redox imbalance: Evidence from in vitro and in vivo studies.

Coenzyme Q (CoQ) analogs with variable number of isoprenoid units have been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study we used CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains), a novel quinone derivative, and investigated its molecular actions against LPS-induced inflammation and redox imbalance in murine RAW264.7 macrophages and mice. In LPS-stimulated macrophages, non-cytotoxic concentrations of CoQ0 (2.5-10 µM) inhibited iNOS/COX-2 protein expressions with subsequent reductions of NO, PGE2, TNF-α and IL-1ß secretions. This inhibition was reasoned by suppression of NFκB (p65) activation, and inhibition of AP-1 (c-Jun., c-Fos, ATF2) translocation. Our findings indicated that IKKα-mediated I-κB degradation and MAPK-signaling are involved in regulation of NFκB/AP-1 activation. Furthermore, CoQ0 triggered HO-1 and NQO-1 genes through increased Nrf2 nuclear translocation and Nrf2/ARE-signaling. This phenomenon was confirmed by diminished CoQ0 protective effects in Nrf2 knockdown cells, where LPS-induced NO, PGE2, TNF-α and IL-1ß productions remained high. Molecular evidence revealed that CoQ0 enhanced Nrf2 steady-state level at both transcriptional and translational levels. CoQ0-induced Nrf2 activation appears to be regulated by ROS-JNK-signaling cascades, as evidenced by suppressed Nrf2 activation upon treatment with pharmacological inhibitors of ROS (N-acetylcysteine) and JNK (SP600125). Besides, oral administration of CoQ0 (5 mg/kg) suppressed LPS-induced (1 mg/kg) induction of iNOS/COX-2 and TNF-α/IL-1ß through tight regulation of NFκB/Nrf2 signaling in mice liver and spleen. Our findings conclude that pharmacological actions of CoQ0 are mediated via inhibition of NFκB/AP-1 activation and induction of Nrf2/ARE-signaling. Owing to its potent anti-inflammatory and antioxidant properties, CoQ0 could be a promising candidate to treat inflammatory disorders.

Differential activation of inflammatory pathways in testicular macrophages provides a rationale for their subdued inflammatory capacity.

Spermatogenic cells express cell-specific molecules with the potential to be seen as "foreign" by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway-specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined. METHODS: The expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment. RESULTS: TNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo. CONCLUSION: Taken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain-containing 3 and induces activator protein 1 activation.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.

miR-195 plays a role in steroid resistance of ulcerative colitis by targeting Smad7.

An imbalance in pro- and anti-inflammation is an important mechanism of steroid resistance in UC (ulcerative colitis), and miRNAs may participate in this process. The present study aimed to explore whether miRNAs play a role in the steroid resistance of UC by regulating gene expression of the inflammation signal pathway. SS (steroid-sensitive) patients, SR (steroid-resistant) patients and healthy individuals were recruited. In vivo miRNA profiles of serum samples showed that miR-195 was decreased significantly in the SR group compared with the SS group (P<0.05). This result was confirmed by qPCR (quantitative real-time PCR) and miRNA ISH (in situ hybridization) in serum and colon tissue samples. Online software was used to identify Smad7 mRNA as a potential target of miR-195. The direct interaction of miR-195 and Smad7 mRNA was investigated using a biotinylated miR-195 pull-down assay. Overexpression of a miR-195 precursor lowered cellular levels of Smad7 protein; conversely, antagonism of miR-195 enhanced Smad7 translation without disturbing Smad7 mRNA levels. A luciferase reporter assay revealed a repressive effect of miR-195 via a single Smad7 3'-UTR target site, and point mutation of this site prevented miR-195-induced repression of Smad7 translation. Furthermore, increased levels of miR-195 led to a decrease in c-Jun and p65 expression. In contrast, transfection with anti-miR-195 led to increased levels of c-Jun and p65 protein. The decrease in miR-195 led to an increase in Smad7 expression and corresponding up-regulation of p65 and the AP-1 (activator protein 1) pathway, which might explain the mechanism of steroid resistance in UC patients.

Syk and IRAK1 Contribute to Immunopharmacological Activities of Anthraquinone-2-carboxlic Acid.

Anthraquinone-2-carboxlic acid (9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid, AQCA) was identified as one of the major anthraquinones in Brazilian taheebo. Since there was no report explaining its immunopharmacological actions, in this study, we aimed to investigate the molecular mechanism of AQCA-mediated anti-inflammatory activity using reporter gene assays, kinase assays, immunoblot analyses, and overexpression strategies with lipopolysaccharide (LPS)-treated macrophages. AQCA was found to suppress the release of nitric oxide (NO) and prostaglandin (PG) E2 from LPS-treated peritoneal macrophages without displaying any toxic side effects. Molecular analysis revealed that AQCA was able to inhibit the activation of the nuclear factor (NF)-κB and activator protein (AP)-1 pathways by direct suppression of upstream signaling enzymes including interleukin-1 receptor-associated kinase 1 (IRAK1) and spleen tyrosine kinase (Syk). Therefore, our data strongly suggest that AQCA-mediated suppression of inflammatory responses could be managed by a direct interference of signaling cascades including IRAK and Syk, linked to the activation of NF-κB and AP-1.

Degraded λ-carrageenan activates NF-κB and AP-1 pathways in macrophages and enhances LPS-induced TNF-α secretion through AP-1.

BACKGROUND: Carrageenan (CGN), a high molecular weight sulfated polysaccharide, is a traditional ingredient used in food industry. Its degraded forms have been identified as potential carcinogens, although the mechanism remains unclear. METHODS: The effects of degraded λ-carrageenan (λ-dCGN) on murine RAW264.7 cells and human THP-1-derived macrophage cells were investigated by studying its actions on tumor necrosis factor alpha (TNF-α) secretion, Toll-like receptor 4 (TLR4) expression, and activation of nuclear factor-κb (NF-κB) and activation protein-1 (AP-1) pathways. RESULTS: We found that λ-dCGN was much stronger than native λ-CGN in the activation of macrophages to secrete TNF-α. Treatment of RAW264.7 cells with λ-dCGN resulted in the upregulation of TLR4, CD14 and MD-2 expressions, but it did not increase the binding of lipopolysacchride (LPS) with macrophages. Meanwhile, λ-dCGN treatment activated NF-κB via B-cell lymphoma/leukemia 10 (Bcl10) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation. In addition, λ-dCGN induced extracellular signal-regulated kinases/1/2/mitogen-activated protein kinases (ERK1/2/MAPK) and AP-1 activation. Interestingly, pretreatment of RAW264.7 cells with λ-dCGN markedly enhanced LPS-stimulated TNF-α secretion. This pretreatment resulted in the enhanced phosphorylation of ERK1/2 and c-Jun N-terminal kinase (JNK) and intensified activation of AP-1. CONCLUSIONS: λ-dCGN induced an inflammatory reaction via both NF-κB and AP-1, and enhanced the inflammatory effect of LPS through AP-1 activation. GENERAL SIGNIFICANCE: The study demonstrated the role of λ-dCGN to induce the inflammatory reaction and to aggravate the effect of LPS on macrophages, suggesting that λ-dCGN produced during food processing and gastric digestion may be a safety concern.

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation.

Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.

Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the ß-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

Expression and regulation of MMP1, MMP3, and MMP9 in the chicken ovary in response to gonadotropins, sex hormones, and TGFB1.

Matrix metalloproteinases (MMPs) are a specific class of proteolytic enzymes that play critical roles in follicular development and luteinization in mammals. However, the role of MMPs in avian ovary remains largely unknown. We found that three MMP genes (MMP1, MMP3, and MMP9) were significantly up-regulated in 23-wk-old (laying phase) chicken ovaries compared with 6-wk-old ovaries (prepubertal phase). In reproductively active chicken ovary, MMP1 expression (both mRNA and protein) remained low in prehierarchical and preovulatory follicles but increased in postovulatory follicles (POFs). Both MMP3 and MMP9 expression levels increased during follicular maturation. MMP3 reached maximal expression in the first largest follicle (F1), while MMP9 levels continued to rise in POF1 and POF2 after ovulation. Immunohistochemistry, Western blot analysis, and zymography experiments indicated that MMP1, MMP3, and MMP9 were synthesized and secreted by granulosa cells of different follicles in the chicken ovary. The mRNA expression of MMP1 and MMP3 in the granulosa cells was stimulated by follicle-stimulating hormone, luteinizing hormone, progesterone, and estrogen but not by transforming growth factor beta 1 (TGFB1). However, the mRNA of MMP9 was induced by TGFB1 but not follicle-stimulating hormone, luteinizing hormone, progesterone, or estrogen. Luciferase reporter and mutagenesis analysis indicated the AP1 and NFkappaB elements located in the promoter region from -1700 to -2400 bp were critical for both basal and TGFB1-induced MMP9 transcription. These data provide the first spatial-temporal expression analysis of MMP system in the chicken ovary.

DIXDC1 increases the invasion and migration ability of non-small-cell lung cancer cells via the PI3K-AKT/AP-1 pathway.

DIX domain containing 1 (DIXDC1), is a human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated with a high cell proliferation index. We demonstrated DIXDC1 overexpression in 55% (92/167) of non-small cell lung cancer (NSCLC) cases, compared to adjacent noncancerous lung tissues (P < 0.01). Overexpression of DIXDC1 was associated with lymph node metastasis and more advanced TNM stage (P < 0.001 and P = 0.001, respectively). Kaplan-Meier survival curves and log-rank testing indicated that overexpression of DIXDC1 correlated with worse overall survival in NSCLC (P = 0.031). DIXDC1 was more abundant in seven NSCLC lines than the bronchial cell line HBE, and modulation of its expression regulated AP-1 activity; MMP2, MMP7, and MMP9 protein and mRNA; and invasion ability. Metalloproteinase induction was reversed by PI3K/AKT and AP-1 inhibition. These results suggest DIXDC1 is associated with stage and prognosis in NSCLC, and may promote invasion and migration through PI3K-AKT/AP-1-dependent activation of metalloproteinases.

Glucocorticoids regulate natural killer cell function epigenetically.

Although glucocorticoids are well known for their capacity to suppress the immune response, glucocorticoids can also promote immune responsiveness. It was the purpose of this investigation to evaluate the molecular basis for this apparent dichotomous immunologic effect. Glucocorticoid treatment of natural killer cells (NK) was shown to reduce NK cell cytolytic activity by reduction of histone promoter acetylation for perforin and granzyme B, which corresponded with reduced mRNA and protein for each. In contrast, glucocorticoid treatment increased histone acetylation at regulatory regions for interferon gamma and IL-6, as well as chromatin accessibility for each. This increase in histone acetylation was associated with increased proinflammatory cytokine mRNA and protein production upon cellular stimulation. These immunologic effects were evident at the level of the individual cell and demonstrate glucocorticoids to epigenetically reduce NK cell cytolytic activity while at the same time to prime NK cells for proinflammatory cytokine production.

Expression and identification of a novel gene Spata34 in mouse spermatogenic cells.

Leucine-rich repeat (LRR) containing proteins play an essential role in signal transduction, cell adhesion, cell development, DNA repair and RNA processing. Here we cloned a novel gene, Spata34, encoding a LRR containing protein of 415 aa. Spata34 gene consisted of 9 exons and 8 introns and mapped to chromosome 3qA3. Spata34 is conserved across species in evolution. The Spata34 gene was expressed at various levels, faintly before first weeks postpartum and strongly from 2 weeks postpartum in adult testes. Western blot analysis showed that Spata34 protein was specially expressed in mouse testis. Immunohistochemical analysis revealed that Spata34 protein was most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferous tubules of the adult testis. Overexpression of Spata34 in COS7 cells inhibited the transcriptional activity of AP-1, p53 and p21 which suggested that Spata34 protein may act as a transcriptional repressor in p53 and p21 pathway.

Effect of the Jianpi Bushen Prescription on the expression of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.

This study investigated the effect of the Jianpi Bushen Prescription (JBP) on the expression of 3 major proteins in chemically damaged model mice. The 3 proteins were the Wnt3a, the SHP-1, and the transcription factors (NF-E2, c-jun, and c-fos) of the AP-1 protein family. Kunming mice were randomly divided into chemically damaged group (N=48), which received an abdominal injection of (100 mg/kg) cyclophosphamide once a day for 3 consecutive days, and control group (N=12), which received the same amount of saline. Then, the chemically damaged mice were randomly divided into chemically damaged model group (N=12), which received 0.2 mL/10 g of saline twice a day for 9 days, positive control group (N=12), which received 0.2 mL/10 g of the e-jiao slurry (EJS) compound twice a day for 9 days, low dose JBP group (N=12), which received 0.1 g/kg suspension JBP (100% concentration) twice a day for 9 days and high dose JBP group (N=12), which received 0.1 g/kg suspension JBP (200% concentration) twice a day for 9 days. The bilateral femur and tibia bone marrow were collected from the mice in all groups. The protein expression of the specified proteins and transcription factors in the bone marrow mononuclear cells were detected by Western blot analysis. The results showed that the protein expression of Wnt3a was significantly downregulated in the chemically damaged model group compared to the control group (P<0.05). The low dose JBP, high dose JBP, and e-jiao slurry treatments significantly upregulated the protein expression of Wnt3a compared to the chemically damaged model group (P<0.05), with the low dose JBP producing the best results. Compared to the control group, the protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly higher in the chemically damaged model group (all P<0.05). The protein expressions of SHP-1, c-fos, c-jun, and NF-E2 were significantly lower in the chemically damaged model+the low dose JBP, chemically damaged model+high dose JBP, or chemically damaged model+EJS group compared to chemically damaged model (all P<0.05), with the low dose JBP producing the best results. These results indicate that JBP regulates the expressions of SHP-1, Wnt3a, and AP-1 proteins in chemically damaged mice.