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Negative Mutant (negative + mutant)
Kinds of Negative Mutant Selected AbstractsDirect role of NF-,B activation in Toll-like receptor-triggered HLA-DRA expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2006Keun-Wook Lee Abstract Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-,B activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-,-induced MHC-II expression depends on CIITA rather than on NF-,B. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-,B with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity. [source] Caspase-8- and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 productionFEBS JOURNAL, Issue 9 2007Norihiko Matsumoto Despite a dogma that apoptosis does not induce inflammation, Fas ligand (FasL), a well-known death factor, possesses pro-inflammatory activity. For example, FasL induces nuclear factor ,B (NF-,B) activity and interleukin 8 (IL-8) production by engagement of Fas in human cells. Here, we found that a dominant negative mutant of c-Jun, a component of the activator protein-1 (AP-1) transcription factor, inhibits FasL-induced AP-1 activity and IL-8 production in HEK293 cells. Selective inhibition of AP-1 did not affect NF-,B activation and vice versa, indicating that their activations were not sequential events. The FasL-induced AP-1 activation could be inhibited by deleting or introducing the lymphoproliferation (lpr) -type point mutation into the Fas death domain (DD), knocking down the Fas-associated DD protein (FADD), abrogating caspase-8 expression with small interfering RNAs, or using inhibitors for pan-caspase and caspase-8 but not caspase-1 or caspase-3. Furthermore, wildtype, but not a catalytically inactive mutant, of caspase-8 reconstituted the FasL-induced AP-1 activation in caspase-8-deficient cells. Fas ligand induced the phosphorylation of two of the three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 MAPK. Unexpectedly, an inhibitor for JNK but not for MAPK/ERK kinase inhibited the FasL-induced AP-1 activation and IL-8 production. These results demonstrate that FasL-induced AP-1 activation is required for optimal IL-8 production, and this process is mediated by FADD, caspase-8, and JNK. [source] Regulation of expression of terminal oxidases in Paracoccus denitrificansFEBS JOURNAL, Issue 8 2001Marijke F. Otten In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1 -negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3 -type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3 -type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3 -type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain. [source] Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cellsGENES TO CELLS, Issue 9 2006Peng Sun Neuronal differentiation in PC12 cells induced by nerve growth factor (NGF) requires sustained activation of ERK/MAP kinase pathway (Raf,MEK,ERK cascade). Although classical Ras (H-Ras, K-Ras, and N-Ras) activated by NGF signaling induces activation of ERK pathway, the activation is transient and not sufficient for PC12 cell differentiation. Instead, it has been widely accepted that NGF signaling-mediated Rap1 activation causes sustained activation of ERK pathway. There has been no direct evidence, however, that Rap1 participates in neuronal differentiation. Here we show that NGF signaling induces sustained activation of M-Ras and subsequent sustained activation of ERK pathway and the transcription factor CREB leading to PC12 cell differentiation. Exogenously expressed constitutively active mutant of M-Ras caused neurite outgrowth in PC12 cells and activating phosphorylation of ERK, whereas activated Rap1 did not. Knockdown of endogenous M-Ras by small interfering RNAs as well as the expression of a dominant,negative mutant of M-Ras interfered with NGF-induced neuritogenesis. Since MEK inhibitors prevented M-Ras-induced neurite outgrowth, ERK pathway participates in this differentiation pathway. Furthermore, M-Ras brought about ERK pathway-mediated activating phosphorylation of CREB and the CREB-mediated transcription. In addition, a dominant,negative mutant of CREB inhibited M-Ras-induced neuritogenesis. Taken together, NGF-induced PC12 cell differentiation requires M-Ras,ERK pathway-mediated activation of CREB. M-Ras was predominantly expressed in the hippocampus and cerebellum of mouse brain and in the gray matter of the spinal cord. All these properties of M-Ras were apparently indistinguishable from those of H-Ras. However, NGF stimulation caused transient activation of classical Ras proteins but sustained activation of M-Ras as well as sustained activating phosphorylation of ERK and CREB. Therefore, M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway. [source] IFN-,-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytesGLIA, Issue 3 2007Hyun Jin Cho Abstract ,-Site APP cleaving enzyme 1 (BACE1) is an essential enzyme for the production of , amyloid. Since we found that injection of interferon-, (IFN-,) into young mouse brains increased BACE1 expression in astrocytes, we investigated molecular mechanisms underlying this process by cloning a putative BACE1 promoter. BACE1 promoter activity was differentially regulated by IFN-, in a region specific manner and down-regulated by an inhibitor of Janus kinase 2 (JAK2). A dominant negative mutant of signal transducer and activator of transcription 1 (STAT1) expression suppressed BACE1 promoter activity, and this was rescued by transfecting wild type STAT1. Electrophoretic mobility shift assay and promoter activity assays indicated that STAT1 binds directly to the putative STAT1 binding sequence of BACE1 promoter. Because IFN-, treatment induced STAT1 phosphorylation, we examined whether the expression of a suppressor of cytokine signaling (SOCS), negative regulator of JAK2, suppresses BACE1 promoter activity. The results show that SOCS1 or SOCS3 expression suppressed BACE1 promoter by blocking phosphorylation of Tyr701 residue in STAT1. Also, because IFN-, treatment specifically potentiated extracellular signal regulated MAP kinase (ERK) 1/2 activation, pretreatment of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, PD98059, significantly attenuated IFN-,-induced BACE1 promoter activity and protein expression through blocking phosphorylation of Ser727 residue in STAT1, suggesting that ERK1/2 is associated with IFN-,-induced STAT1 signaling cascade. Taken together, our results suggest that IFN-, activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes. © 2006 Wiley-Liss, Inc. [source] Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytesHEPATOLOGY, Issue 2 2002Yi-Ping Rao Bile acids have been reported to activate several different cell signaling cascades in rat hepatocytes. However, the mechanism(s) of activation of these pathways have not been determined. This study aims to determine which bile acids activate the Raf-1/MEK/ERK cascade and the mechanism of activation of this pathway. Taurodeoxycholic acid (TDCA) stimulated (+235%) the phosphorylation of p74 Raf-1 in a time (5 to 20 minutes) and concentration-dependent (10 to 100 ,mol/L) manner. Raf-1 and ERK activities were both significantly increased by most bile acids tested. Deoxycholic acid (DCA) was the best activator of ERK (3.6-fold). A dominant negative Ras (N17) construct expressed in primary hepatocytes prevented the activation of ERK by DCA. The epidermal growth factor receptor (EGFR)-specific inhibitor (AG1478) significantly inhibited (,81%) the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased phosphorylation of the EGFR (,2-fold) and Shc (,4-fold). A dominant negative mutant of the EGFR (CD533) blocked the ability of DCA to activate ERK. In conclusion, these results show that DCA activates the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily via an EGFR/Ras-dependent mechanism. [source] Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Huei-Mei Huang Abstract JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells. © 2005 Wiley-Liss, Inc. [source] p38, MAP kinase protects rat mesangial cells from TNF-,-induced apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Yan-Lin Guo Abstract p38 MAP kinases (p38) and c-Jun N-terminal protein kinases (JNK) have been associated with TNF-,-induced apoptosis. However, recent studies indicate that an early but brief activation of JNK and/or p38 may actually protect some cells from TNF-,-induced apoptosis. Whether the activation of JNK and p38 provides a pro- or anti-apoptotic signal for TNF-, has been controversial. In this study, we investigated the role of p38 in the regulation of TNF-, cytotoxicity in rat mesangial cells. Treatment of the cells with TNF-, alone had little effect on their viability, but they became very sensitive to apoptosis when treated with TNF-, in the presence of the p38 inhibitor SB 203580. These results suggested that the p38 pathway is critical for mesangial cells to survive the toxic effect of TNF-,. Using adenovirus-mediated gene transfer technique, we further demonstrated that p38,, but not p38,, is essential to protect the cells from TNF-, toxicity. It has been speculated that there is a synergetic interaction between the p38 and the nuclear factor-,B (NF-,B) pathways in protecting certain cells from apoptosis. However, expression of neither p38, nor its dominant negative mutant in mesangial cells interfered with TNF-,-induced translocation of NF-,B, the initial step of NF-,B activation. While it is unclear whether p38, regulates NF-,B transcription activity at other steps, it is apparent that p38, does not affect TNF-,-induced NF-,B activation at the stage of nuclear translocation. J. Cell. Biochem. 82: 556,565, 2001. © 2001 Wiley-Liss, Inc. [source] Enhanced glycogenesis is involved in cellular senescence via GSK3/GS modulationAGING CELL, Issue 6 2008Yong-Hak Seo Summary Glycogen biogenesis and its response to physiological stimuli have often been implicated in age-related diseases. However, their direct relationships to cell senescence and aging have not been clearly elucidated. Here, we report the central involvement of enhanced glycogenesis in cellular senescence. Glycogen accumulation, glycogen synthase (GS) activation, and glycogen synthase kinase 3 (GSK3) inactivation commonly occurred in diverse cellular senescence models, including the liver tissues of aging F344 rats. Subcytotoxic concentrations of GSK3 inhibitors (SB415286 and LiCl) were sufficient to induce cellular senescence with increased glycogenesis. Interestingly, the SB415286-induced glycogenesis was irreversible, as were increased levels of reactive oxygen species and gain of senescence phenotypes. Blocking GSK3 activity using siRNA or dominant negative mutant (GSK3,-K85A) also effectively induced senescence phenotypes, and GS knock-down significantly attenuated the stress-induced senescence phenotypes. Taken together, these results clearly demonstrate that augmented glycogenesis is not only common, but is also directly linked to cellular senescence and aging, suggesting GSK3 and GS as novel modulators of senescence, and providing new insight into the metabolic backgrounds of aging and aging-related pathogenesis. [source] Biosynthesis of biodegradable polyesters from renewable carbon sources by recombinant bacteria ,POLYMER INTERNATIONAL, Issue 10 2002Seiichi Taguchi Abstract Based on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61-3 strains that synthesize random copolyesters consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units, P(3HB- co -3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB- co -3HA) copolyester were very similar to those of low-density polyethylene. We carried out the molecular cloning and characterization of a PhaGPs encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase of Pseudomonas sp 61-3. It was concluded that the PhaGPs gene product is involved in providing mcl-3HA-CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1Ps gene encoding PHA synthase from Pseudomonas sp 61-3 was performed in the PhbCRe negative mutant (PHB,4) of Ralstonia eutropha. The recombinant PHB,4 strain successfully produced PHA copolyesters consisting of 3HB and mcl-3HA units of 6,12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95,97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbABRe genes from R eutropha encoding ,-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe. © 2002 Society of Chemical Industry [source] Localization of a flavonoid biosynthetic polyphenol oxidase in vacuolesTHE PLANT JOURNAL, Issue 2 2006Eiichiro Ono Summary Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+ -translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower. [source] Opposite effects of overexpressed myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton organization, and cell motility in nonmuscle cellsCYTOSKELETON, Issue 3 2008Robbin D. Eppinga Abstract Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5,). GFP-M5 and GFP-M5, co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5,-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5,-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Oxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytesGLIA, Issue 1 2009Hui-Hsin Wang Abstract Upregulation of matrix metalloproteinases (MMPs), especially MMP-9, by oxidized low-density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP-9 via a MAPK-dependent AP-1 activation in rat brain astrocyte (RBA)-1 cells. Results revealed by gelatin zymography, RT-PCR, and Western blotting analyses showed that oxLDL-induced proMMP-9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA-1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP-1 (i.e., c-Fos/c-Jun) is crucial for oxLDL-induced proMMP-9 expression which was attenuated by pretreatment with AP-1 inhibitor (curcumin). The regulation of MMP-9 gene transcription by AP-1 was confirmed by oxLDL-stimulated MMP-9 luciferase activity which was totally lost in cells transfected with the AP-1 binding site-mutated MMP-9 promoter construct (mt-AP1-MMP-9). These results suggested that oxLDL-induced proMMP-9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP-1 activation. Understanding the regulatory mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases. © 2008 Wiley-Liss, Inc. [source] Modulation of p21-activated kinase 1 alters the behavior of renal cell carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 9 2007Gerald C. O'Sullivan Abstract The p21-activated kinase 1 (Pak1) is a serine/threonine kinase whose activity is regulated by both Rho GTPases and AGC kinase family members. It plays a role in cytoskeletal remodeling and cell motility as well as cell proliferation, angiogenesis, tumorigenesis and metastasis. An involvement of Pak1 in renal cell carcinoma (RCC), which remains highly refractory to chemotherapy and radiotherapy, remains to be investigated. Pak1 expression, phosphorylation and kinase activity were examined in RCC cell lines and human tissue from normal and renal carcinoma. We report increased Pak1 expression and constitutive activity in the membrane and nucleus but not the cytoplasm of resected human RCC. To study a role for Pak1 in RCC, we developed 786-0 clones that expressed either a kinase-active Pak1L83,L86 2 different Pak1 dominant negative mutants, Pak1R299 and Pak1L83,L86,R299 or Pak1 siRNA. The expression of Pak1L83,L86 increased 786-0 proliferation, motility and anchorage independent growth, while the dominant negative mutants and Pak1 siRNA abrogated these effects. In addition, Pak1L83,L86 conferred resistance to 5-fluorouracil with a 40% ± 10% increase in cell viability. Conversely, Pak1L83,L86,R299, Pak1R299 and Pak1 siRNA conferred sensitivity with a 65.2% ± 5.5%, 69.2% ± 3.3% and 73.0% ± 8.4% loss in viability, respectively. Finally, Pak1 plays a role in renal tumor growth in vivo. Only 33% of mice developed tumors in the Pak1L83,L86,R299 group and no tumors developed from Pak1R299 cell challenge. Together these findings point to Pak1 as an exciting target for therapy of renal cancer, which remains highly refractory to existing treatments. © 2007 Wiley-Liss, Inc. [source] Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004Toru Ogasawara Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source] Survivin as a target for new anticancer interventionsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2005Nadia Zaffaroni Abstract Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that has been implicated in both control of cell division and inhibition of apoptosis. Specifically, its anti-apoptosis function seems to be related to the ability to directly or indirectly inhibit caspases. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumour cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed and and attractive target for new anticancer interventions. Several preclinical studies have demonstrated that down-regulation of survivin expression/function, accomplished through the use of antisense oligonucleotides, dominant negative mutants, ribozymes, small interfering RNAs and cyclin-dependent kinase inhibitors, increased the apoptotic rate, reduced tumor-growth potential and sensitized tumor cells to chemotherapeutic drugs with different action mechanisms and ,-irradiation in in vitro and in vivo models of different human tumor types. [source] Ultrasound increased BMP-2 expression via PI3K, Akt, c-Fos/c-Jun, and AP-1 pathways in cultured osteoblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Chun-Han Hou Abstract It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Bone morphogenetic protein (BMP) is a crucial mediator in bone formation during fracture healing. Here we found that US stimulation increased BMP-2 expression but not other BMPs. US induced BMP-2 transcription is mediated by AP-1 element but not estrogen receptor response element and GC-rich Sp1 response element. Pretreatment of osteoblasts with phosphatidylinositol 3-kinase (PI3K) inhibitor (Ly294002) and Akt inhibitor inhibited the potentiating action of US; these results were further substantiated by transfecting with the dominant negative mutants of p85 and Akt. US stimulation increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. Transfection of osteoblasts with c-Fos and c-Jun antisense oligonucleotide also reduced US-increased BMP-2 expression. US-increased the binding of c-Fos and c-Jun to the AP-1 element on the BMP-2 promoter and the enhancement of AP-1 luciferase activity was inhibited by Ly294002 and Akt inhibitor. Our results suggest that US increased BMP-2 expression in osteoblasts via the PI3K, Akt, c-Fos/c-Jun, and AP-1 signaling pathway. J. Cell. Biochem. 106: 7,15, 2009. © 2008 Wiley-Liss, Inc. [source] Interleukin-1, induces MMP-9 expression via p42/p44 MAPK, p38 MAPK, JNK, and nuclear factor-,B signaling pathways in human tracheal smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Kao-Chih Liang Matrix metalloproteinases (MMPs) are responsible for degradation of extracellular matrix and play important roles in cell migration, proliferation, and tissue remodeling related to airway inflammation. Interleukin-1, (IL-1,) has been shown to induce MMP-9 production in many cell types and contribute to airway inflammatory responses. However, the mechanisms underlying MMP-9 expression induced by IL-1, in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we investigated the roles of p42/p44 MAPK, p38 MAPK, JNK, and NF-,B pathways for IL-1,-induced MMP-9 production in HTSMCs. IL-1, induced production of MMP-9 protein and mRNA in a time- and concentration-dependent manner determined by zymographic, Western blotting, and RT-PCR analyses, which was attenuated by inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK (SP600125), and NF-,B (helenalin), and transfection with dominant negative mutants of MEK1/2, p38 and JNK, respectively. IL-1,-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was attenuated by pretreatment with U0126, SB202190, SP600125, or transfection with these dominant negative mutants of MEK, ERK, p38 and JNK, respectively. Furthermore, IL-1,-stimulated translocation of NF-,B into the nucleus and degradation of I,B-, was blocked by helenalin. Finally, the reporter gene assay revealed that MAPKs and NF-,B are required for IL-1,-induced MMP-9 luciferase activity in HTSMCs. MMP-9 promoter activity was enhanced by IL-1, in HTSMCs transfected with MMP-9-Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Taken together, the transcription factor NF-,B, p42/p44 MAPK, p38 MAPK, and JNK that are involved in MMP-9 expression in HTSMCs exposed to IL-1, have now been identified. J. Cell. Physiol. 211: 759,770, 2007. © 2007 Wiley-Liss, Inc. [source] Characterization of G proteins involved in activation of nonselective cation channels by endothelinB receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2002Yoshifumi Kawanabe We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+ -permeable nonselective cation channels (NSCC-1 and NSCC-2) in Chinese hamster ovary cells expressing endothelinB receptors (CHO-ETBR) that couple with Gq and Gi. The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET-1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys402Cys403 Cys405,Ser402Ser403Ser405) ETBR (CHO-SerETBR) and ETBR truncated at the cytoplasmic tail downstream of Cys403 (CHO-ETBR,403). Based on the data obtained from actin stress fibre formation, CHO-ETBR couple with G13. Therefore, CHO-ETBR couple with Gq, Gi and G13. CHO-SerETBR and CHO-ETBR,403 couple with G13 and Gq, respectively. ET-1 activated NSCC-1 in CHO-ETBR preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO-SerETBR. On the other hand, ET-1 failed to activate Ca2+ channels in CHO-ETBR,403. Microinjection of dominant negative mutants of G13 (G13G225A) abolished activation of NSCC-1 and NSCC-2 in CHO-ETBR and that of NSCC-1 in CHO-SerETBR. Y-27632, a specific Rho-associated kinase (ROCK) inhibitor, did not affect the ET-1-induced transient and sustained increase in [Ca2+]i in CHO-ETBR. These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ETBR, but not the palmitoylation site itself, is essential for coupling with G13, (2) the activation mechanism of each Ca2+ channel by ET-1 is different in CHO-ETBR. NSCC-1 activation depends on G13 -dependent cascade, and NSCC-2 activation depends on both Gq/PLC- and G13 -dependent cascades. Moreover, ROCK-dependent cascade is not involved in the activation of these channels. British Journal of Pharmacology (2002) 136, 1015,1022. doi:10.1038/sj.bjp.0704805 [source] Alternative infectious entry pathways for dengue virus serotypes into mammalian cellsCELLULAR MICROBIOLOGY, Issue 10 2009Eliana G. Acosta Summary The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-,-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells. [source] | |||||||||||||||||||||||||