Binding Activity In Nuclear Extracts Research Articles

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis.

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation

The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H2O2 was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines).We found that a pre-treatment with FUM significantly enhanced cell viability compared to H2O2-treated cells, and we selected the optimal concentrations in each model (1 and 25μg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H2O2. It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H2O2-induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders.

Phytochemicals of Aristolochia tagala and Curcuma caesia exert anticancer effect by tumor necrosis factor-α-mediated decrease in nuclear factor kappaB binding activity.

Rationale:The active compounds or metabolites of herbal plants exert a definite physiological action on the human body and thus are widely used in human therapy for various diseases including cancer. Previous studies by our group have reported the anticarcinogenic properties of the two herbal plants extracts (HPE) of Aristolochia tagala (AT) Cham. and Curcuma caesia (CC) Roxb. in diethylnitrosamine-induced mouse liver cancer in vivo. The anticarcinogenic properties of these extracts may be due to the active compounds present in them.Objectives:Our objective was to analyze the phytochemical constituents present in AT and CC, to assay their antioxidant properties and to determine their role in a possible intervention on tumor progression.Materials and Methods:Qualitative and quantitative analysis of constituent with anticancer properties present in the crude methanol extract of the two plants CC and AT was carried out following standard methods. Separation of the phytochemical compounds was done by open column chromatography. The extracts were eluted out with gradients of chloroform-methanol solvents. Ultraviolet-visible spectra of individual fractions were recorded, and the fractions were combined based on their λmax. The free radical scavenging activity of crude extracts and fractions obtained was also determined; the radical scavenging activity was expressed as IC50. High-performance thin layer chromatography (HPTLC) analysis of fractionated compounds was carried out to identify partially the phytochemical compounds. The anti-inflammatory and anticancer activity of AT and CC extracts was studied in DEN induced BALB/c mice by analyzing the tumor necrosis factor-α (TNF-α) levels in serum and the nuclear factor kappaB (NF-κB) binding activity in nuclear extracts of the liver.Results:It was observed that both AT and CC contained compounds such as phenolics, tannins, flavonoids, terpenoids, etc., and both extracts exhibited antioxidant capacity. HPTLC analysis revealed the presence of phenolic compounds in CC and indicated the presence of anthocynidin 3-glycosides, 6-hydroxylated flavonols, some flavones and chalcone glycosides in AT and also confirmed the presence of compounds such as terpenes, phenols, steroids, and other organic compounds in CC and presence of flavonoids in AT. In vivo studies carried out in BALB/c mice showed that exposure to DEN caused an increase in TNF-α and NF-κB binding activity. The HPE (CC or AT) was seen to revert this effect.Conclusions:The current paper documents the antioxidant, anti-inflammatory, and anticancer activity of the two extracts probably through TNF-α-mediated decrease in NF-κB binding activity. The active components of AT and CC may act as the potential anticancer agents in hepatocellular carcinoma and warrants further investigation.

Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB Translocation and Promoter Binding

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite, promotes vascular dysfunction, injury and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p<0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p<0.05) and 2.53-fold (± 0.24; p<0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p<0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

ACTIVATION AND EXPRESSION OF NF-κB IN CIRCULATING MONONUCLEAR CELLS IN APPARENTLY HEALTHY PEOPLE

ObjectivesJUPITER trial demonstrated that administering Rosuvastatin to ‘apparently healthy people’(with normal lipid levels, but hsCRP3 2 mg/L) could decrease the incidences of cardiovascular diseases significantly. Whereas, little is known about...

CREB is an essential transcription factor for prostaglandin E2 induced IL-23p19 (116.18)

Abstract We reported previously that PGE2 promotes IL-23p19 expression in dendritic cells (DC) treated with LPS. The effect is mediated through EP4 through the induction of cAMP. This study is focused on the signaling pathways involved in PGE1OH (an EP4 agonist)-induced p19 expression. EP4 activates adenylate cyclase and increases intracellular cAMP which leads to activation of PKA and EPAC. We found that adenylate cyclase, cAMP, and EPAC play a role in IL-23p19 induction. Using chemical inhibitors of PKA, PI3K, MAPK, and GSK3, we did not see a decrease in IL-23p19 expression. Previous studies reported the involvement of NFkB/c-Rel, ATF-2, SMAD-3 and AP-1 in p19 expression. Since two CRE-binding sites have been identified in the p19 promoter, we focused on CREB as a potential transcription factor activated by PGE1OH. Luciferase experiments including vectors containing mutated CRE sites and expressing dominant negative versions of CREB demonstrated the essential role CREB plays in IL-23p19 expression. In a transcription factor assay we found increased CREB binding activity in nuclear extracts from cells treated with PGE1OH. In addition, a ChIP assay confirmed CREB binding to the p19-promoter in the presence of PGE1OH. Overall, we identified CREB as a new transcription factor required for PGE1OH induction of IL-23p19 expression.

POT1‐independent single‐strand telomeric DNA binding activities in Brassicaceae

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana, and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea, partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.

Effect of CD37 small modular immuno-pharmaceutical (SMIP) on direct apoptosis in chronic lymphocytic leukemia cells via transcriptional up-regulation of the BH3 family member BIM

3035 Background: CD37 is a tetraspan transmembrane family protein selectively expressed on normal and transformed B-cells. A novel CD37SMIP was previously demonstrated to mediate superior direct apoptosis and NK-cell mediated killing of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Methods: Given the superior in vitro apoptosis observed with CD37SMIP treatment and early clinical activity observed in highly refractory CLL patients, we hypothesized that a unique mechanism of cell killing was utilized by CD37SMIP. This was pursued in preclinical studies outlined below. Results: Unlike many other agents utilized to treat CLL, death mediated by CD37SMIP does not depend upon caspase activation. Nonetheless, CD37SMIP treatment of CLL cells promotes time-dependent induction of mitochondrial membrane depolarization, mitochondrial translocation of Bax, and up-regulation of Bim protein. CD37SMIP Bim protein induction occurred concomitantly with an increase in BIM mRNA levels. Electrophoretic mobility shift assay using oligonucleotides of the BIM promoter demonstrated increased protein binding activity in nuclear extracts derived from CD37SMIP treated cells and the physical interaction of FoxO3a transcription factor with the FoxO3a responsive element in the BIM promoter was demonstrated using a “protein pull down” assay and confirmed by chromatin immunoprecipitation assays. Furthermore, CD37SMIP treatment significantly increased BIM promoter regulated luciferase reporter expression in B-CLL cells. Consistent with a primary role of Bim up-regulation in mitochondrial membrane destabilization and apoptosis, transfection of CLL cells with BIM siRNA resulted in inhibition of CD37SMIP-induced mitochondrial membrane depolarization and apoptosis. Conclusions: These studies demonstrate CD37SMIP mediated apoptosis in CLL cells occurs via FoxO3a-dependent transcriptional up-regulation of BIM protein. This distinct mechanism of apoptosis utilized by CD37SMIP contrasts it with other agents used for CLL treatment. Additionally, it provides a mechanism for the promising clinical activity of TRU-016 (humanized CD37SMIP) observed to date in refractory CLL patients. [Table: see text]

Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression

Striking similarities exist between molecular mechanisms driving embryonic liver development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryonic hepatic development. BMP expression has been observed in neoplasia but the expression and biological role of BMP4 in human HCC are unknown. We found increased BMP4 mRNA and protein in HCC cell lines and tissue samples compared to primary human hepatocytes and corresponding non-tumourous tissue. Hypoxia further induced BMP4 expression in HCC cells, which was abolished by transfection of a dominant negative form of HIF-1 alpha (dnHIF-1 alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to a putative hypoxia-response element in the BMP4 promoter. Sequence analysis of the BMP4 promoter revealed two Ets-1 binding sites, and Ets-1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF-1 alpha completely abrogated hypoxia-induced Ets-1 activity as well as BMP4 expression. Overexpression of Ets-1 markedly enhanced BMP4 promoter activity, while antisense Ets-1 almost completely abolished basal as well as hypoxia-induced BMP4 expression. These data demonstrate that Ets-1 activity contributes to baseline expression of the BMP4 gene and is the predominant mediator of the HIF-dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP4 expression, HCC cell lines were treated with antisense BMP4 constructs or siRNA against BMP4. BMP4 suppression resulted in a strong reduction of the migratory and invasive potential and anchorage-independent growth. Furthermore, tube formation assays indicated that BMP4 expressed by HCC cells promotes vasculogenesis. Our findings demonstrate that BMP4 is increased in HCC and promotes HCC progression. Therefore, BMP4 expression may have clinical relevance, and interfering with BMP4 signalling appears as an attractive therapeutic target for this highly aggressive tumour.

498 Autocrine Intestinal Epithelial Cell GM-CSF Signaling Promotes Homeostatic Responses to Gut Injury

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship

497 ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship

Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumor progression

Striking similarities exist between molecular mechanisms driving embryonic liver development and HCC progression. Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryogenic hepatic development. However, BMP expression levels have not been analyzed in HCC. The aim of this study was to determine the expression and biological role of BMP4 inhuman HCC. Methods and Results: BMP4 mRNA and protein are up-regulated in HCC cell lines and tissue compared to primary human hepatocytes and non-tumorous tissue. Hypoxia further induced BMP4 expression in HCC cells and this induction was abolished by transfection of a dominant negative form of HIF-1alpha (dn HIF-1alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to the hypoxia-response element in the bmp4 promoter. Sequence analysis of the bmp4 promoter revealed two Ets-1 binding sites, and Ets-1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF-1alpha completely abrogated hyoxia-induced Ets-1 activity as well as BMP4 expression. Over-expression of Ets-1 markedly enhanced bmp4 promoter activity while anti-sense Ets-1 expression almost completely abolished basal as well as hypoxia induced BMP4 expression. These data demonstrate that Ets-1 activity contributes to baseline expression of the bmp4 gene and is the predominant mediator of the HIF-dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP expression in HCC, HCC cells were treated with BMP inhibitors chordin and noggin. Both inhibitors strongly reduced the migratory and invasive potential of HCC cells in functional assays. Conclusion: BMP4 is strongly expressed in HCC and promotes tumorigenicity. BMP4 expression may serve as a prognostic maker and interfering with BMP4 signaling as an attractive therapeutic target for this highly aggressive tumor.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture

The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

Constitutive Production of NF-κB2 p52 Is Not Tumorigenic but Predisposes Mice to Inflammatory Autoimmune Disease by Repressing Bim Expression

Normal development of the immune system requires regulated processing of NF-kappaB2 p100 to p52, which activates NF-kappaB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-kappaB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-kappaB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-kappaB2 mutants and p52 and suggest a causal role for sustained NF-kappaB2 activation in the pathogenesis of autoimmunity.

Functional Induction of P-glycoprotein in the Blood-Brain Barrier of Streptozotocin-Induced Diabetic Rats: Evidence for the Involvement of Nuclear Factor-κB, a Nitrosative Stress-Sensitive Transcription Factor, in the Regulation

The objective of this study was to investigate the transport kinetics of cyclosporin A, a well known substrate for P-glycoprotein (P-gp), across the blood-brain barrier (BBB), and the expression of the transporter in the brain of streptozotocin-induced diabetic rats. The in vivo transport clearance of cyclosporin A was significantly reduced in diabetic rats compared with that in the control. The decreased transport was associated with the increased level of mRNA and the protein for P-glycoprotein in the rat brain. The functional activity of the efflux transporter in mouse brain capillary endothelial (MBEC4) cells, an in vitro model of the BBB, was also stimulated when slow nitric oxide (NO)-releasing donors were present, whereas the stimulation was absent in the case of rapid NO-releasing donors (e.g., S-nitroso-N-acetyl-dl-penicillamine and diethylenetriamine). The stimulatory effect was highest for sodium nitroprusside (SNP) and the functional induction associated with the increased mRNA and protein level of the transporter. The pretreatment of the cell with SNP along with ascorbate, methylene blue, or superoxide dismutase attenuated the induction of function and expression for P-glycoprotein, suggesting that the reaction product between superoxide and NO is involved in the induction of function and expression. The level of nuclear translocation of nuclear factor-kappaB (NF-kappaB) and DNA binding activity of nuclear extracts to the NF-kappaB consensus oligonucleotide was increased in MBEC4 cells pretreated with SNP. Taken together, these observations suggest that nitrosative stress leads to the up-regulation of the message for the efflux transporter and, ultimately, to the enhanced function, probably via a NF-kappaB-dependent mechanism.

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A upregulates ACE and AT1 receptor gene expression and signaling: role in cardiac hypertrophy

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signaling antagonizes the physiological effects mediated by the renin-angiotensin system (RAS). The objective of this study was to determine whether the targeted-disruption of Npr1 gene (coding for GC-A/NPRA) leads to the activation of cardiac RAS genes involved on the hypertrophic remodeling process. The Npr1 gene-knockout (Npr1(-/-)) mice showed 30-35 mmHg higher systolic blood pressure (SBP) and a 63% greater heart weight-to-body weight (HW/BW) ratio compared with wild-type (Npr1(+/+)) mice. The mRNA levels of both angiotensin-converting enzyme and angiotensin II type 1a receptor were increased by three- and fourfold, respectively, in Npr1(-/-) null mutant mice hearts compared with the wild-type Npr1(+/+) mice hearts. In parallel, the expression levels of interleukin-6 and tumor necrosis factor-alpha were increased by four- to fivefold, in Npr1(-/-) mice hearts compared with control animals. The NF-kappaB binding activity in nuclear extracts of Npr1(-/-) mice hearts was increased by fourfold compared with wild-type Npr1(+/+) mice hearts. Treatments with captopril or hydralazine equally attenuated SBP; however, only captopril significantly decreased the HW/BW ratio and suppressed cytokine gene expression in Npr1(-/-) mice hearts. The ventricular cGMP level was reduced by almost sixfold in Npr1(-/-) mice compared with wild-type control mice. The results of the present study indicate that disruption of NPRA/cGMP signaling leads to the augmented expression of cardiac RAS pathways that promote the development of cardiac hypertrophy and remodeling.

Effect of the α3β1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the α3β1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-κB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-α3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the α3β1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the α3β1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.

Molecular regulation of the PAI‐1 gene by hypoxia: contributions of Egr‐1, HIF‐1 α, and C/EBPα

Hypoxia, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. Plasminogen activator inhibitor-1 (PAI-1) is an important factor suppressing fibrinolysis under conditions of low oxygen tension. We previously reported that hypoxia induced PAI-1 mRNA and antigen expression in murine macrophages secondary to increased de novo transcription as well as increased mRNA stability. We now show in RAW264.7 murine macrophages that the transcription factors early growth response gene-1 (Egr-1), hypoxia-inducible factor-1alpha (HIF-1alpha), and CCAAT/enhancer binding protein alpha (C/EBPalpha) are quickly activated in hypoxia and are responsible for transcription and expression of PAI-1. Murine PAI-1 promoter constructs, including Egr, HIF-1alpha, and/or C/EBPalpha binding sites, were transfected into RAW 264.7 murine macrophages. To identify the relative importance of each of these putative hypoxia-responsive elements, cells were exposed to normobaric hypoxia, and transcriptional activity was recorded. At 16 h of hypoxic exposure, murine PAI-1 promoter deletion constructs that included Egr, HIF-1alpha, and/or C/EBPalpha binding sites demonstrated increased transcriptional activity. Mutation of each of these three murine PAI-1 promoter sites (or a combination of them) resulted in a marked reduction in hypoxia sensitivity as detected by transcriptional analysis. Functional data obtained using 32P-labeled Egr, HIF-1alpha response element (HRE), and C/EBPalpha oligonucleotides revealed induction of DNA binding activity in nuclear extracts from hypoxic RAW cells, with supershift analysis confirming activation of Egr-1, HIF-1alpha, or C/EBPalpha. ChIP analysis confirmed the authenticity of these interactions as each of these transcription factors binds to chromatin under hypoxic conditions. Further, the induction of PAI-1 by Egr-1, HIF-1alpha, or C/EBPalpha was replicated in primary peritoneal macrophages. These data suggest that although HIF-1alpha appears to dominate the PAI-1 transcriptional response in hypoxia, Egr-1 and C/EBPalpha greatly augment this response and can do so independent of HIF-1alpha or each other. These studies are relevant to ischemic up-regulation of the PAI-1 gene and consequent accrual of microvascular thrombus under ischemic conditions.

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-κB, in mouse calvarial osteoblasts

Interleukin-6 (IL-6), and the related cytokines IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM), are potent stimulators of osteoclastic bone resorption. In the present study, we have addressed the possibility that the neuropeptide vasoactive intestinal peptide (VIP) may regulate the production of and/or sensitivity to the IL-6 family of cytokines in mouse calvarial osteoblasts. VIP stimulated IL-6 mRNA expression and protein release in a time- and concentration-dependent manner, whereas mRNA expression of the IL-6 receptor, as well as mRNA expressions of IL-11, LIF, OSM and their cognate receptors, were unaffected by VIP. In cells transfected with the IL-6 promoter coupled to luciferase, VIP increased transcriptional activity. The effects of VIP were shared by the related neuropeptide PACAP-38, belonging to the same superfamily of neuropeptides, whereas secretin did not have any effect, indicating that the effects were mediated by VPAC 2 receptors. The effects of VIP were potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram and mimicked by forskolin, indicating the involvement of the cyclic AMP/protein kinase A pathway. This was further demonstrated by the facts that the stimulatory effect of VIP on luciferase activity could be reversed by the PKA inhibitors H-89 and KT5720 and was mimicked by cyclic AMP analogues selective for PKA, but not by those selective for Epac. In addition, VIP enhanced the phosphorylation of CREB, as assessed by both immunocytochemical analysis and Western blot. The DNA binding activity of nuclear extracts to C/EBP was increased by VIP, whereas binding to AP-1 was decreased. In contrast, DNA binding to NF-κB, as well as nuclear translocation of NF-κB and C/EBP, were unaffected by VIP. The mRNA expressions of C/EBPβ, C/EBPδ, C/EBPγ, c-Jun, JunB, c-Fos, Fra-1 and IκBα and protein level of IκBα were all unaffected by VIP. These observations, together, demonstrate that VIP stimulates IL-6 production in osteoblasts by a mechanism likely to be mediated by VPAC 2 receptors and dependent on cyclic AMP/protein kinase A/CREB activation and also involving the transcription factors C/EBP and AP-1.