Functional Cross-talk between ��-Catenin and NF��B Signaling Pathways in Colonic Crypts of Mice in Response to Progastrin

We recently reported a critical role of NFκB in mediating hyperproliferative and anti-apoptotic effects of progastrin on proximal colonic crypts of transgenic mice overexpressing progastrin (Fabp-PG mice). We now report activation of β-catenin in colonic crypts of mice in response to chronic (Fabp-PG mice) and acute (wild type FVB/N mice) progastrin stimulation. Significant increases were measured in relative levels of cellular and nuclear β-catenin and pβ-cat⁴⁵ in proximal colonic crypts of Fabp-PG mice compared with that in wild type littermates. Distal colonic crypts were less responsive. Interestingly, β-catenin activation was downstream of IKKα,β/NFκB, because treatment of Fabp-PG mice with the NFκB essential modulator (NEMO) peptide (inhibitor of IKKα,β/NFκB activation) significantly blocked increases in cellular/nuclear levels of total β-catenin/pβ-cat⁴⁵/and pβ-cat⁵⁵² in proximal colons. Cellular levels of pβ-cat^33,37,41, however, increased in proximal colons in response to NEMO, probably because of a significant increase in pGSK-3β^Tyr216, facilitating degradation of β-catenin. NEMO peptide significantly blocked increases in cyclin D1 expression, thereby, abrogating hyperplasia of proximal crypts. Goblet cell hyperplasia in colonic crypts of Fabp-PG mice was abrogated by NEMO treatment, suggesting a cross-talk between the NFκB/β-catenin and Notch pathways. Cellular proliferation and crypt lengths increased significantly in proximal but not distal crypts of FVB/N mice injected with 1 nm progastrin associated with a significant increase in cellular/nuclear levels of total β-catenin and cyclin D1. Thus, intracellular signals, activated in response to acute and chronic stimulation with progastrin, were similar and specific to proximal colons. Our studies suggest a novel possibility that activation of β-catenin, downstream to the IKKα,β/NFκB pathway, may be integral to the hyperproliferative effects of progastrin on proximal colonic crypts.Accumulating evidence suggests that gastrins play an important role in proliferation and carcinogenesis of gastrointestinal and pancreatic cancers (1, 2). Progastrin and glycine-extended gastrin (G-Gly)³ are predominant forms of gastrins found in many tumors, including colon (3–5). Progastrin exerts potent proliferative and anti-apoptotic effects in vitro and in vivo on intestinal mucosal cells (6–10) and on pancreatic cancer cells (11). Transgenic mice overexpressing progastrin from either the liver (hGAS) or intestinal epithelial cells (Fabp-PG) are at a higher risk for developing pre-neoplastic and neoplastic lesions in colons in response to azoxymethane (12–15). Treatment with G-Gly similarly increased the risk for developing pre-neoplastic lesions in rats (16). Thus progastrin and G-Gly exert co-carcinogenic effects in vivo (12–16).Under physiological conditions, only processed forms of gastrins (G17, G34) are present in the circulation (17). In certain disease states, elevated levels of circulating progastrin (0.1 to >1.0 nm) are measured (1). Because co-carcinogenic effects of progastrin are measured in Fabp-PG mice, which express pathophysiological concentrations of hProgastrin (<1–5 nm) (12), elevated levels of circulating progastrin measured in certain disease states in humans may play a role in colon carcinogenesis. A curious finding was that pre-neoplastic and neoplastic lesions were significantly increased in proximal, but not distal, colons of Fabp-PG mice, in response to azoxymethane (12, 14), which may reflect an increase in proliferation and a decrease in azoxymethane-induced apoptosis in proximal colons of Fabp-PG mice (18). We reported a critical role of NFκB activation in mediating proliferation and the anti-apoptotic effect of progastrin on pancreatic cancer cells (in vitro) and on proximal colonic crypts of Fabp-PG mice (in vivo) (11, 18). Whereas the Wnt/β-catenin pathway is known to play a role in the proliferation of colonic crypts (19), its role in mediating biological effects of progastrin remains unknown.β-Catenin is regulated by canonical (GSK-3β phosphorylation-dependent) and non-canonical (GSK-3β phosphorylation-independent) pathways. In the canonical pathway, inhibition of GSK-3β protects β-catenin against degradation by protein complexes, consisting of GSK-3β, axin, and adenomatous polyposis coli (20). In a resting cell, β-catenin is not present in the cytoplasm or nucleus because of proteasomal degradation of β-catenin that is not bound to E-cadherin (20). Following inactivation of GSK-3β, β-catenin stabilizes in the cytoplasm and translocates to the nucleus where it cooperates with Tcf/Lef for activation of target genes (20). In the current studies, we examined whether β-catenin is activated in proximal versus distal colonic crypts of Fabp-PG mice. Relative levels of β-catenin and its target gene product, cyclin D1, were significantly increased in proximal versus distal colonic crypts of Fabp-PG mice. We next examined a possible cross-talk between NFκB and β-catenin activation and the role of GSK-3β. Our results suggest the novel possibility that β-catenin activation in response to progastrin is downstream to IKKα,β/NFκB p65 activation, and that phosphorylation of GSK-3β at Tyr²¹⁶ may be critically involved.To examine whether differences measured in the response of proximal versus distal colons in Fabp-PG mice were not an artifact of chronic stimulation, we additionally injected WT FVB/N mice with progastrin, as an acute model of stimulation. Our results confirmed that differences we had measured in Fabp-PG mice are not an artifact of chronic stimulation but represent inherent differences in the response of proximal versus distal colonic crypts to circulating progastrins.We and others (18, 21) have previously demonstrated goblet cell hyperplasia in colonic crypts of transgenic mice overexpressing progastrin. In the current studies, we confirmed a significant increase in goblet cell hyperplasia/metaplasia (?) in proximal colonic crypts of Fabp-PG mice. Importantly, goblet cell hyperplasia was reversed to wild type levels by attenuating NFκB activation (and hence β-catenin activation) in NEMO-treated mice. The results of the current studies thus further suggest that pathways which dictate goblet cell lineage may be modulated by progastrin and may be downstream of NFκB/β-catenin activation. This represents a novel paradigm, which needs to be further examined.     

Oral Supplementation with Non-Absorbable Antibiotics or Curcumin Attenuates Western Diet-Induced Atherosclerosis and Glucose Intolerance in LDLR?/? Mice – Role of Intestinal Permeability and Macrophage Activation

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively) and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1). Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.     

Fish Oil Suppresses Cell Growth and Metastatic Potential by Regulating PTEN and NF-κB Signaling in Colorectal Cancer

Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF) play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP), NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1)&FO∶CO(2.5∶1) respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates apoptosis, diminishes DNA damage and inhibits inflammatory signalling in a dose and time dependent manner so as to inhibit progression of colon cancer.     

Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

Focal adhesion kinase (FAK) is a major signaling molecule which functions downstream of integrins or in conjunction with mitogenic signaling pathways. FAK is overexpressed and/or activated in many types of human tumors, in which it promotes cell adhesion, survival, migration and invasion. In addition to FAK''s ability to regulate signaling through its scaffolding activities, FAK encodes an intrinsic kinase activity. Although some FAK substrates have been identified, a more comprehensive analysis of substrates is lacking. In this study, we use a protein microarray to screen the human proteome for FAK substrates. We confirm that several of the proteins identified are bona fide in vitro FAK substrates, including several factors which are known to regulate the NFκB pathway. Finally, we identify a role for FAK''s kinase activity in both canonical and non-canonical NFκB signaling. Our screen therefore represents the first high throughput screen for FAK substrates and provides the basis for future in-depth analysis of the role of FAK''s kinase activity in the processes of tumorigenesis.     

Comparison of the Safety and Pharmacokinetics of ST-246? after IV Infusion or Oral Administration in Mice,Rabbits and Monkeys

Background

ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (IV) formulation may be required for some hospitalized patients who are unable to take oral medication. An IV formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation.

Methodology/Principal Findings

The pharmacokinetics of ST-246 after IV infusions in mice, rabbits and nonhuman primates (NHP) were compared to those obtained after oral administration. Ten minute IV infusions of ST-246 at doses of 3, 10, 30, and 75 mg/kg in mice produced peak plasma concentrations ranging from 16.9 to 238 µg/mL. Elimination appeared predominately first-order and exposure dose-proportional up to 30 mg/kg. Short IV infusions (5 to 15 minutes) in rabbits resulted in rapid distribution followed by slower elimination. Intravenous infusions in NHP were conducted at doses of 1 to 30 mg/kg. The length of single infusions in NHP ranged from 4 to 6 hours. The pharmacokinetics and tolerability for the two highest doses were evaluated when administered as two equivalent 4 hour infusions initiated 12 hours apart. Terminal elimination half-lives in all species for oral and IV infusions were similar. Dose-limiting central nervous system effects were identified in all three species and appeared related to high C_max plasma concentrations. These effects were eliminated using slower IV infusions.

Conclusions/Significance

Pharmacokinetic profiles after IV infusion compared to those observed after oral administration demonstrated the necessity of longer IV infusions to (1) mimic the plasma exposure observed after oral administration and (2) avoid C_max associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and IV administration. The administration of ST-246 was well tolerated as a slow IV infusion.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Ginkgolide B Suppresses Methamphetamine-Induced Microglial Activation Through TLR4-NF-κB Signaling Pathway in BV2 Cells

Accumulating evidence suggests that microglial cells have altered morphology and proliferation in different brain regions of methamphetamine (Meth) abusers and Meth-abusing animal models. However, the possible mechanisms underlying Meth-induced microglial activation remain poorly understood. Meanwhile, Toll-like receptor4 (TLR4) is closely associated with inflammation. Therefore the aim of the present study was to assess whether Meth treatment affects TLR4 expression; in addition, we evaluated the effects of ginkgolide B (GB), a diterpene lactone extracted from Ginkgo biloba, on Meth-mediated inflammation. BV2 cells were treated with Meth. Interestingly, Meth treatment significantly increased TLR4 expression, activated the NF-κB signaling pathway, and promoted TNF-α, IL-6 and IL-1β excretion. These effects, however, were partially attenuated by GB pre-treatment. To further confirm the role of TLR4 in Meth-mediated inflammation, the siRNA technology was applied to knock down TLR4, which resulted in hampered Meth-mediated inflammatory responses, confirming the important role of TLR4 in this process. Taken together, our findings suggested that Meth exposure results in BV2 cell activation, in association with TLR4 upregulation. GB could attenuate Meth-induced inflammation, at least partially through TLR4-NF-κB signaling pathway, therefore, targeting TLR4 may constitute a potential intervention strategy for Meth mediated neuroinflammation.     

Expanding NFκB and SUMO ties

Comment on: Lee MH, et al. Mol Cell 2011; 43:180-91.     

Effusanin E Suppresses Nasopharyngeal Carcinoma Cell Growth by Inhibiting NF-κB and COX-2 Signaling

Rabdosia serra is well known for its antibacterial, anti-inflammatory and antitumor activities, but no information has been available for the active compounds derived from this plant in inhibiting human nasopharyngeal carcinoma (NPC) cell growth. In this study, we isolated and purified a natural diterpenoid from Rabdosia serra and identified its chemical structure as effusanin E and elucidated its underlying mechanism of action in inhibiting NPC cell growth. Effusanin E significantly inhibited cell proliferation and induced apoptosis in NPC cells. Effusanin E also induced the cleavage of PARP, caspase-3 and -9 proteins and inhibited the nuclear translocation of p65 NF-κB proteins. Moreover, effusanin E abrogated the binding of NF-κB to the COX-2 promoter, thereby inhibiting the expression and promoter activity of COX-2. Pretreatment with a COX-2 or NF-κB-selective inhibitor (celecoxib or ammonium pyrrolidinedithiocarbamate) had an additive effect on the effusanin E-mediated inhibition of proliferation, while pretreatment with an activator of NF-κB/COX-2 (lipopolysaccharides) abrogated the effusanin E-mediated inhibition of proliferation. Effusanin E also significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity, furthermore, the expression of p50 NF-κB and COX-2 were down-regulated in the tumors of nude mice. These data suggest that effusanin E suppresses p50/p65 proteins to down-regulate COX-2 expression, thereby inhibiting NPC cell growth. Our findings provide new insights into exploring effusanin E as a potential therapeutic compound for the treatment of human nasopharyngeal carcinoma.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.