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.     

Statistical Comparison of Carcinogenic Effects and Dose–Response Relationships in Rats and Mice for 2,4-Toluene Diamine to those Ascribed to Toluene Diisocyanate

The U.S. National Toxicology Program (NTP) conducted 2-year bioassays of commercial grade toluene diisocyanate (TDI) (80% 2,4-TDI and 20% 2,6-TDI) and 2,4-toluene diamine (TDA) and concluded that both were carcinogenic in rodents. In the TDI study, there was an unproven but likely formation of TDA either because of flawed test-substance handling and storage conditions and/or the atypical exposure conditions employed. Although the carcinogenic responses in both studies were qualitatively similar, several statistical analyses were performed to substantiate this possibility more rigorously. Seven different statistical approaches combine to yield a robust and consistent conclusion that, if only a small fraction (approximately 5%) of the dose of TDI were hydrolyzed to TDA in the TDI study, then that would be sufficient to explain the observed carcinogenic responses in the TDI study.     

Integrated Model of Metabolism and Autoimmune Response in β-Cell Death and Progression to Type 1 Diabetes

Progression to type 1 diabetes is characterized by complex interactions of environmental, metabolic and immune system factors, involving both degenerative pathways leading to loss of pancreatic β-cells as well as protective pathways. The interplay between the degenerative and protective pathways may hold the key to disease outcomes, but no models have so far captured the two together. Here we propose a mathematical framework, an ordinary differential equation (ODE) model, which integrates metabolism and the immune system in early stages of disease process. We hypothesize that depending on the degree of regulation, autoimmunity may also play a protective role in the initial response to stressors. We assume that β-cell destruction follows two paths of loss: degenerative and autoimmune-induced loss. The two paths are mutually competing, leading to termination of the degenerative loss and further to elimination of the stress signal and the autoimmune response, and ultimately stopping the β-cell loss. The model describes well our observations from clinical and non-clinical studies and allows exploration of how the rate of β-cell loss depends on the amplitude and duration of autoimmune response.     

Response to “Critical Assessment of the Evidence for Striped Nanoparticles”

Stirling et al., (10.1371/journal.pone.0108482) presented an analysis on some of our publications on the formation of stripe-like domains on mixed-ligand coated gold nanoparticles. The authors shed doubts on some of our results however no valid argument is provided against what we have shown since our first publication: scanning tunneling microscopy (STM) images of striped nanoparticles show stripe-like domains that are independent of imaging parameters and in particular of imaging speed. We have consistently ruled out the presence of artifacts by comparing sets of images acquired at different tip speeds, finding invariance of the stipe-like domains. Stirling and co-workers incorrectly analyzed this key control, using a different microscope and imaging conditions that do not compare to ours. We show here data proving that our approach is rigorous. Furthermore, we never solely relied on image analysis to draw our conclusions; we have always used the chemical nature of the particles to assess the veracity of our images. Stirling et al. do not provide any justification for the spacing of the features that we find on nanoparticles: ~1 nm for mixed ligand particles and ~ 0.5 nm for homoligand particles. Hence our two central arguments remain unmodified: independence from imaging parameters and dependence on ligand shell chemical composition. The paper report observations on our STM images; none is a sufficient condition to prove that our images are artifacts. We thoroughly addressed issues related to STM artifacts throughout our microscopy work. Stirling et al. provide guidelines for what they consider good STM images of nanoparticles, such images are indeed present in our literature. They conclude that the evidences we provided to date are insufficient, this is a departure from one of the authors’ previous article which concluded that our images were composed of artifacts. Given that four independent laboratories have reproduced our measurements and that no scientifically rigorous argument is presented to invalidate our STM images, and also given that Stirling et al. do not contest the quality of our recent STM images, we re-affirm that specific binary mixture of ligands spontaneously form features in their ligand shell that we describe as stripe-like domains ~1 nm in width.     

Disruption of α3 Connexin Gene Leads to Proteolysis and Cataractogenesis in Mice

Gap junction channels formed by α₃ (Cx46) and α₈ (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of α₃ connexin in vivo, the α₃ connexin gene was disrupted in mice. Although the absence of α₃ connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted α₃ gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell–cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.     

Clinicopathologic Features and Molecular Characteristics of Glucose Metabolism Contributing to 1?F-fluorodeoxyglucose Uptake in Gastrointestinal Stromal Tumors

Fluorine-18 fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography–computed tomography (PET/CT) is useful in the preoperative diagnosis of gastrointestinal stromal tumors (GISTs); however, the molecular characteristics of glucose metabolism of GIST are unknown. We evaluated ¹⁸F-FDG uptake on preoperative PET/CT of 40 patients and analyzed the expression of glycolytic enzymes in resected GIST tissues by qRT-PCR, western blotting, and immunohistochemistry. Results of receiver operating characteristic curve analysis showed that the maximum standardized uptake value (SUVmax) cut-off value of 4.99 had a sensitivity of 89.5%, specificity was 76.2%, and accuracy of 82.5% for identifying tumors with a high risk of malignancy. We found that ¹⁸F-FDG uptake correlated positively with tumor size, risk grade, and expression levels of glucose transporter 1 (GLUT1), hexokinase 1 (HK1), and lactate dehydrogenase A (LDHA). Elevated HK and LDH activity was found in high-risk tumors. Among the isoforms of GLUT and HK, GLUT1 and HK1 expression increased with higher tumor risk grade. In addition, overexpression of glycolytic enzymes M2 isoform of pyruvate kinase (PKM2) and LDHA was observed in GISTs, especially in high-risk tumors. These results suggest that upregulation of GLUT1, HK1, PKM2, and LDHA may play an important role in GIST tumorigenesis and may be useful in the preoperative prediction of malignant potential.     

Tube Formation Potential of BMSCs and USSCs in Response to HIF-1α Overexpression under Hypoxia

Despite numerous studies on therapeutic effects of mesenchymal stem cells on ischemic tissue regeneration, including angiogenesis, their mechanism of action remains ambiguous. Due to the scarce of investigations based on different stem cell sources with known inherent molecular differences, present study compare tube formation of Bone marrow Mesenchymal Stem Cells and Unrestricted Somatic Stem Cells with known reported different Hox gene expression profile in response to HIF-1α overexpression under hypoxia. This might shed light on some parameters for selection of more responsive source with improved therapeutic effects. Superior in vitro tube formation on Matrigel substratum has been observed by Unrestricted Somatic Stem Cells compared to Bone marrow Mesenchymal Stem Cells which might possibly be due to the discriminating molecular properties of stem cell sources. It may help choosing the appropriate stem cell type for a given therapeutic expectations and also suggests some potential targets for future genetic modification of stem cells.     

Response to: “Response to Different measures of 'genome-wide' DNA methylation exhibit unique properties in placental and somatic tissues

We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues.” The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues” (Price ME, Cotton AM, PeÒaherrera MS, McFadden DE, Kobor MS, Robinson WP. Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues. Epigenetics 2012; 7: 652–63; PMID: 22531475; 10.4161/epi.20221). The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).     

Eukaryotic Initiation Factor 2α - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells'' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.     

TRAF6 Autoubiquitination-Independent Activation of the NFκB and MAPK Pathways in Response to IL-1 and RANKL

The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFκB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFκB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFκB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKγ/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFκB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function.