The transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) regulates induction of an extensive cellular stress response network when complexed with the cAMP-responsive element binding protein (CBP) at antioxidant response elements (ARE) located in the promoter region of target genes. Activating transcription factor 3 (ATF3) can repress Nrf2-mediated signaling in a manner that is not well understood. Here, we show that ATF3-mediated suppression is a consequence of direct ATF3-Nrf2 protein-protein interactions that result in displacement of CBP from the ARE. This work establishes ATF3 as a novel repressor of the Nrf2-directed stress response pathway. [Cancer Res 2008;68(2):364-8]

The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCα, -β1, and -β2 to the membrane, but only CXCR1 activated PKCε. CXCL8 also failed to activate PKCε in RBL-2H3 cells stably expressing CXCR2. ΔCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCε as well as CXCR1. Expression of the PKCε inhibitor peptide εV1 in RBL-2H3 cells blocked PKCε translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization. εV1 also inhibited CXCR1-, CCR5-, and ΔCXCR2-mediated cross-regulatory signals for GTPase activity, Ca2+ mobilization, and internalization. Peritoneal macrophages from PKCε-deficient mice (PKCε−/−) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca2+ mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCε to mediate cross-inhibitory signals. Inhibition or deletion of PKCε decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization, suggesting that cross-regulation is a Ca2+-independent process. Because ΔCXCR2, but not CXCR2, activates PKCε and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of Gi and Gq in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing Gαi3 (PAFR-Gαi3) or Gαq (PAFR-Gαq) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-Gαi3, or PAFR-Gαq was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of β-hexosaminidase. PAFR-Gαi3 and PAFR-Gαq mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-Gαi3, but not PAFR-Gαq, mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated βarrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-Gαi3 activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that Gi exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both Gi and Gq activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by Gi, the data suggest that differential activation of both Gi and Gq by PAFR likely mediate specific as well as redundant signaling pathways.

Cardiopulmonary bypass procedures are one of the most common operations and blood oxygenators are the centre piece for the heart-lung machines. Blood oxygenators have been tested as entire devices but intricate details on the flow field inside the oxygenators remain unknown. In this study, a novel method is presented to analyse the flow field inside oxygenators based on micro Computed Tomography (μCT) scans. Two Hollow Fibre Membrane (HFM) oxygenator prototypes were scanned and three-dimensional full scale models that capture the device-specific fibre distributions are set up for computational fluid dynamics analysis. The blood flow through the oxygenator is modelled as a non-Newtonian fluid. The results were compared against the flow solution through an ideal fibre distribution and show the importance of a uniform distribution of fibres and that the oxygenators analysed are not susceptible to flow directionality as mass flow versus area remain the same. However the pressure drop across the oxygenator is dependent on flow rate and direction. By comparing residence time of blood against the time frame to fully saturate blood with oxygen we highlight the potential of this method as design optimisation tool. In conclusion, image-based reconstruction is found to be a feasible route to assess oxygenator performance through flow modelling. It offers the possibility to review a product as manufactured rather than as designed, which is a valuable insight as a precursor to the approval processes. Finally, the flow analysis presented may be extended, at computational cost, to include species transport in further studies.

Caffeine has been shown to directly increase fatty acid oxidation, in part, by promoting mitochondrial biogenesis. Mitochondrial biogenesis is often coupled with mitophagy, the autophagy-lysosomal degradation of mitochondria. Increased mitochondrial biogenesis and mitophagy promote mitochondrial turnover, which can enhance aerobic metabolism. In addition, recent studies have revealed that cellular lipid droplets can be directly utilized in an autophagy-dependent manner, a process known as lipophagy. Although caffeine has been shown to promote autophagy and mitochondrial biogenesis in skeletal muscles, it remains unclear whether caffeine can increase lipophagy and mitochondrial turnover in skeletal muscle as well. The purpose of this study was to determine the possible contribution of lipophagy to caffeine-dependent lipid utilization. Furthermore, we sought to determine whether caffeine could increase mitochondrial turnover, which may also contribute to elevated fatty acid oxidation. Treating fully differentiated C2C12 skeletal myotubes with 0.5 mM oleic acid (OA) for 24 hr promoted an approximate 2.5fold increase in cellular lipid storage. Treating skeletal myotubes with 0.5 mM OA plus 0.5 mM caffeine for an additional 24 hr effectively returned cellular lipid stores to control levels, and this was associated with an increase in markers of autophagosomes and autophagic flux, as well as elevated autophagosome density in TEM images. The addition of autophagy inhibitors 3-methyladenine (10 mM) or bafilomycin A1 (10 μM) reduced caffeine-dependent lipid utilization by approximately 30%. However, fluorescence and transmission electron microscopy analysis revealed no direct evidence of lipophagy in skeletal myotubes, and there was also no lipophagy-dependent increase in fatty acid oxidation. Finally, caffeine treatment promoted an 80% increase in mitochondrial turnover, which coincided with a 35% increase in mitochondrial fragmentation. Our results suggest that caffeine administration causes an autophagy-dependent decrease in lipid content by increasing mitochondrial turnover in mammalian skeletal myotubes. K E Y W O R D Scaffeine, lipid utilization, mitochondrial turnover, mitophagy, skeletal muscle

No abstract

Colitis-associated colorectal cancer (CAC) is mediated by inflammation-induced tumorigenesis, causing DNA damage in susceptible cell populations. Dysregulation of inflammatory pathways in the colon is attributed to cancer pathogenesis and such signaling is attributed to NF-κB signaling. Transcription factor NF-κB is the master regulator of gene transcription and is activated through one of two pathways. Our focus is on the understudied noncanonical NF-κB signaling pathway, including NF-κB inducing kinase (NIK). NIK must be stabilized for activation of this pathway. Noncanonical signaling is attributed to the production of pro-inflammatory cytokines, recruitment of immune cells, and cell proliferation. We hypothesize that this pathway maintains gut homeostasis and when dysregulated causes cell populations to become tumorigenic. The objective of this study is to validate the clinical relevance of noncanonical signaling. We observe that diminished noncanonical signaling via whole-body Nik knockout mice results in reduced stem cell marker expression, enhanced proliferative capacity, altered microbiome composition, and increased susceptibility towards inflammation-induced tumorigenesis in the colon. Using CAC mouse models, conditional knockout strains show deletion of noncanonical signaling in epithelial cells results in increased susceptibility to colorectal tumorigenesis. Likewise, human colonic biopsy samples collected from CAC patients show significantly decreased expression levels of genes related to the noncanonical NF-kB pathway. This data suggests that the noncanonical NF-κB pathway has a protective role against colorectal cancer by regulating immune system homeostasis in the GI tract.

NF-κB signaling is divided into two distinct pathways, defined as either canonical or noncanonical. Canonical NF-κB signaling is well described and characterized. However, significantly less is known regarding mechanisms regulated by the noncanonical NF-κB pathway, especially outside of the lymphoid system. Recently, interest in the role of noncanonical NF-κB in the mucosal immune system has been increasing, with emphasis on NF-κB inducing kinase (NIK). NIK is an essential kinase in this cascade. Our data shows that lack of noncanonical signaling through the complete knockout of Nik in mice results in an aberrant large intestine phenotype characterized by reduced stem cell marker expression, altered regeneration and differentiation capacity under stress, changes in the microbiome, and predisposition to inflammation-associated tumorigenesis. Subsequent mechanistic studies utilizing novel mice with cell type specific deletions of either RelA/p65 or Nik revealed separate and distinct rolls for canonical and noncanonical NF-κB signaling in attenuating inflammation and tumor progression. RelA/p65 attenuates disease progression through the myeloid compartment in the gut, whereas Nik functions through the intestinal epithelial cell compartment. Dysregulation of noncanonical signaling is also observed in human colorectal cancer patients, which have marked suppression of this pathway in colonic biopsy samples. Together, these findings indicate that noncanonical NF-κB signaling is a critical regulator of immune system homeostasis in the gastrointestinal tract and plays a vital protective role against colorectal cancer.