Regulation of Murine Airway Surface Liquid Volume by CFTR and Ca2+-activated Cl− Conductances

Two Cl− conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca2+-activated Cl− conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl−/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca2+ signal in response to mucosal nucleotides that may contribute to the increased Cl−/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca2+ signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height

Small Theropod Teeth from the Late Cretaceous of the San Juan Basin, Northwestern New Mexico and Their Implications for Understanding Latest Cretaceous Dinosaur Evolution

Studying the evolution and biogeographic distribution of dinosaurs during the latest Cretaceous is critical for better understanding the end-Cretaceous extinction event that killed off all non-avian dinosaurs. Western North America contains among the best records of Late Cretaceous terrestrial vertebrates in the world, but is biased against small-bodied dinosaurs. Isolated teeth are the primary evidence for understanding the diversity and evolution of small-bodied theropod dinosaurs during the Late Cretaceous, but few such specimens have been well documented from outside of the northern Rockies, making it difficult to assess Late Cretaceous dinosaur diversity and biogeographic patterns. We describe small theropod teeth from the San Juan Basin of northwestern New Mexico. These specimens were collected from strata spanning Santonian - Maastrichtian. We grouped isolated theropod teeth into several morphotypes, which we assigned to higher-level theropod clades based on possession of phylogenetic synapomorphies. We then used principal components analysis and discriminant function analyses to gauge whether the San Juan Basin teeth overlap with, or are quantitatively distinct from, similar tooth morphotypes from other geographic areas. The San Juan Basin contains a diverse record of small theropods. Late Campanian assemblages differ from approximately coeval assemblages of the northern Rockies in being less diverse with only rare representatives of troodontids and a Dromaeosaurus-like taxon. We also provide evidence that erect and recurved morphs of a Richardoestesia-like taxon represent a single heterodont species. A late Maastrichtian assemblage is dominated by a distinct troodontid. The differences between northern and southern faunas based on isolated theropod teeth provide evidence for provinciality in the late Campanian and the late Maastrichtian of North America. However, there is no indication that major components of small-bodied theropod diversity were lost during the Maastrichtian in New Mexico. The same pattern seen in northern faunas, which may provide evidence for an abrupt dinosaur extinction

Von Willebrand Factor Type A domain of hCLCA1 is sufficient for U-937 macrophage activation

The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease

Secreted hCLCA1 is a signaling molecule that activates airway macrophages.

The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage-monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation

6% FBS is determined to be the optimal FBS % in growth medium to activate macrophages.

<p>(<b>A</b>) U-937 macrophage cells were activated for 24 h using 66.7 μg/mL or 200 μg/mL of FBS-free eGFP medium or FBS-free wild-type hCLCA1 medium with 3%, 6%, or 10% FBS growth medium. IL-8, IL-6, IL-1β, TNF-α, IL-12a and IL-10 were quantified by their mRNA expression using RT-qPCR. The fold difference at each concentration was compared against eGFP (the control) at the same concentration. Results were the means of 5 samples ± SEM. Each sample was a result of an individual transfection paired with an eGFP transfection. Significant fold differences from corresponding control values (eGFP of the same concentration) are indicated by * (<i>p</i> < 0.05), ** (<i>p</i> < 0.005) or *** (<i>p</i> < 0.001). (<b>B</b>) Representative western blot analysis probing against hCLCA1 N-terminal antibody was utilized in conjunction with Coomassie gel staining to compare the total protein contents between FBS-containing hCLCA1 medium and FBS-free hCLCA1 medium. Both western blot and coomassie gel staining showed that hCLCA1 was one of the major secreted molecules, and there was less impurities in the FBS-free hCLCA1 medium without a major loss in hCLCA1 protein. </p

Activation of macrophages with immuno-purified hCLCA1.

<p><b>A</b>) Western blot analysis of immuno-purified hCLCA1 and eGFP using hCLCA1 N-terminal antibody; and silver stained acrylamide gel containing immuno-purified hCLCA1 and eGFP. (<b>B</b>) Using a standard curve generated from a 2-fold dilution series of lysozyme on a silver stained SDS-PAGE gel, (<b>C</b>) immuno-purified hCLCA1 was determined to be 6.225 ± 0.307 pg/μL. Result was presented as the mean of 3 samples ± SEM. The mRNA expression of cytokines in macrophages stimulated with immuno-purified hCLCA1 for (<b>D</b>) 24 h and (<b>E</b>) 48 h were quantified using RT-qPCR. The fold difference was calculated against the corresponding control (immunoprecipitation of eGFP using hCLCA1-N14 antibody). Results for (<b>D</b>) and (<b>E</b>) were presented as the means of 10 samples ± SEM. Each sample was a result of an individual transfection paired with an eGFP transfection. Significant fold differences from corresponding control values (immuno-purified eGFP) are indicated by * (<i>p</i> < 0.05), ** (<i>p</i> < 0.005) and *** (<i>p</i> < 0.001). </p