Mary McKee

Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed.

Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium

Commensal-associated molecular patterns, the major products of nonpathogenic bacteria, are present at high concentrations at the apical surface of the intestinal epithelium. However, the nature of the interaction of commensal-associated molecular patterns with the lumenal surface of the epithelium has not been defined. We have recently demonstrated that intestinal epithelial cells constitutively express several Toll-like receptors (TLRs) in vitro and in vivo that seem to be the key receptors responsible for immune cell activation in response to various bacterial products. In this study we characterize the subcellular distribution of two major TLRs, TLR2 and TLR4, and their ligand-specific dynamic regulation in the model human intestinal epithelial cell line T84. Immunocytochemical studies indicate that TLR2 and TLR4 are constitutively expressed at the apical pole of differentiated T84 cells. After stimulation with lipopolysaccharide or peptidoglycan, TLRs selectively traffic to cytoplasmic compartments near the basolateral membrane. Thus, we demonstrate that TLRs are positioned at the apical pole where they are poised to monitor the sensitive balance of the lumenal microbial array. The results of this dynamic epithelial surveillance can then be conveyed to the underlying cell populations of the lamina propria via these innate immune pattern recognition receptors.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

Deletion of Cavin/PTRF Causes Global Loss of Caveolae, Dyslipidemia, and Glucose Intolerance

Caveolae are specialized invaginations of the plasma membrane found in numerous cell types. They have been implicated as playing a role in a variety of physiological processes and are typically characterized by their association with the caveolin family of proteins. We show here by means of targeted gene disruption in mice that a distinct caveolae-associated protein, Cavin/PTRF, is an essential component of caveolae. Animals lacking Cavin have no morphologically detectable caveolae in any cell type examined and have markedly diminished protein expression of all three caveolin isoforms while retaining normal or above normal caveolin mRNA expression. Cavin-knockout mice are viable and of normal weight but have higher circulating triglyceride levels, significantly reduced adipose tissue mass, glucose intolerance, and hyperinsulinemia--characteristics that constitute a lipodystrophic phenotype. Our results underscore the multiorgan role of caveolae in metabolic regulation and the obligate presence of Cavin for caveolae formation.

The cellular Na+ pump as a site of action for carbon monoxide and glutamate: A mechanism for long-term modulation of cellular activity

Carbon monoxide (CO) induces a long-lasting alteration in cerebellar alpha 3-Na,K-ATPase independent of [Na+] but linked to cGMP synthesis and localized to Purkinje neurons. The action of CO is absent in Purkinje neuron-deficient mice, mimicked by 8-Br-cGMP, and blocked by inhibition of PKG. Glutamate (Glu) and metabotropic agonists mimic the action of CO, an effect that requires PKC and is associated with CO synthesis. These data suggest that CO regulates Na,K-ATPase through cGMP and PKG, and that Glu regulates CO through mGluRs. This system is also modulated by NMDA agonists and nitric oxide, possibly via Glu release, as well as by free radicals. These findings offer a mechanism by which CO, Glu, and free radicals can exert specific effects on synaptic transmission (relevant to long-term changes in cell excitability), as well as more general actions on energy metabolism (relevant to the pathophysiology of excitotoxicity).

Distinct Expression Patterns of Different Subunit Isoforms of the V-ATPase in the Rat Epididymis

Abstract In the epididymis and vas deferens, the vacuolar H+ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.

ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3−/− pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3−/− lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3−/− mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.

Aquaporin 9 Expression along the Male Reproductive Tract

Abstract Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3–4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogenous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.

Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.

Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.