Pagano, Richard E.

Vascular endothelial growth factor receptor‐2 (VEGFR2) plays a key role in vasculogenesis and pathophysiological angiogenesis. Cell surface levels of VEGFR2 is regulated by endocytosis and secretory transport of newly synthesized receptors to the plasma membrane. The mechanism of VEGFR2 sorting and trafficking along the secretory pathway is not well defined. In the present study, using human endothelial cells we demonstrate that sorting and trafficking of VEGFR2 from the Golgi is regulated by syntaxin 6, a t‐SNARE involved in intra‐Golgi and post‐Golgi membrane fusion events. Loss of syntaxin 6 function results in altered sorting of VEGFR2 from the Golgi resulting in receptor down regulation. Our data support a model in which syntaxin 6 regulates delivery of VEGFR2 from the Golgi apparatus to the cell surface thereby regulating VEGF‐A‐induced endothelial cell proliferation, migration, and morphogenesis. Grant Funding Source NIH

The majority of patients with cystic fibrosis (CF) have a single codon deletion in the CFTR gene that impairs assembly of the multidomain glycoprotein, most of which succumbs to ER quality control and proteasomal degradation. Exit from the ER can be facilitated by specific manipulations i.e. growth of cells at reduced temperature, but the rescued ΔF508 is rapidly cleared from the distal secretory pathway and degraded in lysosomes. Recently, accumulation and redistribution of free cholesterol was observed in CF cells and attributed to the lack of CFTR function (White et al. (2004) Am J Respir Cell Mol Biol. 31:538‐43). Here we show that the presence of several misassembled mutant CFTR proteins including ΔF508 in the distal secretory pathway rather than the absence of a functional CFTR is responsible for lipid alterations. Unesterified cholesterol and BIODIPY‐lactosylceramide accumulate in punctate late endosomal structures and are unable to translocate to the Golgi and ER for esterification. This block can be overcome by Rab9 overexpression resulting in clearance of accumulating intracellular cholesterol. Similar alterations were observed on expression of a temperature‐rescued variant of the related ABC protein MRP1. Thus, on escape from ER quality control, misassembled mutants of CFTR and possibly other proteins may impair sterol and glycosphingolipid homeostasis which involves trafficking through the membranous compartments traversed by the aberrant proteins as they are cleared. Supported by the NIH (DK051870).

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193 → Ala) and H193N (His193 → Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor D - threo - 1 - phenyl - 2 - decanoylamino - 3 - morpholinopropan - 1 - ol, at concentrations which inhibited the wild-type enzyme by > 80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ∼60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated β1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated β1-integrin, pFAK, and pCAS to near control levels and partially restored (∼40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses β1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.

Abstract Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface β-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper–17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-κB transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the β-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent β-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.

The targeting of lysosomal transmembrane (TM) proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of mucolipin‐1 (Mcoln1), the TM protein defective in the autosomal recessive disease, mucolipidosis type IV, was studied by overexpressing full‐length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53‐amino acid C‐terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ∼35‐kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that the co‐expression of full‐length Mcoln1 with kinase‐inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident TM proteins from the Golgi to the lysosomes.

We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% ( P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed.