Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63 + /CD66b + nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to a1-antitrypsin (a1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and a1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

ADP-ribosylation factor (ARF) mediated recruitment of COPI to membranes plays a central role in transport between the endoplasmic reticulum (ER) and the Golgi. The activation of ARFs is mediated by guanine nucleotide exchange factors (GEFs). Although several ARF-GEFs have been identified, the transport steps in which they function are still poorly understood. Here we report that GBF1, a member of the Sec7-domain family of GEFs, is responsible for the regulation of COPI-mediated events at the ER-Golgi interface. We show that GBF1 is essential for the formation, differentiation, and translocation of pre-Golgi intermediates and for the maintenance of Golgi integrity. We also show that the formation of transport-competent ER-to-Golgi intermediates proceeds in two stages: first, a COPI-independent event leads to the formation of an unstable compartment, which is rapidly reabsorbed in the absence of GBF1 activity. Second, the association of GBF1 with this compartment allows COPI recruitment and leads to its maturation into transport intermediates. The recruitment of GBF1 to this compartment is specifically inhibited by brefeldin A. Our findings imply that the continuous recruitment of GBF1 to spatially differentiated membrane domains is required for sustained membrane remodeling that underlies membrane traffic and Golgi biogenesis. INTRODUCTIONTransport between the endoplasmic reticulum (ER) and the Golgi is mediated by transport intermediates generated by three recognizable traffic stages: the formation of transport intermediates at the ER, maturation and movement of such intermediates toward the Golgi, and coalescence at the incoming cis-face of the Golgi. The differentiation of ER-Golgi transport intermediates requires the sequential action of two cytosolic coat complexes, COPII and COPI (Aridor et al., 1995;Rowe et al., 1996;Scales et al., 1997). COPII recruitment is coupled to the differentiation of specialized ER subdomains called endoplasmic reticulum exit sites (ERESs) (Bannykh and Balch, 1997). Protein export from the ER seems to occur exclusively from ERESs. Morphologically, ERESs are characterized by COPII-coated tubular ER elements adjacent to a collection of vesicular tubular clusters (VTCs) coated with COPI (Bannykh et al., 1996). VTCs represent a subsequent stage of transport, and one ERES generates multiple VTCs during its existence (Lippincott-Schwartz et al., 1998). Association of COPI with ERESs occurs after these membranes have been first differentiated by the activity of COPII .COPI binding seems to be required at multiple stages of ER-Golgi transport. Experimental evidence indicates that the initial requirement for COPI association is to differentiate VTCs adjacent to ERES. Specifically, when COPI binding to membranes is inhibited, cargo proteins and resident Golgi proteins fail to exit the ER and be incorporated into ERESs (Dascher and Balch, 1994;Lippincott-Schwartz et al., 1989;Peters et al., 1995;Rowe et al., 1996). Additional COPI function is required after VTCs leave the ERES region and ini...

ADP-ribosylation factor (ARF)-facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine nucleotide exchange factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)-Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP-ribosylation factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP-tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t 1/2 is approximately 17 AE 1 seconds). GBF1 cycles faster than GFP-tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF-GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER-Golgi traffic.

COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation factor (ARF) or the ARF-activating guanine nucleotide exchange factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1. We report that siRNA-mediated depletion of GBF1 causes COPI dispersal but does not lead to collapse of the Golgi. Instead, it causes extensive tubulation of the cis-Golgi. The Golgi-derived tubules target to peripheral ER-Golgi intermediate compartment (ERGIC) sites and create dynamic continuities between the ERGIC and the cis-Golgi compartment. COPI dispersal in GBF1-depleted cells causes dramatic inhibition of the trafficking of transmembrane proteins. Unexpectedly, soluble proteins continue to be secreted from GBF1-depleted cells. Our findings suggest that a secretory pathway capable of trafficking soluble proteins can be maintained in cells in which COPI recruitment is compromised by GBF1 depletion. However, the trafficking of transmembrane proteins through the existing pathway requires GBF1-mediated ARF activation and COPI recruitment.

Protein traffic is necessary to maintain homeostasis in all eukaryotic organisms. All newly synthesized secretory proteins destined to the secretory and endolysosmal systems are transported from the endoplasmic reticulum to the Golgi before delivery to their final destinations. Here, we describe the COPII and COPI coating machineries that generate carrier vesicles and the tethers and SNAREs that mediate COPII and COPI vesicle fusion at the ER-Golgi interface.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Background: GBF1, BIG1, and BIG2 are guanine nucleotide exchange factors (GEFs) that activate ARFs to regulate secretory traffic.Results: GBF1 activity promotes subsequent recruitment of BIG1/2.Conclusion: The three GEFs are functionally coupled in a GEF/ARF/GEF cascade.Significance: Coupling integrates all coating events within the pathway to simultaneously regulate passage of cargo through multiple compartments.

Rationale: Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated.Objectives: We investigated changes to the leukotriene A 4 hydrolase (LTA 4 H)-proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD.Methods: A/J mice were exposed to air or cigarette smoke for 22 weeks followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human cohorts were used to analyze changes to the LTA 4 H-PGP pathway in never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and LTA 4 H aminopeptidase activity were detected by mass spectroscopy, LTA 4 H amounts were detected by ELISA, and acrolein was detected by Western blot.Measurements and Main Results: Mice exposed to cigarette smoke developed emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of LTA 4 H aminopeptidase, which ordinarily degrades PGP. We recapitulated these findings in smokers with and without COPD. PGP and AcPGP are closely associated with cigarette smoke use. Once chronic inflammation is established, changes to LTA 4 H aminopeptidase remain, even in the absence of ongoing cigarette use. Acrolein modifies LTA 4 H and inhibits aminopeptidase activity to the same extent as cigarette smoke.Conclusions: These results demonstrate a novel pathway of aberrant regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately involved in disease progression in the absence of ongoing cigarette smoke exposure. We highlight a mechanism by which acrolein potentiates neutrophilic inflammation through selective inhibition of LTA 4 H aminopeptidase activity. Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).