Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung.

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.

Caenorhabditis elegans Rab escort protein (REP-1) differently regulates each Rab protein function and localization in a tissue-dependent manner.

Rab proteins play a critical role in intracellular vesicle trafficking and require post-translational modification by adding lipids at the C-terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue-specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep-1 gene and found that a rep-1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab-3 or rab-27 suggested that rep-1 functions only in the RAB-27 pathway, and not in the RAB-3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP-1 did not cause defecation defects compared to severe defects in either RAB-27 or RabGGTase disruption, suggesting that REP-1 is not essential for RAB-27 signaling in defection. Some Rab proteins did not physically interact with REP-1, and localization of these Rab proteins was not severely affected by REP-1 disruption. These findings suggest that REP-1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP-1.

Rab3 GTPase-activating protein regulates synaptic transmission and plasticity through the inactivation of Rab3.

Rab3A small G protein is a member of the Rab family and is most abundant in the brain, where it is localized on synaptic vesicles. Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and this change in activity is associated with the trafficking cycle of synaptic vesicles at nerve terminals. Rab3 GTPase-activating protein (GAP) stimulates the GTPase activity of Rab3A and is expected to determine the timing of the dissociation of Rab3A from synaptic vesicles, which may be coupled with synaptic vesicle exocytosis. Rab3 GAP consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150. Recently, mutations in p130 were found to cause Warburg Micro syndrome with severe mental retardation. Here, we generated p130-deficient mice and found that the GTP-bound form of Rab3A accumulated in the brain. Loss of p130 in mice resulted in inhibition of Ca(2+)-dependent glutamate release from cerebrocortical synaptosomes and altered short-term plasticity in the hippocampal CA1 region. Thus, Rab3 GAP regulates synaptic transmission and plasticity by limiting the amount of the GTP-bound form of Rab3A.

Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development.

The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (p< or =0.05) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland.

Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in >80% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UV-inactivated Ad, carbachol-stimulated release of bulk protein and beta-hexosaminidase were significantly (P< or =0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines.

The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre-synaptic vesicles and pancreatic beta-cells.

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line.

The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels.

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells.

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immunoelectron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.