Transforming Growth Factor-ß and Interleukin-1ß Signaling Pathways Converge on the Chemokine CCL20 Promoter.

CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1ß. We have determined that IL-1ß-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-ß. TGF-ß is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvß8 (itgb8). Here we confirm correlative increases in αvß8 and IL-1ß with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1ß- and αvß8-mediated TGF-ß activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5'-flanking region of CCL20 to determine that IL-1ß-driven CCL20 expression is dependent on αvß8-mediated activation of TGF-ß. We identify a TGF-ß-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1ß-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvß8-dependent activation of TGF-ß regulates IL-1ß-dependent CCL20 expression in COPD.

Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation.

Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic ß-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation.

Interleukin-1beta induces increased transcriptional activation of the transforming growth factor-beta-activating integrin subunit beta8 through altering chromatin architecture.

The integrin αvß8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-ß. Through its association with LAP, TGF-ß is maintained in a latent form that must be activated to function. Binding to the integrin αvß8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-ß activation in vivo. Altered expression of the integrin ß8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1ß (IL-1ß) increases ITGB8 expression on lung fibroblasts, which increases αvß8-mediated TGF-ß activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1ß. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvß8 surface expression, and αvß8-mediated TGFß activation.

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells.

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic ß cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in ß cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in ß cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.

High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts.

Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic beta-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28mM glucose resulted in approximately 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.

Mouse and human lung fibroblasts regulate dendritic cell trafficking, airway inflammation, and fibrosis through integrin αvß8-mediated activation of TGF-ß.

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models - conditional deletion in mouse fibroblasts of the TGF-ß-activating integrin αvß8 and neutralization of αvß8 in human COPD fibroblasts - we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvß8-mediated activation of TGF-ß by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.