Since Hsp90 is a known modulator of HSF1 activity, we examined the effects of two pharmacological inhibitors of Hsp90, novobiocin and geldanamycin, on HSF1 DNA-binding activity in the Xenopus oocyte model system. Novobiocin exhibits antiproliferative activity in culture cells and interacts with a C-terminal ATP-binding pocket on Hsp90, inhibiting Hsp90 autophosphorylation. Treatment of oocytes with novobiocin followed by heat shock results in a dose-dependent decrease in HSF1 DNA-binding and transcriptional activity. Immunoprecipitation experiments demonstrate novobiocin does not alter HSF1 activity through dissociation of Hsp90 from either monomeric or trimerized HSF1, suggesting that the effect of novobiocin on HSF1 is mediated through alterations in Hsp90 autophosphorylation. Geldanamycin binds the N-terminal ATPase site of Hsp90 and inhibits chaperone activity. Geldanamycin treatment of oocytes resulted in a dose-dependent increase in stability of active HSF1 trimers during submaximal heat shock and a delay in disassembly of trimers during recovery. The results suggest that Hsp90 chaperone activity is required for disassembly of HSF1 trimers. The data obtained with novobiocin suggests the C-terminal ATP-binding activity of Hsp90 is required for the initial steps of HSF1 trimerization, whereas the effects of geldanamycin suggest N-terminal ATPase and chaperone activities are required for disassembly of activated trimers. These data provide important insight into the molecular mechanisms by which pharmacological inhibitors of Hsp90 affect the heat shock response.

Significance Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease caused by the failure to clear bacteria. Bacterial clearance depends on a layer of airway surface liquid (ASL) covering the airways, rich in antimicrobial compounds and mucins, that removes bacteria from the airway through mucociliary clearance. This study provides the first demonstration that inhalation of bacteria triggers CFTR-dependent ASL secretion. We suggest that this response to inhaled pathogens is an important but previously unknown part of the innate immune response that would be missing in CF patients, resulting in reduced bacterial killing and facilitating infection.

Autonomic dysfunction is a serious complication of diabetes and can lead to cardiovascular abnormalities and premature death. It was recently proposed that autonomic dysfunction is triggered by oxidation-mediated inactivation of neuronal nicotinic acetylcholine receptors (nAChRs), impairing synaptic transmission in sympathetic ganglia and resulting in autonomic failure. We investigated whether the receptor for advanced glycation end products (RAGE) and its role in the generation of reactive oxygen species (ROS) could be contributing to the events that initiate sympathetic malfunction under high glucose conditions. Using biochemical, live imaging and electrophysiological tools we demonstrated that exposure of sympathetic neurons to high glucose increases RAGE expression and oxidative markers, and that incubation with RAGE ligands (e.g. AGEs, S100 and HMGB1) mimics both ROS elevation and nAChR inactivation. In contrast, co-treatment with either antioxidants or an anti-RAGE IgG prevented the inactivation of nAChRs. Lastly, a role for RAGE in this context was corroborated by the lack of sensitivity of sympathetic neurons from RAGE knock-out mice to high glucose. These data define a pivotal role for RAGE in initiating the events associated with exposure of sympathetic neurons to high glucose, and strongly support RAGE signaling as a potential therapeutic target in the autonomic complications associated with diabetes.

The effect of fluoride treatment on the expression of a panel of osteogenic and stress markers in Stage 55 premetamorphic Xenopus larvae was examined at the precise onset of replacement of the larval cartilaginous skeleton with bone. A dosing regimen of 10 mmol/L sodium fluoride over 8 days was followed, during which time larvae developed to Stage 58, when the process of progressive ossification takes place in the vertebral column and membranous bones of the skull, pelvic, and pectoral girdles and portions of the appendicular skeleton. Markers of bone formation, including COL1A1, the transcription factors Osterix, RUNX2-II, and matrix metalloproteinases MMP1 and MMP13, decreased relative to age-matched controls, though the osteoblast marker BGLAP was not significantly altered. Expression of the pro-osteoclastogenic factor RANKL decreased, whereas expression of the anti-osteoclastogenic factor osteoprotegerin increased. Altered expression of oxidative stress markers, with the exception of superoxide dismutase, was generally not observed. These data demonstrate the potent effects of fluoride on the expression of factors required for osteoblast and osteoclast differentiation, as well as on the expression of osteoblast products, including MMP1 and collagen. Importantly, these effects were observed in the absence of significant changes in the expression of oxidative stress markers. The results provide the first molecular insights into the mechanisms underlying skeletal fluorosis in a whole organism developmental model.