Sodium arsenite and cadmium chloride are two widespread environmental toxicants which have deleterious effects on living organisms. At the cellular level, sodium arsenite and cadmium chloride cause oxidative stress, dysregulation of gene expression, apoptosis, and the unfolding of protein. Furthermore, both chemical stressors individually have the ability to induce heat shock protein (HSP) accumulation. HSPs are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. Previously, our laboratory demonstrated that treatment of A6 kidney epithelial cells of the frog Xenopus laevis, with either cadmium chloride or sodium arsenite plus a concurrent mild heat shock resulted in an enhanced accumulation of HSPs that was greater than found with the sum of the individual stressors. To the best of our knowledge, no information is available to date on the effect that these two chemical stressors have in combination on HSP accumulation in aquatic organisms. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in Xenopus A6 cells. Immunoblot analysis revealed that the relative levels of HSP30 and HSP70 accumulation in A6 cells treated concurrently with sodium arsenite and cadmium chloride for 12 h were significantly higher than the sum of HSP30 or HSP70 accumulation from cells subjected to the treatments individually. For instance, the combined 10 µM sodium arsenite plus 100 µM cadmium chloride treatment resulted in a 3.5 fold increase in HSP30 accumulation and a 2.5 fold increase in HSP70 accumulation compared to the sum of the stressors individually. This finding suggested a synergistic action between the two stressors. Pretreatment of cells with KNK437, an HSF1 inhibitor, inhibited the combined sodium arsenite- and cadmium chloride-induced accumulation of HSP30 and HSP70 suggesting that this accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemical analysis employing the use of laser scanning confocal microscopy (LSCM) revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined 10 µM sodium arsenite and 10, 50 or 100 µM cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin-proteasome degradation system. Finally, while incubation of A6 cells with 1 µM sodium arsenite plus 10 µM cadmium chloride did not induce a detectable accumulation of HSPs, the addition of a 30 °C mild heat shock resulted in a strong accumulation of HSP30 and HSP70. This study has demonstrated that concurrent sodium arsenite and cadmium chloride treatment can enhance HSP accumulation. Since HSP accumulation is triggered by proteotoxic stress, these findings are relevant given the fact that aquatic amphibians in their natural habitat may be exposed to multiple chemical stressors simultaneously.

There are sex differences in degenerative disease prevalence in humans. Most models of degenerative disease use male animals. Examining female and male responses to stress may give insight into disease prevalence. Heat shock proteins are chaperones linked to damaged proteins in degenerative diseases and may be expressed differentially in females and males. My goal was to characterize the induced expression of Hsp70 and Hsp27 in the brain and heart of female and male rats. Rats were heat shocked, brains and hearts were removed 24 hours after, and western analyses were done to quantify the expression of these proteins. Immunofluorescence was used to localize Hsp70 and Hsp27 in the hippocampus. Overall, male rats have significantly greater induced expression of both Hsp70 and Hsp27 in the brain. In the hippocampus, Hsp70 was localized in blood vessels and microglia, and Hsp27 was localized in astrocytes, following heat shock.

Gene expression at the level of mRNA translation is critical for cells to respond to external signals; it allows changes in protein synthesis without triggering transcription of a new set of genes. Control of mRNA translation and stability is important in several cellular processes including cell growth and differentiation. Thus regulation of the cellular machinery involved in mRNA translation is crucial. Poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) are important members of the cellular mRNA translation machinery, the mRNAs that encode these proteins belong to the terminal oligo pyrimidine tract (TOP) containing family. Translation of the TOP mRNAs is regulated by growth signals and usually codes for several proteins involved in mRNA translation. Our laboratory has previously reported up regulation of PABP1 mRNA translation during recovery from heat shock. It was also shown that the terminal oligopyrmidine tract (TOP) cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation; however the mechanism for achieving this is unknown. In the studies reported here, we showed that translation of eEF1A and RPS6 expression was similarly enhanced during recovery from heat shock. Analyses of samples of in vivo cross linked RNA– protein complexes, immunoprecipitated by ZNF9 antibody, for the presence of specific mRNAs showed that the cellular nucleic acid binding protein ZNF9 binds not only to TOP mRNAs but also mRNA that lack the TOP element such as to β-actin mRNA. To elucidate the mechanism of activation of TOP mRNA translation, as a candidate trans acting factor, siRNA was used to deplete the cellular level of ZNF9 from heat shocked HeLa cells to examine its potential role in stimulation of TOP mRNA translation during recovery from heat shock. Results show that the knock down of ZNF9 disallowed the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect on the constitutive expression of either β-actin or PABP1, eEF1A and RPS6 in exponentially growing HeLa cells. These results suggest that binding of ZNF9 to TOP mRNAs per se does not inhibit translation, but more likely it acts as a general facilitator of mRNA translation. It is possible that modification of the interaction between ZNF9 with other unknown protein factors is responsible for its preferred effect on all three TOP mRNAs studied here. Additionally, results also suggest that a different TOP sequences amongst the observed TOP mRNAs responds similarly to ZNF9.

Processing information in the nervous system is energetically expensive, constraining the ability of the system to survive disturbances caused by stress. While some organisms compensate for extreme changes in the abiotic features of their environment, the mechanisms underlying this are poorly understood. We used the locust Descending Contralateral Movement Detector (DCMD) neuron to study how the propagation characteristics of action potentials (APs) change following an acute energy stress in control and heat shock (HS) pre-treated animals. We also attempted to determine if Ca2+ is involved in the DCMD AP and the possible changes indicated above. Conduction velocity decreased over an hour of recording in all groups, except those with minimal dissections, and we observed an increase in AP half-width and a decrease in the slope of the rising phase of the AP over time. After HS pre-treatment the response to a standard looming stimulus was delayed, showed significantly fewer APs and a lower peak frequency compared to controls. Brief application of sodium azide (NaN3) as an acute metabolic inhibitor did not subsequently affect DCMD’s conduction velocity or ability to fire at high frequencies during the recording period. There were no significant differences from control animals with extracellular Ca2+ manipulations; however we cannot conclude that Ca2+ does not contribute to DCMD’s AP because Na+ could have flowed through Ca2+ channels in the absence of extracellular Ca2+. Furthermore, examination of possible performance impairments with decreased Ca2+ currents, to indicate if Ca2+ current manipulation may account for the performance impairment, could not be conducted because no differences in AP characteristics were observed with Ca2+ manipulations. We suggest that the slowing of propagation in all groups represents a response to energetic stress and that HS modifies neuronal properties in ways that can be interpreted as saving energy in case of future stressors. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2009-08-11 08:36:26.571

The mechanism and subsequent patterns of Heat shock protein (Hsp) expression in skeletal muscle specific to contraction type was determined. Rat tibialis anterior (TA) muscle was forcibly lengthened (LC) or shortened (SC) in 5 sets of 20 repetitions before being removed at 2, 8, 24, 48, 72, or 168 hours and analyzed for muscle damage and Hsp25 and Hsp72 expression. Isometric peak torque was reduced to 63% and 33% (P<0.001) at 3-minutes after SC and LC, respectively. Muscle fibre damage appeared at 8h and beyond following LCs, but no damage was observed after SCs. Hsp25 content in LC muscle increased by 3.1±0.53 fold (P<0.01) at 48h and remained elevated. Hsp72 content increased by 3.8±0.66 fold at 24h and remained elevated. Neither Hsp25 nor Hsp72 content was elevated following SCs. Muscle damage associated with LCs results in a greater Hsp accumulation than SCs and 100 SCs do not result in increased Hsp content.

The mechanism and subsequent patterns of Heat shock protein (Hsp) expression in skeletal muscle specific to contraction type was determined. Rat tibialis anterior (TA) muscle was forcibly lengthened (LC) or shortened (SC) in 5 sets of 20 repetitions before being removed at 2, 8, 24, 48, 72, or 168 hours and analyzed for muscle damage and Hsp25 and Hsp72 expression. Isometric peak torque was reduced to 63% and 33% (P<0.001) at 3-minutes after SC and LC, respectively. Muscle fibre damage appeared at 8h and beyond following LCs, but no damage was observed after SCs. Hsp25 content in LC muscle increased by 3.1±0.53 fold (P<0.01) at 48h and remained elevated. Hsp72 content increased by 3.8±0.66 fold at 24h and remained elevated. Neither Hsp25 nor Hsp72 content was elevated following SCs. Muscle damage associated with LCs results in a greater Hsp accumulation than SCs and 100 SCs do not result in increased Hsp content.

Skeletal muscle injury results in functional deficits that can take several weeks to fully recover. Ultimate recovery of function is dependent on the muscle’s ability to regenerate, a highly coordinated process that involves transient muscle inflammation and the replacement of damaged myofibers. Instrumental in the inflammatory response, is the pro-inflammatory cytokine TNF-α. Expression of TNF-α is thought to be regulated, in part, by the stress sensing 70 kDa heat shock protein (Hsp70). However, it remains unclear how Hsp70 alters TNF-α following injury, and if so, how these changes affect skeletal muscle repair. Therefore, we up-regulated Hsp70 expression using 17-allylamino-17-demethoxygeldanamycin (17-AAG) prior to and following BaCl2-induced injury, and assessed TNF-α and myogenin content. Regenerating fiber cross-sectional area (CSA) and in vivo isometric torque were also analyzed in the weeks following the injury. Treatment of 17-AAG resulted in a ~5 fold increase in Hsp70 of the uninjured muscle, but did not affect any other biochemical, morphological or functional variables compared to controls. In the days following the injury, TNF-α and myogenin were elevated and directly correlated. At these earlier time points (≤7 days), treatment of 17-AAG increased TNF-α above that of the injured controls and resulted in a sustained increase in myogenin. However, no differences were observed in regenerating fiber CSA or in vivo torque production between the groups. Together, these data suggest that Hsp70 induction increases TNF-α and myogenin content following BaCl2-induced injury, but does not appear to alter skeletal muscle regeneration or attenuate functional deficits in otherwise healthy young mice.

Heat shock proteins (Hsps) are increasingly being seen as having roles other than those of intracellular molecular chaperones, particularly with regard to their potential to act as cytokines, and to stimulate the innate immune system. Hsps have also been found to promote bone resorption and osteoclast formation in vitro, although the mechanism has not been previously identified. The overall aims of this thesis were to determine whether Hsps could stimulate bone resorption by affecting the RANKL/OPG pathway, and to address the hypothesis that Hsps can act as a danger signal to the innate immune system. In order for Hsps to affect either the RANKL/OPG system of bone resorption or act as danger signals they would need to be actively released from cells, ideally in a controlled manner following exposure to the source of stress. Hsp60 and Hsp70 were found to be released from a range of immune cells including the cell lines Jurkat and U937, and also PBMCs, T-cells and B-cells. This release was not due to cell damage. The release of Hsp60 and Hsp70 were downregulated by inhibitors of protein secretion, in particular Hsp70 release was reduced by compounds that inhibited lysosomal pathways and Hsp60 release by classical secretion inhibitors. Hsp60, Hsp70, GroEL and LPS all affected the RANKL/OPG system of bone regulation; OPG production and release was down-regulated in the MG63 and GCT osteoblast-like cell lines following treatment with Hsp60, Hsp70 and LPS, and RANKL expression was upregulated following treatment with Hsp60, Hsp70, GroEL and LPS. This effect on the RANKL/OPG system was found to translate into an effect on osteoclast formation when conditioned media from treated osteoblasts was added to osteoclast precursors in the presence of M-CSF. A range of different factors that affected Hsp release were identified; PHA activation of PBMCs was found to upregulate Hsp60 release from PBMCs. GroEL and LPS caused an upregulation in Hsp70 release from PBMCs and GCT osteoblast like cells, and Hsp70 was found to stimulate Hsp60 release from PBMCs and GCT cells. These responses of Hsp release were used to form a theory of a cascade-like danger signal that may occur when cells are exposed to bacterial infection and which would result in activation of antigen presenting cells via previously identified receptors for Hsps such as CD14/TLR4 or by unidentified pathways. The elevated release of Hsps in response to GroEL and LPS was also identified as a mechanism that could stimulate bone loss during infection or autoimmuniry by affecting the RANKL/OPG system. hi conclusion, Hsp60 and Hsp70 can be released from immune cells under normal conditions, and from both immune and osteoblast-like cells following stimulation with LPS and other Hsps. The observed release responses provide a mechanism through which Hsps can act as danger signals to the innate immune system, and also as promoters of bone resorption via the RANKL/OPG system.

The heat shock response (HSR) is a highly conserved mechanism that enables organisms to survive environmental and pathophysiological stress. In Drosophila, the HSR is regulated by a single transcription factor, heat shock factor (HSF). During stress, HSF trimerizes and binds to over 200 loci on Drosophila polytene chromosomes with only nine mapping to major heat shock (HS) inducible gene loci. The function of HSF binding to the other sites in the genome is currently unknown. Some of these sites may contain yet unidentified “minor” HS genes. Interestingly, the binding of HSF also coincides with puff regression at some sites. Two such sites contain the major developmentally regulated genes Eip74 and Eip75: key regulators in the response to 20-hydroxyecdysone (20E), the main hormone responsible for the temporal co-ordination of post-embryonic development in Drosophila. Previous work in our and other labs indicates that the regression of non-HS puffs during the HSR is dependent on the presence of functional HSF. Using chromatin immunoprecipitation (ChIP) followed by hybridization to genome tiling arrays (Chip), I have identified 434 regions in the Drosophila Kc cell genome that are bound by HSF during HS, and have determined that 57% of these sites are located within the transcribed regions of genes. By examining the transcriptional response to HS in Kc cells and third instar larvae using expression microarrays, I found that only about 10% of all genes within 1250 bp of an HSF binding site are transcriptionally regulated by HS and many genes whose transcript levels change during HS do not appear to be near an HSF binding site. Furthermore, genes with an HSF binding site within their introns are significantly enriched (modified Fisher Exact p-value between 2.0x10-3 and 1.5x10-6) in gene ontology terms related to developmental processes and reproduction. Using expression microarray technology, I characterized the transcriptional response to 20E and its structural analog ponasterone A. I have identified multiple HSF binding sites within Eip74 and Eip75, and show that induction of the HSR correlates with repression of these genes and all other 20E-inducible genes. Taken together, this work provides a basis for further investigation into the role of HSF binding to sites not associated with HS genes and its possible function as a repressor of gene transcription during conditions of stress and as a regulator of developmental genes under stress and non-stress conditions.

Hsp104 and ClpB are hexameric ATPases that resolubilize aggregated proteins in collaboration with the Hsp70 chaperone system. Hsp104/ClpB functionally interact only with their respective Hsp70 system and this specificity is mapped to the Hsp104/ClpB coiled-coil domain (CCD). We hypothesize that the interaction between Hsp70 and Hsp104/ClpB CCD stimulates nucleotide exchange and release of substrate from Hsp70. In the current study, the CCDs of E. coli ClpB and S. cerevisiae Hsp104 have been purified. Isolated domains are monomeric and well folded. They inhibit refolding of aggregated firefly luciferase in a species-specific manner. We found that the ATPase activity of E. coli DnaK is stimulated at low concentrations of the E. coli ClpB CCD but not by yeast Hsp104 CCD. However, in another bacterial system (Thermus thermophilus) we found that the ClpB CCD inhibits The ATPase activity of DnaK suggesting that the interaction may have different consequences in distinct chaperone networks.