Role of HSP70 in cellular thermotolerance
ABSTRACT Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning.
For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C).
A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells.
The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.
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ABSTRACT: Abstract Purpose: The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems. Materials and methods: Human articular chondrocytes were cultured by monolayer or pellet culture system at 32 °C, 37 °C, and 41 °C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S). Results: The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ. Conclusion: The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 °C, 37 °C, and 41 °C in monolayer, pellet, or combined cultures.International Journal of Hyperthermia 05/2014; 30(3):210-6. DOI:10.3109/02656736.2014.906048 · 2.77 Impact Factor
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ABSTRACT: The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the "recovery phase", roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress.06/2013; 2(2):224-43. DOI:10.3390/cells2020224
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ABSTRACT: Heat shock during restorative procedures can trigger damage to the pulpodentin complex. While severe heat shock has toxic effects, fever-range heat stress exerts beneficial effects on several cells and tissues. In this study, we examined whether continuous fever-range heat stress (CFHS) has beneficial effects on thermotolerance in the rat clonal dental pulp cell line with odontoblastic properties, KN-3. KN-3 cells were cultured at 41°C for various periods, and the expression level of several proteins was assessed by Western blot analysis. After pre-heat-treatment at 41°C for various periods, KN-3 cells were exposed to lethal severe heat shock (LSHS) at 49°C for 10min, and cell viability was examined using the MTS assay. Additionally, the expression level of odontoblast differentiation makers in surviving cells was examined by Western blot analysis. CFHS increased the expression levels of several heat shock proteins (HSPs) in KN-3 cells, and induced transient cell cycle arrest. KN-3 cells, not pre-heated or exposed to CFHS for 1 or 3h, died after exposure to LSHS. In contrast, KN-3 cells exposed to CFHS for 12h were transiently lower on day 1, but increased on day 3 after LSHS. The surviving cells expressed odontoblast differentiation markers, dentine sialoprotein and dentine matrix protein-1. These results suggest that CFHS for 12h improves tolerance to LSHS by inducing HSPs expression and cell cycle arrest in KN-3 cells. The appropriate pretreatment with continuous fever-range heat stress can provide protection against lethal heat shock in KN-3 cells.Archives of oral biology 04/2014; 59(7):741-748. DOI:10.1016/j.archoralbio.2014.03.014 · 1.88 Impact Factor