Induction of physiological thermotolerance in MDCK monolayers: contribution of heat shock protein 70

Abstract

Enhanced survival of both individual cells and whole organisms following a heat stress is termed thermotolerance. In organisms, the maintenance of tissue function rather than the survival of individual cells ultimately determines outcome following thermal challenge. We used MDCK kidney epithelial cells to compare alterations in chaperone activity (as a measure of cellular tolerance) and epithelial barrier function (as a measure of physiological tolerance) after thermal challenge. Quercetin, an inhibitor of heat shock factor-dependent transcriptional activity, both potentiated the effects of heat on naive monolayers and blocked conditioning of monolayers following moderate heat shock, suggesting a central role of heat shock protein (HSP) family members in the maintenance of epithelial integrity. We used MDCK cells that constitutively overexpressed HSP70 to demonstrate 2 functionally distinct components of the response of monolayers to thermal stress. The maintenance of epithelial barrier function during exposure to elevated temperatures is regulated by a complex network of processes that involve the actions of HSP70 but that are independent of alterations in chaperone activity as reflected by changes in the thermal inactivation/refolding of luciferase. In contrast, the restoration of barrier function following a heat stress is directly modulated by HSP70 in a manner that can be fully accounted for by changes in chaperone activity. This study demonstrates an important, albeit complex, protective role for heat shock proteins in the modulation of MDCK epithelial barrier function following a thermal stress.

Fig 1.

The effect of HSP inhibitor quercetin on the recovery of epithelial barrier function in heat shock–exposed MDCK monolayers. (A) Filter-grown MDCK cells were heated to 45°C for 45 minutes either with no preconditioning (37°C/45°C) or 24 hours after a preconditioning exposure to 42°C for 2 hours (42°C/45°C) in the absence or in the presence of quercetin (100 μM) (Q). Transepithelial resistance was measured sequentially over the 24-hour experimental period after the exposure to 45°C. Each symbol represents the mean ± SE of sets of 10 filters. ***P < 0.001 and *P < 0.05 represent a significant difference between 42°C/45°C vs Q/42 °C/45°C; ^###P < 0.001 and ^##P < 0.01 represent a significant difference between 37°C/45°C vs Q/37°C/45°C. (B) MDCK cells were maintained at 37°C or were heated to 42°C for 2 hours in the presence of the solvent control (DMSO/42°C) or quercetin (100 μM) (Q/42°C). After a further 24-hour incubation at 37°C, HSP70, HSP90, and HSC70 protein expressions were determined by Western blot analysis as described in Materials and Methods. The numbers below each lane of the HSP autoradiogram represent relative densitometry determined using Adobe Photoshop 7.0 software

Fig 2.

The recovery of epithelial barrier function in heat shock-exposed MDCK monolayers that constitutively overexpress HSP70. (A) HSP70, HSP90, and HSC70 protein expressions were determined in either parent untransfected MDCK cells or in MDCK_pRC.1 that contain the empty vector, or MDCK_p70.2 cells that were transfected with a vector that directs the expression of human HSP70. The numbers below each lane of the HSP autoradiogram represent relative densitometry determined using Adobe Photoshop 7.0 software. In the experiment shown in (B), transfected MDCK cells grown on permeable filters were exposed to heat shock (45°C for 45 minutes). The cells were then returned to control temperature (37°C) and transepithelial resistance was measured sequentially over the 24-hour experimental period. Data represent means ± SE (n = 4). ***P < 0.001 vs MDCK_pRC.1

Fig 3.

The effect of heat preconditioning on changes in HSP expression and the recovery of epithelial barrier function in heat shock– exposed MDCK monolayers that constitutively overexpress HSP70. (A) One set of MDCK_pRC.1 or MDCK_p70.2 cells was maintained continuously at 37°C. The other set of cells was exposed to 42°C for 2 hours followed by 24-hour incubation at 37°C. Cellular levels of HSP70, HSP90, and HSC70 protein expressions were determined by Western blot analysis as described in Materials and Methods. The numbers below each lane of the HSP autoradiogram represent relative densitometry determined using Adobe Photoshop 7.0 software. (B) Filter-grown MDCK_pRC.1 or MDCK_p70.2 cells were exposed to a preconditioning heat stress (42°C for 2 hours) followed by incubation at 37°C. Twenty-four hours later, MDCK monolayers were exposed to 45°C for 45 minutes. The cells were then returned to control temperature (37°C) and transepithelial resistance was measured sequentially over the 24-hour experimental period. Data represent means ± SE (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 vs MDCK_p70.2

Fig 4.

Protection of luciferase activity from thermal degradation in transfected MDCK cells. Either MDCK_pRC.1 or MDCK_p70.2 cells were transfected with the luciferase expression vector pGL3. After 48-hour incubation period, cells were either maintained at 37°C (A) or preconditioned to 42°C for 2 hours (B). Following a further 24-hour incubation at 37°C, cells were exposed to 45°C for 45 minutes in the presence of protein synthesis inhibitor cycloheximide (10 μg/ mL). Luciferase activity was determined either immediately after the heat shock or following a 4-hour recovery period at 37°C. Data represent means ± SE (n = 4). ***P < 0.001 vs MDCK_pRC.1