Heat shock protein 70 (Hsp70) inhibits oxidative phosphorylation and compensates ATP balance through enhanced glycolytic activity

Abstract

To address possible effects of heat shock protein 70 (Hsp70) on energy metabolism, we established a cell line expressing different levels of Hsp70 and evaluated changes in glucose and lactate metabolites, as well as ATP levels accordingly. In addition, activities of enzymes involved in glycolysis [phosphofructokinase (PFK) and lactate dehydrogenase (LDH)], Krebs cycle [citric synthase (CS)], and oxidative phosphorylation {NADH dehydrogenase [complex I (CI)] and ubiquinol:cytochrome-c reductase [complex III (CIII)]} were analyzed. The results show that both glucose consumption and lactate excretion were elevated significantly in cells expressing increased levels of Hsp70. Simultaneously, the activities of glycolytic enzymes PFK and LDH were increased markedly in cells overexpressing Hsp70. Activities of enzymes CI and CIII, both involved in oxidative phosphorylation, decreased upon increased expression of Hsp70. These findings were supported by nonsignificant reductions of CS activities in cells that overexpressed Hsp70, whereas intracellular ATP levels remained constant over a wide range of Hsp70 expression. In conclusion, overexpression of Hsp70 in HeLa cells results in downregulation of oxidative phosphorylation, in particular, multiprotein CIII, the main source of reactive oxygen species. In exchange, upregulation of the glycolytic pathway compensates for the homeostasis of cellular ATP supply.

Fig. 1.

Representative immunoblots of heat shock protein 70 (Hsp70) expression in cells in the presence of distinct concentrations of doxycycline (Doxy; 0, 0.001, 0.1, and 2 μg/ml). Total protein lysates (312.5 ng) were used, followed by SDS-PAGE and immunodetection with Hsp70 MAb. A: samples from double-stable HeLa cells expressing a tetracycline-controlled transactivator protein and transfected with the plasmid pTRE2hyg-HSP70 (hyg = hygromycin). A set of known amounts of standard Hsp70 (50–200 ng) was loaded in parallel. B: samples derived from nontransfected cells treated the same way as transfected cells serving as controls.

Fig. 2.

Expression levels of Hsp70 in different categories of HeLa cells. In addition to the nontransfected baseline-level (BL) group, transfected cells were allocated arbitrarily into low-level (LL) and high-level (HL) groups according to the intensity of their Hsp70 expression. BL, n = 12; LL, n = 10; HL, n = 10.

Fig. 3.

Glucose consumption (A) and lactate excretion (B) of transfected cells expressing different amounts of Hsp70 and linear correlation between glucose and lactate (C). BL, cells with BL of Hsp70 (n = 12); LL, n = 10; HL, n = 10. *P < 0.05 vs. BL. Comparison among multiple groups was assessed by Kruskal-Wallis test. Differences among groups were tested by Wilcoxon test. Correlation significance was calculated using Spearman correlation coefficient (P < 0.01).

Fig. 4.

Activity of glycolytic enzymes in HeLa cells expressing different levels of Hsp70. A: activity of phosphofructokinase (PFK). B: activity of lactate dehydrogenase (LDH). BL, n = 12; LL, n = 10; HL, n = 10. *P < 0.05 vs. BL; #P < 0.05 vs. LL. For details, see Fig. 3. Open circles represent statistical outliers.

Fig. 5.

Activity of mitochondrial aerobic enzymes from HeLa cells expressing different levels of Hsp70. A: citrate synthase (CS); BL, n = 12; LL, n = 9; HL, n = 10. B: NADH dehydrogenase [complex I (CI)]; BL, n = 12; LL, n = 9; HL, n = 10. Open circles represent statistical outliers. C: ubiquinol:cytochrome-c reductase [complex III (CIII)]; BL, n = 12; LL, n = 4; HL, n = 7. For details, see Fig. 3. *P < 0.05 vs. BL.

Fig. 6.

Intracellular ATP levels of transfected cells expressing different amounts of Hsp70 (A). Relationship between ATP level and CS activity (B). BL, n = 12; LL, n = 10; HL, n = 10. For details, see Fig. 3.

Fig. 7.

Simplified flow chart of Hsp70 impact on ATP generation. High expression levels of Hsp70 in heat-shocked HeLa cells led to a shift in energy metabolism with increased anaerobic glycolysis and attenuated oxidative phosphorylation. Therefore, under challenging environmental conditions, the danger of potential, harmful release of reactive oxygen species (ROS) in mitochondria is diminished. F-1,6-P, fructose 1,6-bisphosphate; CoA, coenzyme A.