In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

Abstract

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracyclineregulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.Cells exposed to nonlethal, elevated temperatures become transiently resistant to a subsequent heat shock. Thermotolerance development is paralleled by expression of heat shock proteins, which include members of the heat shock protein 70 (Hsp70) family (21,29). In thermotolerant cells, endogenous proteins such as p68 kinase and exogenous reporter gene products such as ␤-galactosidase and firefly luciferase show increased resistance to heat damage (4,24,27,31).It is likely that Hsp70 contributes to the protection of cellular proteins by functioning as a molecular chaperone. Such a function is implied from in vitro studies in which purified members of the Hsp70 family are found to contribute to the heat protection of several substrate proteins. Hsp70 alone can protect topoisomerase I and DNA polymerase against heat inactivation and facilitate the reactivation of heat-inactivated topoisomerase I in a concentration-dependent fashion (3, 44). For reactivation of DNA polymerase, ATP is required (44). For the recovery of heat-denatured firefly luciferase and chemically denatu...

Hsp72 chaperone function is dispensable for protection against stress-induced apoptosis

“…4b) and due to the expression of co-chaperones. However, Nollen et al (1999) reported that thermotolerant cells were more resistant to heat than cells expressing identical levels of Hsp72.…”

“…Thermotolerant L929 cells that express endogenous Hsp72, showed significant protection against luciferase denaturation. The increased protection of luciferase activity in thermotolerant cells compared to cells expressing transgenic Hsp72 has already been noted (Nollen et al 1999) and is probably related to the presence of additional chaperone and co-chaperone proteins in thermotolerant cells (Craig 1985).…”

Testicular Superoxide Dismutase Activity, Heat Shock Protein 70 Concentration and Blood Plasma Inhibin-.ALPHA. Concentration of Dogs with a Sertoli Cell Tumor in a Unilateral Cryptorchid Testis

“…High temperature or heat stress has been found to induce an increase in HSP protein production in certain tissues [22]. HSP 70 stimulates proliferation of some types of tumor cells [6], and it has been found to be an anti-apoptotic protein that inhibits cell death by apoptosis in human and canine mammary gland tumor cells [18] and human prostatic adenocarcinama cells [20].…”

“…A high ROS concentration in the cryptorchid testis is an important risk factor for the occurrence and development of testicular tumors [29]. Heat shock protein (HSP) 70 is the main protein produced abundantly in some tissues exposed to heat stress [22], and it induces the development of certain tumors [6,18,20]. Inhibin (INH) in male animals is a glycoprotein produced by Sertoli cells [23,24,28] and is composed of two subunits, an α-unit and a β-unit [8,23].…”

Invited Review: Interplay between molecular chaperones and signaling pathways in survival of heat shock

“…15 and 21 for review). In fact, aggregation of nuclear proteins as well as a reporter enzyme (firefly luciferase) after heat shock was reduced in thermotolerant cells expressing HSPs, and these cells demonstrated faster solubilization of aggregated proteins during the recovery period (2,26,42,53). Furthermore, expression of HSP72 (the major inducible member of the HSP70 family) alone was sufficient to reduce nuclear protein aggregation and accelerate refolding of luciferase after heat shock (42, 54).…”