Maintenance of glucocorticoid receptor function following severe heat-shock of heat-conditioned cells

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Abstract

The competence of the glucocorticoid receptor to regulate gene expression is thought to depend on Hsp70-driven continuous reactivation following spontaneous inactivation of its hormone-binding state. We show here that the glucocorticoid-binding capacity of HeLa cells fell with increasing temperature in the range 43–45 °C in a manner that closely paralleled the loss of soluble receptor protein. Receptor activity was maintained during moderate (43 °C) but not severe (45 °C) heat shock. Hsp70 was rapidly rendered insoluble and was replenished by soluble chaperone at 43 but not 45 °C. In heat-conditioned cells expressing different levels of Hsp70, we observed a positive correlation between the concentration of active receptor and the amount of Hsp70 rendered insoluble by heat shock. Much higher amounts of Hsp70 were rendered insoluble and receptor competence to regulate gene expression was preserved after severe heat shock of appropriately heat-conditioned cells. An excess of Hsp90 was found associated with resolubilized heat-inactivated receptor from severely heat-shocked cells. The data indicate that GR activity is maintained, provided that denaturation and/or aggregation of the receptor is prevented by Hsp70; and that the concentration of the chaperone is the limiting determinant of receptor activity in heat-shocked HeLa cells.

Introduction

Heat-induced denaturation of proteins in living cells adversely affects the integrity of the cytoskeleton, the nucleolus and the centrosome, inhibits transcription, pre-mRNA splicing and mRNA translation, reduces nuclear import and export of macromolecules and eventually leads to cell death (Welch, 1992). Heat-denatured proteins have a tendency to form large non-specific aggregates, and proteins in the nucleus show this effect most markedly (Michels et al., 1997). After recovery from a non-lethal heat shock cells can survive an extreme heat shock, which would otherwise be lethal; this phenomenon is known as transiently acquired thermotolerance. Resolubilization of protein aggregates, reassembly of heat-disrupted organelles and relief of inhibition of RNA and protein synthesis are substantially accelerated in heat-conditioned as compared to naı̈ve (not previously stressed) cells after heat shock (Mizzen and Welch, 1988; Welch, 1992).

All living cells respond to heat shock and other forms of physical and chemical stress by synthesising at enhanced rates a set of highly conserved proteins known as heat shock proteins. Prominent among these are the 70 and 90-kDa heat shock proteins (Hsp70 and Hsp90, respectively) (Welch, 1992; Hartl and Hayer-Hartl, 2002). Hsp70 is an ATPase constantly shuttling between the cytoplasm and nucleus, that binds to unfolded proteins in the presence of its co-chaperone partner, Hsp40, to prevent them from aggregating and to assist them in refolding to the native state (Hartl and Hayer-Hartl, 2002). Although the protein-folding activity of the Hsp70–Hsp40 chaperone machine is reportedly inhibited in vitro at temperatures higher than 41 °C, the ability of Hsp70 to hold denatured proteins in a refoldable state is not affected (Freeman and Morimoto, 1996). Partially unfolded proteins in particular are maintained in a refoldable state by Hsp90, an abundant ATPase, capable of providing Hsp70 with substrates for folding, and surmised itself to catalyse protein folding (Jakob et al., 1995; Freeman and Morimoto, 1996; Caplan, 1999). Both chaperones are predominantly located in the cytoplasm under normal growth conditions and relocate to the nucleus upon heat shock, Hsp90 much less readily than Hsp70 (Akner et al., 1992; Welch, 1992). While DnaK, the Hsp70 homologue in Escherichia coli, alone can resolubilize small protein aggregates (Skowyra et al., 1990), the dissolution of large aggregates in yeast has been reported to involve the Hsp104–Hsp70 chaperone machine, with Hsp70 being required to maintain the retrieved proteins in a soluble state (Glover and Lindquist, 1998). Drosophilla cells on the other hand, lack a functional Hsp104 homologue but express high levels of Hsp70, suggesting that survival following severe heart shock is possible solely by preventing denatured proteins from aggregating (Schirmer et al., 1996). Although members of the Hsp100 family of proteins have been identified in mammals (Schirmer et al., 1996), previous studies have implicated Hsp70 in promoting recovery of mammalian cells from heat shock (Lewis and Pelham, 1985).

Steroid hormone receptors (SHRs) are conformationally labile proteins, thought to constantly cycle between a hormone-binding state, capable of transducing hormonal signals into a transcriptional response, and an inactive state which does not bind hormone (Smith et al., 1995). That inactive receptor is absent from normally growing cells is taken to indicate a higher rate of hormone-responsive complex assembly, compared to turnover (Smith et al., 1995). Reactivation of inactive SHRs to the hormone-binding state is thought to proceed by way of Hsp70-driven, ATP-hydrolysis-dependent ‘maturation’ of an intermediate receptor complex with Hsp70, Hsp90 and the Hsp90/Hsp70-organizing protein (Hop) to the active receptor complex, which comprises a single receptor molecule, a dimer of Hsp90, an immunophilin and one molecule of the acidic protein p23 (Smith et al., 1995; Dittmar et al., 1997; Kosano et al., 1998; Pratt and Toft, 1997). This ‘maturation’ is envisaged to follow the conversion of Hsp90 from a holding to a folding conformation upon exchange of bound ADP for ATP (Dittmar et al., 1997; Kosano et al., 1998; Caplan, 1999), whereas turnover of the active complex is believed to follow ATP hydrolysis by Hsp90 (Prodromou et al., 1999). Since Hsp90, Hsp70, Hsp40 and p23 are subject to sequestration by heat-denatured proteins in vitro (Jakob et al., 1995; Bose et al., 1996; Freeman et al., 1996), one would expect that heat shock treatment of cells can adversely affect receptor activity. Accordingly, we and others have previously reported that heat shock inactivates the receptor complement of naı̈ve CHO and HeLa cells and that GR activity is regained 4–6 h after the shock (Anderson et al., 1991; Mitsiou and Alexis, 1995). Recently, Kosano et al. (1998) have shown that reactivation of in vitro heat-inactivated progesterone receptor upon addition of Hsp90, Hsp70, Hsp40, Hop and p23 is possible provided that Hsp70 and Hsp40 were present during heating to protect the receptor from irreversible thermal damage. In this study we set out to examine whether receptor activity can be fully maintained in appropriately heat-conditioned cells and whether it is the level of total Hsp70 or the extent of mobilisation of the chaperone to the insoluble fraction that relates to the maintenance of receptor activity in the heat-conditioned cells.

Section snippets

Materials, plasmids and antibodies

Human glyceraldehyde–phosphate dehydrogenase antisense template (pTRI-GAPDH) was obtained from Ambion and pGEX-2T was from Pharmacia. Plasmid ptkCAT5.1 has been described (Mitsiou and Alexis, 1995). Human GR expression plasmid pGR107 (Hollenberg et al., 1985) was generously provided by Dr. R. Evans (The Salk Institute, La Jolla, CA). Human HDJ-2 cDNA (Oh et al., 1993) was a kind gift of Dr. S. Kato (Sagami Chemical Research Center, Kanagawa, Japan). Plasmid pGST-hGR (304–428) was constructed by

Maintenance of GR activity following moderate but not severe heat shock of naı̈ve cells

To study the effect of heat shock on GR activity we compared the hormone-binding capacity of heat-shocked HeLa cells, as determined using a whole cell hormone binding assay, with the amount of soluble receptor protein. Since glucocorticoids promote receptor docking to nuclear acceptor sites and receptor protein turnover, thermal effects on receptor solubility and hormone binding activity were determined using cells maintained in culture medium supplemented with steroid-stripped serum. In

Discussion

In this study we determined the effect of heat shock on GR binding activity using [3H]TA and a whole cell hormone binding assay. We preferred [3H]TA to [3H]dexamethasone because mammalian ABC transporters have been reported to reduce the intracellular accumulation of dexamethasone but not TA (Kralli and Yamamoto, 1996), and [3H]TA to [3H]cortisol because of the higher affinity and lower non-specific binding of the former compared to the latter steroid (Ruh et al., 1981). Scatchard plot analysis

Acknowledgements

We thank Drs. C.A. Anderson, R.M. Evans, S. Kato, H. Schaller, G. Schütz and D.O. Toft for antibodies and plasmids. This work was supported in part by a PENED grant from the Greek General Secretariat for Research and Technology to M.N. Alexis. D.J.M. was supported by a Marie Curie Fellowship of the European Community programme ‘Training and Mobility of Researchers’ under contract number HPMF-CT-2000-00904.

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    Present address: Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY, USA.

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