Manipulation of protein kinases reveals different mechanisms for upregulation of heat shock proteins in motor neurons and non-neuronal cells
Introduction
A common cellular response to physiological and environmental stress is increased production of heat shock proteins (Hsps) (Bukau and Horwich, 1998, Hartl, 1996, Morimoto and Santoro, 1998). Evidence points to a chaperone-based mechanism controlling transactivation of genes encoding stress-inducible heat shock genes, whereby activation of the major, stress-responsive transcription factor, heat shock transcription factor 1 (Hsf1) is tightly controlled at a series of checkpoints (Voellmy, 2004). The first checkpoint is sequestration of monomeric Hsf1 in the cytoplasm by Hsp90/multichaperone complexes that repress the activity of two nuclear localization signals and several domains that promote trimerization (Baler et al., 1993, Rabindran et al., 1993, Zou et al., 1998). Damaged proteins (e.g., thermally denatured, oxidized, etc.) compete for constitutive, complex-forming Hsps, relieving this inhibition and allowing nuclear accumulation of Hsf1 homotrimers, which bind to heat shock elements (HSE) upstream of genes encoding stress response proteins including Hsps (Pelham, 1982, Wu, 1984, Zuo et al., 1995). The second checkpoint is that DNA binding is not sufficient for initiation of transcription. A subsequent reaction that involves hyperphosphorylation of Hsf1 is required to achieve the transcriptionally active state (Sarge et al., 1993, Sorger et al., 1987, Sorger and Pelham, 1988, Xia and Voellmy, 1997). There is also evidence of another Hsp90/multichaperone complex in the nucleus that binds and inhibits a subset of DNA-bound Hsf1 trimers (Guo et al., 2001). The displacement of these inhibitory complexes from Hsf1 by the nuclear protein Daxx greatly enhanced transactivation of heat shock genes in HeLa and HEK293 cells (Boellmann et al., 2004).
Stress-induced hyperphosphorylation of Hsf1 occurs concomitant with transactivation of heat shock genes and is detected by retardation of the electrophoretic mobility of Hsf1 in SDS-PAGE and Western blotting (Hensold et al., 1990, Xia and Voellmy, 1997). The importance of hyperphosphorylation is also demonstrated by studies showing impairment of Hsp70 induction (Erdos and Lee, 1994, Yamamoto et al., 1994) and Hsf1 activity (Baek et al., 2001, Ohnishi et al., 1999, Xia and Voellmy, 1997) by protein kinase inhibitors and activation by protein phosphatase inhibitors (Chang et al., 1993, Xia and Voellmy, 1997). Several studies have addressed the role of phosphorylation at specific amino acid residues and the kinases involved (Table 1).
Most studies of Hsf1 regulation have been carried out using homogenous, rapidly dividing cell lines. However, many post-mitotic neuronal subtypes fail to induce Hsps in response to stress (Brown and Rush, 1999, Kaarniranta et al., 2002, Manzerra and Brown, 1996, Marcuccilli et al., 1996, Maroni et al., 2003, Tonkiss and Calderwood, 2005). Motor neurons, the primary target cells in diseases such as amyotrophic lateral sclerosis (ALS), have a high threshold for inducing a heat shock response both in vivo (Manzerra and Brown, 1992, Manzerra and Brown, 1996) and in primary culture (Batulan et al., 2003). Non-steroidal anti-inflammatory drugs (NSAIDs), which in non-neuronal cells increase transactivation of heat shock genes in response to stress by enhancing DNA binding of Hsf1 trimers (Cotto et al., 1996, Jurivich et al., 1992, Jurivich et al., 1995), failed to overcome the high threshold for the heat shock response in motor neurons (Batulan et al., 2005). Overexpression of wild-type Hsf1 (Hsf1wt) was also unable to complement the deficient stress response despite its accumulation in the nucleus of motor neurons (Batulan et al., 2003). However, overexpression of a constitutively active form of Hsf1 (Hsf1act), with the regulatory domain deleted (amino acids 203–315), induced robust expression of Hsp70 and Hsp40 in cultured motor neurons (Batulan et al., 2006), suggesting that impaired phosphorylation of key residues in this region may account for the high threshold of activation.
This study examined whether manipulating phosphorylation at residues in the regulatory domain of DNA-bound Hsf1 trimers could trigger heat shock response in motor neurons by testing the previously described activation effect of phosphorylation at Ser230 by calcium/calmodulin-dependent kinase IIα (CaMKIIα) and inhibitory action of Ser303 phosphorylation by glycogen synthase kinase 3β (GSK3β). Protein kinase C (PKC), which had a variable effect on Hsf1 activation in previous studies, was also tested. Activation of PKC by phorbol ester, inhibition of GSK3β by lithium chloride (LiCl), and overexpression of two CaMKIIα splice variants all failed to induce Hsp70 in the presence or absence of heat shock. However, overexpression of a constitutively active form of CaMKIV (CaMKIVact) did induce expression of Hsp70 in motor neurons, but likely through an Hsf1-independent mechanism.
Section snippets
PKC activation induced Hsp70 in fibroblasts, but not motor neurons
Increased expression of Hsp70 following treatment with phorbol ester has been described in many studies (Ding et al., 1997, Holmberg et al., 1997, Holmberg et al., 1998, Jacquier-Sarlin et al., 1995, Xia and Voellmy, 1997). For example, treatment with 1 μM phorbol 12 myristate 13-acetate (PMA) increased nuclear translocation and activation of Hsf1 in human epidermoid A431 cells, resulting in increased Hsp70 expression that was highest after 8 h (Ding et al., 1997). Adapting this protocol, mouse
Discussion
The central nervous system is particularly sensitive to heat stress (Khan and Brown, 2002) and a milder or impaired heat shock response in many types of neurons may contribute to this vulnerability (Batulan et al., 2003, Brown and Rush, 1999, Kaarniranta et al., 2002, Manzerra and Brown, 1996, Marcuccilli et al., 1996, Maroni et al., 2003). Hsf1 is the major transcription factor mediating induction of heat shock genes in response to stress (Morimoto, 1998, Voellmy, 2004). Although neurons can
Spinal cord-dorsal root ganglion (DRG) cultures
Primary cultures of dissociated spinal cord and DRG were prepared from embryonic day 13 CD1 mice (Charles River Laboratories, Wilmington, MA) as previously described (Roy et al., 1998). Following removal from the embryos and dissociation by mincing and incubating with trypsin (Invitrogen Life Technologies, Burlington, ON), cells were plated onto 18 mm glass coverslips (Merlan Scientific Ltd., Georgetown, ON) at a density of 350,000–400,000 in 12-well culture dishes (BD Biosciences, Mississauga,
Acknowledgments
This research was supported by grants to HD from the Canadian Institutes for Health Research (CIHR), CIHR/MDC/ALS Society of Canada neuromuscular partnership, and the ALS Association (ALSA). DT is a recipient of the McGill Faculty of Medicine Internal Studentship. The authors thank Dr. Howard Schulman for the kind gift of all CaMK plasmids; Dr. Richard Voellmy and Alexis Hall for the hsf1 plasmids and valuable discussion; Dr. Richard Morimoto for the pEGFP-N2 reporter plasmid and valuable
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