Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1–HSF2 heterocomplexes

Abstract

Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity.

Evidence for a Functional Role of the Molecular Chaperone Clusterin in Amyloidotic Cardiomyopathy

“…9,10 Studies suggest that the regulation of CLU is directly linked to the heat shock response through HSF1-HSF2 heterocomplex binding to the CLU promoter; thus, modulation of CLU transcription occurs in stress-or disease-induced states. 11,12 Overexpression of CLU has been reported in Alzheimer's disease (AD) studies demonstrating that the chaperone complexes to soluble amyloid ␤ protein (A␤) and is present as a component of the amyloid plaques. In addition, CLU has been linked to cardiovascular diseases; it is a constituent of human atherosclerotic plaques, upregulated at both mRNA and protein levels in myocarditis and ischemia models, and localized to damaged myocardium in myocardial infarction.…”

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability

“…It was found that inactivation of HSF1 does not alter proteasome expression (Taylor et al, 2005) and our data are in accordance with this report as we show that Hsf1 À/À iMEFs exhibit a slight but not significant decrease in proteasome subunit expression. However, this slight decrease could be explained by the interdependence between HSF2 and HSF1 (Loison et al, 2006;Sandqvist et al, 2009). Thus, one could hypothesize that the lack of HSF1 would affect HSF2 binding and consequently reduce HSF2 activity.…”

“…Cell extracts were prepared using osmotic shock methods as previously described (Loison et al, 2006). Protein extracts were separated on polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Bioscience, Little Chalfont, England).…”

“…Proteasome inhibition leads to the accumulation of misfolded proteins and consequently induces expression of all the major HSPs (Bush et al, 1997), but Pirkkala et al (2000) clearly showed that HSF1, but not HSF2, has a key role in this induction. Nevertheless, to add to the complexity of this proteotoxic response, our group showed that an HSF1/HSF2 heterocomplex was present on the promoter of the chaperone gene clusterin, following proteasome inhibition (Loison et al, 2006). The existence of the HSF1/ HSF2 heterotrimer was recently confirmed (Sandqvist et al, 2009) and it was shown that HSF2 can act as a modulator of HSF1 activity in response to proteotoxic insults (Ostling et al, 2007).…”

From Humans to Experimental Models: The Cytoprotective Role of Clusterin in the Kidney

“…The molecular mechanism(s) for either constitutive or inducible expression of CLU have not been well investigated. It has been reported that CLU gene proximal promoter contains a 'clusterin element' (CLE) that is specifically bound by heat-shock factor (HSF) 1 after heat shock, or by HSF1-HSF2 up on proteasome inhibition [31,32], resulting in the induction of CLU gene transcription.…”