To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1.

BackgroundThe heat shock response (HSR) and the unfolded protein response (UPR) are both activated by proteotoxic stress, although in different compartments, and share cellular resources. How these resources are allocated when both responses are active is not known. Insight in possible crosstalk will help understanding the consequences of failure of these systems in (age-related) disease.ResultsIn heat stressed HEK293 cells synthesis of the canonical UPR transcription factors XBP1s and ATF4 was detected as well as HSF1 independent activation of the promoters of the ER resident chaperones HSPA5 (BiP) and DNAJB9 (ERdj4). However, the heat stress activation of the DNAJB9 promoter, a XBP1s target, was not blocked in cells expressing a dominant negative IRE1α mutant, and thus did not require XBP1s. Furthermore, the DNA element required for heat stress activation of the DNAJB9 promoter is distinct from the ATF4 and ATF6 target elements; even though inhibition of eIF2α phosphorylation resulted in a decreased activation of the DNAJB9 promoter upon heat stress, suggesting a role for an eIF2α phosphorylation dependent product.ConclusionsThe initial step in the UPR, synthesis of transcription factors, is activated by heat stress but the second step, transcriptional transactivation by these factors, is blocked and these pathways of the UPR are thus not productive. Expression of canonical ER chaperones is part of the response of heat stressed cells but another set of transcription factors has been recruited to regulate expression of these ER chaperones.

The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction.

Mammalian cells respond to a lack of amino acids by activating a transcriptional program with the transcription factor ATF4 as one of the main actors. When cells are faced with cytoplasmic proteotoxic stress, a quite different transcriptional response is mounted, the heat shock response, which is mediated by HSF1. Here, we show that amino acid deprivation results in the inactivation of HSF1. In amino acid deprived cells, active HSF1 loses its DNA binding activity as demonstrated by EMSA and ChIP. A sharp decrease in the transcript level of HSF1 target genes such as HSPA1A (Hsp70), DNAJB1 (Hsp40), and HSP90AA1 is also seen. HSPA1A mRNA, but not DNAJB1 mRNA, was also destabilized. In cells cultured with limiting leucine, HSF1 activity also declined. Lack of amino acids thus could lead to a lower chaperoning capacity and cellular frailty. We show that the nutrient sensing response unit of the ASNS gene contains an HSF1 binding site, but we could not detect binding of HSF1 to this site in vivo. Expression of either an HSF1 mutant lacking the activation domain (HSF379) or an HSF1 mutant unable to bind DNA (K80Q) had only a minor effect on the transcript levels of amino acid deprivation responsive genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-012-0347-1) contains supplementary material, which is available to authorized users.

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