Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.     

PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response

Background

Under stress, AMP-activated protein kinase (AMPK) plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP) expression under stress is unclear.

Methodology/Principal Findings

We found that heat stress induced dephosphorylation of AMPKα subunit (AMPKα) in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKα under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A) inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKα dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl₂, celastrol, MG132)-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKα expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability.

Conclusions/Significance

These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress.     

HSP27 and HSP70: potentially oncogenic apoptosis inhibitors

Stress or heat shock proteins (HSPs) such as HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including heat, reactive oxygen species or anticancer drugs. Their overexpression allows cells to survive to otherwise lethal conditions. Several different mechanisms may account for the cytoprotective activity of HSP27 and HSP70. First, both proteins are powerful chaperones. Second, both inhibit key effectors of the apoptotic machinery including the apoptosome, the caspase activation complex (both HSP27 and HSP70), and apoptosis inducing factor (only HSP70). Third, they both play a role in the proteasome-mediated degradation of apoptosis-regulatory proteins. HSP27 and HSP70 may participate in oncogenesis, as suggested by the fact that overexpression of heat shock proteins can increase the tumorigenic potential of tumor cells. The down-regulation or selective inhibition of HSP70 might constitute a valuable strategy for the treatment of cancer.     

HSP27 and HSP70: Potentially Oncogenic Apoptosis Inhibitors

Stress or heat shock proteins (HSPs) such as HSP27 and HSP70 are expressed in response to a wide variety of physiological and environmental insults including heat, reactive oxygen species or anticancer drugs. Their overexpression allows cells to survive to otherwise lethal conditions. Several different mechanisms may account for the cytoprotective activity of HSP27 and HSP70. First, both proteins are powerful chaperones. Second, both inhibit key effectors of the apoptotic machinery including the apoptosome, the caspase activation complex (both HSP27 and HSP70), and apoptosis inducing factor (only HSP70). Third, they both play a role in the proteasome-mediated degradation of apoptosis-regulatory proteins. HSP27 and HSP70 may participate in oncogenesis, as suggested by the fact that overexpression of heat shock proteins can increase the tumorigenic potential of tumor cells. The down-regulation or selective inhibition of HSP70 might constitute a valuable strategy for the treatment of cancer.     

Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5′ truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.     

Cell cycle-dependent association of HSP70 with specific cellular proteins

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.