Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs

NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-gamma; stimulation of macrophages with mixtures of IFN-gamma and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.     

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.     

Heat shock proteins: essential proteins for apoptosis regulation

Many different external and intrinsic apoptotic stimuli induce the accumulation in the cells of a set of proteins known as stress or heat shock proteins (HSPs). HSPs are conserved proteins present in both prokaryotes and eukaryotes. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. HSPs have a protective function, that is they allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several of these proteins have demonstrated to directly interact with components of the cell signalling pathways, for example those of the tightly regulated caspase-dependent programmed cell death machinery, upstream, downstream and at the mitochondrial level. HSPs can also affect caspase-independent apoptosis-like process by interacting with apoptogenic factors such as apoptosis-inducing factor (AIF) or by acting at the lysosome level. This review will describe the different key apoptotic proteins interacting with HSPs and the consequences of these interactions in cell survival, proliferation and apoptotic processes. Our purpose will be illustrated by emerging strategies in targeting these protective proteins to treat haematological malignancies.     

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.     

Heat shock proteins HSP70 and GP96: structural insights

Several heat shock proteins (HSPs) act as potent adjuvants for eliciting anti-tumor immunity. HSP-based tumor vaccine strategies have been highly successful in animal models and are undergoing testing in clinical trials. It is generally accepted that HSPs, functioning as chaperones for tumor antigens, elicit tumor-specific adaptive immune responses. HSPs also appear to induce innate immune responses in an antigen-independent fashion. Innate responses generated by HSPs may contribute to anti-tumor immunity. Immunologically active chaperones with anti-tumor activity are referred to as “immunochaperones”. Here, we review the studies that address the role of structural domains or regions of the immunochaperones HSP70 and GP96 that may be involved in the induction of adaptive or innate immune responses. This article forms part of the Symposium in Writing “Thermal stress-related modulation of tumor cell physiology and immune responses”, edited by Elfriede Noessner.     

Identification and induction of hsp70 gene by heat shock and cadmium exposure in carp

A member of the multi-gene family, encoding 70 kD stress proteins, was identified from the common carp (Cyprimus carpio). Homologies, observed at both nucleic acid and amino acid levels, and also the intronless structure of this gene, strongly suggest that it corresponds to a heat-inducible hsp70 gene in carp. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp70 mRNA levels and to follow the inducer-specific expression of this gene in different tissues during in vivo studies. Carp hsp70 mRNA is not detectable in the brain and muscle, and its concentration is around the limit of detection in the kidney and liver of unstressed animals. The expression of hsp70 is induced by elevated temperature and it responds to Cd treatment in a tissue and time-dependent manner.     

Heat shock proteins as potential targets in the therapy of inflammatory arthritis

Whether heat shock proteins (hsp) will be therapeutic targets in arthritis depends on their role in pathogenesis. In this article, three possibilities are considered. Firstly, an excessive immune response to bacterial hsp could be arthritogenic — as may occur in reactive arthritis. In these circumstances therapy would be directed to down-regulating this immune response, or altering the nature of the immune response e.g. by changing cytokine production from interferon-g to IL-4. However this approach depends on the immune response to bacterial hsp not being critical for control of the bacterial infection. Secondly, an immune response to bacterial hsp may induce autoimmunity by cross-reactivity, e.g. with the homologous human. This could also be modulated in the same way with a lower likelihood of interfering with control of the infectious agent, since only a component of the immune response against the bacterial hsp will be cross-reactive with self. Thirdly, recent experiments raise the possibility that joint inflammation might be controlled by T cells which recognizes self hsp, particularly hsp60. Therapies might enhance this response; protection from experimental arthritis by prior immunization with hsp60 is well established. Whether similar approaches will be viable after arthritis is established remains to be seen.     

Heat shock and ecdysterone activation of the Drosophila melanogaster hsp23 gene; a sequence element implied in developmental regulation.

The regulation of the Drosophila melanogaster hsp23 gene by heat shock and ecdysterone has been analysed by measuring activities of hsp--Escherichia coli beta-galactosidase hybrid genes in transfected hormone-sensitive D. melanogaster cells. Mutation analysis identified multiple, distinct promoter elements. A sequence element, which also occurs in the promoters of several other developmentally regulated Drosophila genes, is present in regions of the hsp23 promoter that are essential for its ecdysterone, but not its heat-regulated activity; this element may represent a binding site for an ecdysterone--receptor complex. Mutant promoters that can be activated only by heat shock or by hormone have been constructed. Thus the two types of regulation of the hsp23 gene can function independently of each other.     

Heat shock proteins 27, 40, and 70 as combinational and dual therapeutic cancer targets

The heat shock proteins are essential players in the development of cancer and they are prime therapeutic targets. Targeting multiple hsps in dual therapies decreases the likelihood of drug resistance compared to utilizing mono-therapies. Further, employing an hsp inhibitor in combination with another therapy has proven clinically successful. Examples of efficacious strategies include the inhibition of hsp27, which prevents protein aggregation, controlling hsp40’s role as an ATPase modulator, and inhibiting hsp70 from acting as a molecular chaperone. While hsp40 therapies are just in the beginning stages, hsp27 and hsp70 therapies have been successfully used in dual inhibition treatments with hsp90 inhibitors and in combinational therapy with antineoplastic drugs. Both dual and combinatorial therapies show encouraging results when used in treating chemotherapeutically resistant diseases.