Protective effects of hsp70 in inflammation

Inflammation results from the recruitement to a given tissue or organ and the activation of leucocytes, among which the monocytes-macrophages play a major role. These phagocytic cells produce high levels of reactive oxygen species (ROS) as well as cytokines. Whereas both ROS and cytokines have the potential to regulate the expression of heat shock (HS)/stress proteins (HSP), it appears that these proteins in turn have the ability to protect cells and tissues from the deleterious effects of inflammation. The mechanisms by which such protection occurs include prevention of ROS-induced DNA strand breaks and lipid peroxidation as well as protection from mitochondrial structure and function. In vivo, HS protects organs against a number of lesions associated with the increased production of ROS and/or cytokines. In an animal model for adult respiratory distress syndrome, an acute pulmonary inflammatory condition, HS completely prevented mortality. HSP (hsp70 in particular) may also exert protective effects in the immune system by contributing to the processing and presentation of bacterial and tumoral antigens. The analysis of the expression of hsp70 may prove of diagnostic and prognostic value in inflammatory conditions and therapeutical applications are being considered.     

Heat shock protein gene expression during Xenopus development

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.     

Heat shock proteins and infection: interactions of pathogen and host.

Invasive microorganisms encounter defensive attempts of the host to starve, destroy and eliminate the infection. In experimental model systems aiming to imitate defensive actions of the host, microorganisms respond by the rapid acceleration in the rate of expression of heat shock and other stress proteins. Heat shock proteins (hsp) of most if not all pathogens are major immune targets for both B- and T-cells. Host cells involved in the defensive action cannot avoid exposure to their own reactive compounds, such as oxygen radicals, resulting in premature cell death and tissue damage. Long-term consequences to the host may include cancer. In cells in tissue culture, induction of host-specific hsps occurs upon exposure to oxidants and in viral infections. Drugs that bind to members of the hsp70 family induce peroxisome proliferation and hepatocarcinoma, but may open the way for the development of novel drugs in support of antimetabolite treatment of infections and cancer.     

Heat shock proteins and infection: Interactions of pathogen and host

Invasive microorganisms encounter defensive attempts of the host to starve, destroy and eliminate the infection. In experimental model systems aiming to imitate defensive actions of the host, microorganisms respond by the rapid acceleration in the rate of expression of heat shock and other stress proteins. Heat shock proteins (hsp) of most if not all pathogens are major immune targets for both B- and T-cells. Host cells involved in the defensive action cannot avoid exposure to their own reactive compounds, such as oxygen radicals, resulting in premature cell death and tissue damage. Long-term consequences to the host may include cancer. In cells in tissue culture, induction of host-specific hsps occurs upon exposure to oxidants and in viral infections. Drugs that bind to members of the hsp70 family induce peroxisome proliferation and hepatocarcinoma, but may open the way for the development of novel drugs in support of antimetabolite treatment of infections and cancer.     

Developmental control of heat shock and chaperone gene expression Hsp70 genes and heat shock factors during preimplantation phase of mouse development

Heat shock genes are found in all organisms, and synthesis of heat shock proteins is induced by various stressors in nearly all the cells forming these organisms. However, a particular situation is noticed for hsp70 genes in mouse embryos at the beginning of their development. First, spontaneous expression of hsp70 is observed at the onset of zygotic genome activity. Second, inducible expression is delayed until morula or early blastocyst stages. A better understanding of both these points depends on a more careful analysis of hsp70 expression in relation to their major regulators, the heat shock factors. In this review, we will see how the development of the preimplanta tion embryo highlights the complexity of heat shock gene regulation involving trans-cis interactions and the cellular and nuclear environment.     

Hsp70 in parasites: as an inducible protective protein and as an antigen

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with thehsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.     

Heat shock protein gene expression during embryonic development of the zebrafish

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.     

Polymorphism in the regulatory sequence of the human hsp70-1 gene does not affect heat shock factor binding or heat shock protein synthesis

A bi-allelic polymorphism found in the regulatory region of the human heat shock (HS) protein (HSP) hsp70-1 gene, which comprises an A-->C transversion, 3 bp upstream of the HS element (HSE), has been associated with extended HLA haplotypes. In view of the chaperoning and protective functions of Hsp70, we investigated whether this hsp70-1 bi-allelic polymorphism could modulate the stress response, which may relate to enhanced resistance or susceptibility to certain diseases. We compared the basal and HS-induced HS factor (HSF)-binding activity of the two polymorphic HSEs, hsp70-1 mRNA accumulation and HSP expression in two human Epstein Barr virus (EBV)-transformed B cell lines typed for hsp70-1 promoter alleles. Our results suggest that hsp70-1 promoter polymorphism does not influence HSF-binding activity, hsp70 mRNA accumulation or synthesis in human EBV-transformed B cell lines.     

Role of hsp70 in cytokine production

Interleukin-1 and tumor necrosis factor-alpha are potent, multifunctional cytokine mediators of inflammation and immune responses that are produced primarily by activated monocytes and macrophages. Three published papers by different groups have shown that heat shock and chemical stress with heavy metal salts or sulfhydryl reagents, all of which induce the expression of heat shock protein 70 (hsp70), concomitantly inhibit the production of these cytokines in human monocytes and mouse macrophages activated by lipopolysaccharide. These papers are reviewed and discussed in some detail. Other studies suggest that various anti-inflammatory drugs, including acetylsalicyclic acid, auranofin and dexamethasone, can also facilitate HSP expression in macrophages. However, while these studies are interesting, it is clear that not a great deal of work has been done and/or published in this area. Since many pharmaceutical companies are developing cytokine synthesis inhibitors as potential anti-inflammatory drugs, one aim of this article is to emphasize that understanding the molecular mechanism(s) that lead to increased HSP expression and decreased cytokine biosynthesis may assist in achieving this goal.     

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.     

Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/Hsc70 inducibility

The source of intraspecies variation in the expression of heat shock proteins (HSPs) remains unresolved but could shed light on differential stress tolerance and disease susceptibility. This study investigated the influence of variable basal HSP synthesis on differential inducibility of HSP synthesis. Basal and heat-induced synthesis of the major HSP families in peripheral blood monocytes from healthy donors (n=42) were analysed using biometabolic labelling and densitometry. Basal Hsp70/Hsc70 synthesis and percentage induction of Hsp70/Hsc70 synthesis were significantly correlated (r=−0.57, p<0.0001), and described most accurately by an exponential decay equation (R=0.68, R²=0.46). This regression equation suggests that increasing levels of basal Hsp70/Hsc70 synthesis are accompanied byan exponential decrease in the percentage induction of Hsp70/Hsc70 synthesis. The model fits data from European and non-European population groups independently, although both coefficients in the regression equation were larger for non-Europeans. This implies population group as an additional factor influencing differential HSP expression. The differential inducibility of Hsp70/Hsc70 due to variable basal synthesis of Hsp70/Hsc70 and based upon population group may contribute to differential stress tolerance or disease susceptibility. Received 27 March 2000; received after revision 19 June 2000; accepted 20 June 2000     

The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling

The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations. Received 2 December 1998; received after revision 19 February 1999; accepted 18 March 1999     

Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome

Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by ‘click chemistry’ with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for example, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.     

The humoral immune response to heat shock proteins.

Humoral immune reactions to heat shock proteins (hsp) from microorganisms are one aspect of microbial infections in humans. The production of antibodies which are specific to epitopes present on procaryotic hsp leads also to the appearance of cross-reactive serum antibodies in the host organism that react with human hsp. This article discusses the consequences of such autoreactive antibodies for the host in context with the development of immune tolerance and autoimmune diseases, especially rheumatoid arthritis (RA), and in experimental animal models for arthritis such as adjuvant arthritis in rats. On the basis of epitope cross-reactivity between hsp and other host proteins, a hypothesis is presented for the development of autoimmune disease following the production of hsp-specific antibodies.     

The humoral immune response to heat shock proteins

Humoral immune reactions to heat shock proteins (hsp) from microorganisms are one aspect of microbial infections in humans. The production of antibodies which are specific to epitopes present on procaryotic hsp leads also to the appearance of cross-reactive serum antibodies in the host organism that react with human hsp. This article discusses the consequences of such autoreactive antibodies for the host in context with the development of immune tolerance and autoimmune diseases, especially rheumatoid arthritis (RA), and in experimental animal models for arthritis such as adjuvant arthritis in rats. On the basis of epitope cross-reactivity between hsp and other host proteins, a hypothesis is presented for the development of autoimmune disease following the production of hsp-specific antibodies.     

Heat shock response in the central nervous system

The heat shock response is induced in nervous tissue in a variety of clinically significant experimental models including ischemic brain injury (stroke), trauma, thermal stress and status epilepticus. Excessive excitatory neurotransmission or the inability to metabolically support normal levels of excitatory neurotransmission may contribute to neuronal death in the nervous system in many of the same pathophysiologic circumstances. We demonstrated that in vitro glutamate-neurotransmitter induced excitotoxicity is attenuated by the prior induction of the heat shock response. A short thermal stress induced a pattern of protein synthesis characteristic of the highly conserved heat shock response and increased the expression of heat shock protein (HSP) mRNA. Protein synthesis was necessary for the neuroprotective effect. The study of the mechanisms of heat shock mediated protection may lead to important clues as to the basic mechanisms underlying the molecular actions of the HSP and the factors important for excitotoxic neuronal injury. The clinical relevance of these findings in vitro is suggested by experiments performed by others in vivo demonstrating that pretreatment of animals with a submaximal thermal or ischemis stress confers protection from a subsequent ischemic insult.