Acetylsalicylic acid-induced release of HSP70 from mast cells results in cell activation through TLR pathway

OBJECTIVE: Mast cells are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat shock proteins (HSPs). It has been reported that HSPs are capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system. In the current study, we explored whether the stress response induces HSPs and affects the signaling pathways of mast cells. METHODS: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to exogenous and endogenous HSP70 was monitored by measuring cytokine release. RESULTS: Using BMMC, we show that treatment with heat shock or acetylsalicylic acid results in a selective induction of HSPs, and leads to release of HSP70 into the extracellular environment. The release of HSP70 from mast cells may be of functional importance. We found that after induction of HSP70, the production of TNF-alpha and IL-6 was increased. In a number of experiments, we demonstrated that exogenous/secreted HSP70 is most likely responsible for the activation of mast cells to produce cytokines. Extracellular HSP70 induced production of TNF-alpha and IL-6 through the activation of the TLR4 receptor pathway, which was evidenced by an abrogation of the response in mast cells cultured from TLR4(null) or HSF-1(-/-) mice. CONCLUSION: Our experiments suggest that stress conditions can induce pro-inflammatory cytokine production by mast cells through an autocrine or paracrine stimulation of TLR receptors after a heat shock response. The recognition that heat shock proteins induce mast cell activation suggests an involvement of these cells in the immunological processes induced by heat shock response.     

Regulation of human heme oxygenase-1 gene expression under thermal stress

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.     

Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats.

The effect of aging on the induction of heat shock protein 70 (HSP70)-encoding gene expression by elevated temperatures was studied in cultures of lung- or skin-derived fibroblasts from young (5 mo) and old (24 mo) male Wistar rats. Although the kinetics of the heat shock response were found to be similar in the two age groups, we observed lower levels of induction of HSP70 mRNA and HSP70 protein in confluent primary lung and skin fibroblast cultures derived from aged animals. Additional experiments with freshly excised lung tissue showed a similar age-related decline in the heat-induced expression of HSP70.     

Chaperone-mediated in vitro assembly of Polyomavirus capsids

The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximately 70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.     

A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation.

The 70-kDa heat shock proteins (hsp70s) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate proteins coupled to cycles of ATP hydrolysis. In the prokaryote Escherichia coli, the hsp70 DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp70s and DnaJ-related proteins have been identified in eukaryotes, to our knowledge no GrpE-related proteins have been reported. We report the isolation and characterization of a eukaryotic grpE-related gene, MGE1. MGE1, an essential nuclear gene of the yeast Saccharomyces cerevisiae, encodes a soluble protein of the mitochondrial matrix. Cells with reduced expression of Mge1p accumulate the precursor form of a mitochondrial protein. Since mitochondrial hsp70 is required for translocation of precursors of mitochondrial proteins from the cytosol into the matrix of mitochondria, these data suggest that Mge1p acts in concert with mitochondrial hsp70 in protein translocation.     

Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells

Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.     

Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner

We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.     

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene.

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress.