HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population.

Vibrio cholerae causes a dehydrating diarrheal illness that can be rapidly fatal in the absence of specific treatment. The organism is an historic scourge and, like similar infectious diseases, may have influenced the evolution of the human genome. We report here the results of the first candidate gene association study of cholera. In a family-based study of 76 pedigrees from Dhaka, Bangladesh, we evaluated the association between cholera and five candidate genes-the cystic fibrosis transmembrane receptor; lactoferrin; long palate, lung and nasal epithelium clone 1 (LPLUNC1); estrogen-related receptor alpha and calcium-activated chloride channel 1. We found a significant association with a marker in the promoter region of LPLUNC1 (rs11906665), a member of a family of evolutionarily conserved innate immunity proteins. An earlier microarray-based study of duodenal biopsies showed significantly increased expression of LPLUNC1 in cholera patients compared with healthy control subjects. Our results suggest that variation in host innate immune responses may influence the outcome of exposure to V. cholerae in an endemic setting.

Members of the 70-kilodalton heat shock protein family contain a highly conserved calmodulin-binding domain.

The 70-kilodalton heat shock protein (hsp70) family members appear to be essential components in a number cellular protein-protein interactions. We report here on the characterization of a new functional region in hsp70, a calmodulin-binding site. We have identified a 21-amino-acid sequence within the hsp70 protein that contains a calmodulin-binding domain. The peptide formed a potential amphipathic alpha helix and bound calmodulin with high affinity. Comparison of amino acid homology of this calmodulin-binding sequence with analogous hsp70 sequences from other species showed a high degree of conservation.

Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells.

We have developed a novel permeabilized-cell system to study transcription mechanisms. In permeabilized cells, heat-induced activation of the heat shock factor and transcription of the hsp70 gene require Ca2+. Activation involves at least two steps: Ca(2+)- and heat-dependent activation of heat shock factor binding and a second step, prior to transcription of hsp70, that requires ATP and is sensitive to genistein, a protein kinase inhibitor.

DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells.

The heat shock response is among the most highly conserved examples of regulated gene expression, being present in all cellular organisms. Transcriptional activation of heat shock genes by increased temperature or other cellular stresses is mediated by the binding of a heat shock factor (HSF) to a conserved nucleotide sequence (the heat shock element) present in the promoter of heat-inducible genes. Despite the high degree of conservation of this response, embryonic stages of development are characterized by the absence of a heat shock response. Murine erythroleukemia (MEL) cells also lack this response, and we report here a detailed characterization of this defect for one of the most highly conserved of these genes, hsp70. Surprisingly, heat-induced transcriptional activation of this gene does not occur, despite the induction of a protein with the binding specificity of murine HSF. However, the MEL HSF differs slightly in apparent size from the HSF in 3T3 cells, which exhibit a normal heat shock response. These data suggest that activation of mammalian HSF by heat requires at least two separate steps: an alteration of binding activity followed by further modification that activates transcription. MEL cells do not respond to heat shock because they lack the ability to perform this secondary modification. These cells provide a useful system for characterizing heat shock activation in mammals.

Effects of hyperglycemia and hyperthermia on the pH, glycolysis, and respiration of the Yoshida sarcoma in vivo.

Tissue (extracellular) pH (pHe) and intracellular pH (pHi) were measured together in vivo in the solid Yoshida sarcoma and normal organs (liver, gastrocnemius muscle) of noninbred Wistar rats. pHe was monitored by insertion of a miniature capillary glass electrode, and pHi was measured indirectly by equilibrium partitioning of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione across the cell membrane. Under normal conditions, tumor, liver, and gastrocnemius had a similar pHe of 7.05--7.30; tumor pHi was consistently higher (7.2) than that of the normal tissues (6.8--7.1). Curative hyperthermia (42 degrees C for 1 hr) did not significantly change tumor pHe or pHi. After ip glucose injection [6 g/kg body wt; blood glucose level greater than 400 mg/100 ml (22 mmoles/liter) for 4 hr], tumor pHe decreased markedly to 6.6 within 4 hours and did not return to normal for a further 12--14 hours, whereas tumor pHi was hardly affected. No marked change was noted in pHe or pHi of the normal organs following glucose loading of the host. In tumor slices removed from hyperglycemic hosts, marked reduction of both respiration and glycolysis was observed. Hyperglycemia (4 hr) plus hyperthermia at 40 degrees C (1 hr) had a synergistic inhibitory effect on metabolism that was equivalent to heat alone at 42 degrees C, and respiration and glycolysis almost ceased after 3--4 hours. However, tumor heating at 40 degrees C in hyperglycemic hosts was not equivalent to hyperthermia at 42 degrees C: With the former treatment, tumor regression did not occur, and animal survival did not differ from that of control untreated rats. The data do not support the postulate that the effects of heat on tumor cells are mediated via low pHi or that hyperglycemia leads to a lowered pHi which sensitizes the tumor to destruction at 40 degrees C instead of 42 degrees C.

Effect of hyperglycemia on blood flow, pH, and response to hyperthermia (42 degrees) of the Yoshida sarcoma in the rat.

Hyperglycemia (blood glucose, > 20 mmol/liter) caused a 90 to 100% inhibition of blood flow in the solid Yoshida sarcoma of rat feet, as measured by the fractional distribution of 86Rb and 133Xe clearance. Blood flow through the normal gastrocnemius muscle was increased by 50%, while liver blood flow remained unaltered. Hyperglycemia abrogated the temperature differential (approximately 1 degree) between the heating bath and the tumor, promoting more uniform tumor heating. During the period of reduced blood flow, the pH of the tumor extracellular fluid, measured by miniature glass electrode, declined from 7.19 to 6.63 due to decreased efflux of lactate from the tumor. Tumor intracellular pH, measured by partitioning of dimethyloxazolidinedione across the cell membrane, increased from 7.21 to 7.36. At a very high blood glucose concentration (50 mmol/liter), the tumor was isolated from the host, with almost total blockade of water, chloride, glucose, lactate, and dimethyloxazolidinedione exchange between the tumor and the blood. Hyperglycemia therefore represents a convenient means of isolating the Yoshida sarcoma from the host blood supply to enable more selective treatment with hyperthermia and possibly other modalities.

Gadd45 and Gadd153 messenger RNA levels are increased during hypoxia and after exposure of cells to agents which elevate the levels of the glucose-regulated proteins.

We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GRPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunicamycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels of gadd45 and gadd153 mRNA as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gadd153, and GRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expression of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and H7, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors H7 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein, H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by H7 and 2-aminopurine only.

Effect of hyperthermia (45 degrees C) on calcium flux in Chinese hamster ovary HA-1 fibroblasts and its potential role in cytotoxicity and heat resistance.

Hyperthermia caused a major increase in uptake of 45Ca2+ into Chinese hamster ovary HA-1 cells. Increased permeability to Ca2+ was observed with heating periods as brief as 45 degrees C for 4 min and reached a maximum at 45 degrees C for 30 min. In addition to elevation of Ca2+ influx, heat induced an increase in 45Ca2+ exchange with the extracellular Ca2+ pool. The effect of heat on Ca2+ permeability was transient, and Ca2+ influx returned to normal values by approximately 9 h at 37 degrees C. Comparison of the time courses of increased Ca2+ permeability and cell inactivation at 45 degrees C indicated that the heating time required for maximum permeability to Ca2+ was similar to the initial resistant "shoulder" period of the cell survival curve. This suggests that Ca2+ could play a permissive role in thermal cell inactivation; efficient cell killing may require a threshold concentration of intracellular Ca2+. The kinetics of heat-induced increase in Ca2+ permeability also resembled that for the induction of thermotolerance. This might suggest a messenger role for Ca2+ in thermotolerance induction. Direct increase in cellular Ca2+ levels with Ca2+ ionophore A23187 (5 X 10(-6) M) led to subsequent heat resistance. However, the heat resistance produced by A23187 was of a lesser magnitude than heat-induced thermotolerance. In addition, A23187 did not induce the stress protein species characteristic of thermotolerance (heat shock proteins), but instead led to the synthesis of a related set of proteins (glucose-regulated proteins). The data thus suggest a role for Ca2+ in the cellular effects of hyperthermia. They are also of potential clinical relevance in that cellular responses to heat might be modified pharmacologically, by the judicious use of Ca2+ active agents, such as Ca2+ ionophores and channel blockers.

X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates.

Extracellular signal-regulated kinases (ERKs), also known as mitogen-activated protein (MAP) kinases, are rapidly phosphorylated and activated in response to a number of external factors which promote growth and differentiation (T. G. Boulton, S. H. Nye, D. J. Robbins, N. Y. Ip, E. Radziejewska, S. D. Morgenbesser, R. A. DePinho, N. Panayotatos, M. H. Cobb, and G. D. Yancopoulos, Cell, 65: 663-675, 1991; S. L. Pelech and S. S. Jasbinder, Science (Washington DC), 257: 1355-1356, 1992; G. Thomas, Cell, 68: 3-6, 1992). We have identified two novel stimulators of MAP kinase activity, ionizing radiation and H2O2. Both radiation and H2O2, as well as the known agonist 12-O-tetradecanoylphorbol 13-acetate activate MAP kinase through the production of reactive oxygen intermediates. Our results demonstrate a direct link between the MAP kinase signal transduction pathway and reactive oxygen species and provide a unifying mechanism for activation of early- and late-response genes by inducers of oxidative stress.