Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by the binding of xenobiotic and endogenous ligands. AhR interacts with heat shock protein (Hsp) 90 complexes and can be used as a functional substrate to detect chaperone-dependent processes. Yeast Hsp90 (hsp82) mutants that variably affected AhR signaling were identified using reporter gene assays. Some mutated alleles resided in the p23/adenosine triphosphate (ATP)–binding pocket of Hsp90, so the relationship between the cochaperone Sba1 (yeast p23) and adenosine triphosphatase (ATPase) activity was investigated. Deletion of the p23 gene in the hsp82G170D mutant background had a greater effect on AhR signaling than the individual mutations, suggesting that these 2 mutations have separate actions on AhR signaling. In contrast, p23 overexpression suppressed temperature sensitivity and AhR signaling defects in the hsp82G170D mutant strain, suggesting that there is a relationship between these 2 proteins. The mutated hsp82G170D protein lacked detectable ATPase activity and p23 binding in vitro, which may relate to the weakened AhR signaling observed in mutant cells. Sba1 (p23) suppressed Hsp82 ATPase activity in vitro. These studies implicate the p23 protein and the G170 region of Hsp90 as being important, but not essential, for AhR signaling. Our results are consistent with a model in which p23 inhibits Hsp90 ATPase activity, thereby stabilizing ATP-Hsp90-client protein complexes.

Transcription of mammalian heat shock genes can be regulated by heat shock factors (HSF) 1 and 2. Although it has been shown previously that these factors respond to distinct stimuli, a broad analysis of the induction and function of these factors in living cells has not been performed. In our study, we assayed binding of human HSF1 and HSF2 at the promoters of 32 genes identified through LocusLink as heat shock genes in response to elevated temperature and hemin-induced differentiation in human K562 erythroleukemic cells using the chromatin immunoprecipitation technique. We also measured the induced expression of these genes under these 2 conditions. We found that 17 of the 32 genes were transcriptionally induced during heat shock, and HSF1 binding was detected at 15 of the 17 promoters. Nearly all the genes induced by heat shock were also induced to a lesser degree during hemin treatment. However, some genes were induced significantly more during hemin treatment than during heat shock. A new finding is that HSF1 and HSF2 bind to the same targets, but HSF1 binding is activated more by heat than by hemin treatment, and HSF2 binding is only activated by hemin treatment and not by heat. This technology also identified previously unknown HSF1 binding sites near genes that were previously shown to be heat inducible that may contribute to gene-specific regulation.

Heat shock protein 27 (HSP27) modulates actin-dependent cell functions in several systems. We hypothesized that HSP27 modulates wound contraction. Stably transfected fibroblast cell lines that overexpress HSP27 (SS12) or underexpress HSP27 (AS10) were established, and cell behaviors related to wound contraction were examined. First, fibroblast-populated collagen lattice (FPCL) contraction was examined because it has been studied as a wound-healing model. In floating FPCL contraction assays, SS12 cells caused increased contraction, whereas AS10 cells caused reduced contraction. Because floating matrix contraction is thought to be mediated by the tractional force of the cells, cell behaviors related to tractional force were examined. In collagen matrix, SS12 cells elongated faster and to a greater extent and contained longer stress fibers than control cells, whereas AS10 cells were slower to elongate than control cells. SS12 cells attached to the dishes more efficiently than the control, whereas AS10 cells attached less efficiently. Migration of SS12 cells on collagen-coated dishes was also enhanced, although AS10 cells did not differ from the control cells. In summary, HSP27 regulates fibroblast adhesion, elongation, and migration and the contraction of the floating matrix in a manner dependent on the level of its expression.

Sessile marine invertebrates undergo constant direct exposure to the surrounding environmental conditions, including local and global environmental fluctuations that may lead to fatal protein damage. Induction of heat shock proteins (Hsps) constitutes an important defense mechanism that protects these organisms from deleterious stress conditions. In a previous study, we reported the immunological detection of a 60-kDa Hsp (Hsp60) in the sea anemone Anemonia viridis (formerly called Anemonia sulcata) and studied its expression under a variety of stress conditions. In the present study, we show that the sponge Tetilla sp. from tidal habitats with a highly variable temperature regime is characterized by an increased level of Hsp60. Moreover, we show the expression of Hsp60 in various species among Porifera and Cnidaria, suggesting a general importance of this protein among marine invertebrates. We further cloned the hsp60 gene from A viridis, using a combination of conventional protein isolation methods and screening of a complementary deoxyribonucleic acid library by polymerase chain reaction. The cloned sequence (1764 bp) encodes for a protein of 62.8 kDa (588 amino acids). The 62.8-kDa protein, which contains an amino terminal extension that may serve as a mitochondrial targeting signal, shares a significant identity with mitochondrial Hsp60s from several animals but less identity with Hsp60s from either bacteria or plants.

Repeated mild heat shock (RMHS) has been shown to have several beneficial hormetic effects on human skin fibroblast undergoing aging in vitro. Because an age-related decline in proteasome activity is 1 of the reasons for the accumulation of abnormal proteins during aging, we have investigated the effects of RMHS on the 20S proteasome, which is the major proteolytic system involved in the removal of abnormal and oxidatively damaged proteins. Serially passaged human skin fibroblasts exposed to RMHS at 41°C for 60 minutes twice a week had increased 3 proteasomal activities by 40% to 95% in early- and midpassage cultures. RMHS-treated cells also contained a 2-fold higher amount of the proteasome activator 11S, and the extent of the bound activator was double in early- and midpassage cells only. Furthermore, there was no difference in the content of the 19S proteasome regulator in the stressed and the unstressed cells. Therefore, RMHS-induced proteasome stimulation in early- and midpassage fibroblasts appears to be due to an induction and enhanced binding of 11S proteasome activators. In contrast to this, the proteasomal system in late-passage senescent cells appears to be less responsive to the stimulatory effects of mild heat shock.

Carbon tetrachloride (CCl₄) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide–mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl₄-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3–12 hours after CCl₄ treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6–24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl₄-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl₄ treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl₄ treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl₄-treated rat liver to protect the hepatocytes from the damage being inflicted.

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = −3.708, P < 0.0001) but not with sex (r = −10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical cancer in animal model studies and in human clinical trials. I3C and its physiologic condensation product diindolylmethane (DIM) also induce apoptosis of tumor cells in vitro and in vivo, suggesting that these phytochemicals might be useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic acid microarray studies on transformed keratinocytes and tumor cell lines exposed to pharmacologic concentrations of DIM in vitro are consistent with a cellular response to nutritional deprivation or disruptions in protein homeostasis such as endoplasmic reticulum (ER) stress. In this report we investigate whether specific stress response pathways are activated in tumor cells exposed to DIM and whether the ER stress response might contribute to DIM's cytotoxicity. Induction of the stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78, GRP94, and asparagine synthase was documented by Western blot and real-time reverse transcriptase–polymerase chain reaction in C33A cervical cancer cells, and induction of a subset of these was also observed in cancer cell lines from breast (MCF-7) and prostate (DU145). The results are consistent with activation of more than 1 stress response pathway in C33A cells exposed to 75μM DIM. Phosphorylation eIF2α was rapidly and transiently increased, followed by elevated levels of ATF4 protein. Activation of IRE1α was indicated by a rapid increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional activation dependent on an ATF6–XBP-1 binding site was detected by transient expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT); induction of the GADD153 (CHOP) promoter was also confirmed by transient expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved at later times, coinciding with the onset of apoptosis. The results support the hypothesis that cytotoxic concentrations of DIM can activate cellular stress response pathways in vitro, including the ER stress response. Conversely, DIM was especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing degrees; nutrient limitation had a similar, but even more pronounced, effect. Because DIM toxicity in vitro is enhanced in cells undergoing nutritional deprivation and ER stress, it is possible that stressed cells in vivo, such as those within developing solid tumors, also have increased sensitivity to killing by DIM.

The induction of heat shock proteins (Hsps) serves not only as a marker for cellular stress but also as a promoter of cell survival, which is especially important in the nervous system. We examined the regulation of the constitutive and stress-induced 70-kD Hsps (Hsc70 and Hsp70, respectively) after sciatic nerve (SN) axotomy in the neonatal mouse. Additionally, the prevention of axotomy-induced SN cell death by administration of several preparations of exogenous Hsc70 and Hsp70 was tested. Immunohistochemistry and Western blot analyses showed that endogenous levels of Hsc70 and Hsp70 did not increase significantly in lumbar motor neurons or dorsal root ganglion sensory neurons up to 24 hours after axotomy. When a variety of Hsc70 and Hsp70 preparations at doses ranging from 5 to 75 μg were applied to the SN stump after axotomy, the survival of both motor and sensory neurons was significantly improved. Thus, it appears that motor and sensory neurons in the neonatal mouse do not initiate a typical Hsp70 response after traumatic injury and that administration of exogenous Hsc/Hsp70 can remedy that deficit and reduce the subsequent loss of neurons by apoptosis.

Angiotensin II (Ang II) is a potent vasoconstrictor and induces inflammation and end-organ injury through its activation of the proinflammatory transcription factor, nuclear factor–κB (NF-κB). Heat shock (HS) treatment with subsequent expression of heat shock proteins (Hsps) is an effective strategy for tissue protection against oxidative injuries. Recently, HS and Hsps have been shown to interact with NF-κB in tissue injury. In this study, we investigated whether HS could protect against Ang II–induced hypertension and inflammation by inhibiting NF-κB. Sprague-Dawley rats were divided into control and HS groups. Control and 24-hour post–heat shocked rats were treated with Ang II. At days 1, 3, 5, 7, 11, and 14 after Ang II administration, systolic blood pressures were measured by tail-cuff plethysmography, and aorta tissues were collected. Aorta NF-κB deoxyribonucleic acid–binding activity was measured by electrophoretic mobility shift assay, and NF-κB p65 subunit, Hsp70, Hsp27, and interleukin-6 (IL-6) expressions were measured by Western analysis. HS treatment significantly decreased Ang II–induced hypertension. The activation of NF-κB in aorta by Ang II was suppressed by HS treatment. The elevated expression of IL-6 induced by Ang II treatment was also decreased by HS treatment. Although Ang II treatment induced an increase in Hsp70 and Hsp27, HS treatment induced a greater elevation of Hsp70 and Hsp27 expression. HS treatment protects against Ang II–induced hypertension and inflammation. This protection may relate to the interaction of Hsps and the NF-κB pathway.