The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation.

 Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status, a cellular-adaptive response occurs requiring functional chaperones, antioxidant production, and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes–induced nephropathy and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring advanced glycation end-products (pentosidine), protein oxidation (protein carbonyls [DNPH]), and lipid oxidation (4-hydroxy-2-nonenal [HNE] and F2-isoprostanes) in plasma, lymphocytes, and urine, whereas the lymphocyte levels of the heat shock proteins (Hsps) heme oxygenase-1 (HO-1), Hsp70, and Hsp60 as well as thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. We found increased levels of pentosidine (P < 0.01), DNPH (P < 0.05 and P < 0.01), HNE (P < 0.05 and P < 0.01), and F2-isoprostanes (P < 0.01) in all the samples from type 2 diabetic patients with nephropathy with respect to control group. This was paralleled by a significant induction of cellular HO-1, Hsp60, Hsp70, and TrxR-1 (P < 0.05 and P < 0.01). A significant upregulation of both HO-1 and Hsp70 has been detected also in lymphocytes from type 2 diabetic patients without uraemia. Significant positive correlations between DNPH and Hsp60, as well as between the degree of renal failure and HO-1 or Hsp70, also have been found in diabetic uremic subjects. In conclusion, patients affected by type 2 diabetes complicated with nephropathy are under condition of systemic oxidative stress, and the induction of Hsp and TrxR-1 is a maintained response in counteracting the intracellular pro-oxidant status.

Estrogen (E₂) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2IG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E₂ in estrogen receptor–positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor–negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and anti-estrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E₂- and Cd-induced Hsp22 was incorporated into high–molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E₂ and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor– positive breast cancer cells.

A major problem in autologous stem cell transplantation is the occurrence of relapse by residual neoplastic cells from the graft. The selective toxicity of hyperthermia toward malignant hematopoietic progenitors compared with normal bone marrow cells has been utilized in purging protocols. The underlying mechanism for this selective toxicity has remained unclear. By using normal and leukemic cell line models, we searched for molecular mechanisms underlying this selective toxicity. We found that the differential heat sensitivity could not be explained by differences in the expression or inducibility of Hsp and also not by the overall chaperone capacity of the cells. Despite an apparent similarity in initial heat-induced damage, the leukemic cells underwent heat-induced apoptosis more readily than normal hematopoietic cells. The differences in apoptosis initiation were found at or upstream of cytochrome c release from the mitochondria. Sensitivity to staurosporine-induced apoptosis was similar in all cell lines tested, indicating that the apoptotic pathways were equally functional. The higher sensitivity to heat-induced apoptosis correlated with the level of Bcl-2 protein expression. Moreover, stable overexpression of Bcl-2 protected the most heat sensitive leukemic cells against heat-induced apoptosis. Our data indicate that leukemic cells have a specifically lower threshold for heat damage to initiate and execute apoptosis, which is due to an imbalance in the expression of the Bcl-2 family proteins in favor of the proapoptotic family members.

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.

The heat shock response involves activation of heat shock transcription factor 1 (Hsf1) followed by the rapid synthesis of the protective heat shock proteins (Hsps). To determine if the stress experienced during streptozotocin (STZ)-induced diabetes altered the heat shock response, male Sprague-Dawley rats (n = 33; 280–300 g) were assigned to 4 groups: (1) control, (2) diabetic (30 days after 55 mg/kg STZ i.v.), (3) heat stressed (42°C for 15 minutes), and (4) diabetic heat-stressed group (heat stressed 42°C for 15 minutes, 30 days after 55 mg/kg STZ i.v.). The content of Hsp72, Hsp25, and Hsf1 in skeletal muscles, heart, kidney, and liver was assessed by Western blotting, while electrophoretic mobility shift gel analysis was used to assess Hsf activation. Without heat stress, the constitutive expression of Hsp25, Hsp72, and Hsf1 in tissues from diabetic animals and controls was similar. However, 24 hours following heat stress, the heart, kidney, and liver from diabetic animals showed an increased Hsp72 and Hsp25 content compared to the same tissues from heat-stressed nondiabetic animals (P < 0.05). The white gastrocnemius and plantaris muscles from heat-stressed animals (diabetic and nondiabetic) both showed significant and similar elevations in Hsp72 content. Interestingly, while all muscles from nondiabetic animals showed significant (P < 0.05) increase in Hsp25 content after heat stress, no increase in Hsp25 content was detected in muscles from heat-stressed diabetic animals. As expected, Hsf activation was undetectable in all tissues from non–heat-stressed animals but was detectable in tissues from both diabetic and nondiabetic animals following heat stress with the exception of diabetic skeletal muscle, where it was attenuated. Hsf1 content was unaltered in all tissues examined except in the white gastrocnemius muscles from heat-stressed diabetic animals. where it was undetectable. These results suggest that when tissues from STZ-induced diabetic animals are heat stressed, the Hsp/stress response is altered in a tissue-specific manner. This impaired ability to activate the stress response may explain, at least in part, the selective atrophy of certain muscles or muscle fiber types during diabetes.

Hsp90 is an abundant and highly conserved chaperone that functions at later stages of protein folding to maintain and regulate the activity of client proteins. Using a recently described in vitro system to fold a functional model kinase Chk1, we performed a side-by-side comparison of the Hsp90-dependent chaperoning of Chk1 to that of the progesterone receptor (PR) and show that these distinct types of clients have different chaperoning requirements. The less stable PR required more total chaperone protein(s) and p23, whereas Chk1 folding was critically dependent on Cdc37. When the 2 clients were reconstituted under identical conditions, each client folding was dose dependent for Hsp90 protein levels and was inhibited by geldanamycin. Using this tractable system, we found that Chk1 kinase folding was more effective if we used a type II Hsp40 cochaperone, whereas PR is chaperoned equally well with a type I or type II Hsp40. Additional dissection of Chk1-chaperone complexes and the resulting kinase activity suggests that kinase folding, like that previously shown for PR, is a dynamic, multistep process. Importantly, the cochaperones Hop and Cdc37 cooperate as the kinase transitions from immature Hsp70- to mature Hsp90-predominant complexes.

Heat shock proteins are induced under stress conditions and they act as molecular chaperones to refold denatured polypeptides. Stress resistances including thermotolerance generally are correlated with levels of the heat shock proteins. We investigated a fruit fly gene encoding a small heat shock protein, Hsp27, to determine if it functions in stress response of Drosophila melanogaster. A knockout Hsp27 allele was generated. Flies homozygous for this allele were viable, without obvious defects, and fertile, indicating Hsp27 is not essential for development. In stress-response tests, loss of the Hsp27 gene caused no defects in resistance to heat shock or oxidative treatments. However, a significant reduction in starvation resistance was associated with the genotype without a functional Hsp27 gene. The data suggest that the Drosophila HSP27 protein acts as a chaperone to provide cellular stress resistance, although its function may be limited to a subset of the stress response such as the starvation resistance.

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3δ) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn² or Co² , but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn² /Co² -dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.

The Whitehall Study is a prospective epidemiological study of cardiovascular risk factors in healthy members of the British Civil Service, which has identified psychological distress as a major risk factor for coronary heart disease. The levels of circulating Hsp60 in 860 participants from the Whitehall cohort and 761 individuals diagnosed with diabetes have been measured and related to psychological, biological, and genetic factors. In the Whitehall participants, concentrations of Hsp60 ranged from undetectable to mg/mL levels. Circulating Hsp60 correlated with total and low-density lipoprotein (LDL) cholesterol and was positively associated with a flattened slope of cortisol decline over the day. Levels of this stress protein also correlated with measures of psychological stress including psychological distress, job demand, and low emotional support. Mass spectrometric analysis of circulating immunoreactive Hsp60 reveal that it is predominantly the intact protein with no mitochondrial import peptide, suggesting that this circulating protein emanates from mitochondria. The Hsp60 is stable when added to plasma and the levels in the circulation of individuals are remarkably constant over a 4-year period, suggesting plasma levels are partly genetically controlled. Sequence analysis of the HSP60-HSP10 intergenic promoter region identified a common variant 3175 C>G where the G allele had a frequency of 0.30 and was associated with higher Hsp60 levels in 761 type 2 diabetic patients. The extended range of plasma Hsp60 concentrations in the general population is genuine and is likely to be related to genetic, biological, and psychosocial risk factors for coronary artery disease.

Heat shock protein (Hsp) 70B′ is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B′ and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B′. Flow cytometry was used to assay hsp70B′ promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B′ and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B′ is a secondary responder. Interestingly ZnSO₄ induces Hsp70B′ and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B′. Both Hsp70B′ and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B′ contributes to cell survival.