This minireview provides insight into feedback regulation of heme-dependent metabolism as a defensive cellular response against stress. Interactions among heme-, iron-, porphyrin-, and CO/NO-dependent metabolic pathways during the stress-induced response are emphasized in the context of feedback regulation. The hypothetical model of the latter interactions is presented as tightly controlled feedback cycles.

Exposure of cells to arsenicals activates multiple stress pathways resulting in the induction of specific genes whose identity and role in the adaptation to arsenical-induced cellular stress are poorly understood. We report here the identification of a novel gene encoding an arsenite-inducible, cysteine- and histidine-rich RNA-associated protein, AIRAP, that is conserved among mammals, Drosophila and C elegans. Immunochemistry and cell fractionation experiments indicate that, when induced, AIRAP is present in both the nucleus and the cytoplasm, and cross-linking experiments indicate that it associates with RNA in vivo. The expression of a C elegans homologue of AIRAP, aip-1, is also induced by exposure to arsenite, and expression of an aip-1::gfp transgene is most pronounced in hypodermal cells. RNA-mediated interference (RNAi) of aip-1 lowers the resistance of nematodes to arsenite yet does not appear to affect viability under standard growth conditions. These experiments suggest a role for AIRAP/AIP-1 in protecting cells from the toxic effects of arsenite.

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO₂ and CdCl₂), hypertonic stress (sorbitol and NaCl), or anisomycin, an activator of p38 mitogen–activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)–induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl₂ but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase–activated protein (MAPKAP) kinase- 2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

It has recently been reported that αB-crystallin, a low-molecular-weight heat shock protein, may be released from cells by mechanical stretch. We investigated a physiological role of αB-crystallin in platelet function. αB-crystallin inhibited platelet aggregation induced by thrombin or botrocetin in hamsters and humans. These platelets had specific binding sites for αB-crystallin. Moreover, αB-crystallin significantly reduced thrombin-induced Ca² influx and phosphoinositide hydrolysis by phospholipase C in human platelets. Additionally, plasma levels of αB-crystallin were markedly elevated in cardiomyopathic hamsters. Levels of αB-crystallin in vessel walls after endothelial injury were markedly reduced. Therefore, our results suggest that αB-crystallin, which is discharged from vessel walls in response to endothelial injury, acts intercellularly as a regulator of platelet function.

Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation. To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E coli cells and the composition of the S fraction. We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing. A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction. Fda was purified from E coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro. We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system. The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda. Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ. GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda. It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect.

We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37°C compared to control embryos raised at 27°C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.

Tumor necrosis factor-α (TNFα) could cause apoptosis in hepatic tissue of d-galactosamine sensitized mice, as evidenced by the increase in the extent of DNA fragmentation. The hepatic apoptosis induced by TNFα was associated with hepatocellular damage as assessed by plasma alanine aminotransferase activity. Schisandrin B (Sch B) pretreatment at daily doses ranging from 0.5 to 2 mmol/kg for 3 days caused a dose-dependent protection against TNFα-induced apoptosis in mice. The hepatoprotection was accompanied by a parallel reduction in the extent of hepatocellular damage. The same Sch B pretreatment regimens increased hepatic Hsp70 level in a dose-dependent manner. The relevance of Sch B–induced increase in Hsp70 expression to the prevention of TNFα-triggered hepatic apoptosis remains to be elucidated.

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c–mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42°C for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37°C, those exposed to heat stress (42°C for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate, hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.

Within steroid receptor heterocomplexes the large tetratricopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (Hsp90) and act coordinately with Hsp90 to modulate receptor activity. The reversible nature of the interaction between the immunophilins and Hsp90 suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a 5-kilobase (kb) 5′-flanking region of the human gene and demonstrated that a ∼50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABPα and GABPβ subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GABP is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.