Hsp70B' regulation and function

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.     

Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.     

Cell number-dependent regulation of Hsp70B' expression: evidence of an extracellular regulator

Hsp70B' is a unique member of the human Hsp70 family of chaperones about which information is scarce. Unlike the major inducible Hsp72 protein, Hsp70B' is strictly inducible having little or no basal expression levels in most cells. We observed that Hsp70B' appears transiently in response to heat stress whereas Hsp72 levels persist for many days. Also, Hsp70B' is optimally induced when cell numbers are low, whereas Hsp72 levels are greatest at higher cell number. Hsp70B' promoter activation was measured by flow cytometry using an Hsp70B' promoter-driven GFP construct. In heat stressed cells, promoter activation is cell number independent over a broad range. However, when cell number increases beyond a certain population size, cells are less stress inducible for Hsp70B' and induction becomes highly cell number-dependent. Cell number differences in Hsp70 activation cannot be explained by changes in Hsf-1 DNA-binding activity or hyperphosphorylation. Cells with few or no cell matrix attachments (laminin-coated and low attachment plates, respectively) appear to be more sensitive to cell number-dependent inhibition. Medium conditioned by the low cell number (LCN) populations supports increased Hsp70B' promoter activation in high cell number (HCN) cultures. Likewise, medium conditioned in HCN culture conditions causes decreased activation of Hsp70B' promoter in LCN cultures. As HCN-conditioned medium has all the components necessary for cell growth, two possibilities for the activation of Hsp70B' gene expression exist: an inhibitory component that accumulates in culture medium at HCN, or an activator that accumulates at LCN.     

Hsp70B' and Hsp72 form a complex in stressed human colon cells and each contributes to cytoprotection

Hsp70B′ is the only major human isoform in the hsp70 family that is strictly stress-inducible, and therefore available to function only in stressed cells. Since Hsp70B′ is evolutionarily closely related to human Hsp72, they are thought to function similarly, but direct evidence of Hsp70B′ function in stressed cells has been lacking. Here we showed that both Hsp70B′ and Hsp72 are essential relatively early after heat stress in the acquisition of cytoprotection by two human colon cell lines. Using flow cytometry to count viable cells, we also showed that cytoprotection is more pronounced in cultures grown at low cell number (LCN), where there is an ample amount of both Hsp70s. siRNA knock-down of either Hsp70B′ or Hsp72 severely handicapped the ability of cells to acquire cytoprotection. Hsp70B′ and Hsp72 were found to form a complex following stress that included the co-chaperone HOP. These results taken together support the hypothesis that Hsp70B′ and Hsp72 play cooperative roles in cell survival of proteotoxic stress. In addition there are implications for chemotherapy protocols and for pathological conditions in which the contributions to cytoprotection of both Hsp70B′ and Hsp72 are modulated by cell numbers or density.     

Ability of Insulin and DsRNA to Induce Interferon System and Hsp 70 in Fibroblast and Epithelial Cells in Relation to Their Effects on Cell Growth

We have examined the ability of insulin and dsRNA, a well-known interferon inducer, in relation to their effects on cell growth, to induce the expression of hsp 70 and the synthesis of interferon in epithelial HT-29 and fibroblast Madin-Darby bovine kidney (MDBK) cells. Insulin was mitogenic in both MDBK and HT-29 cells; MDBK cells nevertheless required much higher concentrations. DsRNA stimulated the growth of MDBK but inhibited that of HT-29 cells. Both substances induced a transient synthesis of hsp 70 in HT-29 and MDBK cells with similar kinetics. However, whereas both insulin and dsRNA efficiently induced 2′5′ oligoadenylate synthetase and an antiviral state through interferon synthesis in HT-29 cells, only dsRNA caused these effects in MDBK cells. Thus, insulin cannot, unlike dsRNA, elicit an antiviral state in all cell systems, although, like dsRNA, it can induce hsp 70, thereby suggesting the cell specificity of insulin action. These results reveal that the mitogenic and IFN-inducing effects of insulin and dsRNA are dependent on the cell type and unrelated to hsp 70 expression.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Intracellular localization of heat shock proteins in maize

The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centrifugation was employed to separate organelles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelle associated. Hsp 25 and 72 were in fractions containing closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fractions containing overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better separation of the two sets of closely equilibrating organelles. When a 13,000g centrifugation step to remove mitochondria was employed prior to gradient centrifugation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either maintaining the ribosomes on the membrane or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments.     

Hsp72 interacts with paxillin and facilitates the reassembly of focal adhesions during recovery from ATP depletion

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.     

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.     

Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice

Members of heat shock proteins (Hsp70) family have been considered to respond to a large variety of stressful conditions. But it was suggested that, in pulmonary cells, Hsp response depends more closely on the type of stimulus. The lungs are critical organs potentially subjected to air pollution affecting respiratory function and, therefore, these organs are of particular interest with regard to the stress response. To investigate the stress dependence of Hsp70 response in lungs, we created transgenic mice where the firefly luciferase reporter gene is under the control of the murine hsp70-1 promoter and exposed them to different sublethal toxic conditions. For each condition, the level of transgene induction and pulmonary toxicity were assessed. We found that hsp70-1 promoter was stimulated by heat shock and cadmium but not by ozone, paraquat, and parathion, even if these chemicals induced respiratory distress and lung inflammation. Similar observations were made when expression of the endogenous hsp70-1 gene was analyzed, indicating that our transgenic model was accurately detecting hsp70-1 induction. Thereby, it appeared that hsp70-1 response is selective and depends on signaling pathways triggered by the toxicants rather than by their pathologic toxicity per se. Furthermore, because all the chemicals used in our study have been previously described to increase the level of oxidative stress, it indicates that there is no direct and simple correlation between hsp70-1 response and the level of oxidative stress, but more specific oxidative patterns should be involved in Hsp regulation.