Growth arrest of human B lymphocytes is accompanied by induction of the low molecular weight mammalian heat shock protein (Hsp28).

A large number of protein and molecular markers have been identified that delineate the early stages of human B cell activation and proliferation. In contrast, few if any molecules are transiently expressed precisely as activated B cells stop proliferating and undergo growth arrest. We demonstrate that the low molecular weight heat shock protein (hsp28) exhibits unique induction kinetics that specifically demarcates this interval. After mitogenic activation of unstimulated splenic B cells, hsp28 protein and phosphorylation transiently increase coinciding precisely with the peak of cellular proliferation and the onset of growth arrest. Although most neoplastic B cells constitutively express hsp28, three cell lines were identified that were hsp28-. No differences in phenotype or growth kinetics were detected between hsp28+ and hsp28- neoplastic B cells demonstrating that hsp28 expression is not essential for cell growth. However, when treated with phorbol ester or heat shock, these hsp28- cell lines synthesize hsp28 followed by the onset growth arrest. The consistency with which hsp28 induction transiently delineates the interval from peak proliferation to the onset of growth arrest suggests hsp28 itself is likely to be involved in regulating this process.     

Tumor necrosis factor induces the rapid phosphorylation of the mammalian heat shock protein hsp28.

Tumor necrosis factor alpha was found to rapidly phosphorylate the unique mammalian small heat shock protein hsp28 without impairing its cytoplasmic localization and without inducing the synthesis of the heat shock proteins. In contrast to the C-kinase-dependent phosphorylation of hsp28 in response to the tumor promoter phorbol-12-myristate-13-acetate, the heat- and tumor necrosis factor-mediated phosphorylation of this heat shock protein appears to occur independently of C kinase. These observations suggest that a C-kinase-independent phosphorylation of hsp28 may be an early event in the cellular action of tumor necrosis factor alpha.     

Interleukin 1 and tumour necrosis factor increase phosphorylation of the small heat shock protein. Effects in fibroblasts, Hep G2 and U937 cells

Interleukin 1 alpha and tumour necrosis factor-alpha stimulated phosphorylation of three 27 kDa phosphoproteins in MRC-5 fibroblasts which was sustained for up to 2 h after adding the cytokines. All three phosphoproteins were immunoprecipitated by a specific antiserum to the small mammalian heat shock protein, hsp 27. The three phosphoproteins from stimulated or control cells contained phosphoserine but not phosphothreonine or phosphotyrosine. Similar increases in phosphorylation of immunoprecipitable 27 kDa proteins were seen in U937 cells stimulated by TNF alpha and Hep G2 cells stimulated by IL1 alpha.     

Role of increased basal expression of heat shock protein 72 in colonic epithelial c2BBE adenocarcinoma cells.

Although the expression of heat shock proteins (hsps) can be induced by a variety of stressful stimuli, certain neoplasms, including human intestinal T84, HT-29, and Caco2 adenocarcinoma cell lines, express constitutively high levels even under nonstress conditions. In this study, we examine the functional significance of increased hsp72 in spontaneously differentiating Caco2bbe (C2) cells. The expression of hsp72 in these cells was specifically inhibited by hsp72 antisense transfection. The loss of hsp72 expression did not affect growth rate, contact inhibition, morphological development, or functional differentiation. In contrast, these cells were significantly more sensitive to the injurious effects of oxidants and tumor necrosis factor (TNF) but not doxorubicin. To investigate potential mechanisms of action, a number of steps in the TNF-mediated cell death was measured. Antisense reduction of hsp72 did not alter activation of IkappaB. In contrast, mitochondrial cytochrome c release and activation of caspase 9 were significantly delayed in hsp72 antisense cells stimulated either with TNF or monochloramine. In conclusion, high endogenous expression of hsp72 by intestinal adenocarcinoma cells appears to confer selective survival advantage but does not affect their growth and differentiation.     

Heat Shock Protein 27 Plays Two Distinct Roles in Controlling Human Breast Cancer Cell Migration on Laminin-5

It has recently been reported that phosphorylation of the small heat shock protein 27 (hsp27) enhances p38 MAP kinase dependent migration of bovine and human vascular endothelial cells. We have examined the role of hsp27 in controlling the constitutive migration of human breast cancer cells on the extracellular matrix molecule laminin-5. In a haptotaxis assay, anisomycin- or heat shock-induced phosphorylation of hsp27 enhances migration of MDA-MB-231 breast cancer cells constitutively overexpressing hsp27. Under these conditions, hsp27 redistributes to the nucleus. Unphosphorylated hsp27, which remains in the cytosol, induces resistance to a subset of drugs that inhibit haptotactic migration of these cells. We conclude that hsp27 plays two distinct roles in controlling migration of breast cancer cells: phosphorylated hsp27 enhances migration, while unphosphorylated hsp27 can sustain migration in the presence of inhibitory drugs.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.     

Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity.

Heat treatment and various other stresses render tumor cells resistant to cytotoxicity mediated by tumor necrosis factors (TNFs). Here, we elucidate the molecular basis of this phenomenon by demonstrating that the major heat shock protein, hsp70, protects tumor cells from TNF cytotoxicity even in the absence of stress. The human hsp70 gene was stably introduced into highly TNF-sensitive WEHI-S tumor cells both in the sense and antisense orientation. All clones constitutively expressing the exogenous human hsp70 gene were protected from TNF-mediated killing approximately 1000-fold. Remarkably, the growth of one clone was actually stimulated by low concentrations of TNF. Moreover, a clone expressing antisense hsp70 RNA was rendered extremely sensitive to TNFs. Hsp70-mediated protection from TNF cytotoxicity was confirmed in transient expression experiments employing retroviral vectors. Changes in cellular sensitivity to TNF were not associated with alterations in the binding of TNF to its receptors. Neither the transfection procedure itself nor overexpression of the low molecular weight heat shock protein, hsp27, had any effect on cellular susceptibility to TNFs. Our data suggest that hsp70 may increase the oncogenic potential of some tumor cells by providing them with an escape mechanism from immunological defense.     

Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock

During normal development in D. melanogaster, messenger RNAs for three of the seven heat shock proteins (hsp83, hsp28 and hsp26) accumulate in adult ovaries and are abundant in embryos until blastoderm. The three mRNAs appear to originate in nurse cells and subsequently pass, during stages 10-11, into the oocyte. Little if any of the four other heat shock mRNAs is present in unshocked ovaries or embryos at any time examined. Pre-blastoderm embryos fail to accumulate these heat shock mRNAs even if subjected to heat shock. The accumulation in normal oogenesis of mRNAs for only three of the seven heat shock proteins indicates the existence of differential, possibly multiple controls of heat shock gene expression, and suggests that heat shock proteins hsp83, hsp28 and hsp26 function in the oocyte or early embryo.     

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery

Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38alpha and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30.     

Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.     

Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25 degrees C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37 degrees C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.     

Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution

Tetrahydrobiopterin (BH4) and heat shock protein 90 (hsp90) have been anticipated to regulate endothelial nitric oxide synthase (eNOS)-dependent superoxide anion radical (O2*-) generation in endothelial cells. It is not known, however, whether hsp90 and BH4 increase O2*- in a synergistic manner, or whether this increase is a consequence of downstream changes in eNOS phosphorylation on serine 1179 (eNOS-S1179) and changes in dimer/monomer distribution. Here O2*- production from purified BH4 -free eNOS and eNOS:hsp90 complexes determined by spin-trapping methodology showed that hsp90 neither inhibits O2*- nor alters the requirement of BH4 to inhibit radical release from eNOS. In endothelial cells, O2*- detection with the novel high-performance liquid chromatography assay of 2-hydroxyethidium showed that inhibition of hsp90 did not increase O2*-, while a significant increase in O2*- was detected in BH4 -depleted cells. Radicicol, a hsp90 inhibitor, disrupted eNOS:hsp90 association, decreased eNOS-S1179, but increased biopterin production in a dose-dependent fashion. These changes were followed by an increase in eNOS activity, demonstrating that high biopterin levels offset inhibition of eNOS phosphorylation and diminished interaction with hsp90. In contrast, depletion of biopterin did not affect hsp90 levels or interaction with eNOS or eNOS dimer/monomer ratio in bovine aorta endothelial cells (BAECs). We conclude that low BH4 but not inhibition of hsp90 increases O2*- in BAECs by mechanism(s) that unlikely involve phosphorylation to eNOS-S1179 or eNOS monomerization.     

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II

Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp 90s begin with proline; the initial methionine residue is removed. The alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4 to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form. The purified hsp 90 mixture contains 2 mol of phosphate/mol of polypeptide. Both hsp 90 proteins are phosphorylated at two homologous sites. For the alpha protein, these sites correspond to serine 231 and serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation sites is absent from the beta protein. The sequence between phosphorylation sites of both hsp 90s is predicted to have alpha helical structure. Dephosphorylated hsp 90 is phosphorylated at both sites by casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no other hsp 90 residues were phosphorylated by casein kinase II in vitro.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Acquired thermotolerance following heat shock protein synthesis prevents impairment of mitochondrial ATPase activity at elevated temperatures in Saccharomyces cerevisiae

The complex molecular response of cells to sudden temperature changes is a well-characterized phenomenon. Although it is clear that the induction of heat shock proteins provides protection from heat in all of the organisms so far tested, very little is known about the role that this set of proteins plays in cellular homeostasis. Recently, putative roles for hsp60 and hsp70-like proteins have been proposed in Saccharomyces cerevisiae. hsp70-like proteins have been shown to be necessary for translocation of precursor polypeptides into mitochondria and endoplasmic reticulum, while hsp60 is required for the assembly of precursor polypeptides into oligomeric complexes following incorporation into the mitochondrial matrix. In this paper, we report that a brief temperature shock (44 degrees C) impairs coupling of oxidative phosphorylation in S. cerevisiae as measured indirectly by the Cl-CCP/oligomycin assay. Furthermore, at high temperature oligomycin stimulates rather than inhibits oxygen uptake under nonthermotolerant conditions. Pretreatment of cells for a short period of time at 37 degrees C, prior to exposure to higher temperatures rescues the capacity to maintain coupling between oxidative phosphorylation and electron transport. Inhibition of cytoplasmic RNA or protein synthesis during heat shock prevents the protection of this mitochondrial activity. We propose that one of the roles of the induction of heat shock proteins (or related activities) is to protect mitochondrial ATPase activity under conditions of further increase in temperature.     

Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells.

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca(2+)-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca(2+)/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca(2+)](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca(2+)-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.