©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Expression and Regulation of hsp70 and hsp90 by Phorbol Esters and Heat Shock (*)

Muriel R. Jacquier-Sarlin (1), Lan Jornot (2), Barbara S. Polla (1) (3)(§)

From the (1) Health and Environment Program, Nuclear Medicine Division and the (2) Pulmonary Division, University Hospital, 1211 Geneva 14, Switzerland and the (3) Laboratoire de Physiologie Respiratoire, CHU Cochin Port-Royal, 24, rue du Faubourg Saint-Jacques, 75014 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human peripheral blood monocytes (PBM) produce superoxide anions (O) by a process involving electron transfer from NADPH to O, catalyzed by the respiratory burst enzyme NADPH oxidase. We have previously shown that phagocytosis, while activating NADPH oxidase, induced in PBM the synthesis of heat shock (HS) proteins (HSP). The present study was undertaken to establish whether this increase in HSP expression was related to O and/or to classical second messengers such as protein kinase C (PKC). Thus, the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) were compared with those of heat shock on the expression, in PBM, of the major HSP, hsp70 and hsp90, using biometabolic labeling, Western and Northern blotting, and gel mobility shift assays. PMA induced the accumulation of mRNA and an increased expression of hsp90 and, to a lesser extent, hsp/hsc70 (hsc is the cognate, constitutive form). This induction was also observed in PBM from patients with chronic granulomatous disease, a genetic defect in NADPH oxidase, and was abolished by the PKC inhibitors staurosporine and H-7. PMA did not cause activation of the HS factor, and the PMA-induced overexpression of HSP was not blocked by the transcriptional inhibitor actinomycin D. HSP-specific mRNA stability was increased after PMA exposure as compared with heat shock. These results suggest that O is not involved in the PMA-mediated induction of hsp70 and hsp90 and that, in contrast to HS, PMA increases the expression of HSP as a result of PKC-induced mRNA stabilization rather than of transcriptional activation of HS genes.


INTRODUCTION

Exposure of cells to elevated temperatures or to a wide variety of physical and chemical injuries activates the expression of stress response genes, coding for the heat shock (HS())() /stress proteins (HSP) (1). These proteins are generally classified into families according to their apparent molecular weight and respective inducers and play essential roles in protein chaperoning and cellular protection. HSP expression is not limited to cells undergoing acute stress, and several members of HSP families are constitutively expressed (HS cognates). Furthermore, the expression of several members of the HSP families (hsp/hsc70, hsp90) has been described to be modulated during nonstressful conditions such as cell cycle (2) and differentiation (3) , in response to serum stimulation (4) , and during the early stages of embryogenesis (5) . The expression of HSP has been shown to be regulated by both transcriptional and translational events (6, 7) .

Intracellular accumulation of abnormal or degraded proteins has been proposed as a common signal for the induction of stress protein synthesis (8) , while we have suggested that reactive oxygen species would represent a ubiquitous second messenger for HSP induction. One model to study the effects of endogenous reactive oxygen species production on HSP induction is phagocytosis, during which the activation of the respiratory burst enzyme NADPH oxidase leads to the massive generation of superoxide anions (O) (9) . We found that phagocytosis of inactivated Staphylococcus aureus by human monocytes-macrophages induces the selective synthesis of hsp70, which is enhanced in the presence of iron, while infection with Leishmania major, which does not activate the respiratory burst, fails to induce a host cell stress response (10, 11) . These and other results suggest that reactive oxygen species, among which hydroxyl radicals are generated via the iron-dependent O-driven Fenton reaction, play a specific role in hsp70 induction (12) .

Since, however, many activation events, distinct from reactive oxygen species production, such as calcium mobilization and activation of protein kinases (13) , occur during phagocytosis, we investigated the role of O in HSP regulation using phorbol 12-myristate 13-acetate (PMA), a non-particulate, non-receptor-mediated activator of NADPH oxidase (14) . PMA is a potent activator of protein kinase C (PKC) and may mimic in vitro a number of activation events occurring during inflammation. We thus compared the expression of HSP in human peripheral blood monocytes (PBM) after HS or exposure to PMA. We also compared the inducing effects of HS and PMA on the stress response in cells from normal donors and in cells from patients with chronic granulomatous disease (CGD). CGD is characterized by a genetic defect in NADPH oxidase leading to a selective lack of O production upon phagocytosis or activation with PMA, while PKC activation is unaltered. To further differentiate between the respective roles of O and PKC in the regulation of HSP expression, we analyzed the effects of PKC inhibitors on HSP induction by HS or PMA.

Our results indicate that there is a differential expression of hsp70 and hsp90 in human PBM following exposure to the two stresses considered here, HS and PMA. This differential expression is the consequence of the involvement of both distinct second messengers and distinct mechanisms for molecular regulation of HS gene expression.


MATERIALS AND METHODS

Cells and Media

PBM were isolated from normal volunteers and from two patients with CGD by gradient centrifugation and were purified by adherence as described previously (15) . The freshly isolated cells were cultured at 37 °C in antibiotic-free RPMI 1640 medium (Gibco, Paisley, Scotland) supplemented with 10% fetal calf serum (Gibco) and 1% glutamine (Gibco) in a humidified atmosphere (95% air, 5% CO).

Reagents and Exposure to HS

PBM were incubated for 3.5 h at 37 °C with 50 ng/ml PMA (Sigma). For HS, PBM were incubated in a water bath at 44 °C for 20 min, followed by a recovery period of 2 h at 37 °C. The transcriptional inhibitor actinomycin D (Sigma; 5 µg/ml) was added to PBM 10 min prior to HS or exposure to PMA and was present throughout the experiments. In other experiments, PKC inhibitors were added 30 min prior to HS or exposure to PMA. Staurosporine was used at 200 nM and H-7 (1-(5-isoquinolinyl)-2-methylpiperazine) at 100 µM. Both PKC inhibitors were from Calbiochem.

Superoxide Measurement

PBM were washed and resuspended in buffer containing 138 mM NaCl, 6 mM KCl, 1 mM MgSO7HO, 1.1 mM CaCl2HO, 0.2 mM EGTA, 5.5 mM glucose, and 20 mM HEPES (pH 7.4) at 20 10 cells/ml. 10 PBM were stimulated with PMA (1.6 10M) for 30 min at 37 °C or exposed to HS for 20 min at 44 °C. The extracellular production of O was measured by the superoxide dismutase-inhibitable reduction of cytochrome c as described previously (14) . In parallel experiments, the intracellular production of O was qualitatively determined by nitro blue tetrazolium (NBT) reduction as described previously (16) . Briefly, 4 10 PBM were incubated in phosphate-buffered saline containing 0.04% NBT and 10% fetal calf serum. After PMA (100 ng/ml) or HS stress, PBM were cytocentrifuged on a glass slide, fixed with methanol, and stained with safranin for 1 min. 200 cells/slide were counted under a light microscope.

Analysis of Protein Synthesis

In these experiments, RPMI 1640 medium was replaced by RPMI 1640 medium without methionine (Gibco). PMA-treated PBM were metabolically labeled with [S]methionine (6 µCi/ml; specific activity of >1000 Ci/mmol; Amersham International, Buckinghamshire, United Kingdom) added during the last 90 min of treatment with mitogen. For heat-shocked PBM, labeling was performed for 90 min at 37 °C after the 2 h of recovery. After labeling, aliquots corresponding to equal cell numbers were resolved by SDS-PAGE (10% polyacrylamide) according to Laemmli (17) and revealed by autoradiography. Hsc70, hsp70, and hsp90 were characterized by Western blotting. Proteins were electrophoresed; transferred to nitrocellulose membranes; and probed with mouse monoclonal antibodies against human constitutive hsc70 (SPA820), inducible hsp70 (SPA810), or hsp90 (SPA840; all from Stressgen Biotech Corp., Victoria, Canada). Bound antibodies were revealed with anti-mouse IgG-peroxidase conjugated (Sigma) in the presence of HO and 4-chloro-1-naphthol (Sigma).

RNA Extraction and Northern Blotting

Cells were harvested immediately after HS or PMA treatment and lysed in buffer containing 5 M guanidium isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.5% sodium N-laurylsarcosine, and 0.1 M 2-mercaptoethanol. Total RNA was purified and stored in Tris/EDTA. For Northern analysis, total RNA (5 µg) was denatured and fractionated by electrophoresis on 1% agarose gels in 5% formaldehyde as described by Maniatis et al.(18) . After electrophoresis, the RNA was transferred onto a Biodyne membrane in 20 SSC (1 SSC = 0.15 M NaCl, 0.015 M trisodium citrate (pH 7)). The membranes were UV-cross-linked; backed for 2 h at 80 °C; prehybridized for at least 5 h at 55 °C in 5 SSC, 10 mM NaPO, 50% formamide, 5 Denhardt's solution (1 Denhardt's solution = 0.002% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 0.1% SDS, and 250 µg/ml salmon sperm; and hybridized overnight in the same buffer at 65 °C with a [P]UTP-labeled antisense RNA transcript (2 10 cpm/ml). This RNA probe was prepared as described (19) . For analysis of hsp90 mRNA, the membranes were hybridized with a DNA probe labeled using the multiprime labeling kit from Amersham International in the same buffer as described above at 42 °C for 12-16 h. This probe was an isolated HindIII-restricted fragment derived from an entire human hsp90 gene (kindly provided by R. Morimoto). After hybridization, the membranes were washed sequentially at 65-70 °C in 2 SSC and in 0.2 SSC, 0.1% SDS. The membranes were then subjected to autoradiography on Hyperfilm (Amersham International) at -70 °C using intensifying screens. As an internal control for the loading of RNA, we analyzed the band corresponding to 28 S mRNA.

HSP mRNA Stability after HS and PMA Treatment

After HS (20 min at 44 °C, followed by a recovery period of 30 min at 37 °C) or PMA exposure (50 ng/ml, 6 h), actinomycin D was added to the culture medium at a final concentration of 5 µg/ml to block RNA transcription as described previously (19) . The cells were lysed 2, 4, 6, and 8 h after administration of actinomycin D, and total RNA was extracted and analyzed by Northern blotting as described above. Results were expressed as the percentage of the mRNA values obtained before the addition of actinomycin D (time 0).

Preparation of Nuclear Extracts and Gel Mobility Shift Assays

Preparation of nuclear protein extracts was adapted from the method of Dignam et al. (20). Briefly, cells were collected and kept on ice for 10 min in hypotonic lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each antipain, leupeptin, and pepstatin. The cells were homogenized in a Dounce homogenizer, and the nuclei were pelleted at 3000 rpm and resuspended in extraction buffer (10 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 5% glycerol) with constant mixing at 4 °C for 30 min. The samples were centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was dialyzed against buffer containing 20 mM HEPES (pH 7.9), 75 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 20% glycerol. Samples were quick-frozen in liquid nitrogen and stored at -80 °C. Protein concentration was determined with a bicinchoninic acid protein assay kit (Pierce). Nuclear extracts were then analyzed for HS element (HSE) binding activity by gel mobility shift assays. Binding reactions were performed for 30 min at 25 °C by adding 5 µg of nuclear proteins to a mixture containing 10 cpm of [-P]ATP end-labeled, double-stranded HSE oligonucleotide (5`-GCCTCGAATGTTCGCGAAGTT-3`) in 15 µl of dialysis buffer containing 2 µg of poly(dI-dC) and 10 µg of bovine serum albumin. For the competition experiments, a 100-fold molar excess of nonradioactive HSE or a 100-fold molar excess of a tumor-promoting agent-responsive element oligonucleotide (5`-CTTGTGAGTCATTCC-3`) was added. Samples were electrophoresed on a nondenaturing 4% polyacrylamide gel, dried, and autoradiographed.


RESULTS

Differential Induction of Stress Proteins following Exposure to HS or PMA

The induction of HSP expression in PBM following HS or PMA exposure is shown in Fig. 1A. Exposure of PBM to 44 °C for 20 min induced both hsp70 and hsp90 (Fig. 1A, lane 2) as well as hsp65 and hsp110 (Fig. 3A, lanes 4-6), whereas exposure to PMA essentially induced the expression of hsp90 (Fig. 1A, lane 3). Time course experiments indicated that PMA-induced HSP synthesis was maximal at 3.5 h of incubation before reaching a plateau (data not shown). The differential induction of HSP observed in Fig. 1A was confirmed by Western blot analysis performed with specific monoclonal antibodies against hsc70, hsp70, and hsp90 (Fig. 1B). Both HS and PMA slightly increased the expression of constitutive hsc70 (Fig. 1B, panela); the antibody used to detect hsc70 cross-reacts with inducible hsp70. Using an antibody specific for inducible hsp70, we confirmed that HS strongly increased the expression of this protein, while only a weak induction was observed with PMA (Fig. 1B, panelb). In contrast, both stresses stimulated the expression of hsp90 with a similar intensity (Fig. 1B, panelc).


Figure 1: Differential induction of HSP in human PBM following exposure to HS or PMA. A, SDS-PAGE analysis of protein synthesis by PBM after HS (20 min at 44 °C) (lane 2) or treatment with PMA (50 ng/ml, 3.5 h, 37 °C) (lane 3). Lane 1 is the control. B, Western blot analysis of hsc70 (panela), hsp70 (panelb), and hsp90 (panelc) after exposure to HS (lanes 2) or PMA (lanes 1). Lanes 3 are the controls. While HS induced major classical HSP synthesis, PMA essentially increased the expression of hsp90. a, actin.




Figure 3: PMA-mediated HSP synthesis in human PBM is inhibited by PKC inhibitors. A, SDS-PAGE analysis of protein synthesis by PBM after HS (lanes 4-6) or treatment with PMA (lanes 7-9) in the absence (lanes 4 and 7) or presence of staurosporine (200 nM) (lanes 5 and 8) or H-7 (100 µM) (lanes 6 and 9). PKC inhibitors were added to PBM 30 min before exposure to HS or PMA. B, Western blot analysis of hsp70 (panela) and hsp90 (panelb) protein induction in PBM exposed to HS (lanes 2-4) or incubated with PMA (lanes 5-7) in the absence (lanes 2 and 5) or presence of H-7 (100 µM) (lanes 3 and 6) or staurosporine (200 nM) (lanes 4 and 7). Lanes 1 are the controls (C).



Superoxide Anion Production during Exposure to HS or PMA

As an approach to understand this differential induction of HSP following HS and PMA exposure, we first compared the production of O by PBM exposed to HS or treated with PMA. In are summarized the results of O production as determined by cytochrome c (extracellular O production) and NBT (intracellular O production) reduction. While PMA induced in PBM both extracellular and intracellular O production (38.4 ± 1.9 nmol of O/10 cells, respectively; 152 ± 9.5 NBT-positive cells), HS did not induce O production at least as detected by the methods used. On the other hand, as described previously (21) , pre-exposure of PBM to HS abolished the PMA-induced O production ().

Involvement of PKC in PMA-induced hsp90 Expression

To determine whether the PMA-induced HSP synthesis resulted from the activation of PKC (rather than from the subsequent activation of the respiratory burst), we compared PBM from normal donors and PBM from patients with CGD with respect to the induction of HSP by PMA. In the latter cells, PMA increased hsp90 expression as in control cells (Fig. 2, compare lanes 5 and 2). To provide further arguments in favor of the hypothesis that PMA-mediated hsp90 synthesis relates to PKC activation rather than to O production, we analyzed the effects of the PKC inhibitors staurosporine and H-7 on HSP expression. Control experiments indicated that at the concentrations used, these inhibitors were not toxic for the cells (Fig. 3A, lanes 1-3). The PKC inhibitors had no effect on HS-induced HSP (Fig. 3A, lanes 4-6), while in contrast, they abolished the PMA-induced HSP synthesis (lanes 7-9). Western blot analysis confirmed both the lack of inhibition in HS-mediated HSP expression in the presence of staurosporine and H-7 (Fig. 3B, lanes 2-4) and the inhibition of PMA-mediated hsp90 expression (lanes 5-7). The effect of staurosporine on the inhibition of HSP synthesis was greater than that of H-7, an observation that might relate to the fact that staurosporine inhibits PKC more specifically than H-7.


Figure 2: Comparison of HSP induction in PBM from normal donors and from patients with CGD. Shown is SDS-PAGE analysis of protein synthesis by PBM from normal donors (lanes 1-3) and from patients with CGD (lanes 4-6) exposed to HS (lanes 3 and 6) or to PMA (lanes 2 and 5). Unstressed cells (control (C)) are shown in lanes 1 and 4. PMA induced the expression of hsp90 in both normal and CGD cells.



Differential Kinetics of hsp70 and hsp90 mRNA Levels following Exposure to HS or PMA

We then addressed the possibility of a differential level of regulation in HSP synthesis following HS or PMA exposure. We first investigated the effects of PMA on hsp70 and hsp90 mRNA levels. Cytoplasmic RNA was isolated at different times following PMA treatment, and HSP mRNA levels were determined by Northern blot analysis with specific probes for hsp70 or hsp90 (Fig. 4). In unstressed cells, neither hsp70 nor hsp90 mRNA was detected (Fig. 4, lane1 in A and B). HS induced a rapid increase in hsp70 mRNA levels at 30 min, whereas PMA led to a delayed accumulation of hsp70 mRNA, detectable after 2 h and increasing over time (up to 6 h) (Fig. 4A). Even at 6 h, the levels of hsp70 mRNA were still lower with PMA than with HS. In contrast, both stresses induced hsp90 mRNA accumulation, and in this case, the levels of mRNA at 6 h were higher with PMA than with HS (Fig. 4B). The kinetics of hsp90 mRNA induction by PMA appeared similar to those obtained with the same stressor for hsp70 mRNA. Both hsp70- and hsp90-specific mRNA induced at 6 h by PMA were suppressed by pretreatment of the cells with the PKC inhibitors staurosporine and H-7 (Fig. 4, A and B, compare lane 7 with lanes 8 and 9).


Figure 4: Kinetics of HSP mRNA induction in PBM following exposure to HS or PMA. Total RNA (5 µg/lane) from control PBM (C; lanes 1), heat-shocked PBM (lanes 2), or PBM treated with PMA for the indicated time periods (lanes 3-7) was analyzed using the P-radiolabeled hsp70 (A) and hsp90 (B) cDNA probes as described under ``Materials and Methods.'' Both hsp70- and hsp90-specific mRNA induced by PMA (6 h) were suppressed by pre-exposure of the cells to the PKC inhibitors staurosporine (compare lanes 7 and 8) and H-7 (compare lanes 7 and 9).



Differential Activation of HSF following Exposure to HS or PMA

To determine the effects of PMA on the activation of HSF and its binding to HSE, we performed gel mobility shift assays using a synthetic oligonucleotide containing the consensus HSE-binding site from the human HSP promoter. Gel mobility shift assays were done with nuclear protein extracts prepared from cells treated for different periods of time with PMA. HS induced the appearance of typical HSFHSE complexes (Fig. 5, A, lane 2; B, lanes 2 and 4), while following exposure to PMA, no specific HSFHSE complexes were detected (A, lanes 3-8) (22) . Specificity of HSF binding was tested by competition experiments with a molar excess of 100-fold nonradioactive HSE or tumor-promoting agent-responsive element (Fig. 5B). Under the conditions of our experiments, both the constitutive HSE binding activity (CHBA) (when expressed, which was not the case in all experiments; see Fig. 5A) and the inducible forms of DNA binding activity were suppressed by an excess of cold HSE.


Figure 5: PMA fails to induce HSFHSE binding activity in human PBM. A, PBM were exposed to HS (lane 2) or to PMA for the indicated time periods (lanes 3-8). Five µg of nuclear proteins were subjected to gel mobility shift assay using the HSE sequence from the human hsp70 promoter. As expected, HS induced HSFHSE binding activity. In contrast, after exposure to PMA, no HSFHSE binding activity was detected. Unstressed cells are shown in lane 1 (control (C)). B, the specificity of HSFHSE binding activity was controlled by competition experiments with a 100-fold molar excess of cold HSE (lanes 2 and 6) or tumor-promoting agent-responsive element (TRE; lanes 4 and 7). In this experiment, we also observed constitutive HSFHSE binding activity (CHBA).



Effects of Actinomycin D on HSP Synthesis following Exposure to HS or PMA

Because of the observed lack of HSF activation in PMA-mediated HSP synthesis, we also tested the effects of the transcriptional inhibitor actinomycin D. Western blotting performed with specific antibodies against hsp70 and hsp90 indicated that hsp70 synthesis was inhibited by pretreatment with actinomycin D in heat-shocked cells only, with no effect in PMA-treated PBM (Fig. 6, compare lanes 3 and 4). In contrast to hsp70, both HS- and PMA-induced hsp90 synthesis were unaffected by actinomycin D (Fig. 6, compare lanes 5 and 6), which suggests that during HS-mediated induction of HSP synthesis, different regulatory mechanisms could be involved for different HSP.


Figure 6: Effects of actinomycin D on the expression of HSP induced by HS or PMA. Shown are the Western blots for hsp70 (A) and hsp90 (B) in PBM exposed to HS (lanes 3 and 4) or incubated with PMA (lanes 5 and 6). Actinomycin D (5 µg/ml) prevented the HS-mediated hsp70 expression, but had no effects on hsp90 and PMA-induced hsp70 expression. Lanes 1, control cells; lanes 2, control cells with actinomycin D (AD).



PMA Increases HSP mRNA Stability

To further establish that the increased HSP expression following PMA was caused by a post-transcriptional rather than a transcriptional mechanism, we compared the hsp70 and hsp90 mRNA half-lives in HS- and PMA-treated cells. As shown in Fig. 7, PMA increased HSP mRNA stability compared with HS, an effect more important for hsp70 mRNA than for hsp90 mRNA. In heat-shocked cells, we observed a 95% decrease in hsp70 mRNA levels 8 h after the addition of actinomycin D, compared with a 48% decrease in PMA-treated cells (Fig. 7A). With respect to hsp90 mRNA, which has been described to be more stable than hsp70 mRNA (23) , the difference in the two conditions was less: 8 h after treatment with actinomycin D, there was a 35% decrease following HS, compared with a 20% decrease in PMA-treated cells (Fig. 7B).


Figure 7: Effects of HS and PMA on HSP mRNA stability. After exposure of PBM to HS or PMA, actinomycin D was added (5 µg/ml final concentration), and the culture was continued for 2, 4, 6, or 8 h at 37 °C. At each time point, the cells were lysed, and both hsp70- and hsp90-specific mRNA levels were determined by Northern blot analysis. Results (means ± S.E.) are expressed as the percentage of the mRNA values obtained before the addition of the inhibitor (time 0 (T0)).




Discussion

In this study, we report that HS and PMA lead to differential expression of HSP in human PBM. Our data support the distinct regulation of various members of the HSP families and provide new insight into the respective roles of O and PKC as second messengers in the stress response.

While heat-shocked PBM synthesized all classical HSP (hsp110, hsp90, hsp70, as well as hsc70, hsp65, etc.), PMA-treated cells mostly increased the expression of hsp90 and, to a lesser extent, hsc70 and hsp70. Mitogen-induced HSP synthesis had been described before in human lymphocytes and in the human premonocytic line U937 (24, 25) . While hsc70 was predominantly induced in lymphocytes, both hsc70 and hsp70 were only slightly and transiently induced by mitogens in the U937 cells, which is in agreement with our results in PBM. Hsp90, which also is inducible by mitogens, actually consists of at least two proteins, and , each encoded by its own gene (26) . Both forms of hsp90 are regulated by HS and mitogenic stimuli, with hsp90 being more inducible by HS than by mitogens, while the opposite is true for hsp90. These HSP genes share similar promoter regions; thus, regulatory mechanisms involving a distinct unique secondary messenger or a cascade of messengers must exist to explain the differential HSP expression that we and others observed.

We propose that the PMA-induced hsc70, hsp70, and hsp90 synthesis did not result from the activation of NADPH oxidase and the resulting production of O, but was mediated by the activation of PKC inasmuch as it was prevented by PKC inhibitors. This was not the case for HS-induced HSP synthesis, although protein kinases, mitogen-activated protein kinase, and PKC have been described to be activated by HS (27) . The finding that O alone is neither necessary nor sufficient to induce HSP synthesis was further supported by our observations in cells from patients with CGD. Indeed, these cells, which lack a functional NADPH oxidase, were able to mount a HS response following both elevated temperature and exposure to PMA. The addition of iron during PMA treatment (but not during HS) significantly increased the synthesis of hsp70 in normal PBM (data not shown). These results suggest that O production is not directly responsible for HSP induction during phagocytosis, while its resulting transformation into hydroxyl radicals in the presence of iron may be essential as inducers of HSP synthesis because of the oxidative alteration of non-self (or self) proteins. Only hydroxyl radicals, and not O, may have the ability to oxidatively alter proteins in a way similar to HS, with the presence of altered proteins being the ultimate signal for hsp70 induction. In contrast, the expression of hsc70 and hsp90 appears to be tightly regulated by PKC activity, at least during mitogen stimulation.

On the other hand, our data also indicate that while HS-induced HSP synthesis is principally controlled at the transcriptional level (activation of HSF), the PMA-induced HSP synthesis resulted from a stabilization of HSP mRNA (Fig. 7). The temporal delay in the accumulation of hsp70 and hsp90 mRNA during PMA treatment stands in marked contrast to the rapid increase in hsp70 and hsp90 mRNA levels upon HS. In the latter case, increased steady-state levels of mRNA were detectable within the first 30 min. Furthermore, in contrast to HS, PMA failed to induce activation, translocation, and DNA binding activity of HSF (Fig. 5) (7, 28) . The lack of effect of the transcriptional inhibitor actinomycin D on PMA-mediated HSP synthesis further supports a predominantly post-transcriptional regulation of HSP expression by PMA (Fig. 6). We also found that in contrast to hsp70, hsp90 induction upon HS was not inhibited by actinomycin D, which suggests that the expression of this specific HSP family is regulated post-transcriptionally even during HS, which is in agreement with the increased hsp90 mRNA stability as compared with hsp70 mRNA (Fig. 7).

In eukaryotes, cellular growth, differentiation, and response to environmental stimuli are associated with differential mRNA stability (29). Many transiently expressed genes including cytokines, oncogenes (c-myc and c-fos), and transcriptional activators accumulate in activated cells secondary to enhanced stability of specific mRNA (30, 31, 32) . A number of these mRNAs contains AU-rich sequences as regulatory motifs in their 3`-untranslated region. The presence of such AU-rich sequences modulates the turnover of mRNA by targeting it to the cytoplasm for rapid degradation (33, 34) .

A similar mechanism of degradation could be employed to reduce the constitutive level of HSP mRNA expression under normal conditions. Directly after their synthesis, HSP mRNAs could be targeted to and degraded in the cytosol before accumulation. Indeed, Moseley et al.(35) have demonstrated that in addition to transcriptional regulation, the human gene coding for hsp70 can also be regulated post-transcriptionally through the 3`-untranslated region by HS. The 3`-untranslated region of the hsp70 message is AU-rich and contains an AUUUA-like motif (6, 36) . Furthermore, several groups have recently shown that cell activation by phorbol esters or calcium ionophores stabilizes AU-rich mRNA sequences by controlling, via PKC-mediated phosphorylation, the activation of a cytoplasmic protein called AU-binding factor (37) . AU-binding factor forms in vitro complexes with a variety of labile RNA containing multiple reiterations of the pentamer AUUUA (38) . From data concerning the inhibition by staurosporine and H-7 of HSP synthesis and HSP mRNA accumulation in PMA-treated PBM, we propose that a similar translational mechanism involving cycles of phosphorylation consecutively to the PMA-mediated activation of PKC could lead to stabilization and increased levels of HSP mRNA. However, we cannot exclude that during physiological events such as cell proliferation and differentiation, HSP synthesis results from a growth factor-mediated pathway that would activate serum- and/or growth factor-responsive elements in the promoters of hsp70 and hsp90 genes (39, 40) .

According to the nature of the stimuli (HS, phagocytosis, mitogen, etc.), a given cell could use either genomic 5`-transcriptional regulatory elements or 3`-mRNA post-transcriptional elements, or both, to finely control HSP synthesis. Under conditions associated with a sustained HSP expression such as cell proliferation and differentiation, inflammation, ischemia, or carcinogenesis, stabilization of mRNA could be the essential mechanism for HSP regulation, as we observed in cells stimulated with PMA. In contrast, stresses such as HS that lead to a rapid alteration of cellular proteins induce a more rapid and transient transcriptional regulation of HS genes and subsequent HSP synthesis.

Taken together, our results indicate that regulation of HSP induction after HS principally involves transcriptional events, whereas PMA-mediated HSP synthesis, as a paradigm for HSP induction during inflammation and cell activation and differentiation, results from a post-transcriptional regulatory mechanism.

  
Table: Superoxide production by human monocytes following HS or PMA treatment: colorimetric measurement and NBT assay

Extracellular O production was determined by reduction of cytochrome c monitored at 550 nm. Intracellular O was determined by NBT assay performed as described. Values are means ± S.E. from three distinct experiments.



FOOTNOTES

*
This work was supported by Swiss National Research Foundation Grants 32-37464.93 (to B. S. P.) and 31-37799.93 (to L. J.) and by INSERM, France (to M. R. J. S. and B. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed in Paris. Tel.: 33-1-44-41-23-36; Fax: 33-1-44-41-23-33.

The abbreviations used are: HS, heat shock; HSP, heat shock protein(s); PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PBM, peripheral blood monocytes; CGD, chronic granulomatous disease; NBT, nitro blue tetrazolium; PAGE, polyacrylamide gel electrophoresis; HSE, heat shock element; HSF, heat shock factor.


ACKNOWLEDGEMENTS

We thank I. Maridonneau-Parini for stimulating our interest in PMA regulation of HSP, R. Morimoto for helpful comments and for the hsp90 cDNA probe, P. D. Lew for providing the cells from the CGD patients, and L. Mudespacher for skillful technical assistance.


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