Nomega -nitro-L-arginine inhibits inducible HSP-70 via Ca2+, PKC, and PKA in human intestinal epithelial T84 cells

Juliann G. Kiang1,2,3, Sharon C. Kiang1, Yuang-Taung Juang2, and George C. Tsokos1,2

1 Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring 20910-7500; and Departments of 2 Medicine and 3 Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The nitric oxide (NO) synthase inhibitor Nomega -nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC50 of 0.0471 ± 0.0007 µM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca2+ dependent, we measured [Ca2+]i after treating cells with L-NNA. L-NNA at 100 µM significantly decreased resting [Ca2+]i. Likewise, treatment with 1 µM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca2+]i and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca2+]i and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca2+]i and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.

heat; nitric oxide; heat shock protein; protein kinase C; protein kinase A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is produced by constitutive and inducible NO synthase (cNOS and iNOS, respectively) in many cell types, including endothelial cells, neurons, and macrophages (34). NO is involved in a wide range of physiological processes, including blood vessel relaxation (14), neurotransmitter transduction (26), memory functions (31), and host defense against microbial pathogens (28). Increased levels of NO (16, 41, 43) and NOS activity (1, 16, 30, 43, 47) have been measured during hemorrhagic shock. Inhibition of NO production reduces the damage caused by hemorrhage (2, 16, 54). It appears that activation of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through cNOS activation to alleviate the damaged brain area (for review, see Ref. 3). A number of in vitro studies (11, 13, 48, 49) have demonstrated that heat stress inhibits iNOS gene expression.

Interactions between NO and heat shock protein (HSP) production have been studied previously. Reports have demonstrated that increases in NO production result in elevation of HSP-70 expression in smooth muscular cells (50), Hep G2 cells (32), and hepatocytes (25). In smooth muscle cells, NO-induced HSP-70 expression is mediated by heat shock factor-1 (HSF1) activation (50). HSPs are overexpressed under stress and display cytoprotection against various insults in many different types of organs and cells (23, 24). Our laboratory has found that inhibition of NO production by a NOS inhibitor can protect rat small intestine from injury caused by ischemia-reperfusion and that treatment with NOS inhibitors attenuates HSP-70 expression induced by heat stress. However, the underlying protective mechanism has not been studied.

The mechanism underlying the cytoresistance provided by HSPs is not fully clear. Evidence indicates that overexpression of inducible HSP 70 kDa (HSP-70i; a member of the HSP 70-kDa family) diminishes the intracellular Ca2+ response to stress or toxins (23, 24), downregulates the basal enzymatic activities of protein kinase A (PKA), PKC (7), c-Jun amino-terminal kinase, and p38 kinase (9), upregulates the basal enzymatic activities of protein phosphatase-1 and -2A (7, 29), and enhances the immunogenicity of tumor cells (33). These observations suggest that diminished signal transduction may mediate HSP-70-induced cytoprotection. NOS inhibitors may interrupt the signal pathway so that the cytoprotection is diminished.

Studies of human epidermoid A-431 cells (5, 18), Madin-Darby canine kidney cells (51), rat luteal cells (17), human breast cancer T-47D cells (20), MCF7 cells (21), and MDA-MB-231 cells (22) have indicated that HSP-70 is regulated by intracellular Ca2+ concentration ([Ca2+]i). Only cNOS is Ca2+ dependent (8). However, the interaction between NO generation and [Ca2+]i has not been investigated.

In this study, we found that heat stress increased HSP-70i expression in human colon carcinoma T84 cells. Our experiments provide a link between NOS, PKC, and PKA activities, placing NOS activity upstream of PKC and PKA activity. Blockade of NOS eventually causes decreased HSP-70i expression by inhibiting HSF1 translocation from the cytosol to the nucleus.


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INTRODUCTION
METHODS
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Cell culture. Human colon carcinoma T84 cells (American Type Culture Collection, Rockville, MD) were grown in 75-cm2 tissue culture flasks (Costar, Cambridge, MA) containing DMEM supplemented with 0.03% glutamine, 4.5 g/l glucose, 25 mM HEPES, 10% fetal bovine serum, penicillin (50 µg/ml), and streptomycin (50 U/ml; GIBCO-BRL, Gaithersburg, MD) and incubated in a 5% CO2 atmosphere at 37°C. Cells were fed every 3-4 days.

RNA extraction. T84 cells (5 × 106/sample) were combined with 0.8 ml of TRIzol reagent (GIBCO-BRL), sonicated, and then combined with 0.2 ml chloroform. The mixture was vortexed and centrifuged at 12,000 g for 15 min at 4°C. The clear aqueous liquid containing RNA (450 µl) was transferred to a new Eppendorf tube, and isopropanol (225 µl) was added, vortexed, and centrifuged at 12,000 g for 15 min at 4°C. The RNA pellets were collected and washed with 75% ethanol. The quantity of extracted RNA dissolved in water was determined spectrophotomically.

RT-PCR. The reaction mixture was 1× reverse transcription buffer containing 1 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 U RNasin, 0.5 µg oligo(dT)15 primer, 1 µg total RNA, and 15 U avian myeloblastosis virus RT (Promega, Madison, WI) in a final volume of 20 µl. The mixture was incubated at 37°C for 10 min and then at 42°C for 20 min. The transcription reaction was terminated by heating the mixture at 95°C in a water bath for 10 min before chilling on ice. The primers specific for HSF1, HSP-70, and beta -actin were as follows: HSF1, 5'-CTG GCC ATG AAG CAT GAG AA-3' and 5'-GAG AAC TGC CGG CTA TAC TA-3'; HSP-70, 5'-AAG GTG GAG ATC ATC GCC AA-3' and 5'-GCG ATC TCC TTC ATC TTG GT-3'; and beta -actin, 5'-ATG GAT GAT GAT ATC GCC GC-3' and 5'-AGG AAT CCT TCT GAC CCA TG-3'. Each length of the single-stranded primer was 20 bp and had a ratio of 1:1 for guanine-to-cytosine and adenine-to-thymine to maximize specificity.

Subsequently, 1× PCR buffer (80 µl) containing 3 mM MgCl2, 400 µM deoxy nucleosine triphosphate, 1 µM of each upstream and downstream primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA) was added to the above mixture. Mineral oil (100 µl; Sigma, St. Louis, MO) was layered on top of the mixture to prevent evaporation during the thermal cycling. Thirty PCR cycles were run (at 95°C for 1 min, 54°C for 1.5 min, and 72°C for 1.5 min). Identical quantities (10 µl) of each PCR product were loaded onto 1% agarose gels prepared with Tris-borate-EDTA (TBE) buffer. The gel was stained with 2 µl ethidium bromide (stock concentration, 1 µg/µl) in 100 ml TBE buffer and photographed. The interested bands were quantitated densitometrically (19).

Western blots. To investigate the synthesis of HSPs, we incubated cells in medium at different temperatures for different periods of time, returning them to 37°C for different periods of time. Cells were then removed from the culture plate with trypsin-EDTA (GIBCO-BRL) and pelleted by centrifugation at 750 g for 10 min. The pellet was lysed in Tris buffer (pH 6.8) containing 1% SDS and 1% 2-mercaptoethanol. Aliquots containing 20 µg protein were resolved on SDS-polyacrylamide slab gels (Novex precasted 10% gel, San Diego, CA). Protein was blotted onto a nitrocellulose membrane (type NC, 0.45 µm, Schleicher & Schuell), using a Novex blotting apparatus according to the manufacturer's suggested protocol. After the nitrocellulose was blocked by incubation for 90 min at room temperature in PBS containing 5% nonfat dried milk, the blot was incubated for 60 min at room temperature with mouse polyclonal antibody against cNOS, HSP-72, and HSF1 (Stressgen, Calgary, AB, Canada) at a 500× dilution in PBS-5% BSA containing 0.1% thimerosal. The blot was then washed three times (for 10 min each) in PBS-0.1% Tween 20 before being incubated for 60 min at room temperature with a 1,000× dilution of rabbit anti-mouse IgG peroxidase conjugate (Amersham Life Science, Arlington Heights, IL) in PBS-1% gelatin. The blot was washed six times (for 5 min each) in PBS-0.1% Tween 20 before detection of the peroxidase activity using enhanced chemiluminenscence (Amersham).

NO measurements. NO production indicated as nitrite was measured using the Griess reagent system with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride under acidic conditions (Promega).

[Ca2+]i measurements. Suspended cells were loaded with 5 µM fura 2-AM plus 0.2% Pluronic F-127 at 37°C for 60 min. Cells were then washed with Na+ Hanks' solution before fluorescence measurements. The method used to determine [Ca2+]i was as described previously (18).

Activity assays of PKC and PKA. The samples were extracted from both cytosol and membrane-bound fractions according to the manufacturer's suggested protocol (Life Technologies, Gaithersburg, MD). The cells were homogenized in extraction buffer without detergent and centrifuged at 100,000 g for 30 min. The supernatant contained the cytosolic enzyme, and the pellet contained the membrane-bound enzyme. The amount of protein in the samples was measured with Bio-Rad protein assay dye (Bio-Rad, Richmond, CA). The activities of PKC and PKA were measured using commercial assay kits (Life Technologies). The PKA activity was measured by phosphorylation of a specific substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly, and the enzymatic activity of PKC was measured by phosphorylation of Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, as described by Yasuda et al. (53). [gamma -32P]ATP (6,000 Ci/mmol stock solution) was used in this study. The substrate for PKA and PKC was incubated with protein samples at 30°C for 5 min and then transferred to the phosphocellulose disc. The disc was washed twice for 3-5 min with phosphoric acid solution and dried. The radioactivity was measured with an automatic liquid scintillation counter. The activities of enzymes were calculated according to the manufacturer's suggested protocol and expressed as the percentage of untreated cells.

Statistical analysis. All data are expressed as means ± SE. One-way ANOVA, two-way ANOVA, Studentized-range test, Bonferroni's inequality test, and Student's t-test were used for comparison of groups with P < 0.05 considered significant.

Chemicals. Chemicals used in this study were albumin, Nomega -nitro-L-arginine (L-NNA), aminoguanidine, L-arginine, Nomega -nitro-L-arginine methyl ester (L-NAME), L-N6-(1-iminoethyl)lysine (L-NIL), S-nitroso-N-acetylpenicillamine (SNAP; Sigma), H-89, GF-109203X (Calbiochem, La Jolla, CA), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), fura 2-AM, and Pluronic F-127 (Molecular Probes, Eugene, OR).


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
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L-NNA inhibits heat stress-induced increases in HSP-70i. Previously, we (18, 20-22) have established that human carcinoma T84 cells behave similarly to many other types of cells in producing HSP-70i in response to heat stress (38). Therefore, the following experiments were conducted with T84 cells exposed to 45°C for 10 min followed by recovery at 37°C for 6 h to induce optimal levels of HSP-70i.

In preliminary experiments, we found that intravenous administration of L-NNA but not aminoguanidine (an inhibitor of iNOS) to rats 1 h before exposure to heat stress effectively inhibited HSP-70i expression in rat small intestine (unpublished data). Hence, we treated T84 cells with L-NNA at different concentrations prior to heat stress. Figure 1 shows that L-NNA inhibited the heat stress-induced increase in HSP-70i production in a concentration-dependent manner with an IC50 of 0.0471 ± 0.0007 µM. Similar to the results observed with the in vivo studies, aminoguanidine did not alter the heat stress-induced increase in HSP-70i production in T84 cells (data not shown). These results suggest that L-NNA regulates HSP-70i expression.


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Fig. 1.   Nomega -nitro-L-arginine (L-NNA) inhibits inducible heat shock protein 70 kDa (HSP-70i) induction by heat stress. A: T84 cells were treated with different concentrations of L-NNA for 60 min before and during heat stress at 45°C for 10 min and then allowed to recover for 6 h at 37°C. HSP-70i production was detected by immunoblotting and quantitated by densitometry (n = 3). The IC50 of L-NNA calculated using the GraphPad Inplot program is 0.0471 ± 0.0007 µM. B: a representative immunoblot against HSP-70i antibody to lysates extracted from untreated cells (C) and cells exposed to heat stress (HS), 100 µM L-NNA alone (L), or L-NNA + heat stress (L + HS).

To determine whether cNOS specifically regulates HSP-70i expression, we treated cells with L-NAME (an irreversible inhibitor of cNOS and a reversible inhibitor of iNOS) or L-NIL (a selective inhibitor of iNOS). Like L-NNA, L-NAME also effectively inhibited the heat stress-induced increase in HSP-70i [Fig. 2, A (lane 4 vs. lane 2 and lane 6 vs. lane 2) and B], whereas L-NIL failed to do so [Fig. 2, A (lane 8 vs. lane 2) and B]. To determine whether NO production is associated with HSP-70i expression, we used the NO donor SNAP and NO substrate L-arginine. Cells were treated with SNAP or L-arginine in the presence of L-NNA. In this case, L-NNA failed to inhibit the heat stress-induced increase in HSP-70i [Fig. 2, A (lane 12 vs. lane 10 and lane 16 vs. lane 14) and B]. These results suggest that cNOS rather than iNOS is involved in the regulation of HSP-70i expression via NO production.


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Fig. 2.   L-NNA and Nomega -nitro-L-arginine methyl ester (L-NAME) inhibit HSP-70i expression. T84 cells were treated with 100 µM L-NNA, L-NAME, L-N6-(1-iminoethyl)lysine (L-NIL), S-nitroso-N-acetylpenicillamine (SNAP), L-arginine (L-Arg), L-NNA + SNAP, or L-NNA + L-arginine for 60 min before and during heat stress at 45°C for 10 min and then allowed to recover for 6 h at 37°C. HSP-70i production was detected by immunoblotting and quantitated by densitometry (n = 3). A: a representative immunoblot using an antibody against HSP-70i in lysates extracted from untreated cells or cells treated with respective chemicals before exposure to heat stress. Lanes 1, 3, 5, 7, 9, 11, 13, and 15: unheated; lanes 2, 4, 6, 8, 10, 12, 14, and 16: heated. B: HSP-70i was quantitated by densitometry. Open bars, unheated; filled bars, heated; C, untreated cells. * P < 0.05 vs. unheated cells (treated and untreated); ** P < 0.05 vs. untreated and heated cells; *** P < 0.05 vs. untreated and unheated, untreated and heated, treated and unheated, and treated and heated cells (determined by 2-way ANOVA and Studentized-range test).

To further determine the involvement of NO in heat stress-induced HSP-70i expression, we determined the presence of cNOS in T84 cells through Western blot analysis (Fig. 3A) and estimated NO production as indicated by nitrite. Exposure of cells to heat stress and recovery for 20h increased NO production, which was inhibited by L-NNA treatment (Fig. 3B), suggesting a close relationship between HSP-70i expression and NO production.


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Fig. 3.   L-NNA inhibits nitric oxide (NO) production. T84 cells were treated with 100 µM for 60 min before and during heat stress (HS) at 45°C for 10 min and then allowed to recover for 20 h at 37°C. The supernatant was collected, and NO production, indicated as nitrite, was measured with the Griess reagent system (n = 3). A: a representative Western blot of constitutive NO synthase (cNOS) presence in T84 cells. B: L-NNA treatment significantly inhibited NO production. * P < 0.05 vs. other groups (determined by chi 2-test).

L-NNA inhibits increase in HSP-70i at mRNA level. It has been shown (23, 24) that the heat stress-induced HSP-70i increase in other cell types occurs at the mRNA level. To determine whether the increase in HSP-70i in T84 cells exposed to heat stress occurred at the mRNA or translational level, RNA was collected at different time points after cells were exposed to heat stress at 45°C for 10 min. Using RT-PCR, we observed that the HSP-70i mRNA levels started increasing within 5 min after exposure to heat stress, reached the maximum at 2 h, and remained above the baseline 8 h later (data not shown).

Treatment with actinomycin D (an inhibitor for transcription) or cycloheximide (an inhibitor for translation) but not genistein (an inhibitor for protein tyrosine kinase) 10 min before heat stress effectively blocked heat stress-induced HSP-70i production (Fig. 4A). Using RT-PCR, we found that actinomycin D but not cycloheximide inhibited increases in HSP-70 mRNA caused by heat stress (Fig. 4B), supporting the concept that the heat stress-induced HSP-70i increase occurs at the mRNA level. Treatment with 100 µM L-NNA 1 h before heat stress significantly inhibited HSP-70 mRNA, indicating that L-NNA inhibits HSP-70i at the mRNA level (Fig. 4C).


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Fig. 4.   L-NNA inhibits HSP-70i at the mRNA level. T84 cells were treated with 5 µg/ml actinomycin D (ACD), 60 µg/ml cycloheximide (CHM), or 100 µM genistein (GEN) for 10 min before and during heat stress at 45°C for 10 min and then allowed to recover at 37°C for 2 h to determine HSP-70 mRNA using RT-PCR or for 6 h to determine HSP-70i production (n = 3). A: both actinomycin D and cycloheximide inhibited heat stress-induced HSP-70i production (n = 3). * P < 0.05 vs. vehicle unheated and ACD- and CHM-treated groups. ** P < 0.05 vs. vehicle-, ACD-, GEN-, and CHM-treated unheated groups. B: actinomycin D but not cycloheximide inhibited heat stress-induced increase in HSP-70 mRNA (n = 3). * P < 0.05 vs. vehicle unheated, ACD-treated, and CHM-treated unheated groups. ** P < 0.05 vs. vehicle unheated and ACD- and CHM-treated and unheated groups. C: cells were treated with 100 µM L-NNA 60 min before exposure to heat stress (HS) at 45°C for 10 min and then allowed to recover for 2 h at 37°C. HSP-70 mRNA was quantitated by densitometry (n = 3). * P < 0.05 vs. other groups (determined by chi 2-test).

L-NNA decreases resting [Ca2+]i. Our laboratory (5, 6, 18) reported previously that the heat stress-induced increase in HSP-72 in human A-431 cells was regulated by [Ca2+]i. We aimed to determine whether the heat stress-induced increase in HSP-70i production in T84 cells was also Ca2+ dependent. Cells treated with 10 mM EGTA 30 min before exposure to heat stress exhibited greatly diminished levels of HSP-70i production, whereas cells treated with 100 µM BAPTA displayed slightly attenuated HSP-70i expression. Treatment of cells with both EGTA and BAPTA caused an additive reduction of HSP-70i expression (Fig. 5).


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Fig. 5.   Ca2+ dependence of heat stress-induced HSP-70i production. T84 cells were incubated in Ca2+-free medium containing 10 mM EGTA or treated with 30 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for 10 min before exposure to 45°C for 10 min. These cells were then returned to 37°C for 6 h. A: immunoblotting was used to detect HSP-70i production (representative blot shown). C, unheated; HS, heat stress. B: HSP-70i production was quantitated by densitometry (n = 3). * P < 0.05, ** P < 0.05, *** P < 0.05, **** P < 0.05 vs. other groups (determined by 2-way ANOVA).

Because the heat stress-induced increase in HSP-72 was inhibited by removal of external Ca2+ or L-NNA treatment, we assessed the effect of L-NNA on [Ca2+]i in cells loaded with fura 2. The resting [Ca2+]i in T84 cells was 201 ± 4 nM (n = 6) in the presence of 1.6 mM external Ca2+, whereas in the absence of external Ca2+ the resting [Ca2+]i was 128 ± 2 nM (n = 9). This suggests that Ca2+ entry from external sources contributes to the maintenance of resting [Ca2+]i. The presence of L-NNA at 100 µM for 1 h decreased resting [Ca2+]i to 175 ± 3 nM (n = 3).

L-NNA inhibits heat stress-induced translocation of HSF1 from cytosol to nucleus. HSP-70i expression is mediated by the phosphorylation, trimerization, and translocation of HSF1, which normally resides in the cytosol (24), from the cytosol to the nucleus. To determine whether L-NNA inhibition of heat stress-induced HSP-70i overexpression is preceded by inhibition of HSF1, we separated cytosolic extracts from nuclear extracts from cells treated with 100 µM L-NNA 1 h before heat stress. We found that heat stress increased total HSF1 protein to 220 ± 18% and mRNA to 195 ± 5% (n = 3). L-NNA treatment blocked this increase (Fig. 6, A and B). Heat stress also elevated cytosolic HSF1 translocation to the nucleus, whereas treatment with L-NNA blocked this translocation (Fig. 6, C and D). These results suggest that L-NNA inhibits heat stress-induced increase in HSP-72 by inhibiting HSF1 synthesis and translocation.


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Fig. 6.   L-NNA inhibits heat shock factor-1 (HSF1) synthesis and its translocation from the cytosol to the nucleus. T84 cells were treated with 100 µM L-NNA for 60 min before exposure to 45°C for 10 min. RNA extraction was followed for HSF1 mRNA measurement using RT-PCR. In a parallel experiment, cytosolic extract was separated from the nuclear extract. Immunoblotting was used to detect HSF1 presence. HSF1 mRNA and protein were quantitated by densitometry. A: HSF1 mRNA; B: total HSF1 presence in the cell; C: presence of cytosolic HSF1; D: presence of nuclear HSF1. HS, heat stress. * P < 0.05 vs. respective control (determined by chi 2-test).

L-NNA inhibits PKC and PKA activities. Our laboratory (20-22) has reported previously that PKC and PKA regulate HSP production in human breast cancer cells. Because PKC and PKA are known to be involved in HSP synthesis, we studied the interactions between HSP-70i, L-NNA, PKC, and PKA. We found that cells treated with GF-109203X (a PKC inhibitor) or H-89 (a PKA inhibitor) failed to increase HSP-70i levels in response to heat stress. L-NNA also failed to further inhibit the expression of HSP-70i in cells pretreated with GF-109203X or H-89 (Fig. 7).


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Fig. 7.   HSP-70i inhibition by L-NNA is mediated by protein kinase A (PKA) and PKC pathways. T84 cells were treated with 1 µM H-89 or GF-109203X (GF) 30 min before 100 µM L-NNA for 1 h. Cells were then exposed to 45°C for 10 min and allowed to recover at 37°C for 6 h. Immunoblotting was used to detect HSP-70i levels, which were quantitated by densitometry (n = 3). * P < 0.05 vs. respective control (determined by 2-way ANOVA).

Likewise, treatment with GF-109203X or H-89 significantly decreased [Ca2+]i. L-NNA did not further decrease [Ca2+]i in GF-109203X- or H-89-treated cells (Fig. 8). These data suggest that it is highly likely that L-NNA, GF-109203X, and H-89 use the same pathway to inhibit HSP-70i production. Therefore, we measured the activities of PKC and PKA in cells subjected to similar experimental conditions. Figure 9 shows that the activities of cytosolic and membrane-bound PKC and PKA were decreased after treatment with L-NNA. Heat stress increased the activities of PKA and PKC, but the presence of L-NNA caused a significant inhibition (Fig. 9).


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Fig. 8.   Intracellular Ca2+ concentration ([Ca2+]i) decrease by L-NNA is mediated by PKA and PKC pathways. T84 cells were treated with 1 µM H-89 or GF-109203X 30 min before 100 µM L-NNA. [Ca2+]i was measured using fluorescent probe fura 2. A-C: representative tracings are presented; the initial tracing indicates resting [Ca2+]i.



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Fig. 9.   L-NNA inhibits PKC and PKA activities. T84 cells were treated with 100 µM L-NNA for 60 min before exposure to 45°C for 10 min. The cytosolic and membrane-bound proteins were extracted, and the activities of PKC and PKA were measured. The experiment was conducted 3 times independently. The data are presented as %total enzymatic activities in the cell. * P < 0.05 vs. respective control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we observed that exposure of human intestinal epithelial T84 cells to heat stress increases HSP-70i levels in a time- and temperature-dependent manner. Like other cells, the increase occurs at the mRNA level and is regulated by [Ca2+]i (5, 6, 18). Furthermore, we also found that the NOS inhibitor L-NNA blocked HSP-70i production at the mRNA level. The IC50 of L-NNA was 0.0471 ± 0.0007 µM, which is similar to that reported (27) in the nervous system, suggesting that L-NNA exerts its action on cNOS. Data from the experiments in which L-NAME and L-NIL were used further reinforce the view that cNOS regulates HSP-70i expression. In addition, the results from the experiments involving treatment with NO donors strongly suggest that NO production is involved in HSP-70i expression.

L-NNA downregulates HSP-70i overexpression, which has been associated with protection against ischemia-reperfusion injury (3, 40). It is known (18, 35, 36) that the increase in [Ca2+]i promotes the phosphorylation of cytosolic HSF1 and its translocation to the nucleus followed by its binding to heat shock elements located on the promoter region of the HSP-70 gene. These results are in agreement with findings (5) in human epidermoid A-431 cells. Therefore, the ability of L-NNA to block HSP-70i expression induced by heat stress is probably mainly due to its ability to lower [Ca2+]i. Data obtained from experiments showing that L-NNA failed to additionally reduce [Ca2+]i and HSP-70i expression in cells pretreated with inhibitors of PKC or PKA further reinforce this view. Moreover, the inhibitory effects of L-NNA on the enzymatic activities of PKC and PKA are highly likely to be the result of its ability to decrease [Ca2+]i since these two enzymes are known to be Ca2+ dependent.

On the basis of data from the current study and findings reported previously (18), we propose a model to explain the mechanisms involved in L-NNA-induced inhibition of HSP-70i expression (Fig. 10). At first, heat stress promotes Ca2+ entry via the reversed mode of the Na+/Ca2+ exchanger located at the cell membrane (18). The increases in [Ca2+]i stimulate the Ca2+-dependent cNOS to generate more NO as well as other Ca2+-dependent enzymes such as PKC and PKA. The generation of NO is also promoted by activation of nuclear factor-kappa B (NF-kappa B), which is initiated by increases in free radicals due to heat stress. Activation of PKC and PKA phosphorylates HSF1 in the cytosol, which afterward trimerizes, enters the nucleus, and initiates the process of transcription and translation. L-NNA alone decreases [Ca2+]i. This decrease is thought to attenuate the enzymatic activities of cNOS, PKC, and PKA, which leads to diminished HSF1 phosphorylation, trimerization, and translocation, resulting in a blockade of HSP-70i production.


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Fig. 10.   Proposed mechanism of cNOS regulating HSP-70i expression induced by heat stress. Heat stress increases [Ca2+]i via activation of the reversed mode of Na+/Ca2+ exchangers. The increased [Ca2+]i activates the Ca2+-dependent cNOS, subsequently generating more NO. The increased [Ca2+]i activates the Ca2+-dependent PKC and PKA that phosphorylate cytosolic HSF1. After phosphorylation, the cytosolic HSF1 forms trimers and enters the nucleus to proceed with transcription. L-NNA decreases [Ca2+]i and probably inhibits cNOS as well, thereby leading to inhibition of following cascaded reactions. NF-kappa B, nuclear factor-kappa B; +, increase; -, decrease.

This is the first published study showing that treatment of cells with L-NNA attenuates the heat stress-induced increase in HSF1 expression. This attenuation also represents a reduction of NO production because L-NNA but not L-NIL inhibits cNOS. Currently, it is not clear how NO interacts with HSF1 synthesis. It is documented that heat stress (5) and treatment of cells with a PKC stimulator (5) cause increased HSF1 expression, which is found here in T84 cells as well. Because NO is required to regulate the SP1 binding site of the tumor necrosis factor-alpha (TNF-alpha ) promoter in U-937 cells treated with TNF-alpha (45), it is likely that NO can also regulate the SP1 binding site on the promoter region of the HSF1 gene. The inhibitory effect of L-NNA on cNOS results in decreased NO production, therefore leading to downregulation of the HSF1 gene.

It has been reported (12) that enhanced NO production contributes to the production of proinflammatory molecules such as the cytokines interleukin-6, granulocyte colony-stimulating factor, NF-kappa B, and signal transducer and activator of transcription 3 in rat liver and lungs. In the ischemia-reperfusion-induced tissue injury model, NO displays a dual function. In local tissues, NO appears to combine with superoxide to form peroxynitrite (ONOO-), a powerful oxidant that can be cleaved into free radicals such as OH- and NO<UP><SUB>2</SUB><SUP>+</SUP></UP> and cause local tissue injury (52), although systemically NO is required to sustain vascular integrity and its reduction causes vascular leak (46). In this model, polymorphonuclear neutrophil (PMN) infiltration and leukotriene B4 (LTB4) generation increase. Inhibition of NO production by L-NNA results in significant reduction of local tissue damage, PMN infiltration, and LTB4 generation (42). It has been shown (35) that overexpression of HSP-70i reduces organ injury, PMN infiltration, and LTB4 generation. However, the cytoprotection observed with L-NNA in in vivo experimental models has not been observed in cultured cells. Our data (unpublished observations) indicate that cells treated with L-NNA blocking HSP-70i production are more vulnerable to assaults, whereas cells overexpressing HSP-70i are still cytoprotected. This discrepancy suggests that the protective mechanisms via HSP-70i and NOS inhibitors are different.

It has been reported (4, 10, 37, 38, 44) that tumor cells exposed to sublethal heat shock before chemotherapy are less responsive to treatment. Under such conditions, the presence of HSPs is not desired in cancer patients, and remedies that enable the reduction of HSP production are targeted. Additionally, some HSPs are known to act as molecular chaperones in the cell. The understanding of this link between NOS, PKA, PKC, and HSPs may offer insight into the ability of NO to regulate the activities of molecular chaperones in the cell. HSPs are expressed in many infectious diseases and autoimmune diseases and act as the immunodominant antigen (15, 24). Therefore, the ability of L-NNA, GF-109203X, and H-89 to downregulate HSP-70i may be of therapeutic use for treating patients with cancer, infectious diseases, or autoimmune diseases.

In conclusion, we have shown that cNOS is involved in the induced expression of HSP-70i by affecting the required transcription factor HSF1. cNOS is involved in the regulation of [Ca2+]i and therefore in regulating the activities of PKC and PKA, which are also involved in regulating the activities of HSF1 and HSP-70i.


    ACKNOWLEDGEMENTS

We thank R. L. Collins for making the graphs.


    FOOTNOTES

This work was supported by Department of Defense Research Area Management II Science Technology Objective C and R. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense.

Address for reprint requests and other correspondence: J. G. Kiang, Dept. of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Glen Ave., Silver Spring, MD 20910-7500 (E-mail: Juliann.Kiang{at}na.amedd.army.mil).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00138.2001

Received 29 March 2001; accepted in final form 8 November 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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