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aw Woli
ski1
bieta Grzesiuk2
1 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110, Jabonna, Poland
2 Department of Molecular Biology, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawiskiego 5a, 02-106 Warsaw, Poland
3 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-766 Warsaw, Poland
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Abstract |
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ylicz et al. 2001). In all organisms the Hsp expression under stress conditions is regulated at transcriptional level, e.g., in humans by the heat shock transcription factor Hsf1 (Morimoto, 1998; Wu, 1995), while in Escherichia coli by replacement of the sigma factor 
70 in RNA polymerase by the sigma factor 32 (Gross, 1987). The Hsps allow cell survival under stress conditions by renaturating of denaturated proteins, protecting of stress-labile proteins, preventing protein aggregation (chaperone functions), and by degradation of damaged proteins (protease activities) (Lindquist & Craig, 1988; Morimoto, 1998; Jolly & Morimoto, 2000). They have also many housekeeping functions under non-stressful conditions during the cell cycle, growth, development, and differentiation (Morimoto, 1998). Among a number of plausible inducing factors already studied, extremely low artificial electromagnetic fields have been shown to induce stress response in various cells, such as expression of 32 mRNA (Cairo et al. 1998) and induction of DnaJ and DnaK proteins in Eschericha coli (Chow & Tung, 2000); expression of hsp-16 gene in Caenorhabditis elegans (Miyakawa et al., 2001); induction of heat shock transcription factor Hsf1 and Hsp70, Hsp90 and Hsp27 in human cells (Lin et al. 1997; Lin et al. 1998; Goodman & Blank, 1998; Pipkin et al. 1999). Nevertheless, the role of endogenous electromagnetic fields, i.e., generated by electrically active cells within a body remains controversial. Heat shock proteins (Hsps) protect cells against various environmental and endogenous stressors. Cytoprotection caused by Hsps involves tolerance induced by one agent against other, more severe agents. We have found that exposure of prokaryotic (Escherichia coli) and eukaryotic (Caco-2) cells to an electrical field (EF) connected with a myoelectrical migrating complex (MMC) generated by the small intestine smooth muscle induces the heat shock response. Using Western blot analysis, we have detected an elevated level of sigma factor 32 in E. coli cells exposed to MMC-related EF, and confocal microscopy indicated an increased level of the inducible form of Hsp70 protein in EF-stimulated Caco-2 cells. Additionally, we have found that this induced level of Hsp70 protected the Caco-2 cells against apoptosis caused by camptothecin. Our observations suggest that the myoelectrical activity of the gut may induce heat shock mechanisms in the cells of gut epithelium as well as in gastrointestinal micro-organisms.
(Received 30 January 2006;
accepted after revision 15 May 2006; first published online 25 May 2006)
Corresponding author E. Grzesiuk: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawiskiego 5a, 02-106 Warsaw, Poland. Email: elag{at}ibb.waw.pl
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Introduction |
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The aim of the present study was to determine the effect of induction of the sigma factor 32 in E. coli cell extracts and the Hsp70 protein (an inducible form, often referred to as Hsp72; Welch, 1992) in human colon adenocarcinoma Caco-2 cells after their exposure to the MMC-related EF using our experimental model (Grzesiuk et al. 2001). In both biological model systems, the induction of the heat shock response, as a reaction to EF stimulation, was observed. Moreover, induction of Hsp70 by EF partly protected the Caco-2 cells against camptothecin-induced apoptosis.
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Methods |
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The duodenal MMC recording, MMC characteristics and the electrical stimulation of bacterial cells were described in detail previously (Grzesiuk et al. 2001). Briefly, the myocyte action potentials representing the duodenal MMC signal in a healthy preruminant Friesian calf (4 weeks old) traced by bipolar serosal electrodes were digitally recorded (extracted frequency band of 10–50 Hz; amplitude range, ± 500 µV; PowerLab/4e, ADInstruments, Castle Hill, Australia). One MMC cycle was selected from a control recording made for another study (Puchaa et al. 1998) upon the following criteria: the completeness of MMC phases, the duration and amplitude of MMC cycle phases close to the average values for calves of this age and lack of recording artefacts. The MMC cycle parameters were as follows: cycle duration, 39 ± 8 min; contribution of subsequent MMC phases, phase I of no spiking activity (NSA), 35%; phase II of irregular spiking activity (ISA), 55%; and phase III of regular spiking activity (RSA) 10% (Fig. 1). The digital recording was transmitted into the memory of an electric field-generating device (SGP-generator, ESCO, Warsaw, Poland). E. coli AB1157 (Bachmann, 1987) cells were exposed to MMC-related EFs via two platinum electrodes (o.d. 0.3 mm, length 120 mm) located 2 cm apart and fixed in 30 ml Corex tube (for bacterial cells) or 4 cm apart and fixed in Petri dish (for Caco 2 cells) and placed in a Faraday cage in the CO2 incubator. The range of an alternating electrical field was from Emin
=
–177 µV cm–1 to Emax
= 251 µV cm–1; average medium conductance
= 20 µS cm–1, average sample resistance R
= 0.7 M
, and, as a result, the current density varied from jmin
=
–3.5 x 10–9 A cm–1 to jmax
= 5 x 10–9 A cm–1). During our experiments, the reconstructed in vitro MMC signal was applied by the generator repetitively: in bacterial studies for 2 h (i.e. the entire MMC cycle was repeated ca 4 times) and in Caco-2 cell studies for 24 h (i.e. the MMC cycle was repeated ca 38 times). For E. coli the time of stimulation/number of MMC cycles was chosen according to our previous studies on the bacterial growth rate (Grzesiuk et al. 2001) and on the protective effect of MMC-related EF against UVC irradiation, which was found to be dose independent, at least in the range of one to four complete MMC cycles (Wójcik-Sikora et al. 2001). Taking into consideration that the time of division of Caco-2 cells is about 36 times longer than that of E. coli (ca 40 min for E. coli versus 24 h for Caco-2 cells), the stimulation of these cells was extended to 24 h.
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Expression of 32 in bacteria using Western blot analysis
Overnight cultures of E. coli in Luria-Bertani (LB) broth (Miller, 1972) were diluted 1:20 in C-salts 0.02% MgSo4
x 7H2O, 1%K2HPO4, 0.2% citric acid, 0.35% NH4NaHPO4
x 4H2O (Vogel & Bonner, 1956) supplemented with glucose (0.5%), casamino acids (0.2%) and thiamine (10 µg ml–1), and grown for 2 h with shaking at 37°C in the presence of the MMC-related EF. Bacteria grown without EF (identical conditions except that the electric field-generating device was switched off) served as our control. For heat stimulation, bacteria were incubated at 45°C for 15 or 40 min. Bacteria incubated at 28°C for 40 min served as a negative control. Bacterial cell lysates were prepared immediately after the treatment according to the standard method (Maniatis et al. 1982). The concentration of protein in lysates was determined according to the Lowry method (Lowry et al. 1951), modified to be performed on 96-well plates with BSA (Sigma), as a standard. A total of 25 µg of protein was loaded on each lane for TRICINE SDS-PAGE gel electrophoresis (Schagger & von Jagow, 1987). Protein bands were transferred onto 0.45 µm nitrocellulose membrane (OSMONICS, GE Osmonics Inc, Minnetonka, MN, USA). The level of 32 was determined by incubation of the membrane for 60 min with primary rabbit polyclonal anti-32 antibodies diluted 1:250. Sigma 32 protein purified according to Cooper & Ruettinger (1975) as well as the 32 antibodies were a generous gift from Dr K. Liberek (University of Gdask, Poland). Membranes were blocked with 1% Blot-Qualified BSA (Promega, Madison, WI, USA) and visualization was done using secondary antibodies, antirabbit IgG (Fc) alkaline phosphatase conjugate (dilution 1:5000, incubation time 30 min, as recommended in a technical protocol of ProtoBlot II AP System with Stabilized Substrate, Promega). Densitometry analysis was performed using VILBER LOURMAT System (Marne-la-Vallée Cedex, France) with a digital camera coupled to the computer with software for quantitative data analysis. The separate experiments were repeated six times.
Detection of Hsp70 in Caco-2 cells with confocal microscopy
Caco-2 cells (between 80 and 100 passages) were grown in plastic bottles in a standard Dulbecco's modified Eagle's medium (Sigma) consisting of 2 mM L-glutamine (Sigma), 1% non-essential amino acids (Gibco, Invitrogen Corporation, Carlsbad, CA, USA), 10% heat inactivated fetal bovine serum (Gibco, Invitrogen Corporation, USA), 10 IU ml–1 penicillin G, 100 µg ml–1 streptomycin sulphate and 250 ng ml–1 amphotericin B (Sigma). The cultures were kept in 5% CO2 and 95% O2, 90% relative humidity at 37°C. The experiments with EF stimulation were performed in the CO2 incubator. For determination of Hsp70 expression, the Caco-2 cells (at a density of 5 x 105 cells ml–1) were grown on coverslips in six-well plastic plates for 6–8 days. Subsequently, the coverslips were transferred to Petri dishes containing fresh medium and placed between two electrodes connected to the SGP-generator for 24 h. For heat stimulation, the cells were incubated at 56°C for 30 min, prior to staining. The EF-treated and/or heat-treated and the untreated (control) cells were fixed on coverslips in 4% buffered paraformaldehyde for 20 min. The level of Hsp70 was detected using monoclonal mouse antihuman Hsp70 antibodies (dilution 1:100, incubation time 60 min, Calbiochem, EMD Biosciences Inc, Darmstadt Germany) Because Hsp70 is not expressed constitutively in most cells, this antibody is suitable for ascertaining whether a stress response has occurred in the cell. Increased levels of Hsp70 expression occur following stress even in cells that constitutively express Hsp70 (Calbiochem Protocol, Data Sheet 386032). The Hsp70 antibodies were directly labelled with Zenon Mouse IgG Labeling Kit (Alexa Fluor 555, wavelenth of absorption/emission, 555/565 nm; Molecular Probes, Eugene, OR, USA) and immediately used. The cells were examined by a confocal microscope (LSM 5 PASCAL, Zeiss, Jena, Germany) while a He–Ne (543 nm) laser was employed for excitation. The fluorescence intensity was measured and calculated using AxioVision Release 4.1 software (Zeiss).
Apoptosis assay
To obtain Caco-2 cell suspension, cultures were treated with 0.5% trypsin in 0.2% EDTA. Then the cells were diluted to 2 x 105 cells ml–1, grown overnight for adherence to coverslips and transferred to the Petri dishes to be incubated with or without EF for 24 h. Camptothecin (Sigma) dissolved in DMSO (0.25% v/v solution) was added to a final concentration of 50 µM and the cells were incubated for 24 h with or without EF. Samples treated exclusively with camptothecin or EF, and those without any stimulation were used as respective controls. Three separate experiments were performed to obtain 7–21 slides; in each slide, at least 10 visual areas were analysed. The percentage of apoptotic cells was examined using the Apoptosis Detection Kit (BD Biosciences Pharmingen, San Jose, CA, USA). The method is based on the affinity of Annexin V–FITC (Ca2+-dependent phospholipid-binding protein conjugated to FITC fluorochrome; wavelength of absorption/emission, 488/530 nm) for the phospholipid phosphatidylserine (PS). Additionally, tested cells were stained by propidium iodide (PI; wavelength of absorption/emission, 535/617 nm). Thus, the kit allowed as to detect the viable cells (Annexin V–FITC negative, PI negative), early apoptotic cells (Annexin V–FITC positive, PI negative; green) and late apoptotic cells (Annexin V–FITC positive, PI positive; green and red) or dead cells (Annexin V–FITC negative, PI positive; red)
Statistics
Data were analysed by one-way ANOVA followed by the Tukey–Kramer post hoc test (GraphPad Prism v4.0, GraphPad Software, San Diego, CA, USA). In all statistical analyses, P < 0.05 was taken as the level of significance.
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Results |
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We examined the expression of the sigma factor 32 in whole cell extracts obtained from E. coli cells treated or non-treated with the EF prior to lysis. The results of Western blot analysis and densitometry shown in Fig. 2 indicate an increasing amount of the 32 protein in extracts from cells incubated for 40 min at 45°C (condition for heat shock induction; Fig. 2, lane 2), but ever more in the extracts from cells growing at 37°C, but additionally treated with the EF for 2 h (Fig. 2, lane 5). It is worth notifying that the level of 32 in the extract from cells incubated at 37°C without EF (Fig. 2, lane 4) was much lower and similar to that observed in the sample incubated at 45°C for 15 min (Fig. 2, lane 1). We do not know the origin of the band above the 32 that was always present in our Western blots. Supposedly, it might represent any 32 complexes with other proteins; however, the use of SDS gel excludes this possibility, leading to an alternative suggestion of cross-reaction of anti-32 antibodies with any other Hsps.
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Induction of the Hsp70 protein was investigated in Caco-2 cells using antihuman Hsp70 antibodies conjugated with Alexa Fluor 555. Pretreatment of the Caco-2 cells with the MMC-related EF led to a strong fluorescence of whole cells observed in our confocal microscope studies (Fig. 3). A similar strong fluorescence was also obtained with cells incubated at 56°C (positive control) but not with those incubated at 37°C (negative control). This result indicates that the MMC-related EF stimulates induction of the Hsp70 protein. Moreover, the Caco-2 cells incubated at 56°C were significantly smaller (13 ± 2 µm) and with a more concentrated cytoplasm compared to the control cells (20 ± 2 µm) incubated at 37°C (P < 0.05; Fig. 3). Using the same approach, 21 day cultures of polarized Caco-2 cells were examined and the results were similar to those observed in the 7 day cultures (data not shown).
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Apoptotic cells were detected using the annexin V–FITC test, in which cells with green fluorescence (positive for annexin V) were assumed to be apoptotic. No significant effect of EF treatment on the apoptosis in the Caco-2 cells was detected (Fig. 4). However, when apoptosis was induced with camptothecin and reached a value of 43.5%, the pretreatment of the cells with EF for 24 h led to a significant (36.1%, P = 0.05) decrease of apoptosis (Fig. 4). In contrast, no significant effect of EF treatment on late apoptosis or necrosis (cell death) was detected (Fig. 4), suggesting a protective effect of the MMC-related EF solely on the early stages of apoptosis.
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Discussion |
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32 factor and in Caco-2 cells, it significantly increased expression of the Hsp70 protein. The enterocyte-like Caco-2 cell line of human colon carcinoma is a useful in vitro model of the intestinal epithelium (Malago et al. 2003). Importantly, both results (in bacterial and Caco-2 cells) were achieved at a temperature of 37°C, within the physiological range for mammalian species. It is known that in E. coli, the induction of Hsps reaches its maximum at about 5 min after the temperature shift from 30 to 42°C. It was also shown that the level of 32 and the dnaK gene transcription run simultaneously following the temperature change (Straus et al. 1987; Yura et al. 1993). Previously, we have found that EF, like high temperature, protects E. coli cells against the effects of UV light (Wójcik-Sikora et al. 2001). This protective effect appeared to involve the heat shock response, since it was not observed in bacterial cells deleted for dnaK and dnaJ genes encoding DnaK and DnaJ proteins, members of the Hsp70 and Hsp40 family, respectively. Recently, Kruszewska et al. (2005) demonstrated that the EF stimulation may gently increase the synthesis of peptidoglycan hydrolases in lactic acid bacteria, thereby regulating their growth in the gut. However, according to Qoronfleh et al. (1998), induction of Hsp is known to damage complex or inactivate autolysin production in Staphylococcus aureus. This latter discrepancy needs to be clarified. Katsuki et al. (2004) showed that feeding induces the heat shock response regulated by a heat shock transcription factor 1 (Hsf1) in the small intestine and liver of mice but not in the colon, stomach, brain, muscle or lungs. The authors suggested a mechanism that protects the digestive tract against xenobiotic chemicals that cause oxidative stress. Since the small intestine and liver are the primary regions exposed to xenobiotics, the greatest stress response was found in these organs. In the porcine intestinal epithelium, Hsp70 expression was mainly found in the intestinal crypts (Laubitz D, Jankowska A & Zabielski R, unpublished observations), a preferential place of bacterial colonization, but not in the villi, where absorption takes place. Crypts are closer to the EF-producing muscular layer than the intestinal villi, so it is probable that part of the heat shock response observed in mice by Katsuki et al. (2004) was associated with the elevated myoelectrical activity of the gut during the postprandial period. Following their study, it may be suggested that the prandial pattern characterized by irregular bursts of action potentials (with similar signal characteristics to that observed during phase II of the MMC) may produce the heat shock response. Among the MMC phases, the myoelectric quiescence associated with phase I is not relevant for inducing the heat shock response, since no EF-producing action potentials are present. This was also confirmed in our control experiments without the EF treatment. In contrast, the irregular spiking activity associated with phase II is expected to induce the heat shock response, and phase III might produce the strongest response. Thus, in pigs, calves and humans, in which the contribution of phases II and III to the entire duration of the duodenal MMC is substantial (> 80, > 65 and > 60%, respectively), we may expect more influence on the heat shock response than in dogs, in which phase I comprises more than 65% of the entire duodenal MMC cycle (Zabielski & Naruse, 1999). Yamaguchi et al. (1992) reported that the changes in electric properties of the cell membranes in vitro may be related to the duration of electromagnetic field fluxes. Thus, we may speculate that alterations of EF in phase with duodenal MMC (from quiescence phase I to myoelectrically vigorous phase III) might be the stimulus to induce the heat shock response. Harmful substances present in food, such as irritating chemicals and bacterial toxins, are known to enhance gut motility, in particular the duration and migration of phase III (Bhutta et al. 1999; Yao et al. 2004); thus, presumably, they may further increase the heat shock response. In conclusion, a novel cytoprotective role is suggested for gut motility, which involves induction of heat shock proteins in the epithelial cells.
The pathways leading to Hsp70 induction by the EF and by heat in Caco-2 cells are different (Goodman & Blank, 2002). The first pathway is similar to that of heat shock, but involves a different domain binding Hsf1 in the region of the Hsp70 gene promoter. This domain contains three nCTCTn sequences necessary for response to electric fields. The second pathway involves Hsf1 phosphorylation by members of the mitogen-activated protein kinase (MAPK) subfamilies and results in binding of the activation protein 1 (AP-1) transcription factor (Goodman & Blank, 2002). The possibility that EF induces the heat shock response by affecting oxidative processes has also been considered (Zmyslony et al. 2004). In the present studies, we have shown that the MMC-related EF is a strong inducer of 32 in bacteria and Hsp70 in Caco-2 cells. Moreover, in the Caco-2 cells, induction of Hsp70 with EF occurs at a physiological temperature of 37°C, which seems to be less harmful, at least to cell morphology, than 56°C. At 56°C, the morphometric analysis showed a significant reduction in Caco-2 cell size, concomitant with the increased concentration of cytoplasm, which may suggest the induction of apoptosis (Fig. 3).
Heat shock proteins play an important role in the control of apoptosis by blocking the apoptotic pathway at several levels (Jolly & Morimoto, 2000; Ravagnan et al. 2001; Schmitt et al. 2003). The initiation of apoptosis is associated with activation of regulator caspases and proapoptotic proteins belonging to the Bcl-2 family, such as BAX and BID, and release of a mitochondrial cytochrome c. In the cytosol, the cytochrome c triggers oligomerization of the apoptosis protease activating factor-1 (Apaf-1), which recruits pro-caspase 9 and pro-caspase 3 into the apoptosome, the executor caspase activation multiprotein complex. Hsp70, by interaction with Apaf-1, prevents its interaction with pro-caspase 9 (Saleh et al. 2000; Beere et al. 2000; Beere & Green, 2001). As already mentioned, a stress factor through induction of Hsps may ensure the development of cross-tolerance to another, more severe stress agent. This normally results in extensive apoptosis (Mosser et al. 2000). In our experiments, we have not observed any influence of the MMC-related EF on the intensity of apoptosis in our Caco-2 cells (Fig. 4). However, the camptothecin-induced apoptosis was significantly reduced by EF pretreatment, which, as we have shown, involved Hsp70 induction.
In conclusion, induction of Hsp70 by the myoelectric component of the intestinal MMC may help to maintain proper functioning of the gastrointestinal tract and to restrain cell apoptosis. We postulate that an additional cytoprotection pathway involving Hsp70 is switched on in response to cytotoxic agents that increase apoptosis. In this particular case, the MMC-related EF protects the gut epithelial cells against excessive apoptosis via heat shock proteins.
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ski is a recipient of the Foundation for Polish Science (FNP) scholarship. This article has been cited by other articles:
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  L. Eckmann, T. Nebelsiek, A. A. Fingerle, S. M. Dann, J. Mages, R. Lang, S. Robine, M. F. Kagnoff, R. M. Schmid, M. Karin, et al. Opposing functions of IKK{beta} during acute and chronic intestinal inflammation PNAS, September 30, 2008; 105(39): 15058 - 15063. [Abstract] [Full Text] [PDF]
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