Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1

Shigetada Teshima1, Hiromu Kutsumi2, Tsukasa Kawahara1, Kyoichi Kishi1, and Kazuhito Rokutan1

1 Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima City, Tokushima 770-8503; and 2 Gastrointestinal Unit, Kyoto Red Cross Hospital, Kyoto City, Kyoto 605-0981, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O2-). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [3H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O2- and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH2F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappa B, and inhibition of this activity by nuclear factor-kappa B decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O2- produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.

NADPH oxidase; superoxide anion; hydrogen peroxide; antiapoptosis; cell growth


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PHAGOCYTE NADPH OXIDASE-DERIVED superoxide anion (O2-) and related oxygen metabolites play a crucial role in microbial and tumoricidal activities (1, 27). These oxygen metabolites are now considered to be an important signal for initiation of inflammatory and immune responses. The phagocyte NADPH oxidase consists of a membrane-bound cytochrome b558 heterodimer (gp91-phox and p22-phox) and three cytosolic components (p67-phox, p47-phox, and p40-phox) and is modulated by Rac1/2 (see Ref. 1 for review). On activation, the cytosolic components translocate to the plasma membrane, where the functional NADPH oxidase is assembled.

There is increasing evidence that distinct types of nonphagocytic cells, such as vascular smooth muscle cells (18), glomerular mesangial cells (12), endothelial cells (13), and fibroblasts (15), express p22-, p67-, and p47-phox and can produce small amounts of O2-. These cells were suggested to have a low potential cytochrome b in their plasma membranes whose spectroscopic characteristics were similar to those of the phagocyte cytochrome b558 (12). Furthermore, Meier et al. (15) showed that fibroblasts isolated from a patient with X-linked chronic granulomatous disease causing genetic defects in gp91-phox are able to generate O2- similarly to fibroblasts from a healthy donor. On the basis of these observations, it has been proposed that a genetically distinct isoenzyme of gp91-phox must exist in the nonphagocytic cells having O2--releasing activity (12). Recently, Suh et al. (28) molecularly identified a novel gp91-phox isoenzyme, termed a mitogenic oxidase (Mox1). They also demonstrated that Mox1-derived O2- regulated serum-dependent growth of aortic vascular smooth muscle cells and that overexpression of Mox1 in NIH-3T3 cells enhanced O2- production and induced abnormal cell growth and transformation (28).

We previously reported that primary cultures of guinea pig gastric pit cells express all of the essential components of the phagocyte NADPH oxidase and can release abundant O2- through the oxidase (30, 31). However, the pit cell oxidase displays features different from those of the phagocyte oxidase. Pit cells in culture spontaneously and continuously produce O2- for up to several days and do not exhibit respiratory burst-like production. Potent stimulators for the phagocyte NADPH oxidase, such as phorbol diester, do not increase the rate of O2- production by pit cells (30). O2- generated by gastric pit cells is not lethal to the cells themselves, as similarly observed in endothelial cells (13) and vascular smooth muscle cells (18). However, the physiological implications of O2- production by gastric pit cells remain to be elucidated.

We demonstrate here that guinea pig gastric pit cells express Mox1 but not gp91-phox. Furthermore, we suggest that O2- derived from the Mox1 oxidase may regulate spontaneous apoptosis of pit cells through modulating the caspase- and nuclear factor-kappa B (NF-kappa B)-dependent pathways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and culture of gastric mucosal cells. All procedures involving animals were approved by the Animal Care and Use Committee of the University of Tokushima. Male guinea pigs, weighing ~250 g, were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Gastric mucosal cells were isolated aseptically from guinea pig fundic glands and cultured in RPMI 1640, supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 50 µg/ml gentamicin, as previously described (20, 21, 23, 30). The cell populations were determined by cytochemical and immunocytochemical analyses as well as transmission electron microscopic examination, as previously described (24, 30). The cultured cells consisted of pit cells (~90%), parietal cells (5%), mucous neck cells (<1%), fibroblasts (<1%), and pre-pit cells (5%). Among these cell populations, only mature pit cells expressed detectable levels of p47-phox and p67-phox, and their O2- production was confirmed by nitroblue tetrazolium staining (30). Cell growth was estimated by counting cell number and by measuring the [3H]thymidine uptake, as previously described (17).

Preparation of guinea pig neutrophils. Guinea pig peritoneal neutrophils were obtained by flushing the peritoneal cavity with saline 12 h after an intraperitoneal injection of 25 ml of 3% thioglycolate broth as described previously (30). Microscopic observation after Giemsa staining revealed that >90% of peritoneal exudate cells were neutrophils.

Measurement of O2- release from gastric pit cells. O2- release from gastric pit cells was assayed by measuring the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c as described previously (30). The reduction of ferricytochrome c was spectrophotometrically determined at 550 nm, and the amount of O2- release was expressed as nanomoles per milligram of protein per hour.

Analysis of DNA fragmentation. Cells were washed with PBS and then lysed by treatment for 30 min at 4°C with lysis buffer consisting of 10 mM Tris · HCl (pH 8.0), 10 mM EDTA, and 0.5% Triton X-100. Intact chromatin DNA was pelleted by centrifugation of the lysate at 20,000 g for 30 min at 4°C. The amounts of intact chromatin DNA in the pellets and soluble, fragmented DNA in the supernatants were spectrophotometrically determined at 600 nm by using the diphenylamine reagent (5). Percentage of fragmented DNA recovered in the supernatant was calculated.

The DNA in the supernatant was extracted twice with an equal volume of phenol-chloroform mixture (1:1) and then precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. After treatment with 40 µg/ml of RNase A for 1 h at 37°C and then with 50 µg/ml pronase K for 30 min at 37°C, the extracted DNA was subjected to 2% agarose gel electrophoresis to detect nucleosome-sized DNA ladders. The fragmented DNA was stained with ethidium bromide and visualized by ultraviolet transillumination.

Assessment of nuclear morphology. Chromosomal condensation and fragmentation were assessed by staining with the fluorescent dye Hoechst 33342. Cells were fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. After fixation, they were washed twice with PBS and then exposed to 5 µg/ml of Hoechst 33342 in PBS for 30 min at room temperature. After being washed, samples were mounted with glycerol-PBS (1:1 vol/vol) and subjected to fluorescence microscopic examination.

Measurement of caspase 3-like activity. After cells were washed with ice-cold PBS, they were lysed with 100 mM HEPES buffer (pH 7.5), containing 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml pepstatin, and left on ice for 30 min. After centrifugation at 15,000 g for 30 min at 4°C, the supernatant (100 µg protein) was mixed with 150 µl of 100 mM HEPES buffer (pH 7.5), containing 10% sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, and 400 µM of the synthetic substrate N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (BIOMOL Research Laboratories, Plymouth, PA). The mixture was incubated at 37°C for 1 h. Caspase 3-like activity was assayed by measuring the increase in absorbance at 405 nm. The reaction mixture lacking N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide or cellular proteins was used as a negative control. Enzyme activity was expressed as increase in the absorbance per milligram of protein per hour.

Decoy oligodeoxynucleotide experiments. Double-stranded NF-kappa B decoy and mutant decoy oligodeoxynucleotides (ODNs) were prepared from the complementary single-stranded phosphorothioate-bonded ODN by annealing. The sequence of each decoy ODN was as follows: NF-kappa B decoy, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; mutant decoy, 5'-AGTTGAGCTCACTTTATCAGGC-3'. One nanomole of double-stranded decoy ODN was mixed with 5 µg of LipofectAMINE (Life Technologies, Rockville, MD) in 0.1 ml of serum-free OPTI-MEM (Life Technologies), and then the mixture was incubated for 20 min at room temperature to form liposome complexes. After 0.9 ml of serum-free OPTI-MEM was added to the mixture, cultured gastric mucosal cells were incubated with the liposome complexes for 8 h at 37°C. Thereafter, the decoy ODN-containing medium was replaced by fresh serum-free RPMI 1640, and cells were further incubated for 12 h to assess their apoptosis.

Gel mobility shift assay. Nuclear proteins were prepared from cultured gastric mucosal cells, and the activation of NF-kappa B was examined by gel mobility shift assay, as described previously (20, 23).

RT-PCR and Northern blot analyses. Total RNA was isolated with an acid guanidinium thiocyanate-phenol-chloroform mixture (6). RT reaction and PCR were performed using a TaKaRa RT-PCR kit (TaKaRa, Tokyo, Japan). The sequences of primers used were as follows: human gp91-phox, 5'-CATCATCTCTTTGTGATCTTCT-3' (sense) and 5'-CTTAGGTAGTTTCCACGCATC-3' (antisense) (25); human mox1, 5'-GCTCATTTTGCAGCCGCA-3' (sense) and 5'-AGAATGACCGGTGCAAGG-3' (antisense) (28). Amplified products were separated in a 6% polyacrylamide gel, stained with ethidium bromide, and visualized by ultraviolet transillumination. The resultant PCR products were purified from the gel, ligated into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), and transformed into JM109 cells. Transformed plasmids containing the appropriate insert DNA were selected and sequenced with a DNA sequencer (model ABI 373; PE Biosystems Japan, Tokyo, Japan).

The amplified 589-bp cDNA for guinea pig mox1 described above was used as a probe for Northern blotting. The cDNA probe for guinea pig mox1 or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prelabeled with [alpha -32P]dCTP to a specific activity of >1 × 108 cpm by a random primer labeling kit (Amersham Japan, Tokyo, Japan). Total RNA (15 µg) was subjected to electrophoresis in a 1% agarose gel containing 0.6 M formaldehyde and transferred to a Hybond N-plus filter (Amersham). After prehybridization, the filter was hybridized for 12 h at 60°C by incubating with the labeled cDNA probe for guinea pig mox1 or human GAPDH in the presence of 100 µg/ml heat-denatured salmon sperm DNA and 10% dextran sulfate. The filter was washed three times with 2× SSC (1× SSC: 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at 60°C and then twice with 0.2× SSC containing 0.1% SDS at 60°C. Bound probes were autoradiographed by exposing the filter to Kodak X-Omat films for 2 days at -85°C.

Statistical analysis. ANOVA and Scheffé's test were used to determine statistically significant differences. Differences were considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of Mox1 transcript in gastric mucosal cells. In a previous report (31), we showed that cultured guinea pig gastric mucosal cells had immunoreactive proteins to an antibody raised against synthetic peptide corresponding to human gp91-phox (residues 508-526). However, the molecular masses of the pit cell gp91-phox (51-53 kDa) were somewhat different from those of the neutrophil gp91-phox (51-66 kDa) (31). The amino acid sequence of the epitope of human gp91-phox is 48% identical to that of human Mox1 (28); therefore, it is possible that the antibody recognized the Mox1 but not gp91-phox.

To address this issue, primer sets were designed to specifically hybridize the gp91-phox or mox1 mRNA, respectively. RT-PCR with the primer set for gp91-phox did not amplify any products in gastric mucosal cells (Fig. 1A), whereas RT-PCR for mox1 resulted in the generation of a single product corresponding to the predicted base pair size for the mox1 transcript in gastric mucosal cells, as well as Caco-2 cells, which were reported to express the mox1 mRNA (28). The mox1 transcript could not be detected in guinea pig neutrophils. The amplified PCR product from gastric mucosal cells was confirmed to be a guinea pig mox1 by DNA sequencing; the sequence of the product showed 84% identity to the human mox1 cDNA (454-1042 bp; data not shown). Furthermore, Northern blot analysis with the PCR product as a probe showed that gastric mucosal cells expressed a significant amount of 5-kb mox1 mRNA that was not detected in neutrophils (Fig. 1B).


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Fig. 1.   Expression of mitogenic oxidase (mox1) transcript in gastric mucosal cells. Total RNA was prepared from guinea pig gastric mucosal cells (GMC), guinea pig neutrophils (PMN), and Caco-2 cells. A: extracted RNA (1 µg) was reverse transcribed into cDNA with oligonucleotide antisense primer specific for gp91-phox (left) or mox1 (right). Generated cDNA was used as a template in PCR for gp91-phox (lanes 1 and 3) and mox1 (lanes 4, 6, and 7). Lanes 2 and 5 contain molecular weight markers. B: Northern blot analysis with the cDNA probes for mox1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as described in MATERIALS AND METHODS.

Effects of antioxidants on growth and death of gastric mucosal cells. To reveal the physiological role of O2- in gastric mucosal cells, we at first examined the relationship between O2--producing capacity and cell growth (Fig. 2). After starting culture, isolated cells attached to culture plates within several hours and began to grow, with a doubling time of ~24 h (17). As shown in Fig. 2A, O2--producing activity was observed in the cells that were already attached and grown on the culture plates within 24 h. The activity continued to increase and reached a maximum at 48 h when growing cells became confluent. Since O2--producing activity appeared to be associated with growth of gastric mucosal cells, we examined the effects of SOD and/or catalase on the cell growth. Inclusion of SOD did not change the [3H]thymidine uptake, but catalase or catalase plus SOD significantly suppressed the uptake during the proliferation periods (36-48 h after cultivation) by 50 or 45%, respectively (Fig. 2B), suggesting that hydrogen peroxide dismutated from O2- might function as a signal molecule for cell proliferation.


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Fig. 2.   O2--producing activity, proliferation, and death of gastric mucosal cells. A: O2--producing activity and cell number of gastric mucosal cells during cultivation. Isolated gastric mucosal cells were cultured in 24-well culture plates in RPMI 1640 containing 10% FCS. Adherent cell number was counted by using a hemocytometer every 12 h after cultivation (means ± SD; n = 4). The amount of O2- release was measured as described in MATERIALS AND METHODS, and the results are expressed as nanomoles O2- per milligram protein per hour (means ± SD; n = 8). B: effect of scavenging reactive oxygen intermediates (ROI) on serum-dependent cell growth. After isolated cells were cultured for 36 h in 24-well culture plates in RPMI 1640 containing 10% FCS, floating cells were removed, and attached cells were incubated for 12 h in RPMI 1640 containing 10% FCS and 1 µCi/ml [3H]thymidine, in the absence or presence of 200 U/ml superoxide dismutase (SOD) and/or 350 U/ml catalase. [3H]thymidine uptake was measured as described in MATERIALS AND METHODS. Values are means ± SD (n = 8). # Significantly decreased vs. vehicle-treated cells (P < 0.05 by ANOVA and Scheffé's test). C: suppression of cell death by hydrogen peroxide. After culturing for 48 h, cells were untreated or treated with 50 µM hydrogen peroxide every 12 h in RPMI 1640 containing 10% FCS. Both floating and adherent cells were collected and stained with a Hoechst 33342 dye, as described in MATERIALS AND METHODS. Apoptotic cells having condensed and fragmented chromatin were counted. Results are expressed as %apoptotic cells (means ± SD; n = 4). # Significantly decreased vs. untreated cells at the respective time point (P < 0.05 by ANOVA and Scheffé's test).

After being cultured for 48 h, a majority of the cells (~90%) were mature pit cells (24). When these cells were further cultured, they remained confluent for at least 36 h (Fig. 2A), and the number of floating cells gradually increased. The floating cells had condensed chromatin and fragmented nuclei, as determined by staining with Hoechst 33342 dye. Thus mature pit cells spontaneously underwent apoptotic cell death. As shown in Fig. 2A, after cells became confluent on day 2, O2- production began to gradually decrease. When the confluent cells were treated with a low concentration of hydrogen peroxide (50 µM) every 12 h, the rate of spontaneous cell death was significantly suppressed (Fig. 2C).

Effects of antioxidants on apoptosis. The decline of O2- production by mature pit cells seemed to be related to spontaneous apoptotic cell death, and externally supplemented hydrogen peroxide significantly inhibited their death, leading us to consider that the Mox1 oxidase might regulate the cell death. To confirm this, we examined whether scavenging of O2- and its related oxygen intermediates affected the cell viability. FCS could stimulate proliferation of small numbers of immature cells (<5%) to compensate for the cell loss caused by apoptosis. To correctly evaluate the apoptotic cell loss, cells cultured for 48 h were incubated in FCS-free RPMI 1640 supplemented with saline as a vehicle, SOD plus catalase, or N-acetyl cysteine (NAC) for the indicated hours, and cell viability was monitored by trypan blue exclusion test (Fig. 3). Vehicle-treated cells slowly lost their viability, and ~6% of the cells were dead at 16 h (Fig. 3A). On the other hand, inclusion of SOD plus catalase or NAC significantly accelerated the death, and 10% of the cells died at 8 h. Percentage of DNA ladder increased in proportion with the increase in number of dead cells in the presence of SOD plus catalase or NAC (Fig. 3B).


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Fig. 3.   Effect of ROI on cell viability and DNA fragmentation. After cultivation for 48 h in RPMI 1640 containing 10% FCS, cells were treated with saline (vehicle), 200 U/ml SOD plus 350 U/ml catalase (SOD + catalase), or 5 mM N-acetyl cysteine (NAC) in serum-free RPMI 1640 for the indicated hours. A: cell viability was measured by trypan blue exclusion test, and results are shown as %viable cells (means ± SD; n = 4). B: %DNA fragmentation was assayed using the diphenylamine test. Values are means ± SD for three separate experiments. # Significantly different vs. vehicle-treated cells at the respective time points (P < 0.05 by ANOVA and Scheffé's test). * Significantly different vs. vehicle-treated control cells at time 0 (P < 0.05 by ANOVA and Scheffé's test).

The acceleration of apoptosis by the antioxidants was confirmed by plasma membrane integrity and morphological alterations of nuclear chromatin after staining of cells with Hoechst 33342 dye (Fig. 4, A-C). In vehicle-treated cells, the Hoechst 33342 fluorescence was homogeneously distributed throughout the cell nuclei, and no chromatin condensation or nuclear fragmentation was evident at 12 h (Fig. 4A). In contrast, condensed or fragmented nuclei appeared in the cells treated with SOD plus catalase (Fig. 4B) or NAC (Fig. 4C). In addition, when cells were treated with SOD plus catalase (Fig. 4D) or NAC (Fig. 4D), oligonucleosome-sized DNA fragments appeared within 8 h, and the level increased in proportion with the increase in percentage of DNA ladder (Fig. 3B). Either dismutation of O2- by SOD (Fig. 4D) or reduction of hydrogen peroxide by catalase, but not the enzymes inactivated by boiling, could accelerate the DNA fragmentation. Inclusion of both SOD and catalase additionally enhanced the DNA fragmentation, suggesting that O2- and hydrogen peroxide may function as endogenous inhibitors of spontaneous apoptosis.


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Fig. 4.   Effect of ROI on apoptosis. A-C: nuclear condensation and fragmentation. Isolated cells were cultured for 48 h in RPMI 1640 containing 10% FCS, and they were treated with vehicle (A), 200 U/ml SOD plus 350 U/ml catalase (B), or 5 mM NAC (C) in serum-free RPMI 1640 for 12 h. These cells were fixed and stained with Hoechst 33342 dye as described in MATERIALS AND METHODS. D: DNA ladder formation. After 48-h culture, cells were treated with vehicle (lanes 1-3 and 10), SOD plus catalase (lanes 4-6 and 15), NAC (lanes 7-9), heat-inactivated SOD (lane 11) or catalase (lane 12), SOD (lane 13), and catalase (lane 14) in serum-free RPMI 1640 for the indicated hours. Heat inactivation of the enzymes was performed by boiling for 30 min. Fragmented DNA was isolated and subjected to 2% agarose gel electrophoresis, as described in MATERIALS AND METHODS. The separated DNA was visualized by ethidium bromide staining. Results were similar in 3 separate experiments.

Effects of antioxidants on caspase 3-like activity. We examined the mechanism by which Mox1-derived reactive oxygen intermediates (ROI) suppressed pit cell apoptosis. As shown in Fig. 5A, a general caspase inhibitor, z-Val-Ala-Asp-CH2F (z-VAD-fmk), completely blocked the apoptotic DNA fragmentation induced by the antioxidants, suggesting that O2- and/or hydrogen peroxide may interfere with the caspase-dependent apoptosis pathway. To confirm this, we measured caspase 3-like activity by calorimetric assay. In vehicle-treated cells, caspase 3-like activity slightly increased at 8 h (Fig. 5B). SOD plus catalase or NAC significantly increased the activity at 4 h before the appearance of DNA fragmentation (Fig. 4D). Furthermore, when O2- production was inhibited by diphenylene iodonium, a potent inhibitor for the pit cell Mox1 oxidase (30), the caspase 3-like activity was further increased (Fig. 5B).


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Fig. 5.   Effect of Mox1-derived ROI on caspase 3-like activity. A: inhibition of the antioxidant-induced DNA fragmentation by z-Val-Ala-Asp-CH2F (z-VAD-fmk). After cultivation for 48 h in RPMI 1640 containing 10% FCS, cells were incubated in serum-free RPMI 1640, supplemented with 200 U/ml SOD plus 350 U/ml catalase or 5 mM NAC for 12 h in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 50 µM z-VAD-fmk. DNA analysis was performed as described in Fig. 4 legend. B: caspase 3-like activity. After 48-h culture, cells were treated with vehicle, 200 U/ml SOD plus 350 U/ml catalase, 5 mM NAC, or 1 µM diphenylene iodonium (DPI) for the indicated hours. Cytosolic extracts were prepared from the cells, and caspase 3-like activity was measured by a colorimetric assay using a synthetic substrate, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide. Values are means ± SD (n = 6). # Significantly increased vs. vehicle-treated cells at the respective time points (P < 0.05 by ANOVA and Scheffé's test). * Significantly increased vs. vehicle-treated cells at time 0 (P < 0.05 by ANOVA and Scheffé's test).

NF-kappa B activation. Gastric pit cells possess oxidant-sensitive transcription factor NF-kappa B (23), which plays a critical role in protection from apoptosis in many types of cells (7, 32). Gel mobility shift assay showed that untreated cells growing in RPMI 1640 supplemented with 10% FCS constitutively contained low levels of NF-kappa B binding activity (Fig. 6). When cells were incubated in FCS-free RPMI 1640 supplemented with vehicle for 2 h, this binding activity was pronounced. This NF-kappa B activation was almost completely blocked by inclusion of SOD plus catalase or NAC, suggesting that production of O2- and hydrogen peroxide by pit cells in FCS-free conditions might stimulate NF-kappa B-dependent antiapoptotic pathways.


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Fig. 6.   Activation of nuclear factor (NF)-kappa B. After culturing for 48 h in RPMI 1640 containing 10% FCS, cells were maintained in RPMI 1640 containing 10% FCS (lane 1) or treated with vehicle (lane 2), 200 U/ml SOD plus 350 U/ml catalase (lane 3), or 5 mM NAC (lane 4) in serum-free RPMI 1640 for 2 h. Nuclear extracts were prepared from the cells and analyzed by gel mobility shift assay, as described in MATERIALS AND METHODS. Lanes 5-7 show competition experiments with 100-fold molar excess of unlabeled NF-kappa B (lane 5), mutant NF-kappa B (lane 6), or activator protein-1 oligonucleotide (lane 7). Lanes 8-12 show supershift experiments with nonimmunized rabbit IgG (lane 8), anti-p50 antibody (lane 9), antibody against the amino terminal of p65 (lane 10) or the carboxyl terminal of p65 (lane 11), and anti-c-Rel antibody (lane 12). Interactions shown by arrows are specific NF-kappa B binding activities. Results were similar in 3 separate experiments.

The DNA binding activity was further characterized by competition assay and supershift experiments. The protein-DNA complex competed for unlabeled self oligonucleotide (Fig. 6) but not for unlabeled mutant NF-kappa B and nonself oligonucleotide coding the activator protein-1 binding site. Supershift analyses using antibodies against major components of NF-kappa B (p50, p65, and c-Rel) showed that pit cell-derived ROI activated the p50/p50 homodimer and the p50/p65 heterodimer.

We examined whether inhibition of NF-kappa B activity enhanced spontaneous apoptosis. For this purpose, NF-kappa B decoy ODN experiments were done. First, we performed competition assay to examine whether the NF-kappa B decoy ODN could actually block the binding activity of NF-kappa B induced by Mox1-derived ROI. As shown in Fig. 7A, the NF-kappa B decoy ODN competitor, but not mutant decoy ODN, inhibited the NF-kappa B binding activity.


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Fig. 7.   Inhibition of NF-kappa B activity and acceleration of apoptosis by NF-kappa B decoy oligodeoxynucleotide (ODN). A: competition assay of NF-kappa B decoy ODN. Nuclear extracts were prepared from the cells incubated in serum-free RPMI 1640 for 2 h as described in Fig. 6 legend. The nuclear extracts were incubated with 32P-labeled oligonucleotide probe containing the NF-kappa B binding motif without any competitor (lane 1), with 50- (lane 2) and 100- (lane 3) fold molar excess of unlabeled mutant decoy ODN competitor, or with 50- (lane 4) and 100- (lane 5) fold molar excess of NF-kappa B decoy ODN competitor. NF-kappa B binding activity was analyzed by gel mobility shift assay. B: effect of NF-kappa B decoy ODN on spontaneous apoptosis. Cells were transfected with NF-kappa B decoy ODN (1 µM), mutant (mt) decoy ODN (1 µM), or vehicle alone as described in MATERIALS AND METHODS. Then the cells were incubated in serum-free RPMI 1640 for 12 h. Apoptotic cells were detected by Hoechst 33342 staining. Values are means ± SD in 4 separate experiments. # Significantly increased vs. vehicle-treated cells (P < 0.05 by ANOVA and Scheffé's test).

Next, gastric mucosal cells were transfected with the NF-kappa B decoy ODN, the mutant decoy ODN, or LipofectAMINE vehicle alone. Cell viability was monitored throughout the transfection period, which was assessed by morphology, adherence, and trypan blue exclusion. After transfection, cells were incubated in the fresh medium without serum for 12 h, and spontaneous apoptosis was assessed by Hoechst 33342 staining. Figure 7B shows that the rate of apoptosis significantly increased in the cells transfected with NF-kappa B decoy ODN but not mutant decoy ODN.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous studies (30, 31) showed that the O2--generating system in gastric pit cells shared functional and structural similarities with the phagocyte NADPH oxidase by cell-free reconstitution experiments and immunoblot analysis with antibodies against the human neutrophil phox components (gp91-, p22-, p67-, p47-, and p40-phox). After our reports, a nonphagocyte-specific isoenzyme of gp91-phox (Mox1) was molecularly identified (28). The human Mox1 protein consists of 564 amino acids and has 56% sequence identity to the 569 residues for human gp91-phox. The flavin and pyridine nucleotide binding sites are conserved between gp91-phox and Mox1. However, Mox1 lacks asparagine-linked glycosylation sites that are present in gp91-phox (25, 28). This may be the reason why Mox1 was detected as an immunoreactive protein to the anti-gp91-phox antibody with a somewhat smaller molecular mass than that of gp91-phox (31). In this study, we could confirm that gastric pit cells express the transcript of mox1, but not gp91-phox, by DNA sequencing the RT-PCR product and by Northern blot analysis.

We provide here a possible physiological role of Mox1-derived ROI in gastric pit cells. The activity of the pit cell Mox1 oxidase increased in association with their proliferation. [3H]thymidine uptake experiments suggested that hydrogen peroxide, possibly dismutated from the Mox1 oxidase-derived O2-, might stimulate pit cell growth during the proliferation period, as in fibroblasts (16) and vascular smooth muscle cells (28). Furthermore, the Mox1-derived ROI modulated spontaneous apoptosis of mature pit cells. After being cultured for 48 h, ~90% of the cells matured into final differentiated cells having large secretory granules in the ectoplasm, which is characteristic of mature pit cells or surface mucous cells (24). The Mox1 oxidase activity of these cells gradually reduced in association with the progression of spontaneous apoptosis. During this stage, treatment of cells with a low concentration of hydrogen peroxide partially suppressed their spontaneous apoptosis. Alternatively, scavenging O2- and related oxidants significantly accelerated their apoptosis. These findings suggest that O2- produced by pit cells may not only stimulate cell growth but may also suppress spontaneous apoptosis of mature pit cells. There is growing evidence that ROI are key molecules for regulation of apoptosis and survival. Exposure of cells to sublethal doses of ROI, such as O2- and hydrogen peroxide, is known to exert stimulatory effects on their proliferation (3, 4, 29) and to induce tolerance against several apoptogenic stimuli (2, 14). Furthermore, several types of cells, particularly tumor cells, spontaneously produce O2- to escape from apoptosis induced by NO (14) and Fas (8).

We also provide a possible mechanism(s) for antiapoptotic actions of Mox1-derived ROI in gastric pit cells. The DNA fragmentation initiated by scavenging ROI was completely abolished by z-VAD-fmk. On the other hand, removal of ROI or inhibition of the Mox1 oxidase activity significantly increased caspase 3-like activity, suggesting that Mox1-derived ROI could interfere with the apoptotic processes of activation of group II caspases (caspases 2, 3, and 7). These caspases are cysteine-dependent proteases and sensitive to the cellular reduction/oxidation status. It has been reported that ROI, including O2-, can inhibit the activation of caspase 3-like enzymes and also directly inactivate them (11, 19).

ROI produced by the Mox1 oxidase could constitutively activate NF-kappa B in gastric mucosal cells, which may be an important signal for proliferation and/or survival. In fact, the O2--dependent activation of NF-kappa B was shown to attenuate nitric oxide- and tumor necrosis factor-alpha -induced apoptosis (10, 14). In addition, NF-kappa B is now known to upregulate transcription of several antiapoptotic genes, including cellular inhibitor of apoptosis (cIAP2) (7), Bcl-2 homolog Bfl-1/A1 (32), and cyclooxygenase-2 (14). The present experiments did not identify the antiapoptotic genes downstream of NF-kappa B activation in gastric pit cells. However, the activation of NF-kappa B by Mox1-derived ROI appeared to play a crucial role in the suppression of spontaneous apoptosis of pit cells, since inhibition of NF-kappa B-binding activity by NF-kappa B decoy ODN significantly enhanced their apoptosis, and NAC, a potent inhibitor of NF-kappa B activation (26), could induce apoptosis, similarly to the way that SOD plus catalase did.

To our knowledge, this is the first time that gastric pit cells in culture have been shown to express a potent Mox1 oxidase, and we suggest that O2- derived from the oxidase acts as an important signal for proliferation and survival of the cells. Considering the close relationship between the activity of the pit cell Mox1 oxidase and mitogenic activity or death, the strictly regulated production of O2- may directly or indirectly determine whether pit cells proliferate, inhibit growth, or enter the apoptotic pathway, at least partially in vitro. The ROI-dependent regulation of proliferation and cell death is known to depend on the concentration and the flux rate of its formation (9). If their O2- production is dysregulated, the balance between cell growth and death may be damaged. In fact, overexpression of Mox1 in NIH-3T3 cells enhanced O2--producing ability, resulting in abnormal cell growth and transformation in vitro and aggressive growth of the implanted tumor cells in vivo (28). In addition to the potent mitogenic activity, tumor cells harboring O2--producing ability show a resistance to apoptosis induced by Fas, tumor necrosis factor-alpha , and anticancer drugs (8, 10, 19).

We already examined the expression of the Mox1 oxidase components in human gastric glands and found that pit cells, but not other types of gastric epithelial cells, expressed immunoreactive materials to antibodies against p47-phox and p67-phox and to an antibody recognizing both gp91-phox and Mox1 (22). At present, we do not know whether the Mox1 oxidase regulates rapid proliferation of a pit cell lineage and continuous cell loss by apoptosis in vivo. To reveal the physiological meaning of the O2--generating system in vivo, more detailed examinations of the expression of the Mox1 oxidase in human gastric glands are needed.


    ACKNOWLEDGEMENTS

A part of this work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (to S. Teshima and K. Rokutan).


    FOOTNOTES

Address for reprint requests and other correspondence: K. Rokutan, Dept. of Nutrition, School of Medicine, The Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan (E-mail: rokutan{at}nutr.med.tokushima-u.ac.jp).

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.

Received 17 March 2000; accepted in final form 12 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 279(6):G1169-G1176
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