Vol. 284, Issue 2, G255-G262, February 2003
Heat shock induces intestinal-type alkaline phosphatase in
rat IEC-18 cells
Tsuyoshi
Harada1,
Iwao
Koyama1,
Toshihiko
Kasahara1,
David H.
Alpers2, and
Tsugikazu
Komoda1
1 Department of Biochemistry, Saitama Medical
School, Saitama 350-0495, Japan; 2 Division
of Gastroenterology, Washington University School of Medicine, St.
Louis, Missouri 63110
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ABSTRACT |
We demonstrate a previously unknown
regulation for intestinal-type alkaline phosphatase (IAP) as a heat
shock protein (HSP). Heat shock to rat intestinal epithelial cells
(IEC)-18 at 43°C induced the expression of IAP-I and HSP72 mRNAs time
dependently (<60 min) but did not induce expression of IAP-II, tissue
nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by
the RT-PCR method. To confirm the identity of the IAP-I gene, we
sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in
IEC-18 cells, but it became detectable as a 62-kDa band on a Western
blot after heat shock. IAP-I was also induced by sodium arsenite, which
generates reactive oxygen species and is an inducer of members of the
HSP family. Glutathione suppressed activating protein-1 and cAMP
response element-binding protein activation caused by heat shock but
did not suppress the expression of IAP-I. These results suggest that
cellular stress induces the elevation of IAP-I mRNA and protein
synthesis. IAP-I may play an important role as a dephosphorylating
enzyme under stress conditions.
isozyme; heat shock protein; sodium arsenite; glutathione
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INTRODUCTION |
ALKALINE PHOSPHATASE
(AP; EC 3.1.3.1) is an ectoenzyme anchored to the membrane by a
glycan-phosphatidylinositol (GPI) anchor moiety and hydrolyzes a
variety of monophosphate esters at alkaline pH (35). In
primates, the AP gene family consists of four distinct loci types,
i.e., the tissue nonspecific AP (TNAP), which is expressed mainly in
liver, bone, and kidney; the intestinal AP (IAP); the placental AP
(PLAP); and the germ cell AP (GCAP) (11). It has been
proposed that, during the evolution of the AP gene family, the first
duplication from an ancestral AP gene produced TNAP, and subsequent
duplications gave rise to further modifications resulting in the IAP,
PLAP, and GCAP genes (21, 26). TNAP has been shown to be
heat labile and IAP, PLAP, and GCAP to be heat stable, with IAP
resisting temperatures 
56°C and PLAP and GCAP resisting
temperatures 70°C (12, 28). On the basis of these
findings, we hypothesized that the evolution of AP isozymes has
been associated with the acquisition of heat resistance; however, the
physiological function(s) and substrate(s) of heat-stable AP isozymes
and the significance of their heat-resistant nature remain uncertain.
LPS, which is a pyrogen, has recently been proposed as a candidate
substrate of AP, and two phosphate groups of lipid A, its toxic core,
have been found to be dephosphorylated by AP at physiological pH
(43, 44). LPS has been shown to induce AP in the duodenum, lungs, and liver and in a small intestinal epithelial cell (IEC) line
(16, 34, 43); and an increase in the levels of
intracellular cAMP, mediated by inflammatory factors, such as cytokines
and activation of protein kinase A, has been shown to be involved in
the regulation of AP expression (4, 23, 27, 41). However, little is known about the effect of fever itself on the expression of
heat-stable AP.
Because no PLAP and GCAP isozymes have ever been detected in rat
tissues, IAP is the most heat-resistant isozyme known in rats. IAP is
mainly distributed in the intestines, but exigous synthesis has been
observed in kidney, liver, and lung tissue (14, 18). Rat
intestine produces two distinct isozymes of IAP, IAP-I, and IAP-II,
which have 79% amino acid identity and differ markedly at their
COOH-terminal end (10, 47, 53). These isozymes appear at
different times during postnatal development, have different substrate
specificities, and respond differently to cortisone or cortisone plus
thyroxine, 1,25-dihydroxyvitamin D3, fat feeding, and LPS
(47, 49, 55).
All organisms respond to heat by inducing the rapid synthesis of heat
shock proteins (HSPs). Response is the most highly conserved genetic
system known, existing in every organism in which it has been sought,
from archaebacteria to eubacteria, from plants to animals. Among rat
IEC lines, the heat shock response of HSP has been studied in IEC-18
cells derived from the ileum of a rat in the suckling stage (2,
40, 50, 52). Despite the large amount of information on the
genetic regulation of HSPs and their role as chaperones, little is
known about the regulation and role of AP isozymes in relationship to
the heat shock response. In the present study, we used nontransformed
IEC-18 cells to investigate whether nonlethal thermal stress influences
the expression of AP isozymes in the cells.
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MATERIALS AND METHODS |
Cell culture and heat/chemical shock treatment.
IEC-18 cells (passage 30-34) were routinely cultured in
plastic culture flasks containing DMEM supplemented with 10% fetal calf serum, 10 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 5%
CO2-95% air. Cells were subcultured weekly by using 0.05%
trypsin-0.02% EDTA in PBS without Ca2+ and
Mg2+.
Once the cells became confluent in the flasks (25 cm2 in
size), lids of the flasks were quickly fastened tightly to avoid any loss of CO2 just before heat shock. To induce HSPs, we
subjected IEC-18 monolayers to the nonlethal temperature of 43°C in a
water bath for various lengths of time. At the completion of heat
shock, total RNA was immediately extracted from the cells, or the
flasks were lifted from the water bath, their lids were loosened, and they were promptly returned to the incubator at 37°C for a
predetermined time.
Some IEC-18 cells were pretreated with DMEM containing 10 µg/ml
actinomycin D for 2 h, 200 µM sodium arsenite for 1 h, or 30 mM glutathione (GSH) for 1 h. After this preincubation, the medium was replaced with fresh DMEM, and the cells were subjected to
the heat shock procedure described above.
RNA preparation and PCR.
Total cellular RNA was isolated with a commercial kit (Isogen; Nippon
Gene, Tokyo, Japan) according to the protocol provided by the
manufacturer. cDNAs were reverse transcribed from total RNA (4 µg)
with a Qiagen Omniscript reverse transcriptase kit by using the
oligo(dT)15 primer (Roche, Mannheim, Germany). PCR amplification of the IAP-I, IAP-II, TNAP, HSP72, and HSP90 transcripts was performed with a KOD-Taq polymerase kit (Toyobo, Osaka,
Japan). Sequences of the PCR primers for these transcripts were
derived from the following published sequences: IAP-I, sense
5'-CCTGGAGCCCTACACCGACT-3' and antisense 5'-GCCAGCGTTGAGACCCTTGG-3'
designed to amplify a fragment corresponding to nucleotides
4487-4768, (53); IAP-II, sense
5'-CCTGGAGCCCTACACCGACT-3' and antisense
5'-GGACCCTGGCTGAGTTGGAG-3' for nucleotides 4221-4476,
(53); TNAP, sense 5'-AAGTCCGTGGGCATCGTGAC-3' and antisense
5'-GTGGGAGTGCTTGTGTCTAG-3' for nucleotides 477-803, (36); HSP72, sense 5'-TCGAGGAGGTGGATTAGAG-3' and antisense
5'-GGGATGCAAGGAAAAAAC-3' (1); and HSP90, sense
5'-ACATCATCCCCAACCCTC-3' and antisense 5'-TCCACCAGCAGAAGACTCC-3'
(1). PCR primers for rat 
-actin were purchased from
Clontech Laboratories (Palo Alto, CA). The optimum number of cycles for
the PCR of each primer pair was determined by serial dilutions of the
cDNA in the PCR reactions until a linear response was obtained. The
optimum number of cycles for each primer pair was: 38 for IAP-I, 40 for
IAP-II, 34 for TNAP, 28 for HSP72, 23 for HSP90, and 23 for -actin.
PCR parameters for all cDNAs were denaturation at 94°C for 30 s,
annealing at 57°C for 30 s, and extension at 72°C for 45 s. The PCR products were separated by electrophoresis on 2% agarose
gels. DNA was visualized by ethidium bromide staining. Intensity of the
bands was evaluated with a charge-coupled device camera system (Atto,
Tokyo, Japan).
Sequencing.
Cycle sequencing was performed on a GeneAmp PCR System 9600 (PerkinElmer, Norwalk, CT) utilizing a PRISM Ready Dye Terminator Cycle
Sequencing kit with AmpliTaq DNA polymerase, FS
[Taq-FS; PerkinElmer/Applied Biosystems Division, Foster
City, CA], according to the manufacturer's recommendations. Briefly,
the gel-purified DNA (10.4 µl) was added to a MicroAmp reaction tube
(PerkinElmer) containing 3.2 pmol of sequencing primer and 8.0 µl of
premix [containing buffer, 2-deoxynucleotide 5'-triphosphates (dNTPs), dye-labeled ddNTPs, and Taq-FS/pyrophosphatase]. After the
initial denaturation at 96°C for 2 min, the reaction mixture was
incubated for 25 cycles of 96°C for 10 s, 50°C for 5 s,
and 60°C for 4 min. Excess dye-labeled terminators were removed from
the extension products by spin-column purification (CentriSep spin
column; Princeton Separations, Adelphia, NJ) according to the
manufacturer's directions. Once separated, the extension products were
evaporated to dryness under reduced pressure (SpeedVac; Savant
Instruments, Farmingdale, NY). Each sample was resuspended in 2 µl of
loading buffer (5:1, 1% deionized formamide/50 mM EDTA, pH 8.0),
heated for 2 min at 90°C, and loaded as a 0.5 µl aliquot onto an
Applied Biosystems PRISM 377XL sequencer.
Western blotting.
After heat shock for 60 min, IEC-18 cells were cultured at 37° C for
3 h, washed 2 times with PBS, and then scraped with a rubber
policeman into 1 ml of lysis buffer (10 mM
Tris · HCl buffer, pH 7.8, containing 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine
chloride). An equal volume of n-butanol was added to the
collected cell lysates, and the mixture was stirred at room temperature
for 15 min and then centrifuged at 4,000 g for 30 min. The
aqueous phase was collected, cold acetone (
20°C) was added to a
final concentration of 60% (vol/vol), and the solution was stored at
20°C overnight. Precipitate was collected by centrifugation and
dried to an acetone powder. The acetone powder was solubilized with
sample buffer (60 mM Tris · HCl buffer, pH 6.8, containing 1% SDS, 10% glycerol, and 5% 2-mercaptoethanol) and
boiled for 5 min. These samples were subjected to electrophoresis on a
SDS-PAGE (8% acrylamide) gel under reducing conditions. Separated
proteins were transferred to immobilon-P membranes (Millipore, Bedford, MA) at 0.4 mA for 1 h at 4°C and blocked overnight in
Tris · HCl-buffered saline, pH 7.8, containing
5% nonfat dry milk and 0.1% Tween 20. Membranes were then washed with
Tris · HCl-buffered saline plus 0.1% Tween, and
IAP bands were detected by using the rabbit anti-rat IAP antiserum
characterized previously (54). The membranes were incubated for 1 h at room temperature with this antiserum at a 1:5,000 dilution in the buffer. After being washed with
Tris · HCl-buffered saline plus 0.1% Tween, the
membranes were incubated for 1 h at room temperature with
anti-rabbit horseradish peroxidase-linked antibody at a 1:10,000
dilution as the secondary antibody. IAP bands detected by the
antibodies were visualized by using an enhanced chemiluminescence kit
(Amersham Pharmacia Biotech, Little Chalfont, UK).
Preparation of nuclear extracts and transcription factor
activation assay.
Cells were harvested by removing the incubation medium, rinsing the
cells twice with 10 mM PBS, pH 7.5 containing 150 mM NaCl, 2.7 mM KCl,
and 10 mM of a cocktail of phosphatase inhibitors [(in mM) 5 NaF, 10 -glycerophosphate, 10 paranitrophenyl phosphate, 1 NaVO3], scraping the cells off the substratum with a
rubber policeman, and pelleting them by centrifugation at 1,000 g for 5 min at 4°C. The pellet was then resuspended in 300 µl of 20 mM HEPES buffer, pH 7.5, containing (in mM) 350 NaCl, 1 MgCl2, 0.5 EDTA, 0.1 EGTA, and 20% glycerol, 1% NP-40,
and a protease inhibitor cocktail (Roche, Germany). After 10 min on
ice, the lysate was centrifuged for 20 min at 100,000 g. The
supernatant constituted the total protein extract and was kept frozen
at 80°C. The protein concentration of the nuclear extracts was
measured with a bicinchoninic acid kit (Pierce, Rockford, IL).
The activity of activating protein-1 (AP-1) and cAMP response
element-binding protein (CREB) DNA-binding activities were determined with ELISA-based assay kits (TransAM) obtained from Active Motif (Carlsland, CA). In brief, the nuclear extracts were added to microwells coated with a cold oligonucleotide containing the
consensus-binding site for activator protein-1 (AP-1) or CREB. After
1-h incubation at room temperature, the microwells were washed three
times with washing solution. Antibodies directed against phosphorylated
c-Jun or phosphorylated CREB were used to label the AP-1 or CREB dimers bound to the oligonucleotide and followed by a secondary antibody conjugated to horseradish peroxidase. Finally, the results were quantified by a chromogenic reaction (46).
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RESULTS |
Effect of heat shock on expression of the mRNA of AP isozymes and
HSPs.
To investigate whether exposure of IEC-18 cells to nonlethal heat would
stimulate the expression of AP isozymes and, if so, to determine the
optimal time for induction of their expression, we exposed the cells to
a temperature of 43°C for various lengths of time from 0 to 60 min.
RT-PCR analysis of IAP-I, IAP-II, TNAP, HSP72, HSP90, and -actin
expression was then performed. RT-PCR with both IAP-I and IAP-II
primers before heat stimulation did not yield their amplification
products, which were 282 and 256 bp, respectively (Fig.
1A). Amplification products of
TNAP, HSP72, HSP90, and -actin were observed as single bands of 327, 307, 269, and 764 bp, respectively, on gels loaded with samples from the untreated cells (37°C). The number of amplification cycles used
for these genes was lower than that used for IAP-I or IAP-II (Fig. 1,
A and B).
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Fig. 1.
Effect of heat shock on alkaline phosphatase (AP)
isozymes, heat shock protein (HSP)72, and HSP90-targeted amplification
of RNA from intestinal epithelial cell (IEC)-18 cells. A:
representative profile of the agarose gel electrophoresis of the RT-PCR
products. B: level of each product is expressed as the
relative intensity. The final photocounts measured in the sample
subjected to heat shock for 60 min was used as the reference control.
Each value represents the mean of the results of 3 separate
experiments. TNAP, tissue nonspecific AP; IAP, intestinal AP.
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In the samples from the heat-shocked IEC-18 cells, the amplification
products of IAP-I and HSP72 dramatically increased <60 min in a
time-dependent manner during the observation period; but no band for
IAP-II was detected at 256 bp in this experiment.
Cloning of the PCR product of IAP-I.
The IAP-I PCR product was cloned by the direct sequence method (Fig.
2). The nucleotide sequence of the PCR
product in this experiment had 99.0% homology with the sequence
reported by Xie and Alpers (53). Three sites of
single-nucleotide polymorphism were detected, which reflected the open
reading frame of the IAP-I protein at nucleotide 4601, but the
corresponding codon was the same degeneracy.
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Fig. 2.
Nucleotide sequence of the PCR product from heat-shocked
IEC-18 cells determined by using IAP-I primers compared with the
previously reported rat intestine IAP-I sequence, indicated by
lower-case letters (see Ref. 53). Mismatches between the 2 sequences are marked by asterisks. Positions of PCR primers are
underlined.
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To reconfirm the induction of the IAP-I gene, we sequenced the PCR
product amplified from the exon 8 region (nucleotide position: 3282-3671) (53) with another set of primers. This
product was also induced by heat shock and was 99.9% homologous to the
rat IAP-I gene (data not shown).
IAP-I gene transcription.
As supportive evidence that the induction of IAP-I by thermal stress
was mediated by transcriptional activation of the IAP-I gene, the cells
were exposed to the RNA polymerase inhibitor actinomycin D (10 µg/ml). As shown in Fig. 3, the
time-dependent increases in the expression of IAP-I and HSP72 were
completely or partially inhibited by exposure to the inhibitor. These
findings indicate that the increase in IAP-I or HSP72 expression was
due to an increase in synthesis rather than to a decrease in the rate
of IAP-I mRNA degradation.
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Fig. 3.
Effect of actinomycin D on heat-induced expression of
IAP-I and HSP72. IEC-18 cells were treated or not treated with
actinomycin D (10 µg/ml) for 1 h and then heat shocked, as
described in MATERIALS AND METHODS. Total RNA was extracted
from the cells and converted to cDNA, and RT-PCR was performed by using
targeted primers. The RT-PCR profile shown is representative of the
profiles from 3 separate experiments.
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Production of IAP protein by heat shock.
To assess whether the IAP-I expression was also elevated at the
translational level, we performed Western blot analysis with antiserum
raised against specific IAP protein (Fig.
4), and the 62-kDa IAP band was detected
in the blot of the gel lane containing a sample from the heat-shocked
IEC-18 cells.
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Fig. 4.
Effect of heat shock treatment on the translation of IAP.
Western blot analysis of IAP after heat shock for 1 h and recovery
for 3 h (HS) and in the control (C). The blot shown is
representative of the blots from 3 separate experiments.
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Effect of arsenite and GSH on expression of the IAP-I gene.
Arsenite is known to be capable of inducing stress proteins, including
HSP70, as well as inducing thermotolerance (32, 51). Exposure of IEC-18 cells to arsenite increased the levels of expression of the mRNAs of HSP72 and IAP-I but not of TNAP (Fig.
5). Expression of HSP72 and IAP-I mRNAs
in the arsenite-exposed IEC-18 cells was greater than that in the heat
shock-treated IEC-18 cells. In contrast to the nontreated cells, heat
shock did not dramatically induce the expression of HSP72 and IAP-I in
arsenite-pretreated cells. These results suggest that the regulation of
IAP-I is related to oxidative stress, because arsenic generates
reactive oxygen species.
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Fig. 5.
Effect of arsenite on the gene expression levels of HSP72
(A), IAP-I (B), and TNAP (C). IEC-18
cells were subjected to the following conditions: Control (C), heat
shock at 43°C for 1 h (HS), 200 µM sodium arsenite for 1 h (SA) and 200 µM sodium arsenite for 1 h followed by 43°C for
1 h after removal of the arsenite (SA+HS). Total RNA was prepared
and analyzed by RT-PCR. Gene expression levels were calculated with the
-actin level as an internal control and are shown as a percentage of
the corresponding heat-shock group values. The amount of each RT-PCR
product was assayed in 3 individual experiments, and data are shown as
means ± SD.
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It has been suggested that heat shock impairs the cellular redox
balance and that AP-1-DNA binding activity is increased through activation of c-Jun after heat shock (8, 48). Because AP-1 and CREB/ATF-DNA binding motifs are present as enhancer elements within
the sequenced 5'-flanking region of IAP-I (53), we then sought to determine whether activation of these transcriptional factors
in response to heat shock is involved in the regulation of the
heat-induced increase in IAP-I gene expression. Fig.
6A shows
that, compared with their activity in nuclear extracts from uninduced
cells, AP-1 and CREB-DNA binding activity was induced by heat shock as
well as by arsenite exposure. Exposure of cells to 30 mM GSH for 60 min
before heat shock reduced the heat shock induction of AP-1 and CREB
binding activity to the control levels but not the arsenite-induced
activity.
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Fig. 6.
Effect of glutathione (GSH) on activating protein-1
(AP-1) binding activity, cAMP response element-binding protein (CREB)
binding activity, and the expression levels of heat-induced genes.
IEC-18 cells were incubated at 43°C for 1 h after 1 h
preincubation with 30 mM GSH. A: nuclear extracts (5 µg of
proteins) were incubated in microwells previously coated with
double-stranded oligonucleotides containing a consensus binding
sequence for AP-1 or CREB as described in MATERIALS AND
METHODS. In competition assays (filled columns), the nuclear
extract was mixed with 20 pmol of soluble double-stranded
oligonucleotide (AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3' and CREB,
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') and the DNA binding assay was then
performed. B: total RNA was prepared and analyzed by RT-PCR.
Expression levels of the genes were calculated with the -actin level
as an internal control and are shown as percentages of the
corresponding heat-shock group values. The data represent the
means ± SD of 3 values.
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Having found that GSH prevented the heat shock-induced activation of
AP-1 and CREB-DNA binding, we then examined the effect of GSH on the
expression of IAP-I in heat-treated cells. Exposure of cells to GSH did
not induce expression of IAP-I or HSP72 (Fig. 6B). After
being heat shocked, cells were pretreated with GSH; however, the
expression of IAP-I was increased to the level observed in the
heat-shocked cells without GSH pretreatment.
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DISCUSSION |
The heat shock response is mediated by increased expression of
genes encoding a group of proteins referred to as the HSP family or
stress proteins (33, 38). HSPs are crucial for the
maintenance of cell integrity during normal cell growth as well as
under certain pathophysiological conditions, and they are thought to
support the transportation, folding, and rearrangement of other
proteins by acting as chaperones (9). Members of the HSP
family have been classified according to their apparent molecular
weights, functions, and inducers, respectively. However, IAP has never been previously reported to be a heat shock responder. The present study demonstrates for the first time, to our knowledge, that heat
treatment is capable of inducing IAP production in rat IECs.
Instead of being regulated by increased levels of a transcriptional
activator, HSP gene transcription is regulated by the activation of a
preexisting pool of heat shock transcription factors (HSF) that bind to
the HSP promoter element [HSE; (Ref. 38)]. For example,
HSF1 is folded and maintained in a non-DNA-binding state as a monomer
under normal physiological conditions, and activation of HSF1 is
mediated by disruption of intramolecular interactions, which results in
a homotrimeric form that binds to HSE (37). Xie and Alpers
(53) sequenced the 5'-flanking region (~1.7 kbp) of the
rat IAP-I gene but found no typical HSE within this region. Recently,
several reports have indicated that HSP itself influences the
expression of other genes. Exogenous HSP70 acts as a cytokine by
stimulating a proinflammatory signal transduction cascade that results
in an upregulation of IL-1, IL-6, and tumor necrosis factor-
expression through both CD14-dependent and -independent pathways
(3), and these cytokines or transcriptional factors
activated by HSP70 may be associated with the expression of
heat-induced IAP-I. In the present study, we confirmed that the
response of the elevated level of IAP-I mRNA caused by heat shock was
linked to that of HSP72 and was due to synthesis, because pretreatment
with actinomycin D blocked the elevation, indicating transcriptional
activation of the IAP-I and HSP72 genes. We also confirmed that
arsenite has the ability to induce expression of IAP-I mRNA as well as
HSP72 mRNA. Arsenicals have been shown to generate reactive oxygen
species and cause the induction of a number of major stress proteins
(7). It has been suggested that heat shock impairs the
cellular redox balance, resulting in generation of reactive oxygen
species (6, 13). Alternations in intracellular
oxidation/reduction reactions have been shown to activate signal
transduction cascades that regulate early response genes (19,
25), and AP-1-DNA binding activity is also activated through the
activation of these genes after heat shock (8, 48).
Moreover, the enhancer motifs of AP-1 and CREB/ATF have been found
within the sequenced 5'- flanking region of the rat IAP-I gene
(53). However, we showed that heat treatment of IEC-18 cells after pretreatment with GSH at the relatively high concentration of 30 mM still induced expression of IAP but did not activate AP-1 and
CREB/ATF DNA binding. These findings indicate that heat shock can
induce the expression of IAP-I, even when reactive oxygen species-related signal pathways are suppressed. It is therefore likely
that the regulatory mechanism of the heat shock response of the IAP-I
gene involves the HSF-HSE system upstream of the certified 5'-flanking
region. However, it is unclear at the present time whether the
regulation of heat-induced IAP-I is mediated by some other
transcription factor indirectly associated with HSF or by some other
unknown mechanism.
It has been reported that TNAP is the predominant isozyme in IEC cell
lines and can be induced by retinoic acid,
1,25-dihydroxycholecalciferol, and butyrate (15, 24, 42).
Expression of IAP genes had not been detected previously in IEC cell
lines, because the cell lines were derived from neonatal rat small
intestines and retained features characteristic of immature, fetal-like
crypt cells, as judged by immunologic and morphological criteria
(24, 45). In the fetal rat intestine, TNAP is expressed
primarily in the single layer of cells lining the primitive gut during
the first phase of gestation, and the cells lining the newly developed
crypt cells during the second phase also express TNAP. However, TNAP
expression changes to IAP expression during the third gestational phase
(26). After the postnatal surge of IAP production, the
intestinal epithelium becomes the tissue containing the largest amount
of this enzyme, and this conversion completes the maturation by
morphogenesis and function (56). IAP is a late
evolutionary development in the AP gene family, having appeared first
in mammals and before PLAP appeared in primates (17, 26).
The emergence of IAP may be of significance in relationship to the
hydrolysis and metabolism of phosphorylated substances during the
development of rat intestine.
Two distinct IAP isoenzymes are expressed in the rat intestine and are
encoded by different mRNAs. IAP-I is a 65-kDa AP isozyme in the rat
intestine and is the product of a 2.7-kbp mRNA, whereas IAP-II is a
90-kDa AP isozyme encoded by a 3.0-kbp mRNA (53, 56).
Regulatory differences among IAP isozymes have been demonstrated by the
fact that IAP-II, but not IAP-I, is stimulated by fat feeding, cortisone, and 1,25-dihydroxycholecalciferol. On the other hand, IAP-I
emerges earlier than IAP-II in the neonatal development of rat
intestine (55), and its expression is stimulated by
LPS-inoculation of rat lung (20). We confirmed that AP
activity and 70-kDa IAP protein, probably originating from the IAP-I
gene, were induced in the rat intestine by oral administration of LPS
(29). The lipid A of LPS contains two phosphate groups and
is known to be dephosphorylated by IAP (43, 44), resulting
in its detoxification. IAP-I is therefore constitutive in the rat
intestine and is a more primitive isoenzyme than IAP-II, and
heat-inducible IAP-I as a dephosphorylating enzyme may play an
important role in the host defense system against pathological stress
(inflammation, infection, fever, or metals) or physiological stress
(cell differentiation or tissue development).
Crystal structures of human AP isozymes, PLAP, and TNAP have been
modeled and evolved a number of functional elements and properties not
present in the Eschericiha coli AP, on
NH2-terminal -helix, crown domain, and
metal-binding domain (30, 31, 39). In TNAP, the loop of
crown domain amino acids 405-435 is directly involved in the
binding of collagen (5, 22, 39). From the structural
analysis of active site and active valley, the target of PLAP is
considered to be a phosphorylated protein (31). When the
amino acid sequences of human AP isozymes are homologized to that of
rat IAP-I, the high frequency of mismatches is recognized in the bottom
of the active site valley region and the loop region but not the active
site. However, it is necessary to build a reliable rat IAP-I model to
discuss the specificity of substrate and the role of stress condition.
However, we do not yet know the physiological function(s) of IAP-I as a
stress protein or the mechanism of IAP-I regulation. A study is
currently underway to determine whether the coding sequence upstream of
the sequenced IAP-I gene promoter contains some HSEs that act functionally.
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ACKNOWLEDGEMENTS |
We thank Dr. Yoshimasa Hamada for his encouragement, help, and advice.
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FOOTNOTES |
Address for reprint requests and other correspondence: T. Komoda, Dept. of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0451, Japan (E-mail address: tkalp1lp{at}saitama-med.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.
First published October 23, 2002;10.1152/ajpgi.00244.2002
Received 24 June 2002; accepted in final form 30 September 2002.
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REFERENCES |
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