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Canadian Institutes of Health Research Group on the Functional Development and Physiopathology of the Digestive Tract, Department of Anatomy and Cell Biology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Daniel Ménard, Ph.D., Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, 3001 12e avenue N, Sherbrooke, Québec, Canada J1H 5N4. E-mail: dmenard{at}courrier.usherb.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9 weeks), and adult-type
compartmentalization of fundic (oxyntic) glands involved in mucus,
acid, and zymogen secretion becomes rapidly established
(1, 2, 3). Mucous epithelial cells differentiate from mitotic
progenitors during their migration from the isthmus toward the foveolus
and the surface, whereas parietal, endocrine, and chief zymogenic cells
appear in the forming glands, similar to those in normal renewing adult
mucosa (2, 4). Moreover, recent data argue in favor of a
precocious appearance of gastric secretory functions during ontogeny.
We have reported ultrastructural and biochemical evidence of
significant mucus/glycoprotein production (5) and revealed
immunoreactive fundic pepsinogen-5 (Pg5) and human gastric lipase (HGL)
as well as pepsin and lipase enzyme activities around 10–13 weeks
(6, 7). In fact, coexpression of Pg5 and HGL in chief
cells in the human fetus (7) and adult (8)
represents a unique feature, a gastric lipase enzyme that is absent in
rodents and distinctively localized in other mammals (9).
Although a comprehensive view of the human gastric epithelial functions
involved in restitution and cancer is emerging, our actual knowledge
concerning the factors involved in the control of cell renewal and
zymogen expression/synthesis in chief cells is only fragmentary. It is
also of prime importance to delineate the specific mechanisms
regulating the expression of HGL in fetal, neonatal, and adult gastric
tissue, as initial digestion of dietary fat in stomach of infants and
adults seems to be a prerequisite for efficient intestinal lipolysis
(9, 10). HGL even assumes a more crucial role in
physiological (term and preterm infants) and pathological conditions
associated with pancreatic insufficiency and a low level of pancreatic
lipase (8). Several lines of evidence suggest that insulin-like growth factors (IGF-I and IGF-II) and insulin may be involved in the development and maintenance of gastric functions. These factors are widely expressed in embryonic and adult tissues (11), and null mutations of IGF-I, IGF-II, or IGF-I receptor (IGF-I-R) genes cause retardation of fetal growth in mice (12). The IGF-I-R (also termed type I IGF-R) and insulin receptor (insulin-R) are present at the epithelial level in all segments of the gastrointestinal tract in rodents (13, 14, 15), and both ligands are mitogenic for primary cultures of canine gastric epithelial cells (16). It is noteworthy, however, that IGF-II is the most abundant insulin-related substance detected in human blood cord vessels (17) and in culture medium of human gut-derived epithelial cell lines (18, 19, 20). IGFs are additionally complexed in blood and extracellular fluids to high affinity binding proteins (IGFBP-1 to IGFBP-6), which display sequence homology to a new group of IGFBP-related proteins, but not the IGF-I-R (11, 21, 22). IGFBP-1 to -6 proteins specifically modulate the bioactivity of IGFs, and their respective affinities for each ligand are known to vary (23, 24). Finally, it was reported that human fetal mesenchymal cells synthesize IGF messenger RNA (mRNA) (25), whereas gastric mucosal cells express IGFBP-2 to -6 mRNAs (26). However, whether the mucosa is able to translate these IGFBP transcripts and to secrete the different IGFBPs is unknown, and whether IGFs are able to modulate human gastric epithelial cell proliferation and specific digestive functions independently of IGFBPs remains to be investigated.
A serum-free organ culture methodology has been recently applied to
human fetal gastric mucosa in our laboratory (5). Compared
with strategies based on short-term culture of adult biopsies, this
technique offers many advantages for investigating the roles of growth
factors in gastric physiology such as a prolonged survival time and the
maintenance of tissue under completely defined conditions. It enabled
us to demonstrate that epidermal growth factor and transforming growth
factor-
exert a direct influence on epithelial cell proliferation
and HGL expression in chief cells (27, 28). In our ongoing
effort to identify the regulators of cell proliferation and zymogen
synthesis in human stomach, the present study was undertaken 1) to
establish the cellular and glandular localizations of IGFBPs, 2) to
investigate their secretion patterns, and 3) to verify the specific
influence of IGF-I and IGF-II on cell proliferation and digestive
functions (Pg5 and HGL) by comparing native factors and truncated IGFs
[R3IGF-I; Del(1, 2, 3, 4, 5, 6)IGF-II], which do not
interact with IGFBPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum-free organ culture
Gastric tissue was prepared as previously described
(5). Briefly, the glandular mucosa (body/fundus) was cut
into 5 x 5-mm2 explants and maintained in
organ culture dishes (Falcon Plastics, Los Angeles, CA) at the
interface of 95% air-5% CO2 gas mixture and
culture medium for up to 5 days (37 C). Medium was renewed after 1 day
and every 2 days thereafter. Human recombinant IGF-I, IGF-II
(Collaborative Biomedicals, Bedford, MA), or insulin (Novo Nordisk
Canada, Mississauga, Ontario, Canada) were added at respective
concentrations of 50–100 ng/ml (6.5–13 nM), 100–200
ng/ml (13–26 nM), and 30–300 µU/ml (1.2–12 ng/ml or
0.2–1.6 nM). These dosages correspond to circulating
levels measured in fetal cord blood in normal term and preterm infants
(17, 30). Two truncated IGF peptides lacking the
IGFBP-binding domain (purchased from Upstate Biotechnology, Inc., Lake Placid, NY), namely R3IGF-I
(50–100 ng/ml) and Del(1, 2, 3, 4, 5, 6)IGF-II (100–200 ng/ml), were also tested
to evaluate the modulatory effect of endogenous IGFBPs on IGF-induced
responses.
Antibodies
The following rabbit or mouse primary antibodies raised against
human proteins were shown previously to be highly specific: clone
IR3 for IGF-I-R (31) (Oncogene Science, Inc., Cambridge, MA), clone cII 25.3 for insulin-R
(32) (Oncogene Science, Inc.), polyclonal
antibodies to IGFBP-1 to -5 (33, 34) (Upstate Biotechnology, Inc.), and polyclonal antibody to IGFBP-6
(35) (Austral Biologicals, San Diego, CA). The latter
probe is recommended for enzyme-linked immunosorbent assay and
immunohistochemistry, and it is known to cross-react with other IGFBPs
in Western assay conditions.
Indirect immunofluorescence
Detection of tyrosine kinase IGF-I-R and IGFBP isoforms on
tissue cryosections was performed as described previously
(27). After fixation in either 1% formaldehyde diluted in
100 mM sodium phosphate buffer, pH 7.4 (45 min at 4 C), or
acetone/chloroform (1:1; 5 min at -10 C), specimens were incubated in
100 mM glycine in PBS if quenching of aldehyde residues was
necessary; blocked in Blotto, BSA, or fish gelatin solutions; and then
incubated for 1 h at room temperature with primary antibodies
diluted in 2% BSA-PBS. Fluorescein-conjugated secondary antibodies
were then added for 45 min. Control experiments were performed by
omitting or replacing the primary antibody with the appropriate
nonimmune serum. Primary antibodies were used at the following
dilutions: IGF-I-R, insulin-R, IGFBP-1, IGFBP-3, IGFBP-4, and IGFBP-5,
1:100; IGFBP-2, 1:1000; and IGFBP-6, 1:25. Antimouse (1:30) and
antirabbit (1:50) secondary antibodies were obtained from Roche Molecular Biochemicals Canada (Laval, Québec, Canada).
Western and Far Western immunoblotting
SDS-PAGE was performed as described previously for fetal stomach
(28) on 12% and 15% acrylamide gels. Total proteins from
gastric explants were extracted in 2 x sample buffer [Tris-HCl
(pH 6.8), 4% SDS, and 2% ß-mercaptoethanol]. Proteins in culture
medium were concentrated approximately 30- to 50-fold by
ultrafiltration using Centricon-10 concentrators (Amicon Canada,
Oakville, Ontario, Canada), then mixed with 2 x sample buffer.
Aliquots of tissue proteins (180–200 µg) and medium proteins
(80–100 µg) assayed by the method of Lowry (36) were
separated, transferred, incubated with primary antibodies after
blocking in 0.2% highly purified casein, and then processed with the
Western-Light Plus Chemiluminescent Detection System (Tropix, Bedford,
MA). Autoradiograms exposed in a linear range were quantified by
densitometric analysis with an LKB XL Ultroscan
(Pharmacia Biotech, Baie d’Urfé, Québec,
Canada), and signals were normalized to a keratin-18 control.
Antibodies were used at the following dilutions: IGFBP-1, 1:1000;
IGFBP-2, 1:2000; IGFBP-3, 1:800; IGFBP-4, 1:1000; IGFBP-5, 1:750;
IGFBP-6, 1:100–1000; and keratin-18 (monoclonal CY-90,
Sigma, St. Louis, MO), 1:5000.
For Far Western blots, SDS-PAGE and immunodetection were performed as described above, except that all of these steps were carried out under nonreducing conditions to allow the binding of IGF to IGFBPs (37). After electrotransfer, membranes were incubated with a relatively high concentration of human IGF-I (200 ng/ml) and probed with a monoclonal antibody against this peptide (2 µg/ml; from Upstate Biotechnology, Inc.).
Tritiated thymidine incorporation
To determine the rate and site of DNA synthesis, 2 µCi
[3H]thymidine (80 Ci/mmol; Amersham Pharmacia Biotech Canada, Oakville, Ontario, Canada) were added
per ml medium during the last 6 h of culture (2, 27).
The level of radiopercursor incorporated into trichloroacetic
acid-precipitable material was expressed as disintegrations per min/mg
tissue and reported as stimulation percentage vs. the
control value. For radioautography of incorporation sites, some
explants were fixed in 2.8% glutaraldehyde in 0.1 mol/L cacodylate for
16–24 h at 4 C, postfixed in 2% osmium tetroxide in cacodylate for 30
min, dehydrated, and embedded in epon resin. One-micron sections were
mounted on glass slides, stained with aldehyde fuschin, dipped in
Kodak NTB2 emulsion (Eastman Kodak Co.,
Rochester, NY), and then exposed in the dark for 6 weeks. The
[3H]thymidine incorporation labeling index was
established at the epithelial level and calculated as the percentage of
labeled nuclei (5 or more grains overlying the nucleus) over the total
number of nuclei counted in the entire epithelium. For each fetus, 2–3
explants were processed, and 1000 nuclei were counted/explant.
Lipase and pepsin enzymatic assays
Lipolytic activity was measured using a long-chain triglyceride
substrate, i.e. glycerol
tri-[14C]oleate (Amersham Pharmacia Biotech Canada; SA, 59 mCi/mmol) as detailed previously
(6). Released free [14C]oleic
acid was separated by liquid-liquid partition in
methanol/chloroform/heptane, collected in the supernatant upon
precipitation with carbonate-borate buffer, pH 10.5, and quantitated by
liquid scintillation spectrometry. Activity was expressed as nanomoles
of FFA produced per min/mg tissue. Pepsin activity was assayed by a
modification of the method of Anson and Mirsky (38), using
dialyzed 2% hemoglobin (H-2625, Sigma) as substrate. The
free amino acids generated by pepsin were measured in the supernatant
by spectrometry (280 nm) using an L-tyrosine
standard. Data were expressed in pepsin units, i.e. µmol
of tyrosine-containing peptides released per 10 min.
Statistics
Numeric values are reported as the mean ± SD.
Statistical significance of differences between experimental conditions
was established at 95% and determined by ANOVA followed by Student’s
t test when significance was indicated.
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Results |
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, A and B). A similar distribution of
insulin-R was visualized (not shown), and no significant immunostaining
was observed in all cases when the primary antibody was omitted or
replaced by the appropriate nonimmune serum (Fig. 1C).
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). In three
different specimens, seven bands were visualized in the 28–47 kDa
weight range that could correspond to previously characterized IGFBP
proteins: IGFBP-1, 32 kDa; IGFBP-2, 34 kDa; IGFBP-3 doublet, 39–47
kDa; IGFBP-4, 28 kDa; IGFBP-5, 31 kDa; and IGFBP-6, 36 kDa. No
quantification was performed, however, because immunoreactive signals
are proportional not only to the protein level, but also to the
affinity of the respective IGFBPs for exogenous IGF-I in this assay.
Therefore, specific IGFBP isoforms were identified by classical Western
blot using polyclonal antibodies in either uncultured tissue or
explants cultured for 24 h and their corresponding media.
Anti-IGFBP-6 was tested at various dilutions and was judged
inappropriate for Western blot analysis; it reacted with a 36-kDa
protein (a diffuse band, possibly corresponding to IGFBP-6) and with
other forms (34 kDa, IGFBP-2; results not shown), as previously
reported for intestinal cells (35). In these assays
however, IGFBP-1 to -5 proteins were successfully detected (Fig. 3, A–E), and immunoreactivity for
IGFBP-3 was particularly intense, suggesting that the latter carrier is
the most abundant in intact gastric tissue, followed by IGFBP-1,
IGFBP-2, and IGFBP-5 (compare left lanes). The staining
intensities may vary slightly according to the labeling properties of
each probe. In organ culture, the total amounts (tissue plus medium) of
IGFBP proteins all increased, except for IGFBP-1 (Fig. 3F). Indeed,
IGFBP-2 to -5 were actively synthesized during the first day of
culture, as their total levels increased by up to 3-fold compared with
that in uncultured tissues, as seen for IGFBP-2. Although
immunoreactive IGFBP-1 to -4 were efficiently released in the culture
fluid, no IGFBP-5 was detected. This suggests that either IGFBP-5 is
delivered by a different cellular mechanism, or it is cleaved into
proteolytic fragments that are not recognized by the antibody used. In
this regard, previous studies have demonstrated that IGFBP-5 is very
sensitive to the actions of serine proteases and is only present in
cell culture medium in the form of 20-, 22-, or 24-kDa fragments
(39, 40, 41). Thus, a second set of Far Western ligand blot
experiments was performed on 15% acrylamide gels to verify whether
IGFBP fragments of lower molecular mass were present in the culture
medium of gastric explants (Fig. 2B). One binding protein of
approximately 22 kDa that was not found in the corresponding tissue
homogenate was successfully visualized.
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). IGFBP-1 immunostaining was visualized
at the level of epithelium as well as in several elements of the lamina
propria (mesenchyme and blood vessels; Fig. 4A). No evidence of
restricted synthesis site was found. By contrast, other isoforms were
preferentially associated with epithelial cells. IGFBP-3, IGFBP-4, and
IGFBP-5 (Fig. 4, B–D) were detected in the entire epithelium,
generally with increased reactivity in surface epithelial cells. The
expression of IGFBP-2 protein was more rigorously compartmentalized. An
intense cytoplasmic and basolateral staining was seen in epithelial
cells of the foveolus/neck region, including the proliferative
compartment, whereas the base of glands was negative. This observation
was made in either early forming (Fig. 4E) or maturing units (Fig. 4F).
The distribution of IGFBP-6, consistent with endogenous expression in
epithelial cells, also varied greatly among the compartments. Compared
with superficial epithelium, the bases of primitive (Fig. 4G) and
differentiated glands (Fig. 4H) were mildly positive, and staining was
redistributed to the apical cell region. Again, no significant
immunoreactivity was observed in controls (not shown).
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). At the 50 ng/ml concentration, IGF-I
increased the rate of total DNA synthesis compared with control value
in 10 of 13 cultures (mean stimulation, 45.19 ± 12.70%;
P < 0.01). This effect was already maximal (similar at
100 ng/ml) and was maintained after 48 h (results not shown). By
contrast, neither IGF-II nor insulin significantly stimulated
[3H]thymidine incorporation after the same time
intervals (results for 100 ng and 30 µU/ml dosages are illustrated,
respectively). Explants from three different specimens were processed
for radioautography to determine the sites of precursor incorporation
at the level of the epithelium. The labeled nuclei were found mainly in
the foveolus/neck region of gastric glands, as previously reported
(2). IGF-I supplementation did not alter their overall
morphology and induced a significant increase (42%) in the epithelial
labeling index compared with that in untreated explants (control,
17.9 ± 2.0% of total cells labeled; IGF-I, 25.6 ± 2.9%;
P < 0.006). In a second set of experiments, the
effects of native IGFs were compared with those of truncated analogs
that do not interact with IGFBPs (Fig. 5B).
R3IGF-I added at the same concentration as native
IGF-I, i.e. 50 ng/ml, induced a similar and stronger
stimulation of [3H]thymidine incorporation into
total DNA (R3IGF-I, 68.22 ± 14.23%; IGF-I,
44.68 ± 14.89%; P < 0.04). Addition of 100
ng/ml Del(1, 2, 3, 4, 5, 6)IGF-II caused a strong 1-fold increase in the
incorporation level (114.04 ± 11.60%; P <
0.0005), whereas native IGF-II remained without effect.
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) as well as in culture medium. For
lipase, although the amount of enzyme secreted did not vary (not
shown), there was a significant decrease in tissue activity in explants
treated with IGFs [Fig. 6A; 50 ng/ml IGF-I, -31,6%
(P < 0.027 vs. control); 100 ng/ml IGF-II,
-42,3% (P < 0.022 vs. control)]. In
comparison, both truncated R3IGF-I and
Del(1, 2, 3, 4, 5, 6)IGF-II decreased tissue levels of lipase activity slightly
more than IGFs [Fig. 6A; R3IGF-I (-57.9%)
vs. IGF-I (-31.6%), P < 0.029;
Del(1, 2, 3, 4, 5, 6)IGF-II (-62.9%) vs. IGF-II (-42.3%),
P < 0.08, marginally significant]. As opposed to
lipase, pepsin activity resulting from activation of fundic-type
pepsinogen (Pg5) at acid pH was not modulated by these factors (Fig. 6B), and insulin did not influence lipase or pepsin activities during
the same culture period (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current investigation establishes that immunoreactive IGF-I-R and
insulin-R are expressed at the level of basolateral membranes of human
gastric epithelial cells, corroborating data mainly obtained through
binding studies in rodents (13, 14, 15) and the intestinal
HT-29-D4-Gal cell line (35). Both surface and glandular
epithelia were found to be positive, suggesting that all epithelial
cells, including mitotic precursors, would be competent to respond to
IGF-I, IGF-II, and/or insulin. Organ culture experiments indeed
demonstrate that IGF-I is able to stimulate epithelial cell
proliferation in intact gastric tissues whereas both IGFs reduce HGL
activity in fetal chief cells, similar to epidermal growth factor and
transforming growth factor- in the same model (27, 28).
The mitogenic effects of IGF-I on normal human gastric epithelial cells
would be consistent with those reported with monolayer cultures of
human gastric cancer cells (43). Also, this would agree
with the enhanced growth of the small bowel observed in transgenic mice
overexpressing IGF-I (44). However, the lack of IGF-II
mitogenic activity contrasts with its stimulatory effect on
enterocyte-like Caco-2 cells constitutively expressing the IGF-II
transgene (45). The current study also shows that both
IGFs repress HGL activity without affecting pepsin, an observation that
further supports the existence of a functional uncoupling between the
expression of the two gastric zymogens either during in
utero development (6) or in organ culture (5, 27). The fall in HGL activity induced by IGFs is reminiscent of
the down-regulation of intestinal sucrase-isomaltase activity by IGF-II
(45).
To assess whether IGFBPs have the capacity to modulate IGF biological actions either negatively or positively, we extended previous analyses establishing the presence of IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6 mRNAs in human fetal stomach (26). Indeed, the intrinsic capacity of human gastric epithelial cells to translate these mRNAs and to secrete the proteins remained uncertain. Far Western blotting experiments helped to identify seven IGFBPs by the affinity-bound IGF-I technique (37) that were tentatively associated to known IGFBP-1 to –6, including the 39–47 kDa doublet of IGFBP-3. Western blot analyses using specific antibodies (33, 34) confirmed the presence of IGFBP-1 to -5 with their expected molecular masses in intact tissues as well as in cultured gastric explants and their corresponding media. These experiments clearly established the capacity of the human gastric mucosa to translate IGFBP mRNAs (IGFBP-1 excepted) and to efficiently secrete the various IGFBPs. Incidentally, the total amount of IGFBP-1 remained constant compared with that in uncultured gastric explants, indicating that the gastric pool of IGFBP-1 probably derives from another source, the fetal liver, as the corresponding mRNA is expressed only in this organ (26). This study also enlightens different secretion patterns. Intact IGFBP-1 to -4 proteins were all released into culture medium. IGFBP-2 was the most secreted protein, whereas a 22-kDa IGFBP form, probably corresponding to a proteolytic fragment of IGFBP-5, was detected. Keeping in mind that IGFBP-5 remains preferentially associated in vivo and in vitro with extracellular matrix proteins (46) and that IGFBP-5 fragments isolated from the medium of cultured cells exhibit a reduced, but significant, IGF binding capacity (39, 40, 41), it can be suggested that intact and cleaved IGFBPs together contribute to the modulation of IGF activity through specific mechanisms. According to a new hypothesis, IGFBP-5 fragments may even stimulate the phosphorylation of a putative 420-kDa receptor and mediate IGF-independent effects (40).
To determine whether these IGFBPs were strategically located in specific functional compartments along the foveolus-gland unit, immunohistochemistry was performed with specific antibodies. IGFBP-1 was revealed at the level of epithelium and all components of the lamina propria, consistent with its extragastric origin (26). IGFBP-3, IGFBP-4, and IGFBP-5 were, instead, concentrated in gastric epithelium and associated with all epithelial cell types, although with variable staining intensities. Most importantly, IGFBP-2 exhibited a restricted pattern of distribution; an intense immunoreactivity was detected in the surface mucous epithelium and the junctional proliferative zone. The basal half of forming glands, which contains zymogen- and acid-secreting populations (chief, parietal) (2, 7), was negative. Although it was visualized at the base of glands, anti-IGFBP-6 staining also increased greatly toward the surface. This study clearly illustrates that IGFBP-2 to -6 are either restricted or evenly distributed in the different functional compartments, reinforcing the concept that they are strategically located to act as autocrine/paracrine modulators of IGFs actions. This is particularly evident for IGFBP-2 and IGFBP-6, which are highly concentrated in surface mucous cells and proliferative cells, are known to bind IGF-II with a greater affinity than IGF-I, and are considered IGF-II carriers (21, 24).
To establish whether these IGFBPs have the capacity to positively or negatively modulate native IGFs actions, we compared their effects with those of truncated analogs that do not interact with IGFBPs (47). The observation that the analogs were slightly more efficient than native IGFs for reducing HGL activity at physiological concentrations (50–100 ng/ml) suggests that the binding proteins marginally influence the response of zymogenic chief cells to added IGFs. A similar conclusion could be drawn concerning the effects of IGF-I on cell proliferation, as synthetic R3IGF-I was slightly more potent than the native growth factor. However, the use of synthetic Del(1, 2, 3, 4, 5, 6)IGF-II revealed the existence of an important negative down-modulation exerted by IGFBPs on the mitogenic effects of IGF-II. Although the addition of native IGF-II remains without any significant effect on gastric cell proliferation (even at the 200 ng/ml dosage), the synthetic analog was very potent. Therefore, these observations clearly establish that the influence of IGF-II as a growth-promoting agent is tightly regulated by local expression and/or secretion of IGF-II carriers. The use of an organ culture system preserving the morphological integrity of gastric tissues and mucosal compartments further allows us to propose that the sequestering mechanism determining the local amounts of free and bioactive IGF-II represents the summation of two complementary events: the binding of IGF-II to IGFBPs secreted in the culture fluid as well as its binding to carriers present in the microenvironment of a given epithelial population, i.e. precursor cells (proliferative compartment) or chief cells (base of glands). In this regard, the relative abundance of IGFBP-2 and IGFBP-6 in the precursor cell environment (visualized by immunofluorescence) would explain why exogenous IGF-II does not stimulate mucosal growth in contrast to its truncated analog. Interestingly, two independent studies have shown that IGF-II, IGFBP-2, and IGFBP-6 are normally expressed in cultures of intestinal cells (48) and that their reentry in the rapid growth phase correlates with down-regulation of IGFBP-2 synthesis (49). The physiological relevance for expressing two endogenous IGFBPs may be related to their different intracellular sorting and delivery mechanisms (IGFBP-2, basolateral, vs. IGFBP-6, apical), as observed herein and reported for intestinal cells (35). Taken together, these observations suggest that a fine balance between the latter three peptides may be a critical determinant regulating the local amount of free IGF-II and the slow or rapid proliferation state of dividing populations of gastric epithelial cells. The less abundant IGF-I peptide, mainly synthesized by the fetal liver and gut stromal cells (25, 50), would represent the endocrine/paracrine component of the gastric IGF system that is regulated less rigorously than local IGF-II and is involved in basal stimulation of proliferation.
In conclusion, this investigation provides new evidence for the presence and direct implication of an intragastric IGF/IGFBP system in the fine regulation in epithelial cell division and in the control of zymogen synthesis at the level of maturing chief cells. The mitogenic influence of IGF-II appears to be tightly regulated by IGFBP-2 and IGFBP-6 isoforms preferentially associated with this growth factor and proliferative cells. A working model for future studies regarding the induction of rapid cell division (regeneration) in human gastric epithelium by progression factors would consider the involvement of locally produced IGF-II dissociated from IGFBP-2 and IGFBP-6 complexes after the action of IGFBP proteases released during injury or stress (kallikreins, cathepsins, and matrix metalloproteinases) (51). The novel finding that the mucosal content of IGF-II specifically increases after intestinal resection (52) suggests that a common regulatory mechanism may apply to different gastrointestinal tract segments.
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Acknowledgments |
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Footnotes |
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Received September 27, 2000.
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References |
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and insulin-like growth factor I. J Clin Invest 87:1716–1723
down-regulate human gastric lipase gene expression. Gastroenterology 116:831–841[CrossRef][Medline]
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) or native
(
) IGFs compared with control, P < 0.027 (*).
Statistically significant difference between truncated and native
IGF-I, P < 0.029. Marginally significant
difference between truncated and native IGF-II, P
< 0.08.