Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0711
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ABSTRACT |
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Insulin-like
growth factor-I (IGF-I)-mediated growth of cells can be modulated by
specific IGF binding proteins (IGFBPs) that inhibit or augment IGF-I
ligand-receptor interaction. IGFBP expression and production by human
intestinal muscle cells in culture was characterized in rapidly growing
cells (day
3 of culture), in confluent cells
(day
7), and in postconfluent cells
(day
14). RT-PCR analysis identified
IGFBP-3, IGFBP-4, and IGFBP-5 mRNA during all three phases of growth.
The production of IGFBP-3 and IGFBP-5 was regulated in reciprocal
fashion. IGFBP-5 production was high on
day 3 and decreased two- to fivefold by day
14, and IGFBP-3 production was low on
day 3 and increased five- to eightfold by
day
14. IGFBP-4 production remained
constant. IGFBP-3 inhibited and IGFBP-5 augmented IGF-I-induced
proliferation. IGFBP-3 and IGFBP-5 production was regulated in
reciprocal fashion by transforming growth factor-1 (TGF-
1).
Immunoneutralization of endogenous TGF-
1 decreased the production of
IGFBP-3 and increased the production of IGFBP-5. Addition of exogenous
recombinant human TGF-
1 had the opposite effect. We conclude that
the expression and time-dependent production of IGFBP-3, IGFBP-4, and
IGFBP-5 and their regulation by endogenous TGF-
1 represent
mechanisms by which human intestinal muscle cells regulate autocrine
IGF-I-mediated growth.
smooth muscle cells; insulin-like growth factor-1; proliferation; insulin-like growth factor binding proteins; transforming growth
factor-1
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INTRODUCTION |
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SIX HUMAN INSULIN-LIKE growth factor-I (IGF-I) binding proteins (IGFBPs) have been identified, sequenced, and cloned (13, 14, 17, 29, 30). This family of proteins, which have no sequence homology with the IGF-I receptor, functions as modulators of IGF-I actions. Human IGFBP-1 through IGFBP-6 share ~50% homology within the family, and they each share ~80% sequence homology with IGFBPs of other mammalian species (13, 14, 15, 17, 29, 30). IGFBP-1 through IGFBP-6 are secreted proteins and have been identified in the circulation and the extracellular space; in addition, all but IGFBP-4, which has been identified in soluble form only, are present also on the cell surface or in association with the extracellular matrix (13, 14, 15, 17, 29, 30). Several mechanisms account for the modulatory effects of IGFBPs on IGF-I actions. Secreted IGFBPs, by binding and sequestering IGF-I, inhibit ligand-receptor interaction. IGFBPs on the cell surface or associated with the extracellular matrix, can either inhibit ligand-receptor interaction (IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-6) or promote ligand-receptor interaction (IGFBP-3 and IGFBP-5) (1, 4, 6, 15, 22, 28). Two recent lines of investigation suggest that this system is more complex than originally thought. Although IGFBPs modulate IGF-I actions, emerging evidence suggests that IGF-I may, in turn, modulate the actions of IGFBP-3, IGFBP-4, and IGFBP-5 through regulation of proteolysis of the binding proteins (6, 7, 28). In addition, IGF-I-independent effects have been reported for IGFBP-1, IGFBP-3, and IGFBP-5 (11, 19, 30).
The expression of IGFBPs by vascular and visceral smooth muscle cells is both species and tissue specific. Porcine vascular and aortic smooth muscle cells express IGFBP-2, IGFBP-4, and IGFBP-5 (9). Rat vascular and aortic smooth muscle cells express IGFBP-2, IGFBP-3, and IGFBP-4 (12) and human aortic smooth muscle cells express IGFBP-3, IGFBP-4, and IGFBP-6 (3). Rabbit airway smooth muscle cells express IGFBP-2 exclusively (24). Human myometrium expresses IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 (32). Smooth muscle cells of the rat colon express IGFBP-3, IGFBP-4, and IGFBP-5 (34, 36). An important role for the IGF-I/IGFBP system in the rat intestine is suggested by the observation that the induction of colitis in rats by treatment with trinitrobenzene sulfonate or the induction of enterocolitis by peptidoglycan-polysaccharride resulted in the upregulation of IGFBP-4 and IGFBP-5 expression (34-36). Exogenous IGF-I is capable of upregulating IGFBP-5 expression in cultures of rat colonic muscle cells (36). The expression and regulation of IGFBP production in the human intestine have not been investigated.
Our previous work (16) has shown that human intestinal smooth muscle
cells in culture secrete IGF-I and transforming growth factor-1
(TGF-
1). Although the levels of total IGF-I production remain
constant in culture as cells are proliferating
(day
3 of culture), attain confluence
(day
7 of culture), and become
postconfluent (day
14 of culture), the levels of free
IGF-I decline with time in culture. This is paralleled by the
decreasing ability of exogenous IGF-I to stimulate growth. These
findings suggest that changes in IGFBPs may be responsible for the
changes in IGF-I levels and its effects on growth. An opposite pattern
is seen for TGF-
1: secretion is low early in culture and increases
as cells attain postconfluence (16).
In the present study, we examined the expression of IGFBPs by human
intestinal smooth muscle cells in culture by RT-PCR. The production of
IGFBPs was examined by Western immunoblot analysis of conditioned
medium and cell lysates. The effects of IGFBPs on IGF-I-induced
proliferation were measured by
[3H]thymidine
incorporation. The results show that IGFBP-3, IGFBP-4, and IGFBP-5 are
expressed by human intestinal smooth muscle cells. The secretion of
IGFBP-3 and IGFBP-5 is regulated in a time-dependent reciprocal
fashion. Production of IGFBP-3 increases in confluent and postconfluent
cells, whereas secretion of IGFBP-5 decreases in confluent and
postconfluent cells. IGFBP-3 inhibits and IGFBP-5 augments
IGF-I-induced proliferation. Endogenously produced TGF-1 inhibits
IGFBP-5 production and stimulates IGFBP-3 production.
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METHODS |
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Preparation of isolated muscle cells from human jejunum. Muscle cells were isolated from the circular muscle layer of human jejunum, as described previously (16). Briefly, 4- to 5-cm segments of normal jejunum were obtained from patients undergoing surgery for morbid obesity according to a protocol approved by the Institutional Committee on the Conduct of Human Research. The segments were opened along the mesenteric border, the mucosa was dissected away, and the remaining muscle layer was cut into 2 × 2 cm strips. Slices were obtained separately from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated for two successive 60-min periods at 37°C in 25 ml of medium containing 0.2% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The medium consisted of (in mM): 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% Eagle's essential amino acid mixture. After the second incubation, the partially digested muscle strips were washed and incubated in enzyme-free medium for 30 min and the cells were allowed to disperse spontaneously.
Culture of human intestinal muscle cells. Primary cultures of human intestinal muscle cells were initiated and maintained as described previously (16). Briefly, muscle cells dispersed from the circular layer were harvested by filtration through 500-µm Nitex mesh and centrifugation at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min and resuspension in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamicin, and 2 µg/ml amphotericin B. After washing, the muscle cells were resuspended in DMEM containing 10% fetal bovine serum (DMEM-10) and the same antibiotics. The cells were plated at a concentration of 5 × 105 cells/ml as determined by counting in a hemocytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 medium was replaced every 3 days until the cells reached confluence, at which time they were passaged. All subsequent studies were performed in first passage-cultured cells after 3 days in culture, when cells were proliferating, after 7 days, by which time the cells were confluent, and after 14 days, when the postconfluent cells had attained the "hill-and-valley" configuration characteristic of cultured smooth muscle (16).
Preparation of conditioned medium.
Conditioned medium was prepared from muscle cells growing in 100-mm
plates after 3, 7, or 14 days. At each time point, the cells were
washed free of serum and incubated in DMEM in the absence of serum for
48 h. In some experiments the effect of TGF-1 was examined by
incubation of the cells with either recombinant human TGF-
1
(rhTGF-
1) (10 pM to 1 nM) or neutralizing antibody to hTGF-
1 (100 and 1,000 ng/ml) during the 48-h period. The requirement for new IGFBP
synthesis was investigated by addition of cycloheximide (1 µg/ml). At
the end of the incubation, the conditioned medium was removed and the
protease inhibitors phenylmethylsulfonyl fluoride (PMSF) (0.1 mM) and
aprotinin (0.1 µg/ml) were added to the conditioned medium. Cellular
debris was removed by centrifugation at 350 g for 5 min at 4°C. Aliquots of
the resulting supernatant were concentrated 10-fold in Centricon-10
tubes at 4°C. The resulting concentrated conditioned medium was
added to sample buffer containing 62.5 mM Tris (pH 6.8) with 2% SDS,
25% glycerol, 0.01% bromphenol blue, and 5%
-mercaptoethanol to
provide samples derived from equal amounts of total cellular protein
(200 µg/30 µl).
Preparation of whole cell lysates.
Cell lysates were also prepared from the same cultures from which the
conditioned medium was prepared. The cultured cells were washed three
times in ice-cold PBS and then lysed in PBS (pH 7.4) containing 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 µg/ml PMSF,
1% aprotinin, and 1 mM sodium orthovanadate. The cells were incubated
for 20 min at 4°C and then DNA sheared by passage through a
21-gauge needle. Cellular debris in the lysates was precipitated by
centrifugation at 12,000 g for 20 min
at 4°C. Total cellular protein in each lysate was measured using
the Bio-Rad protein assay (Hercules, CA). The resulting whole cell
lysates were added to a volume of sample buffer containing 62.5 mM Tris (pH 6.8) with 2% SDS, 25% glycerol, 0.01% bromphenol blue, and 5%
-mercaptoethanol to provide samples of equal total cellular protein
(30 µg/30 µl).
Preparation of RNA and RT-PCR analysis of IGFBPs. The expression of IGFBPs was investigated by RT-PCR analysis by modification of previously described methods (33). Briefly, total RNA was isolated from cultures of muscle cells growing in 100-mm culture dishes on days 3, 7, and 14 of culture, using Ultraspec RNA isolation reagent. Two micrograms of total RNA from each preparation were reverse transcribed in a reaction volume of 20 µl containing 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3.0 mM MgCl2, 10 mM dithiotreitol, 0.5 mM dNTP, 2.5 µM random hexamers, and 200 U of SuperScript II RT. The reaction was carried out in a thermal cycler for 10 min at 25°C, for 50 min at 42°C, and terminated by heating to 70°C for 15 min. The reverse-transcribed cDNA (3 µl) was amplified in a final volume of 50 µl by PCR under standard conditions: 2 mM MgCl2, 125 µM dNTP, and 2.5 U Taq polymerase using specific primer pairs for IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 based on the known sequences of the six human IGFBPs (23, 26) and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (18) as internal standard (Table 1). Sense and antisense primers were designed from different exons so that cDNA amplification resulted in PCR products of specific length, whereas genomic DNA would give longer amplified fragments. For each experiment, a parallel control without RT was processed. The amplified PCR products were separated on a 1% agarose gel containing 0.1 µg/ml ethidium bromide. The visualized bands were analyzed semiquantitatively using image scanning densitometry.
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Measurement of IGFBP production in conditioned medium and in whole cell lysates by Western blot analysis. Samples prepared from either whole cell lysates or from conditioned medium were boiled for 5 min and placed on ice before centrifugation for 5 min at 12,000 g. After centrifugation for 5 min at 12,000 g, samples, each derived from equal amounts of total cellular protein, were separated by SDS-PAGE on 12% polyacrylamide gels. The separated proteins were electrotransferred to 0.2-µm nitrocellulose membranes overnight at 4°C in a buffer containing 25 mM Tris (pH 8.3) and 192 mM glycine, with added 20% methanol and 0.1% SDS. The blotted nitrocellulose membranes were washed twice in water and incubated in a blocking buffer consisting of PBS with 3% milk protein. The nitrocellulose membrane was then incubated overnight at 4°C in blocking buffer containing a 1:1,000 dilution of antibody to either IGFBP-3, IGFBP-4, or IGFBP-5. The antibodies against IGFBP-3 and IGFBP-5 display <1% cross-reactivity with other IGFBPs. The antibody against IGFBP-4 displays 50% cross-reactivity with IGFBP-2; however, intact IGFBP-2 has a known molecular mass of 36 kDa, which is dissimilar to the ~24-kDa protein identified in these Western immunoblots. The nitrocellulose was washed in water and incubated in a 1:1,000 dilution of a goat anti-rabbit IgG coupled to horseradish peroxidase for 90 min at room temperature. The resulting protein bands were visualized with enhanced chemiluminescence and quantitated by image scanning densitometry.
[3H]thymidine proliferation
assay.
Proliferation of smooth muscle cells in culture was measured by the
incorporation of
[3H]thymidine, as
described previously (16). Briefly, the cells were washed free of serum
and incubated for 24 h in DMEM. After 24 h of incubation
in the absence of serum, the cells were incubated for an additional 24 h with a submaximal concentration of IGF-I (1 nM). The effects of
IGFBP-3 and IGFBP-5 on IGF-I-induced proliferation were examined by
addition of increasing concentrations of either recombinant human
IGFBP-3 or IGFBP-5 (0.5 to 50 nM). During the final 4 h of this
incubation period, 1 µCi/ml
[3H]thymidine was
added to the medium. The incubation was terminated by washing cells
twice with 1 ml HBSS at 4°C and then incubating for two 10-min
periods with 1 ml of 10% TCA at 4°C. TCA was removed, and 0.5 ml
of 2 N perchloric acid was added to each well and the cells incubated
for 30 min at 60°C. After cooling, 400-µl aliquots of the
supernatant were removed to scintillation vials and counted on a
-scintillation counter. The cell residue was dissolved in 0.3 N NaOH
and neutralized with 0.3 N HCl, and protein was measured spectrophotometrically with the Bio-Rad assay system.
[3H]thymidine
incorporation was expressed as counts per minute per microgram of protein.
Statistical analysis. Values represent means ± SE of n experiments where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired data as was appropriate. Densitometric analysis was performed using computerized densitometry and ImageQuant NT software (Molecular Dynamics). Densitometric values for the bands of interest were determined in areas of equal size and are reported in arbitrary units above background values.
Materials.
Collagenase and soybean trypsin inhibitor were obtained from
Worthington Biochemical (Freehold, NJ). HEPES was obtained from Research Organics (Cleveland, OH). DMEM and HBSS were obtained from
Mediatech (Herndon, VA). Fetal bovine serum was obtained from
BioWhittaker (Walkersville, MD). Ultraspec RNA reagent was obtained
from Biotecx Laboratories (Houston, TX), SuperScript II RNase
H RT, dNTPs,
X174 RF
DNA/Hae III fragments and agarose were
obtained from GIBCO BRL (Gaithersburg, MD). Random primers
and Taq polymerase were obtained from
Perkin Elmer (Foster City, CA). Western blotting materials and the
protein assay kit were obtained from Bio-Rad. Recombinant human
IGFBP-3, recombinant human IGFBP-5, and rabbit polyclonal antibodies to
IGFBP-3, IGFBP-4, and IGFBP-5 were obtained from Upstate
Biotechnology (Lake Placid, NY). Neutralizing antibody to hTGF-
1
(IgY) was obtained from R & D Systems (Minneapolis, MN). rhTGF-
1 was
obtained from Collaborative Biomedical Products (Bedford, MA). Plastic
cultureware was obtained from Corning (Corning, NY). All other
chemicals were obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Characterization of IGFBPs by RT-PCR. The expression of IGFBPs by human intestinal smooth muscle cells during the three phases of culture was examined using semiquantitative RT-PCR. Total RNA was obtained from rapidly growing cells (on day 3 of culture), confluent cells (on day 7 of culture), and postconfluent cells (on day 14 of culture). RNA (2 µg) was reverse transcribed, and the resulting cDNAs were amplified using PCR with specific primers for each human IGFBP and for GAPDH as a control.
RT-PCR identified transcripts of the predicted size for IGFBP-3 (439 bp) and GAPDH (555 bp) during all three phases of culture (Fig. 1). Transcripts of the predicted size for IGFBP-4 (310 bp) and IGFBP-5 (377 bp) were also identified during all phases of culture (Fig. 1).
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Characterization of secreted IGFBPs by Western blot analysis. The presence and relative amounts of IGFBP-3, IGFBP-4, and IGFBP-5 secreted by human intestinal smooth muscle cells during various phases of culture were examined using Western immunoblot analysis. Conditioned medium was obtained from rapidly growing cells, confluent cells, and postconfluent cells.
Western blot analysis of proteins in conditioned medium using an antibody directed against human IGFBP-3 confirmed the presence of two proteins corresponding to the 43- and 45-kDa glycosylated forms of IGFBP-3 (Fig. 2). Densitometric measurement was used to compare the relative amounts of IGFBP-3 secreted into the culture medium during various phases of culture. The production of IGFBP-3 was time dependent with lower levels secreted by rapidly growing cells and four- to fivefold higher levels secreted by confluent and postconfluent cells (Fig. 3).
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Characterization of IGFBPs in cell lysates. Well-defined roles for cell surface-associated IGFBP-3 and IGFBP-5 in regulating the actions of IGF-I have been described previously (15). The effects of cell-associated IGFBP typically parallel those of soluble binding protein and act in concert with soluble protein to regulate IGF-I ligand-receptor interaction. Accordingly, the presence of IGFBP-3 and IGFBP-5 was examined also in cell lysates derived from cultured cells during all three periods of growth. Cell-associated IGFBP-3, as identified by Western immunoblot analysis, had a similar pattern to that secreted by the cells. Levels of IGFBP-3 were lowest in rapidly growing cells, increasing by threefold in confluent cells and by eightfold in postconfluent cells (Fig. 3).
Similarly, the levels of IGFBP-5 associated with the cells paralleled the levels of secreted IGFBP-5. Levels of IGFBP-5 were highest in rapidly growing cells and were three- to fivefold lower in confluent cells and postconfluent cells, respectively (Fig. 4).Modulation of IGF-I-induced proliferation by IGFBP-3 and IGFBP-5. The effects of IGFBP-3 and IGFBP-5 on IGF-I-induced proliferation were identified by measurement of [3H]thymidine incorporation. Incubation of confluent (day 7) human intestinal muscle cells for 24 h with a submaximal concentration of IGF-I (1 nM) increased [3H]thymidine incorporation by 147 ± 18% above basal levels. The ability of IGF-I to induce proliferation was significantly inhibited in a concentration-dependent fashion by recombinant human IGFBP-3 (38 ± 5% inhibition from control with 50 nM IGFBP-3; Fig. 6). In contrast, the ability of IGF-I to induce proliferation was significantly enhanced in a concentration-dependent fashion by recombinant human IGFBP-5 (37 ± 5% above control with 50 nM IGFBP-5; Fig. 6).
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Reciprocal regulation of IGFBP-3 and IGFBP-5 production by
TGF-1.
The pattern of TGF-
1 secretion parallels that of IGFBP-3 in cultured
human intestinal muscle cells (16). Overall, the effects of TGF-
1
are predominantly growth inhibitory in these cells. Two complementary
methods were used to examine the interplay between TGF-
1 and IGFBP-3
and IGFBP-5. In the first method, the ability of TGF-
1 to alter the
production of IGFBP-3 and IGFBP-5 was examined by incubation of the
cells with exogenous TGF-
1. In the second method, an autocrine role
for endogenous TGF-
1 in the regulation of IGFBP-3 and IGFBP-5
production was examined by immunoneutralization of endogenous TGF-
1.
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DISCUSSION |
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In human intestinal smooth muscle cells, the proliferative effects of IGF-I represent the net effects of IGF-I itself and the effects of various IGFBPs acting either as stimulatory or inhibitory modulators of IGF-I ligand-receptor interaction. Three mechanisms then exist by which these cells can regulate the autocrine growth effects of IGF-I: alteration of IGF-I secretion, alteration in IGFBP expression and/or production, or alteration of IGF-I receptor expression (15). Our previous work has measured levels of total and free IGF-I produced by these cells. Although levels of free IGF-I (unbound to IGFBPs) fell nearly 40-fold from day 3 (proliferating cells) to day 14 (postconfluent cells), total IGF-I production remained constant during all periods of growth (16). Changes in IGF-I receptor expression or in its coupling to intracellular signaling cascades may also occur, but this has not been examined.
The current study shows that human intestinal smooth muscle cells in
culture express IGFBP-3, IGFBP-4, and IGFBP-5. Similar mRNA
levels for individual IGFBPs, IGFBP-3, IGFBP-4, and IGFBP-5, were
detected in cells during all phases of culture: in proliferating cells
(day
3 of culture), in confluent cells
(day
7 of culture), and in postconfluent
cells (day
14 of culture). The levels of IGFBP-3
and IGFBP-5 protein produced by the muscle cells, however, depended on
the time in culture: IGFBP-3 levels were low in proliferating cells and
increased fivefold as cells attained confluence, whereas IGFBP-5 levels
were high in proliferating cells and decreased twofold as cells
attained confluence. The levels of IGFBP-4 protein, in contrast, did
not change with time in culture. The levels of IGFBP-3 and
IGFBP-5 production are regulated in reciprocal fashion, at
least in part, by endogenous TGF-1.
IGFBP-5 promotes IGF-I-mediated proliferation in human intestinal
muscle cells. In addition to cellular mechanisms regulating IGFBP-5
mRNA and protein abundance, the ability of IGFBP-5 to bind IGF-I and to
facilitate IGF-I ligand-receptor interaction can also be regulated by
the proteolysis of IGFBP-5 (7, 22). Proteolysis of
IGFBP-5 yields fragments that are capable of binding IGF-I, but due to
the reduced affinity of the fragments for IGF-I, proteolysis, in
effect, acts to promote IGF-I ligand-receptor interaction (1). The
binding of IGF-I to intact IGFBP-5 also protects the binding protein
from proteolysis. The highest levels of intact IGFBP-5 production by
human intestinal muscle cells were detected in rapidly proliferating
cells with lower levels in confluent and postconfluent cells, as the
rate of growth slowed. Increase in the levels of TGF-1 production by
postconfluent human intestinal muscle cells (16) results, in part, in
the concomitant decrease in the production of growth stimulatory
IGFBP-5. This may represent one mechanism by which human intestinal
muscle cells augment IGF-I-mediated growth during periods of rapid
growth and diminish the growth effects as the rate of growth declines.
IGFBP-3 can act to either augment or inhibit IGF-I-mediated
proliferation depending on the cell type. When it acts as an inhibitor of IGF-I-mediated proliferation, as in human intestinal muscle cells,
soluble IGFBP-3 is a potent competitive inhibitor by virtue of its
affinity for IGF-I being higher than that of IGF-I for the IGF-I
receptor, thereby effectively inhibiting ligand-receptor interaction
(28). Membrane- and extracellular matrix-associated IGFBP-3 also
contribute to the modulation of IGF-I actions; the affinity of
membrane-associated IGFBP-3 for IGF-I, however, is 10-fold lower than
that of soluble IGFBP-3, which, in net, is twofold lower than that of
IGF-I for the IGF-I receptor (21). Similar to IGFBP-5,
the activity of IGFBP-3 is also regulated by proteases. Although intact
IGFBP-3 inhibits IGF-I actions, the proteolytic fragments of IGFBP-3,
because of their reduced affinity for IGF-I, act to potentiate its
actions (6). IGFBP-3-specific proteolytic enzymes and mechanisms to
regulate their activity have been identified in a number of cell types
(5, 8). Production of intact IGFBP-3 protein was lowest in rapidly
proliferating human intestinal muscle cells. As cells attained
confluence and postconfluence and the rates of growth declined, the
levels of the growth inhibitory IGFBP-3 increased. In cultured human
fibroblasts, addition of exogenous TGF- has been shown to increase
IGFBP-3 production (20). Increase in the levels of TGF-
1 production by postconfluent human intestinal muscle cells also results, in part,
in the concomitant increase in the production of growth inhibitory
IGFBP-3. This may represent a second complementary mechanism by which
these cells minimize the growth-stimulatory effects of IGF-I as cells
reach confluence and the rate of growth declines.
In contrast to IGFBP-3 and IGFBP-5, IGFBP-4 has been identified only in a soluble form that inhibits IGF-I ligand-receptor interaction (4). The effects of IGFBP-4 are also regulated by the production of specific IGFBP-4 proteases in a number of cells, including vascular smooth muscle cells (6). The resulting fragments of IGFBP-4 are virtually incapable of binding IGF-I and provide a method to increase free IGF-I levels and potentiate IGF-I actions. Human intestinal smooth muscle cells express IGFBP-4 during all phases of culture, but the levels of protein secretion do not appear to depend on the phase of culture as do those of IGFBP-3 and IGFBP-5.
Time-dependent production of IGFBPs by human intestinal muscle cells in
culture may in part explain the previous findings of constant total
IGF-I levels but time-dependent changes in free IGF-I levels. High
levels of free IGF-I were measured in cultures of proliferating cells;
levels of free IGF-I were decreased in cultures of confluent and
postconfluent cells. The net effect of growth stimulatory IGFBP-5 and
growth inhibitory IGFBP-3 in cultures of proliferating cells, when
IGFBP-5 levels are high and IGFBP-3 levels are low, is to promote the
autocrine growth effects of IGF-I. In contrast the net effect of
IGFBP-3 and IGFBP-5 in cultures of confluent and postconfluent muscle
cells, when growth inhibitory IGFBP-3 levels are high and growth
stimulatory IGFBP-5 levels are low, is to reduce the autocrine growth
effects of IGF-I. The pleotrophic growth factor, TGF-1, contributes
to the regulation of the relative levels of IGFBPs produced. The increasing levels of TGF-
1 produced as cells attain confluence and
postconfluence can augment the production of growth inhibitory IGFBP-3
and inhibit the production of growth stimulatory IGFBP-5. The net
effect of these events would be to decrease the growth-stimulating effects of IGF-I in these cells as they become confluent and postconfluent.
IGF-I-independent effects of IGFBP-3 and IGFBP-5, which parallel their
individual effects on IGF-I ligand-receptor interaction, have also been
described (19, 22). Specific receptors for IGFBP-3 have been identified
in Hs 578T breast cancer cells (25), in prostatic adenocarcinoma cells
(PC-3) (27), and in mink lung epithelial cells (19), which mediate an
IGF-I-independent inhibition of growth. This receptor appears to be the
TGF-V receptor (19). IGFBP-5 binds to specific receptors in a
variety of bone cells and stimulates proliferation through an
IGF-I-independent mechanism (1). The IGF-I-independent effects of
IGFBP-1 appear to be mediated by binding to the
5
1-integrin
receptor (11).
In the normal rat colon, IGF-I receptors and IGFBP-3, IGFBP-4, and IGFBP-5 have been identified (34, 36). Infusion of IGF-I in neonatal rats increases growth of the intestine, including the muscularis propria, in a tissue-specific manner (31). IGF-I is capable of upregulating IGFBP-5 expression (36). Inflammation caused by either trinitrobenzene sulfonate instillation or subserosal injection of peptidoglycan-polysaccharide results in increased levels of IGF-I binding and IGFBP-5 and IGFBP-4 in the muscularis propria (35, 36). These findings suggest that the IGF-I system and its regulatory binding proteins serve an important function in the response of the colon to inflammation and in the development of fibrosis and stricture formation. The findings of the current study demonstrate both the role and the interplay of endogenous IGF-I binding proteins that serve to regulate the growth of human intestinal muscle in culture. Taken together, the existing evidence suggests mechanisms by which the IGF-I system may regulate not only the initial response to inflammation but also the limitation of the intestinal response to prevent excessive growth and stricture formation.
In summary, the production of individual IGFBPs, as well as the
interplay of IGFBPs having either stimulatory or inhibitory effects on
proliferation, contribute to the regulation of IGF-I-mediated growth of
human intestinal muscle cells. The endogenous growth factor, TGF-1,
acting in an autocrine fashion contributes to the regulation of IGFBP
production and thereby to the modulation of IGF-I actions in these cells.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: J. F. Kuemmerle, Division of Gastroenterology, Medical College of Virginia Campus, Virginia Commonwealth Univ., PO Box 980711, Richmond, VA 23298-0711.
Received 28 April 1998; accepted in final form 14 August 1998.
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