Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298
Submitted 9 January 2004 ; accepted in final form 26 May 2004
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ABSTRACT |
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insulin-like growth factor-I; Smad2; short interfering RNA
IGFBP-3, like IGFBP-1 and IGFBP-5, is capable of regulating cell growth independently of its effects on IGF-I-stimulated growth (13, 26). Two distinct mechanisms mediating the IGF-I-independent effects of IGFBP-3 have been identified. The first mechanism involves interaction of IGFBP-3 with transforming growth factor- (TGF-
) cell surface receptors. In mink lung epithelial cells and other cells, IGFBP-3 binds to TGF-
receptor (TGF-
R) type V (TGF-
RV) (23, 24). A direct inhibitory effect on growth mediated by this receptor has been proposed, but a mechanism of action has not been fully elucidated. Recent evidence (10) implicates the low-density lipoprotein receptor-related protein-1 in this pathway. IGFBP-3 also binds to and activates intracellular signaling via the TGF-
RII and TGF-
RI heteromeric complex (7, 8, 26). In the T47D breast cancer cell line, this mechanism requires the presence of TGF-
1 and both TGF-
RI and TGF-
RII and results in Smad2 [Homo sapiens mothers against decapentaplegic homolog 2 (Drosophila) (MADH2)] activation and inhibition of growth (8). A distinct, nonreceptor-based mechanism mediating IGF-I-independent inhibition of growth by IGFBP-3 has also been elucidated. IGFBP-3 possesses a consensus nuclear translocation sequence in its COOH terminus (29, 30). After nonreceptor-mediated nuclear translocation of IGFBP-3 via the
-importin pathway, IGFBP-3 binds to the nuclear retinoid X receptor-
(RXR
) and directly inhibits growth of opossum kidney cells, A549 lung cancer cells, T47D breast cancer cells, and various other cells (25, 29, 30).
We (2, 15, 19) have previously shown that human intestinal smooth muscle cells secrete IGF-I and IGFBP-3, -4, and -5. IGFBP-3 acts to inhibit IGF-I-stimulated proliferation (2). Intestinal smooth muscle cells also secrete TGF-1, which inhibits growth directly and increases IGFBP-3 expression (14). Little is known, however, regarding the IGF-I-independent effects of IGFBP-3 on growth of smooth muscle, whether TGF-
1 is required for IGFBP-3 to have these effects as it does in breast cancer cells, or what mechanisms might mediate these effects. These mechanisms may play an important role in the altered growth regulation of intestinal smooth muscle during the intestinal inflammation of Crohns disease in which levels of IGFBP-3 and TGF-
1 are altered and may contribute to muscle hyperplasia and stricture formation.
The present study shows that IGFBP-3 directly inhibits human intestinal smooth muscle growth. IGFBP-3 binds to the TGF- receptor types expressed by intestinal muscle cells. Binding of IGFBP-3 to TGF-
RII is followed by serine phosphorylation of TGF-
RI and Smad2, the primary substrate of activated TGF-
RI receptors. Activation of Smad2 mediates IGFBP-3-dependent inhibition of proliferation. Immunoneutralization of secreted IGFBP-3 increased basal proliferation. The ability of IGFBP-3 to inhibit proliferation does not require the presence of TGF-
1. The effects of IGFBP-3 were abolished in cells after Smad2 gene knockdown with short interfering RNA (siRNA). The results provide evidence that, in addition to the ability of IGFBP-3 to inhibit IGF-I-stimulated growth, endogenous IGFBP-3 also causes direct inhibition of human intestinal smooth muscle cell proliferation by activating TGF-
receptors and Smad2.
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MATERIALS AND METHODS |
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Culture of smooth muscle cells isolated from normal human intestine.
Muscle cells were isolated and cultured from the circular muscle layer of human intestine as described previously (2, 14, 15, 1820). 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 Virginia Commonwealth University Institutional Review Board. The segments were opened along the mesenteric border, the mucosa was dissected away, and the remaining muscle layer was cut into 2 x 2-cm strips. Slices were obtained from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated overnight at 37°C in 20 ml DMEM plus 10% fetal bovine serum (DMEM-10) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin, and 2 µg/ml amphotericin B (DMEM-10) to which was added 0.0375% collagenase (type II), and 0.1% soybean trypsin inhibitor. 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. After resuspension in DMEM-10 containing the same antibiotics, the cells were plated at a concentration of 5 x 105 cells/ml as determined by counting in a hemocytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 was replaced every 3 days until the cells reached confluence. Primary cultures of muscle cells were passaged on reaching confluence. All subsequent studies were performed in first-passage cultured cells after 7 days, at which time the cells are confluent. We (14) have previously shown that these cells express a phenotype characteristic of intestinal smooth muscle as determined by immunostaining for intestinal smooth muscle markers and expression of -enteric actin. Epithelial cells, endothelial cells, neurons, and interstitial cells of Cajal are not detected in these cultures (37, 38).
[3H]thymidine incorporation assay. Proliferation of smooth muscle cells in culture was measured by the incorporation of [3H]thymidine as described previously (2, 14, 1820). Briefly, the cells were washed free of serum and incubated for 24 h in serum-free DMEM in the presence or absence of various test agents. During the final 4 h of this incubation period, 1 µCi/ml [3H]thymidine was added to the medium. [3H]thymidine incorporation into the perchloric acid-extractable pool was used as a measure of DNA synthesis.
Measurement of Smad2 phosphorylation. The levels of phospho-Smad2(Ser465/467) and total Smad2 were measured by immunoblot analysis using standard methods (2, 15, 18, 19, 21). Briefly, confluent muscle cells were washed free of serum and stimulated with various test agents. The reaction was terminated by two rapid washes in ice-cold PBS after which lysates were prepared from the cells. Lysates were separated by SDS-PAGE under denaturing conditions. After the proteins were electrotransferred to nitrocellulose, the membranes were incubated overnight with a 1:1,000 dilution of antibodies recognizing Ser465/467-phosphorylated Smad2 or total Smad2. Bands of interest were visualized with enhanced chemiluminescence on a FluorChem 8800 (Alpha Innotech, San Leandro, CA), and the resulting digital images were analyzed by using AlphaEaseFC version 3.1.2 software.
Measurement of TGF-RI phosphorylation.
Serine phosphorylation (activation) of TGF-
RI was measured by immunoblot analysis after immunoprecipitation of TGF-
RI from whole cell lysates by modification of previously described methods (20, 21). Briefly, confluent muscle cells were washed free of serum and stimulated with various test agents. The reaction was terminated by two rapid washes in ice-cold PBS after which lysates were prepared from the cells. Cell lysates were prepared in a buffer consisting of (in mM): 50 Tris·HCl (pH 7.5), 150 NaCl, 50 NaF, 1 Na orthovanadate, 1 dithiothreitol, 1 PMSF, and 0.5% Nonidet P-40 to which was added 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. The resulting lysates were clarified by centrifugation at 14,000 g for 10 min at 4°C. The lysates were precleared by incubation with protein A agarose beads for 1 h at 4°C. Samples containing equal amounts of protein (0.5 mg) were incubated for 2 h at 4°C with 2 µg of rabbit anti-TGF-
RI after which 10 µl of protein A/G agarose beads were added, and the incubation was continued overnight. The immune complex-agarose beads were washed three times with ice-cold lysis buffer. After the final wash, the beads were resuspended in 25 µl of sample buffer. The samples were boiled for 5 min after which 20 µl of each sample was loaded onto a 15% polyacrylamide gel, and the proteins were separated by SDS-PAGE under denaturing conditions. After the proteins were electrotransferred to nitrocellulose, the membranes were incubated overnight with a 1:2,500 dilution of a monoclonal antibody (22a) recognizing serine-phosphorylated proteins. Bands of interest were visualized with enhanced chemiluminescence on a FluorChem 8800, and the resulting digital images were analyzed by using AlphaEaseFC version 3.1.2 software. Values for serine phosphorylation were normalized to total TGF-
RI levels after the membranes were stripped and reblotted for TGF-
RI.
Ligand binding analysis of TGF- receptors.
Binding of IGFBP-3 to TGF-
receptors was measured by 125I-labeled TGF-
1 affinity cross-linking and ligand blot analysis according to the methods of Cheifetz, et al. (3). Briefly, muscle cells growing in 60-mm dishes were washed twice in binding buffer consisting of (in mM): 50 HEPES (pH 7.5), 128 NaCl, 5 KCl, 1.2 CaCl2, and 1.2 MgSO4 with added 5 mg/ml (wt/vol) BSA. The cells were incubated for an additional 1 h at 37°C in binding buffer and equilibrated for 10 min at 4°C. The cells were then incubated with 100 pM 125I-labeled TGF-
1 at 4°C for 2 h. Nonspecific binding was determined in the presence of unlabeled 100 nM TGF-
1. Unbound 125I-labeled TGF-
1 was removed by washing the cells three times with ice-cold binding buffer. The cells were incubated for an additional 15 min in binding buffer without BSA and containing the bifunctional cross-linking reagent disuccinimidyl suberate (DSS, 25 mM). The cross-linking reaction was terminated by washing the cells in buffer consisting of (in mM): 10 Tris (pH 7.0), 250 sucrose, 1 EDTA, and 0.1 PMSF, with added pepstatin (1 µg/ml) and leupeptin (1 µg/ml). The cells were solubilized in sample buffer, and proteins were separated on 415% gradient polyacrylamide gels under reducing conditions. Gels were dried and then visualized by using autoradiography. The resulting autoradiograms were imaged on a FluorChem 8800. The resulting digital images were then analyzed by using AlphaEaseFC version 3.1.2 software. Bands corresponding to specific TGF-
receptor types were identified by their apparent molecular weight compared with known molecular weight standards.
Transfection of Smad2 siRNA. Protein levels of Smad2 in human intestinal muscle cells were inhibited by transfection of a Smad2 siRNA (6, 36). siRNA sequences were determined based on the sequence of human Smad2. Briefly, muscle cells growing in six-well plates were transfected with 2 µg of Smad2 siRNA or 2 µg on control RNA (Upstate Biotechnology) using TransMessenger transfection reagent according to the manufacturers directions (Qiagen, Valencia, CA). Cells were incubated for 3 h at 37°C with the transfection reagent-RNA complexes. The cells were washed free of transfection reagent-RNA complexes with PBS, and 2 ml of fresh DMEM-10 were added to each well. After 24-h incubation, the ability of Smad2 siRNA, but not control RNA, to inhibit Smad2 levels was determined by Western blot analysis as described above. Initial experiments were performed by using a range of RNA amounts (0.54 µg) and transfection times (2448 h) to determine the optimal conditions. Optimal inhibition of Smad2 was obtained by using 2 µg of RNA after a 24-h incubation period (data not shown).
Measurement of protein content. The protein content of cell lysates was measured by using the Bio-Rad DC protein assay kit according to the manufacturers directions. Samples were adjusted to provide aliquots of equal protein content before SDS-PAGE.
Statistical analysis. Values given represent the means ± SE of the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Students t-test for either paired or unpaired data as appropriate. Analysis of relative densitometric values for Western blots was performed by using AlphaEaseFC version 3.1.2 software. Densitometric values for protein bands were reported in arbitrary units above basal values. Values for Smad2(Ser465/467) were normalized to total Smad protein levels after membranes were stripped and reblotted.
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RESULTS |
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Incubation of muscle cells for 15 min with 500 ng/ml IGFBP-3 increased serine phosphorylation of TGF-RI levels by 110 ± 30% above basal levels (Fig. 2). In control experiments, incubation of muscle cells for 15 min with 1 nM TGF-
1 also increased TGF-
RI serine phosphorylation by 130 ± 20% above basal levels (Fig. 2).
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Smooth muscle cells were incubated for 15 min with increasing concentrations of IGFBP-3 (5500 ng/ml) or with 1 nM TGF-1 as a positive control. IGFBP-3 elicited concentration-dependent Smad2(Ser465/467) phosphorylation with 500 ng/ml, causing a 91 ± 5% increase above basal levels within 15 min (Fig. 3). In T47D breast cancer cells, the effects of IGFBP-3 on TGF-
receptor signaling were dependent on the presence of TGF-
1 (8). This possibility was examined by repeating the experiment in the presence of a neutralizing antibody to TGF-
1. We have previously used this antibody (50 ng/ml), shown it fully neutralizes endogenous TGF-
1 production in these cells, and shown that endogenous TGF-
1 exerts growth-inhibitory effects in human intestinal smooth muscle cells (2, 14). In the presence of the immunoneutralizing antibody to TGF-
1 (50 ng/ml), IGFBP-3 retained the ability to cause concentration-dependent Smad2(Ser465/467) phosphorylation (500 ng/ml: 73 ± 10% above basal) (Fig. 3). The results implied that IGFBP-3 does not require the presence of TGF-
1 to activate the TGF-
receptors and initiate intracellular signaling via Smad2 in human intestinal smooth muscle.
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The role of endogenous IGFBP-3 in regulating smooth muscle cell growth was also examined in the muscle cells. Our previous work (2, 14) has shown that endogenous IGF-I stimulates growth and that IGF-I-stimulated growth is inhibited by IGFBP-3. The direct effects of IGFBP-3 on growth were examined by immunoneutralization of endogenous IGFBP-3 and in the presence of the IGF-I receptor antagonist so that the modulatory role of IGFBP-3 on IGF-I-stimulated growth was eliminated. Muscle cells were incubated for 24 h in the presence of a neutralizing antibody to IGFBP-3 (125 µg/ml). Basal [3H]thymidine incorporation was increased in a concentration-dependent manner in the presence of increasing concentrations of IGFBP-3-neutralizing antibody (Fig. 4B). The results implied that endogenous IGFBP-3 directly inhibited muscle cell proliferation.
IGFBP-3-induced proliferation is Smad2-dependent. The role of Smad2 in IGFBP-3-dependent direct regulation of smooth muscle cell growth was examined in cells in which Smad2 was eliminated by using a siRNA approach (6, 36). Initial experiments were performed to confirm that transfection of Smad2 siRNA resulted in a decrease in Smad2 protein levels. Transfection of muscle cells with 2 µg of control RNA did not alter the levels of Smad2 protein from that present in naive, untransfected cells. In cells transfected with 2 µg of Smad2 siRNA, Smad2 protein was decreased by 69 ± 4% (Fig. 5).
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DISCUSSION |
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This paper shows that endogenous IGFBP-3 directly inhibits normal human intestinal smooth muscle cell proliferation. The effects of IGFBP-3 on proliferation are mediated by binding to TGF- receptors, activation of TGF-
RI receptors, and initiation of intracellular signaling via Smad2 phosphorylation. The evidence that IGFBP-3 directly inhibits growth via TGF-
RI-dependent, Smad2-dependent pathways can be summarized as follows: 1) IGFBP-3 binds to several TGF-
receptor types in a concentration-dependent fashion, 2) IGFBP-3 stimulates serine phosphorylation (activation) of TGF-
RI receptors, 3) IGFBP-3 stimulates concentration-dependent phosphorylation (activation) of Smad2 on Ser465/467, 4) IGFBP-3 causes concentration-dependent inhibition of [3H]thymidine incorporation, 5) the effect of IGFBP-3 on proliferation is abolished when Smad2 expression is diminished, and 6) immunoneutralization of endogenous IGFBP-3 increases basal [3H]thymidine incorporation implying that endogenous IGFBP-3 inhibits proliferation.
Two distinct mechanisms by which IGFBP-3 directly regulates cellular growth have been identified (1). The first involves the interaction of IGFBP-3 with TGF- receptors and TGF-
-dependent signaling mechanisms; the second involves the interaction of IGFBP-3 with nuclear RXR
(8, 23, 29). Via the first mechanism, described in mink lung epithelial cells, IGFBP-3 binds to the
400 kDa TGF-
RV (23, 24). This serine/threonine kinase is widely expressed in normal cells and is also expressed in some transformed cells. TGF-
1 is unable to inhibit growth in the human colorectal carcinoma cell lines HCT-116 and RII-37, which do not express TGF-
RV receptors but do express the lower molecular weight type I TGF-
(TGF-
RI) and type II TGF-
(TGF-
RII) receptors (24). Although IGFBP-3-dependent inhibition of growth coupled to the TGF-
RV receptor has been shown (24), a signaling mechanism activated by this receptor has yet to be identified. Recently, the bovine TGF-
RV receptor was shown to be identical to the human low-density lipoprotein receptor-related protein-1 involved in endocytosis (10) and may provide some insight into its mechanisms of action.
In the Hs578T, MCF-7, and T47D breast cancer cells lines, IGFBP-3 binds directly to TGF-RII receptors. In a fashion similar to TGF-
1, binding of IGFBP-3 to the constitutively active TGF-
RII activates the TGF-
RI receptor serine/threonine kinase (7, 8, 26, 27). Studies performed in T47D cells have been particularly useful in elucidating the distinct TGF-
receptor-dependent mechanisms activated by IGFBP-3. In naive T47D cells, which lack TGF-
RII receptors, IGFBP-3 has no direct effect on growth because, at least in these cells, expression of TGF-
RII and the presence of TGF-
1 peptide are required in order for IGFBP-3 to inhibit growth (8). Under the experimental conditions of TGF
-RII transfection and exogenous TGF-
1 addition, the ability of IGFBP-3 to activate TGF-
RI and Smad2 and inhibit growth of T47D cells is restored (8). Human intestinal smooth muscle cells, in contrast to T47D cells, express both type I and II TGF-
receptors and secrete TGF-
1. Immunoneutralization of endogenous TGF-
1, however, does not diminish IGFBP-3-dependent Smad2 activation or IGFBP-3-dependent inhibition of [3H]thymidine incorporation, implying that IGFBP-3 does not require TGF-
1 to directly inhibit proliferation in human intestinal smooth muscle cells.
One mechanism by which TGF-1 inhibits growth is through the R-Smad signaling pathway (5). Once activated by TGF-
1-dependent association with TGF-
RII, TGF-
RI phosphorylates its primary R-Smad substrates, Smad2 and/or Smad3 (36). Initial phosphorylation of Smad2 on Ser467 in the COOH terminus is followed by phosphorylation of Ser465 (34). Dual serine phosphorylation of Smad2 is required in order for it to associate with the next signaling protein in this pathway, Smad4. The Smad2/3-Smad4 complex translocates to the nucleus in which it acts as a transcriptional regulator of TGF-
- responsive elements. One such target of IGFBP-3-activated Smad signaling is the promoter region of the TGF-
-responsive gene plasminogen activator inhibitor-1 (PAI-1). PAI-1 is involved in maintaining IGFBP-3 homeostasis by blocking the activation of plasmin (7). Plasmin degrades IGFBP-3; this pathway in effect delays IGFBP-3 degradation and augments IGFBP-3-dependent responses. In human intestinal smooth muscle cells, blockade of the Smad2 pathway using Smad2 siRNA abolished the ability of IGFBP-3 to directly inhibit growth. The results implied that although IGFBP-3 binds TGF-
RV receptors, the type V receptor is not coupled to inhibition of proliferation in these cells. This is not to say that this pathway might not affect cell growth, because IGFBP-3 regulates apoptosis in a variety of cells (25, 29, 30).
A distinct mechanism that mediates IGF-I-independent inhibition of growth by IGFBP-3, which does not involve TGF- or other cell surface receptors, has also been elucidated. The COOH terminus of IGFBP-3 possesses a consensus nuclear translocation sequence. This region is distinct from the regions that activate TGF-
receptors and Smad (30). Direct nuclear translocation of IGFBP-3 via the
-importin pathway allows IGFBP-3 to bind to the nuclear RXR
, regulate transcriptional signaling, and directly inhibit growth in opossum kidney cells, A549 lung cancer cells, T47D breast cancer cells, and a number of other cells (7, 25, 29, 30). This mechanism has been shown to regulate not only cellular proliferation but the complementary aspect of growth as well as apoptosis. The ability of IGFBP-3 to regulate apoptosis and the mechanisms involved were not investigated directly in the present study. However, the ability of Smad2 siRNA to fully abolish the effects of IGFBP-3 on [3H]thymidine incorporation suggests that TGF-
RI receptor activation by IGFBP-3 accounts fully for its effects on cellular proliferation. The possibility that IGFBP-3 affects apoptosis via
-importin-dependent nuclear translocation and activation of RXR
was not examined in the present study.
The IGF-I-independent, direct effects of IGFBP-3 have potential clinical relevance in the setting of the intestinal inflammation of Crohns disease, muscle hyperplasia, and stricture formation. Circulating levels of IGF-I and IGFBP-3 are decreased in the setting of intestinal inflammation in Crohns disease (11). The muscularis propria, however, is known to be regulated by IGF-I and IGFBPs produced endogenously. Both IGF-I and TGF-1 expression within the intestinal smooth muscle layer, for example, are increased in regions of muscle inflammation and stricture formation (22, 42). Although evidence suggests that IGF-I overexpression in intestinal muscle does not alter IGFBP-3 expression (39), it is not known what effect intestinal inflammation or TGF-
1 might have on local smooth muscle IGFBP-3 levels. Preliminary studies suggest that IGFBP-3 mRNA levels, measured by real-time PCR, are not altered within intestinal smooth muscle in patients with Crohns disease (J. F. Kuemmerle and J. G. Bowers, unpublished results). We (2) have previously shown, however, that IGFBP-3 protein levels in human intestinal smooth muscle are regulated largely by posttranslational mechanisms rather than by gene transcription. Whereas IGFBP-3 levels are increased by TGF-
1 (2), it is not known whether IGFBP-3 influences TGF-
1 levels in return. The effect of altered IGFBP-3 levels in the inflamed intestinal muscle may be to alter the net growth-regulatory signals, both IGF-I-dependent growth and IGF-I independent growth, as well as both TGF-
1-dependent and possibly IGFBP-3-dependent (via the TGF-
receptor) effects.
In summary, this study shows that endogenous IGFBP-3 directly inhibits the proliferation of human intestinal smooth muscle cells. The mechanism mediating this effect is distinct from the ability of IGFBP-3 to inhibit IGF-I-stimulated growth. After binding of IGFBP-3 to TGF-RII receptors, type I TGF-
receptors are activated and intracellular signaling via Smad2 is initiated. This pathway is coupled to inhibition of proliferation. Although IGFBP-3-dependent inhibition of growth is mediated via TGF-
receptors, these effects are independent of endogenous TGF-
1.
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GRANTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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