Division of Surgery, Department of Hospital Medicine, Bristol Royal Infirmary, Bristol, BS2 8HW, UK
* Author for correspondence (e-mail: claire.m.perks{at}bristol.ac.uk)
Accepted 6 August 2002
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Summary |
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Cell attachment to a general ECM gel was unaffected by IGFBP-1 and -6 but was significantly increased by IGFBP-4 and -5 and decreased by IGFBP-2 and -3. Similar results were obtained for attachment to laminin or collagen type IV. Attachment to fibronectin, however, was increased by IGFBP-3 and decreased by IGFBP-5. The actions of IGFBP-3 and -5 on cell attachment to ECM were lost in the presence of a soluble Arg-Gly-Asp (RGD)-containing fibronectin fragment. Thrombospondin reversed the actions of IGFBP-3 on cell attachment, but IGFBP-5 still increased cell attachment.
On plastic, neither IGFBP-3 nor -5 alone affected cell viability; although ceramide-induced apoptosis was enhanced by IGFBP-3 but reduced by IGFBP-5. The presence of RGD reversed the action of IGFBP-5 on cell death but attenuated that of IGFBP-3. With cells grown on fibronectin, the action of IGFBP-3 was reversed, and it conferred cell survival, whereas the survival effect of IGFBP-5 was lost.
In summary we have demonstrated that IGFBP-3 and -5 both have intrinsic effects on cell survival. In each case the presence of fibronectin or fibronectin fragments determines whether susceptibility to apoptosis is increased or decreased. These effects on cell survival are paralleled by acute effects on integrin receptor function; IGFBP-3 and -5 were able to either enhance or inhibit cell attachment in the presence of fibronectin. Cell survival is tightly controlled by cues from the ECM and from growth factors, particularly the IGFs. Our findings indicate that, in addition to being crucial modulators of IGF actions, the IGFBPs have direct actions on cell attachment and survival that are specific and dependent upon the matrix components present.
Key words: IGFBP, Cell adhesion, ECM, Breast epithelia, Apoptosis, Integrin, Fibronectin
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Introduction |
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The Hs578T breast cancer cell line produces negligible amounts of IGF-I and IGF-II, and it does not possess a functional IGF-I receptor. The addition of IGF-I to these cells does not induce cell growth or survival; thus, Hs578T cells are an ideal model in which to investigate the IGF-independent effects of the IGFBPs (Oh et al., 1993a; Gill et al., 1997
). We have demonstrated previously that the IGFBPs, although they have no effect alone on Hs578T cell death, each have differential effects on apoptosis induced by the two triggers ceramide (C2) and an Arg-Gly-Asp-containing fibronectin fragment (RGD). We showed that IGFBP-1, -2 and -6 had no effect, IGFBP-4 and -5 conferred survival on both C2- and RGD-induced apoptosis, and IGFBP-3 accentuated C2-induced apoptosis but had no effect on RGD-induced cell death (Gill et al., 1997
; Perks et al., 1999a
). We also demonstrated that IGFBP-3 similarly enhanced apoptosis induced by radiation (Hollowood et al., 2000a
; Williams et al., 2000
) and paclitaxel (Fowler et al., 2000
) in a variety of epithelial cell lines.
Cell adhesion to the surrounding extracellular matrix (ECM) is mediated by cell-surface integrin receptors. Integrins modulate cell proliferation and apoptosis, as well as mediating cell attachment, migration and spreading. These receptors are made up of an and a ß subunit, of which there are 18 known
subunits and eight ß subunits, producing at least 24 different receptors. Integrins have no intrinsic enzyme activity, but their activation can lead to the recruitment of signalling proteins to form focal adhesion complexes. It is now clear that there are at least 50 different proteins, including focal adhesion kinase (FAK) and paxillin, that associate at sites of focal adhesions and transduce signals that mediate changes in cell shape or gene expression (Cary and Guan, 1999
; Clark and Brugge, 1995
; Clezardin, 1998
; Zamir and Geiger, 2001
; Berditchevski, 2001
).
Particular integrin receptors recognise specific ECM components such as fibronectin and vitronectin. Many ECM proteins, such as fibronectin, contain an amino-acid sequence, RGD, that activates several integrin receptors and modulates cell attachment and motility.
Within their C-terminal domains, IGFBP-1 and IGFBP-2 each possess an RGD sequence (Jones and Clemmons, 1995). Jones et al. demonstrated that IGFBP-1, but not IGFBP-2, stimulated Chinese hamster ovary (CHO) cells to migrate on plastic in response to a wound. Furthermore, they demonstrated that this action of IGFBP-1 was mediated through its RGD sequence via the
5ß1 integrin receptor (Jones et al., 1993
). We also demonstrated that T47D and Hs578T breast cancer cells have
5ß1 integrin receptors and that the addition of IGFBP-1 to these cells promoted the dephosphorylation of FAK (Perks et al., 1999b
). In addition, Schutt et al. demonstrated that IGFBP-2 binds to the surface of Hs578T breast cancer cells and Ewing sarcoma (A673) cells via the
5ß1 integrin receptor (Schutt et al., 2000
). They also reported an associated decrease in FAK phosphorylation. We have further demonstrated that IGFBP-3 promotes the dephosphorylation of FAK, despite not possessing an RGD sequence (Perks and Holly, 1999c
). It was also reported that IGFBP-5 stimulates the migration of rat mesanglial cells in an IGF- and RGD-independent manner (Abrass et al., 1997
; Berfield et al., 2000
).
Having demonstrated that the IGF-binding proteins have differential effects on apoptosis, along with the accumulating evidence implying that the IGF-binding proteins can interact with either integrin receptors or modulate their signalling, we examined whether the IGF-binding proteins have differential effects on one of the major functions of integrin receptors, that of cell adhesion. Cell adhesion assays have demonstrated the modulation of cell attachment using a variety of peptides and ECM-coated wells (Yeh et al., 1998; Ilic et al., 1998
). They have also been useful for determining the compliment of integrin receptors expressed on different cell types (Meyer et al., 1998
) and demonstrating the effects of IGF-I receptor signalling on cell attachment (Reiss et al., 2001
).
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Materials and Methods |
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Cell culture
Human breast cancer cells (Hs578T) were purchased from ECACC (Porton Down, Wiltshire, UK) and grown in humidified 5% carbon dioxide atmosphere at 37°C. The cells were cultured in Dulbecco's modified Eagles medium (DMEM) with glutamax-1 (L-Alanyl-L-Glutamine) (Gibco, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS), penicillin (500I U/ml), streptomycin (5 mg/ml) and L-glutamine (2 nM) growth media (GM). Experiments were performed in phenol-red-and serum-free DMEM and Hams Nutrient Mix F12 (Gibco) with sodium bicarbonate (0.12%), bovine serum albumin (BSA) (0.2 mg/ml), transferrin (0.1 mg/ml) added (SFM).
Adhesion assay protocol
Cell adhesion assays were undertaken using a protocol modified from one described previously (Yeh et al., 1998; Meyer et al., 1998
). Hs578T cells were grown to confluency in T75 flasks in GM and switched to SFM 24 hours prior to dosing. Twenty-four well plates were coated in 500 µl of either ECM solution (30 µg/ml; additive free DMEM), laminin (5 µg/ml; PBS), collagen type IV (0.25 µg/ml; 0.25% acetic acid) or fibronectin (0.25 µg/ml; PBS) for 1 hour at 37°C. Wells were then washed in phosphate-buffered saline (PBS) before non-specific binding was blocked with 500 µl of PBS containing 0.1% BSA for at least 2 hours at 37°C. Meanwhile, cells were trypsinised and collected in SFM. Pellets were resuspended in 1 ml of SFM, and 50 µl of the cell solution was counted to determine cell number. Cells were further diluted, using SFM to 0.3x106 cells/1.5 ml, to which the binding proteins and other treatments as described in results were added. The cells were placed on a shaker and incubated for 1 hour at room temperature. Wells were washed twice with PBS before control and pre-treated cells were applied at 0.1x106 cells/well and incubated at 37°C for 30 minutes. After 30 minutes, the percentage of cell attachment to ECM for controls was between 40% and 60%; this level of attachment allowed for either an increase or decrease in cell attachment by the various treatments to be observed. In these experiments, increasing the amount of ECM coated on to the plates or the length of time that cells were exposed to the plate resulted in increasing attachment up to a maximum approaching 100%. Assessment of changes in attachment is difficult when the maximal attachment is achieved and therefore conditions were selected in order to facilitate detection of increases or decreases in attachment induced by the various treatments. Unattached cells were collected, and the wells were washed with PBS. Cell pellets were collected and resuspended in 100 µl PBS. Adherent cells were trypsinised and collected. Cell pellets were again resuspended in 100 µl PBS. Fifty microlitres of each solution was counted following trypan blue cell staining, from which the percentage of cells attached was determined. IGFBP-1 and -2 dose responses (0-800 ng/ml) were performed because we had previously shown that IGFBP-1 has additional biological effects at higher doses, including dephosphorylation of FAK. In contrast to the others, these two IGFBPs possess RGD sequences enabling them to act as direct classic ligands for integrin receptors.
Apoptosis assay protocol
We showed previously that the amount of apoptosis present in any given sample, as quantified by flow cytometry, is directly comparable to the amount of cell death measured by trypan blue cell counts in this model of cell death (Perks et al., 1999a). Cells were prepared for the assay in one of two ways. (1) Cells were seeded in six-well plates and grown in GM for 24 hours prior to switching to SFM for a further 24 hours. Cells were pre-incubated with either IGFBP-3 (100 ng/ml) or IGFBP-5 (100 ng/ml) with or without RGD (10 µg/ml) for 24 hours followed by co-incubation of the binding proteins with or without RGD, with an apoptotic dose of C2 ceramide. (2) Cells were seeded in six-well plates coated with or without fibronectin (0.25 µg/ml; PBS) and grown in GM for 24 hours prior to switching to SFM for a further 24 hours. Cells were pre-incubated with either IGFBP-3 (100 ng/ml) or IGFBP-5 (100 ng/ml) for 24 hours followed by a co-incubation of the IGFBPs with an apoptotic dose of C2 ceramide. A 10 µg/ml dose of RGD was used, as it had been shown previously that it did not induce apoptosis (Perks et al., 1999b
), whereas the dose of ceramide used was between 10-15 µM in order to achieve 40-60% cell death. The optimal dose both of IGFBP-3 and -5 to produce an effect on cell death was determined previously to be 100 ng/ml (Gill et al., 1997
; Perks et al., 1999a
).
Trypan blue dye exclusion
Aliquots of cells were loaded on to a haemocytometer (1:1) with trypan blue. Viable cells exclude the dye. Both living and dead cells were counted, and this information was used to calculate the percentage of dead cells or the percentage of cells attached relative to the control.
Flow cytometry
This technique was used to determine the amount of apoptosis in any given sample. The fragmented DNA of an apoptotic cell has less capacity to stain than that in normal cells and appears as a pre-G1 peak on a DNA cell cycle histogram. Cells (1-2x106) were washed in PBS and fixed in 70% ethanol for a minimum of 30 minutes prior to analysis. The fixed cells were pelleted (3873 g, 5 minutes) and washed with PBS. The cells were resuspended in 500 µl of reaction buffer (propidium iodide, 0.05 mg/ml; sodium citrate, 0.1%; RNase A, 0.02 mg/ml; NP-40, 0.3%, pH 8.3) and then incubated for 30 minutes at 4°C prior to measurement using a FACS Calibur Flow Cytometer (Becton Dickinson, Plymouth, UK) with an argon laser at 488nm for excitation. Analysis was performed using a Cell Quest software package (Becton Dickinson, Plymouth, UK).
Statistical analysis
The data were analysed using Statview version 5 software package. Data analysis was performed using ANOVA, and statistically significant differences were considered to be present at P<0.05. The ANOVA post hoc test performed was Bonferroni/Dunn.
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Results |
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Only two of the IGFBPs, IGFBP-1 and -2, possess the classic integrin recognition sequence, RGD. At concentrations of 100 ng/ml and above, IGFBP-2 significantly decreased cell attachment in a dose-dependent manner, with a maximum decrease from control levels of 55.9% to 35.6% achieved using 500 ng/ml (Fig. 1B). At a concentration of 100 ng/ml, IGFBP-1 had no effect on cell attachment (Fig. 1F). When the concentration was increased to 800 ng/ml, IGFBP-1 still did not affect cell attachment (data not shown). At a concentration of 100 ng/ml, IGFBP-3 significantly decreased cell attachment from basal levels of 45% to 33.8% (Fig. 1C). By contrast, increasing doses of IGFBP-5 (0-100 ng/ml) significantly increased cell adhesion, with a maximal increase from control levels of 63.8% to 81.4% achieved using 100 ng/ml (Fig. 1D). IGFBP-4 (100 ng/ml) (Fig. 1E) also significantly increased cell adhesion from basal levels of 62.8% to 69.8%, whereas IGFBP-6 (100 ng/ml) had no effect (Fig. 1F). In all subsequent experiments the binding proteins were used at a concentration of 100 ng/ml.
The effects of the IGFBPs on the adhesion of Hs578T cells to laminin, collagen type IV and fibronectin
The major component of the general ECM gel used in previous experiments was laminin; lower concentrations of collagen type IV, proteoglycan, heparin sulphate and entactin were also present (Sigma datasheet). The results of cell adhesion to laminin indicated that IGFBP-1 and -6 each had no effect (Fig. 2A,C), whereas IGFBP-2 and -3 each significantly decreased cell attachment from 70.9% and 63.8% to 64.4% and 56.8%, respectively (Fig. 2A,B). By contrast, IGFBP-4 and -5 significantly increased cell adhesion from basal levels of 63.8% and 70.9% to 73.8% and 82.1%, respectively (Fig. 2B,C).
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The results of cell adhesion to collagen type IV indicated that IGFBP-1 and -2 each had no effect (Fig. 2D), whereas IGFBP-3 significantly decreased cell adhesion from basal levels of 68.9% to 63.7% (Fig. 2E). Meanwhile both IGFBP-4 and -5 each significantly increased cell attachment from control levels of 68.9% and 71.2%, respectively, to 74.1% and 81.0% (Fig. 2E,F). On this substratum, IGFBP-6 significantly decreased cell adhesion of Hs578T cells to 65.3% from basal levels of 71.2% (Fig. 2F).
We also investigated the effects of the binding proteins on cell adhesion to fibronectin, as there have been reports that IGFBP-3 can bind to fibronectin (Gui and Murphy, 2001). In these experiments the control RGD peptide reduced adhesion to fibronectin more than that to laminin or collagen. This was expected, as the RGD peptide corresponded to a fibronectin sequence and therefore competed better with fibronectin. As shown in Fig. 2G, both RGD and IGFBP-2 significantly decreased cell attachment from control levels of 57.0% to 44.2% and 46.7%, respectively, whereas IGFBP-1 had no effect. IGFBP-3 and -4 each significantly increased cell adhesion from basal levels of 64.7% to 72.1% and 74.7%, respectively (Fig. 2H), whereas IGFBP-5 and -6 each significantly decreased cell adhesion from control levels of 57.0% to 48.8% and 44.2%, respectively (Fig. 2I).
The effects of IGF-I on the actions of IGFBP-3 and -5 on the adhesion of Hs578T cells to ECM
Consistent with these cells being unresponsive to IGFs, IGF-I alone (50 ng/ml) did not affect cell adhesion. However, the presence of IGF-I blocked the ability of IGFBP-3 to inhibit cell adhesion. The level of cell attachment returned to control levels (Fig. 3A).
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The addition of IGF-I to IGFBP-5 reversed its action on cell adhesion, resulting in a decrease in cell adhesion to below basal levels (Fig. 3B).
The effects of an RGD-containing fibronectin fragment or soluble thrombospondin on the actions of IGFBP-3 and -5 on the attachment of Hs578T cells to ECM
The effects of RGD and IGFBP-3 on cell adhesion were not additive, so that in the presence of an RGD-containing fibronectin fragment, IGFBP-3 had no further effect (Fig. 4A). Similarly, in the presence of RGD, IGFBP-5 was no longer able to enhance attachment (Fig. 4B).
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The effects of soluble TSP on the actions of IGFBP-3 and -5 on cell attachment to ECM gel are shown in Fig. 4C,D. TSP alone, at the dose used (0.01 µg/ml), had no effect on cell attachment. Although in the presence of TSP, IGFBP-3 no longer decreased cell adhesion but cell attachment was increased above control levels (Fig. 4C). The ability of IGFBP-5 to increase cell attachment in the presence of TSP was unaffected (Fig. 4D).
The effects of an RGD-containing fibronectin fragment on the actions of IGFBP-3 and -5 on C2-induced apoptosis
Consistent with its well established ability to induce apoptosis, it is apparent in Fig. 5A that C2 significantly increased cell death from basal levels of 15.4% to 26.9%. IGFBP-3 alone had no effect on basal cell death but markedly increased cell death induced by C2 alone from 26.9% to 35%. Either alone or in the presence of IGFBP-3 or C2, RGD (10 µg/ml) had no effect on apoptosis. However, in the presence of RGD, IGFBP-3 was unable to accentuate C2-induced cell death. The accentuation of cell death by IGFBP-3 with this particular experiment did not reach statistical significance, although the P value (P=0.003) was approaching the significance level (P<0.0018) as stipulated by the Bonferroni/Dunn test. The data obtained using flow cytometry, where the amount of apoptosis was determined as a percentage of cells in pre-G1, did, however, show that the accentuation of C2-induced apoptosis by IGFBP-3 was significant. The ceramide analogue significantly increased cell death to 19.4% compared with control levels of 6.3%. IGFBP-3 had no effect on basal levels, but upon co-incubation with C2, it significantly increased cell death to 25.6% compared with C2 alone. The presence of RGD negated the ability of IGFBP-3 to accentuate C2-induced apoptosis and the levels of apoptosis induced were significantly reduced to 20.1% compared with C2 and IGFBP-3 together.
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Co-incubation with IGFBP-5 reduced cell death induced by C2 alone from 31% to 22% (Fig. 5B). Either alone or in the presence of either IGFBP-5 or C2, RGD, 10 µg/ml, had no effect on cell death compared with the control. In the presence of RGD, IGFBP-5 could no longer confer survival against C2-induced apoptosis. The data obtained from flow cytometry, where apoptosis was determined as the percentage of cells in pre-G1, also showed that IGFBP-5 negated C2-induced apoptosis. The ceramide analogue significantly increased apoptosis to 25.0% compared with control levels of 9.9%. IGFBP-5 had no effect alone, although it significantly negated C2-induced apoptosis to 18.4%. On co-incubation with RGD, IGFBP-5 was no longer able to confer survival on C2-induced apoptosis, and the levels of apoptosis were significantly increased to 29.7% compared to 18.4% with IGFBP-5 and C2 together.
The effects of growing Hs578T cells on fibronectin and the ability of IGFBP-5 and -3 to modulate C2-induced death
We determined that the actions of IGFBP-3 and -5 on cell adhesion to fibronectin were opposite to those observed on the ECM gel. Furthermore, we determined that a sub-apoptotic dose of RGD altered the actions of IGFBP-3 and -5 on C2-induced cell death. We therefore investigated the effects of IGFBP-3 and -5 on C2-induced apoptosis when cells were plated on fibronectin. On cells grown on plastic, ceramide significantly increased cell death from 10.6% to 31.2% (Fig. 6A), whereas upon co-incubation with IGFBP-5 there was a significant decrease in cell death to 20.5% compared with C2 alone. When cells were grown on fibronectin, ceramide induced a comparable increase in cell death. Interestingly, IGFBP-5 alone significantly increased cell death but was unable to confer survival on C2-induced apoptosis, in contrast to its effects on plastic.
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Consistent with our previous findings, IGFBP-3 significantly accentuated C2-induced cell death when the cells were grown on plastic (Fig. 6B), but on fibronectin, IGFBP-3 acted in the opposite manner and significantly decreased C2-induced cell death.
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Discussion |
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Cells attach to the ECM through different integrins. The ECM is made up of proteins such as laminin, collagen and fibronectin. Different tissues express various specific combinations of the individual ECM proteins. Of the 20 possible integrin combinations there are only 15 or so receptors that have been described in situ. The integrin receptors themselves have been shown to have affinity for multiple ECM proteins. For example the `vitronectin receptor', vß1, has affinities for vitronectin, fibronectin, TSP and osteopontin. Furthermore the affinities that the integrins display for different ECM proteins can be modulated. Similarly, the ECM proteins can also bind to different integrin receptors, for example fibronectin binds to the
3ß1,
4ß1,
8ß1 and the
vß1 integrins (Clark and Brugge, 1995
; Clezardin, 1998
; Boudeau and Jones, 1999
). Upon examining cell attachment to the main components present in the ECM, laminin and collagen type IV, we found that the actions of the binding proteins were similar to that on ECM gel, with a few exceptions (summarised in Table 1). In contrast to its effects on cell attachment to laminin, ECM and fibronectin, IGFBP-2 did not have any effect on cell attachment to collagen type IV. Although IGFBP-6 was unable to affect cell adhesion to ECM and laminin, it actually decreased cell attachment to collagen and fibronectin. Most notably the actions of IGFBP-3 and -5 on cell attachment to fibronectin were the reverse of those observed on the general ECM gel, laminin or collagen type IV; IGFBP-3 promoted attachment to fibronectin as opposed to inhibiting it, whereas IGFBP-5 decreased adhesion as opposed to promoting it. These data suggest that the binding proteins may interact with common components (such as FAK) that interact with a number of integrin receptors and differentially alter their activation states and thus their affinity for specific ECM proteins. While IGFBP-3 may activate integrin receptors that can bind to fibronectin, it can also reduce activation of the integrins that bind to laminin and collagen; IGFBP-5 may have opposite effects. It is now clear that there are at least 50 different molecules that can accumulate at sites of focal adhesions (Zamir and Geiger, 2001
; Berditchevski, 2001
). We demonstrated previously that one of the key mediators of integrin signalling, FAK, is dephosphorylated in the presence of IGFBP-3 (Perks and Holly, 1999c
). Furthermore the serine/theronine kinases protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) may also be involved in IGFBP-5 and -3 signalling, respectively. Protein kinase C was shown to be involved in the trafficking of the ß1 subunit, activation of the integrins and cell spreading (Ng et al., 1999
; Disatnik and Rando, 1999
; Fahraeus and Lane, 1999
), whereas MAPK to alters integrin activation and affinities (Cary and Guan, 1999
; Hansen and Bissell, 2000
). We have shown that the inhibition of PKC blocked the ability of IGFBP-5 to promote cell adhesion (McCaig et al., 2002
), whereas inhibition of MAPK negated IGFBP-3 actions on cell adhesion (McCaig et al., 2001
). More work is clearly required to ascertain the exact molecules and mechanisms involved in IGFBPs alteration of integrin receptor affinity. The in vitro effect of IGFBPs on cell attachment that we observed were very reproducible but relatively modest; the physiological significance of these results remains to been determined. These studies clearly establish, however, that the IGFBPs have very rapid actions at the cell surface, affecting integrin functions much earlier than the biological effects on cell proliferation and survival that have been defined to date.
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The RGD-containing fibronectin fragment and TSP also modulated IGFBP actions. In the presence of RGD, the effects of IGFBP-3 on cell adhesion and C2-induced cell death were blocked. The effect of IGFBP-5 on cell adhesion was also blocked but in contrast IGFBP-5 accentuated rather than reduced C2-induced apoptosis. TSP can interact directly or indirectly with several integrin receptors. In the presence of TSP, the IGFBP-3 actions on cell attachment to the ECM were reversed, whereas IGFBP-5 still increased cell attachment. These data suggest that while the binding proteins can modulate cell adhesion through direct or indirect interactions with integrins, these actions can be further altered in the presence of other ligands for these receptors. The IGFBPs have been reported to bind with high affinity to a number of proteins that may be involved in integrin interactions at the cell surface including fibronectin (Gui and Murphy, 2001), TSP, osteopontin (Nam et al., 2000
), PAI-1 (Nam et al., 1997
) and ADAMs-12 (Loechel et al., 2000
; Shi et al., 2000
). It is unlikely that the actions that we have described are due to the direct interactions of the IGFBPs with fibronectin or other matrix components because in the attachment assay the cells were exposed to IGFBPs in suspension following detachment and removal from their ECM before examining acute attachment to fresh ECM-coated surfaces. It is feasible that the IGFBPs could have adhered to the cell surface and then affected subsequent interactions with the ECM. These acute actions in cell attachment always paralleled the effects on cell survival observed over a longer time frame. It has been shown that there are specific cell-surface binding sites for both IGFBP-3 (Oh et al., 1993b
) and -5 (Andress, 1998
), and although intracellular signalling pathways have not been defined, it remains feasible that these putative receptors interact with integrin receptors.
In addition to their basic function in mediating cell attachment, the integrin receptors have a well characterised role in maintaining cell survival (Boudeau and Jones, 1999; Ruoslahti and Reed, 1994
). Having established that IGFBP-3 and IGFBP-5 affect the attachment function of integrin receptors, it is very feasible that the actions of these binding proteins on cell survival were also effected by modulating the survival signals transduced via integrin receptors. The effects of IGFBP-3 and -5 on cell attachment to fibronectin were the reverse of those to ECM. Similarly, whereas IGFBP-3 accentuated C2-induced apoptosis of cells grown on plastic, it acted as a survival factor and reduced apoptosis when the cells were grown on fibronectin. By contrast, the survival effect of IGFBP-5, which reduced C2-induced apoptosis of cells grown on plastic, was completely lost when the same cells were grown on fibronectin.
The role of integrin receptors in the actions of IGFBPs is also supported by the observations that other ligands, which activate or inactivate integrin receptors, can dramatically alter the effect of these binding proteins not only on cell attachment but also on cell survival. In addition to the six high-affinity IGFBPs, there are a larger group of proteins that share more limited homology but clearly form more distant relatives of a superfamily (Hwa et al., 1999). Within this superfamily are another group of proteins referred to as CCN proteins, and it is of interest that classic cell receptors have yet to be identified for these proteins although many of their actions on cells have been linked to effects on integrin receptors (Perbal, 2001
) despite the CCN proteins also not containing classic RGD integrin recognition sequences.
These actions of the IGFBPs are clearly not dependent upon their interactions with IGFs as the Hs578T cells do not respond to stimulation by IGFs. We have, however, demonstrated previously that IGF-I negated the ability of IGFBP-3 to accentuate C2-induced apoptosis by inhibiting IGFBP-3 binding to the cell surface in this cell line (Maile et al., 1999). In the presence of IGF-I, IGFBP-3 actions on cell attachment were also negated. Interestingly in the presence of IGF-I, IGFBP-5 did not return to control levels as observed with IGFBP-3, but IGFBP-5 decreased cell attachment relative to the control. It has been well documented that IGF-I actions on cells can be modulated by the IGFBPs and can vary according to conditions. IGFBPs can inhibit the binding of IGF-I to its receptor and also potentiates IGF-I actions when bound to the cell surface and ECM (Jones and Clemmons, 1995
). Our data suggest that, similarly, in the presence of IGF-I, the IGF-independent actions of the binding proteins may also be modulated. Cell survival is tightly controlled by cues from the ECM and from soluble survival factors, with the IGFs being the most potent survival factors for many cell types. Our new findings indicate that, in addition to being crucial modulators of such IGF actions, the IGFBPs also have direct ECM-dependent actions on cell attachment and survival. The IGFBPs may therefore have important effects on both the soluble and the ECM-mediated signals that govern cell survival.
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Acknowledgments |
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References |
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