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Endocrinology Vol. 138, No. 2 683-690
Copyright © 1997 by The Endocrine Society

Articles

Role of Insulin-Like Growth Factor Binding Protein-2 and Its Limited Proteolysis in Neuroblastoma Cell Proliferation: Modulation by Transforming Growth Factor-ß and Retinoic Acid1

Mouna Menouny, Michel Binoux and Sylvie Babajko

INSERM U.142, Hôpital Saint Antoine, 75571 Paris Cedex 12, France

Address all correspondence and requests for reprints to: Sylvie Babajko, INSERM U.142, Hôpital Saint Antoine, 184, rue du Faubourg St. Antoine, 75571 Paris Cedex 12, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate IGF action at cellular level through inhibition or, alternatively, potentiation, where their limited proteolysis is a contributory mechanism.

Under basal conditions, neuroblastoma cells secrete IGFs (essentially IGF-II), IGFBPs (IGFBP-4 and predominantly IGFBP-2 that is partially proteolysed), and proteases, including tissue-type plasminogen (PLG) activator, whose activity is inhibited by PLG activator inhibitor-1.

Neuroblastoma cells were used to investigate the influence of the plasmin system, transforming growth factor-ß and retinoic acid on cell growth and the IGF system. In cells treated with 5 µg/ml PLG, proliferation was stimulated, an effect that was inhibited in the presence of either {alpha}IR-3 (which blocks the type 1 IGF receptor) or anti-IGF-II antibodies. There was a parallel increase in IGFBP-2 proteolysis, which resulted in a 5-fold loss of affinity for IGF-II.

In the presence of 1 ng/ml transforming growth factor-ß, PLG-induced mitogenesis and IGFBP-2 proteolysis were reduced, and Northern blot analysis revealed increased PLG activator inhibitor-1 mRNA. Conversely, with 2 µM retinoic acid, the mitogenic effect of PLG, IGFBP-2 proteolysis, and tissue-type PLG activator mRNAs were increased.

Therefore, IGF-II mediates autocrine proliferation in neuroblastoma cells under the control of IGFBPs secreted by the cells, its bioavailability being enhanced as a result of plasmin-induced IGFBP-2 proteolysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors I and II (IGF-I and -II) play a role in growth regulation in many cell types, including the neuronal and glial cells of the nervous system (1, 2). They are known to participate in the control of proliferation and differentiation of neuroblastoma-derived cell lines via an autocrine mechanism involving the type 1 IGF receptor (3, 4, 5, 6). They also modulate the expression levels of the myc oncogene, which determine the proliferation/differentiation balance in most cells (7, 8).

In all biological fluids, IGFs are noncovalently bound to high-affinity binding proteins. Six molecular species of these IGFBPs have been identified to date (9). IGFBPs are capable of either potentiating or inhibiting the effects of the IGFs, although they also have intrinsic activities that are independent of their binding to IGFs that modulate cell proliferation (10, 11, 12, 13, 14, 15). For instance, IGFBP-3, whose expression is induced by the tumor suppressor gene, p53, seems to be involved in the arrest of proliferation in certain cell types (16).

Under basal conditions, neuroblastoma cells secrete essentially IGFBP-2, smaller amounts of IGFBP-4, and (depending on the cell line) traces of IGFBP-6 (17, 18). When such cells are treated with retinoic acid (RA), IGFBP-6 expression is strongly induced, whereas expression of IGFBP-2 and -4 is reduced and proliferation is arrested. This suggests that the ratio of IGFBP-2 and -4 to IGFBP-6 influences cell growth (18).

IGFBP-2 is produced in various regions of the central nervous system, including the choroid plexuses (19, 20). Together with IGFBP-6, it is the major IGFBP in cerebrospinal fluid (21), where immunoblot analysis reveals proteolysed forms of IGFBP-2 but not of IGFBP-6 (22). A serine protease for IGFBP-2 recently has been identified in porcine aortic smooth muscle cells (23). Most IGFBPs do, in fact, undergo limited proteolysis by a variety of extracellular proteases, a mechanism that now is recognized as essential in the regulation of IGF bioavailability (24). Among the proteases known, plasmin (PL) is one that is involved in the proteolysis of IGFBP-3 produced by osteoblast-like cells (25) and prostate carcinoma cells in culture (26). PL is generated by transformation of plasminogen (PLG) by the activators, tissue-type PLG activator (t-PA) and u-PA, whose action is modulated by two inhibitors, PLG activator inhibitor-1 (PAI-1) and PAI-2. PLG activation is one of the major mechanisms accounting for extracellular proteolysis during development of the nervous system (27), as also in the process of tumor cell invasion (28). Neuroblastoma cells are known to secrete t-PA, which seems to play a role in regulating their differentiation (8, 29).

The aim of this study was to examine the relationships between the PL system and the IGFBPs secreted by human neuroblastoma cell lines and the putative role of IGFBP proteolysis in neuroblastoma cell proliferation. We also have studied the effects of RA and transforming growth factor-ß (TGF-ß), factors known to have antagonistic effects on the expression of genes, both of the PL system (29, 30, 31) and of the IGF system (17, 18, 32), as well as cell growth and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The neuroblastoma cell line, SK-N-SH (33), was kindly provided by J. Bénard (Institut Gustave Roussy, Villejuif, France). Cells were grown in DMEM (GIBCO-BRL, Scotland, UK) supplemented with 10% heat-inactivated FCS in the presence of 100 IU/ml penicillin, 2 mM glutamine, 10 µg/ml gentamicin and 1 µg/ml amphotericin. The neuroblastoma cell line, SH-SY5Y, derived from the SK-N-SH line (34), was grown in RPMI 1640 (GIBCO-BRL) supplemented with 12% heat-inactivated FCS in the presence of 100 IU/ml penicillin and 2 mM glutamine. Cultures were maintained in a humidifed incubator at 37C, with 5% CO2 atmosphere. At the end of the exponential growth phase, cells were trypsinized using 0.05% trypsin-EDTA (DIFCO, Detroit, Michigan) and seeded either in 10-cm-diameter petri dishes at 2 x 106 cells/dish (30,000 cells/cm2) for analysis of proteins, mRNA, and PL activity or in 96-well plates at 15,000 cells/well (45,000 cells/cm2) for analysis of cell proliferation. After 24 h, the medium was discarded and culture continued in serum-free medium for an additional 24 h.

Thereafter, medium was renewed [time 0 (T) of the experiment] with medium containing 2 µM all-trans-RA (Sigma Chemical Company, St. Louis, MO), 1 ng/ml TGF-ß1 (Sigma), 5 µg/ml monoclonal antitype 1 IGF receptor antibody {alpha}IR-3 (Oncogene Sciences, Uniondale, NY), or 10 µg/ml monoclonal antirat-IGF-II antibody specific for rat and human IGF-II (Upstate Biochemical Corporation, Lake Placid, NY). Incubation under these conditions was continued for 8 h, after which the media were supplemented or not with either 5 µg/ml PLG, the previously determined optimal concentration for osteosarcoma cells (25) and neuroblastoma cells, or 0.075 mM Pefabloc (Pc)-SC [4-(2-aminoethyl)-benzene-sulfonyl fluoride] (Chromogenix, Mölndal, Sweden), a serine protease inhibitor previously shown to be innocuous to prostate carcinoma (26) and neuroblastoma cells at this concentration (treated cells cultured in the presence of serum proliferate at the same rate as control cells). Thereafter, culture was pursued for 15 h (T + 24 h) to 3 days (T + 72 h).

14C-thymidine uptake
For the cells cultured in the 96-well plates, 1 µCi 14C-thymidine was added for the final 15 h of culture. Cells then were rinsed five times with PBS and lysed using 100 µl 0.6 N NaOH per well for 3–4 h at 37 C. Liquid scintillation counting was used to determine the amount of radioactivity incorporated into DNA. For each time, results for treated cells were corrected for controls (untreated cells).

MTT test
The number of viable cells was estimated as previously described (35) using a colorimetric assay in which mitochondrial dehydrogenase reduction of tetrazolium salt (MTT), soluble in dimethyl sulfoxide, is detected. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide; thiazolyl blue]), obtained from Sigma, was added to a final concentration of 0.5 mg/ml for a 3-h incubation at 37 C, followed by addition of 100 µl dimethyl sulfoxide. The plates were shaken for 5 min and absorbance at 540 nm for each well measured using a J Bio S.A. 12–505 enzyme-linked immunosorbent assay reader. For each time, results for treated cells were corrected for controls (untreated cells).

Assay for t-PA activity
The assay was performed as previously described (25) using 60 µl conditioned culture medium without phenol red, in the presence of 240 µg S-2251 (a PL substrate that yields a yellow product when cleaved; Chromogenix) and 1.25 µg PLG in a final vol of 150 µl. Reaction product was measured as a function of time over 24 h at 37 C by OD readings at 410 nm.

Western ligand blotting
The conditioned media were desalted on Sephadex G25 columns, lyophilized, and analyzed by Western ligand blotting (36) as previously described (37). Briefly, 1.5 ml eq of each sample were submitted to 11% SDS-PAGE under nonreducing conditions. The secreted proteins were electrotransferred onto nitrocellulose membranes, which were then rinsed for 45 min in TBS (5 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 0.2% Tween, incubated for 48 h at 4 C with a mixture of 125I-IGF-I and 125I-IGF-II (200 000 cpm each) in TBS, 1 mg/ml gelatin, rinsed, and finally autoradiographed at -80 C.

Immunoblotting
Immunoblotting was carried out as previously described (37). Nitrocellulose membranes were prepared as for ligand blotting. After transfer, the membranes were saturated and incubated at 37 C for 1 h with either antihuman (anti-h) IGFBP-2 antibody (kindly provided by J. Schwander, Basel, Switzerland) at 1:2000 dilution or anti-h-IGFBP-6 antibody (kindly provided by Chiron Corporation, Emeryville, CA) at 1:400 dilution. The nitrocellulose membranes were rinsed, then incubated for 45 min with goat polyclonal antirabbit IgG antibody coupled to horse radish peroxidase (Sigma) at 1:10,000 dilution. Horse radish peroxidase oxidation of luminol (ECL Western blotting detection system, Amersham, Aylesbury, UK) gives chemiluminescence, from which the specific IGFBP-antibody complexes can be visualized.

All Western- and immunoblot data shown are representative of at least three separate experiments.

Isolation of RNA and Northern blotting
Total RNAs were extracted from frozen cells using the standard CsCl/guanidine isothiocyanate method (38).

Thirty micrograms of total RNA were loaded onto 1.2% agarose/2.2 M formaldehyde gels, submitted to electrophoresis, stained with ethidium bromide, transferred to Hybond-N nylon membranes (Amersham), and covalently bound to the nylon by baking of the membranes at 80 C for 2 h. After 4 h of prehybridization at 50 C in 5 x SSC, 50% formamide, 5 x Denhardt, 50 mM sodium phosphate, pH 6.5, and 250 µg/ml sonicated salmon sperm DNA, the blots were hybridized to 3 x 106 cpm/ml of 32P-labeled complementary DNA (cDNA) probe (either h-IGFBP-2, h-u-PA, h-t-PA, h-PAI-1, or h-PAI-2 cDNA) (Multiprime DNA labeling system, Amersham) for 24 h at 50 C in the same buffer plus 20% dextran sulphate.

Partial h-IGFBP-2 cDNA [nucleotides 148-1298, according to the sequence published by Binkert et al. (39), 1989] was obtained in our laboratory from a human neuroblastoma (SH-SY5Y) cDNA library (S. Hardouin, unpublished). The cDNA was inserted into the EcoRI site of pT7T3 18U (Pharmacia, Uppsala, Sweden).

The 1.5-kb u-PA, 2.3-kb t-PA, and 1.9-kb PAI-2 cDNAs were inserted into pEMBL8, pBR322, and PUC19 vectors, respectively (ATCC, Rockville, MD). The 1.2-kb PAI-1 cDNA was cloned into pGEM-3 (a gift from D. J. Loskutoff, La Jolla, CA) (40).

All Northern blot data shown are representative of at least three separate experiments.

Competitive binding studies
The methods used have been reported in detail elsewhere (24). Thirty milliliters of conditioned culture media were lyophilized and gel filtered in 1 M acetic acid on a column of Ultrogel AcA-54 (IBF, Villeneuve-la-Garenne, France) to separate IGFs and IGFBPs. The eluates containing the IGFBPs were lyophilized, desalted through a Sephadex G25 column (PD 10, Pharmacia) in 0.1 M sodium phosphate buffer, pH 7.5, 0.1% BSA, then incubated at increasing concentrations in the same buffer at 4 C with 125I-IGF-II (2,500 cpm) in a total vol of 0.4 ml/tube. IGF-II was provided by Ciba Geigy Ldt. (Basel, Switzerland) and iodinated in the laboratory using the chloramine T method. After 18–24 h, free and bound IGF were separated using albumin-coated charcoal. Bound, labeled IGF was determined after subtraction of the blank supernatant (tracer without IGFBP), which was 6–8% of the total counts.

The concentration of IGFBP yielding 20–25% binding of labeled IGF then was used in a competitive binding experiment using 125I-IGF-II (2,500 cpm/tube) and increasing concentrations of unlabeled IGF-II. Conditions of incubation and separation were the same as above. Equilibrium association constants were calculated from Scatchard analyses. The preparations of recombinant human IGF-II used were a gift from Ciba Geigy Ltd.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PL stimulates neuroblastoma cell proliferation
In SH-SY5Y cells submitted to 16, 40, or 64 h of treatment with 5 µg/ml PLG, cell number increased by 50% after 48 h, and 70% after 72 h, and 14C-thymidine incorporation into DNA increased by 100% after 48–72 h, as compared with controls (Fig. 1Go, A and B). The effect in SK-N-SH cells was similar, but weaker: a 20–30% increase in 14C-thymidine uptake and cell number (Fig. 1Go, C and D).



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  		Figure 1. Effect of PLG on human neuroblastoma cell proliferation. At the end of the exponential growth phase, SH-SY5Y (A and B) or SK-N-SH (C and D) cells were trypsinized and seeded in 96-well plates at 45,000 cells/cm2. After 24 h, the medium was discarded and culture continued in serum-free medium for an additional 24 h. Thereafter, medium was renewed (time 0 of the experiment) and 8 h later, PLG and/or Pc added. A and C, Viable cell number was determined at T + 24, T + 48, and T + 72 h using the MTT test as described in Materials and Methods. The actual number of control cells was around 15,000/well. B and D, 1 µCi 14C-thymidine was added 1 h, 25 h, and 49 h after addition of PLG and/or Pc. Radioactivity incorporated into DNA was counted 15 h later, i.e. at T + 24 h, T + 48 h, and T + 72 h. The number of counts incorporated in control conditions was around 500 cpm. Anti-IGF-II (10 µg/ml) or {alpha}IR-3 (5 µg/ml) antibodies were added 1 h before treatment with Pc or PLG. Results are expressed as percentage modulation (means (±SEM) of three separate experiments), corrected for values obtained for controls (untreated cells), which are represented by the zero line. Data were analyzed using the Student’s t test. *, P < 0.01; and **, P < 0.005 compared with PLG-stimulated condition.

 
The specificity of these results was confirmed by experiments using the serine protease inhibitor, Pc (see Materials and Methods). Treatment of SK-N-SH and SH-SY5Y cells with 0.075 M Pc resulted in 15–50% reduced 14C-thymidine uptake (not shown). In addition, preincubation with Pc totally blocked the effect of PLG on cell proliferation (Fig. 1Go). Cell number was below that of controls, indicating a basal proteolytic activity that was inhibited by Pc.

When cells were incubated for 1 h with either 5 µg/ml {alpha}IR-3 monoclonal antibody, which blocks the type 1 IGF receptor, or anti-IGF-II monoclonal antibody, then treated with 5 µg/ml PLG, there was a 60% depression of the influence of PLG (Fig. 1Go). Simultaneous treatment with the two antibodies failed to increase inhibition of the mitogenic effect of PLG (not shown). These results demonstrate that the effects of IGF-II are mediated solely by the type 1 IGF receptor and suggest that other growth factors are activated.

Because neither antibody has any effect on basal proliferation in these cells (18), the present findings are consistent with the IGF system being involved in the mitogenic effects of PLG.

The IGFBPs secreted into the culture media are proteolysed by PL
Western ligand- and immunoblot analyses showed that, under basal conditions, the neuroblastoma cells secreted essentially IGFBP-2, which migrated around 34 kDa as either a triplet (SH-SY5Y cells) or a doublet (SK-N-SH cells), together with smaller quantities of IGFBP-4, migrating around 24 kDa (Fig. 2Go). Apparent concentrations of both IGFBPs were markedly reduced when cells were cultured with PLG but were more or less maintained in the presence of Pc.



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  		Figure 2. Effects of PLG on IGFBP-2 in cultured neuroblastoma cells. Cells were cultured in serum-free medium for 24 h and, 8 h after removal of the medium, treated or not with 5 µg/ml PLG and/or 0.075 mM Pc, then harvested after 40 h of treatment. Western ligand blotting: 1.5 ml eq medium per slot was submitted to electrophoresis and the IGFBPs revealed by their binding to radiolabeled IGFs. Immunoblotting: the same conditions were used for analysis of IGFBP-2 using specific polyclonal antibody. Northern blotting: 30 µg total RNA were separated by electrophoresis and transferred to nitrocellulose. The mRNAs then were hybridized to 32P-labeled IGFBP-2 cDNA probe. Ethid- ium bromide staining of 18S and 28S ribosomal RNA controls confirmed integrity of bound RNAs and homogeneity of gel loading.

 
Immunoblotting, using a specific anti-IGFBP-2 antibody, revealed, in addition to the intact protein, a fragment around 20 kDa not detected by Western ligand blotting. When cells were treated with PLG for 40 h, the intensity of the band corresponding to the 20-kDa fragment was markedly increased, and in some cases, an additional band appeared around 16 kDa, whereas intact IGFBP-2 became difficult to detect. This indicated that the two fragments arose from limited proteolysis of IGFBP-2 (Fig. 2Go). Pc, by contrast, inhibited both IGFBP-2 proteolysis and the effect of PLG, and the 20-kDa fragment became difficult to detect, thus confirming that the band corresponded to a proteolytic fragment.

To check that IGFBP-2 was indeed a PL substrate, cell-conditioned medium was incubated in vitro at 37 C with 1 µg/ml PL. Immunoblot analysis of the reaction product showed that IGFBP-2 was completely proteolysed (Fig. 3AGo). The same experiments could not be performed for IGFBP-6 because neuroblastoma cells secrete very little or no IGFBP-6 under basal conditions. We did, however, examine culture media conditioned by SK-N-SH cells transfected with a plasmid vector, promoting strong expression of IGFBP-6. Here, immunoblotting revealed no additional bands suggestive of IGFBP-6 proteolysis (Fig. 3BGo).



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  		Figure 3. In vitro effects of PL on IGFBP-2 and IGFBP-6 secreted by neuroblastoma cells. A, SK-N-SH cells were cultured in serum-free medium for 24 h, after which the conditioned media were collected and then incubated at 37 C for 1–24 h with 5 µg/ml PLG or 5 µg/ml PL. B, The same procedure was used for SK-N-SH cells transfected with h-IGFBP-6 cDNA. Conditioned media were incubated for 24 h with 5 µg/ml PLG or 1–5 µg/ml PL. IGFBPs were analyzed by Western immunoblotting using specific polyclonal anti-h-IGFBP-2 and anti-h-IGFBP-6 antibodies.

 
Finally, we checked that PLG failed to affect the amounts of mRNA coding for IGFBP-2 (Fig. 2Go), IGFBP-4, or IGF-II (not shown), confirming that PLG acts extracellularly, increasing the limited proteolysis of IGFBPs.

Proteolysis of IGFBPs reduces their affinities for IGFs
The affinity for IGF-II of the IGFBP-2 secreted into the culture media was determined in competitive binding experiments using IGFBPs (primarily IGFBP-2) extracted from conditioned media. From Scatchard analysis of the data obtained, the affinity of intact IGFBPs in control media was 1.26 x 1010l/M, whereas that of proteolysed forms extracted from media conditioned in the presence of PLG was 0.26 x 1010l/M, representing a 4–5-fold reduction (Fig. 4Go).



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  		Figure 4. Competitive inhibition at equilibrium at 4 C by recombinant human IGF-II of 125I-IGF-II binding to IGFBPs extracted from the conditioned media of SK-N-SH neuroblastoma cells. Cells were cultured in the absence (A) or presence (B) of 5 µg/ml PLG (see Materials and Methods). The concentrations of the IGFBP preparations used were selected on the basis of approximately 20% binding of 125I-IGF-II in the absence of unlabeled IGF-II. These corresponded to 300 (A) and 600 (B) µl eq culture medium. The graphs are Scatchard plots of the data. Similar results were obtained in two different experiments.

 
Cell proliferation is modulated by RA and TGF-ß1
Cell growth was altered in cells treated for 48 h with factors known to influence neuroblastoma cell proliferation and/or differentiation. Cell number increased by approximately 20% in SH-SY5Y cells and by 5–10% in SK-N-SH cells when treated with 2 µM RA but decreased by approximately 25% in both cell lines when treated with 1 ng/ml TGF-ß1. In cells subsequently incubated with 5 µg/ml PLG, proliferation was depressed in the TGF-ß1-treated cells but tended to increase in those treated with RA (Fig. 5Go).



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  		Figure 5. Modulation by RA and TGF-ß1 of PL activity: effects on cell proliferation. The conditions used were the same as those described in Fig. 1Go. When medium was renewed (time 0 of the experiment), 1 ng/ml TGF-ß1 or 2 µM RA were added. PLG was added 8 h later. Cell number and radioactivity incorporated into DNA were then counted at T + 48 h. Results represent the means (± SEM) of three separate experiments. Data were analyzed using the Student’s t test. *, P < 0.01; and **, P < 0.005 compared with PLG-stimulated condition.

 
RA and TGF-ß1 modulate IGFBP-2 proteolysis
In cells submitted to 40 h of treatment with 5 µg/ml PLG (T + 48 h), t-PA activity and the amounts of PL generated in the culture media were increased by 50–100% (depending on the cell line and the experimental series), compared with control media, whereas both were depressed in the presence of Pc. t-PA activity as increased by about 30% by 2 µM RA and 1 ng/ml TGF-ß1 decreased it by the same amount (Fig. 6Go).



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  		Figure 6. Effects of TGF-ß1 and RA on t-PA activity. Cells were cultured in serum-free medium for 48 h with or without 1 ng/ml TGF-ß1, 2 µM RA, or 0.075 mM Pc and the t-PA assay carried out as described in Materials and Methods. Reaction product was measured as a function of time by OD readings at 410 nm. Results shown were obtained in two separate experiments, each comprising four different points.

 
Immunoblot analysis of secreted IGFBP-2 revealed no change in proteolysis in the presence of either RA or TGF-ß1 alone. In contrast, when cells incubated for 8 h with either RA or TGF-ß1 were subsequently treated with PLG for 40 h, PLG-induced IGFBP-2 proteolysis was suppressed by TGF-ß1 and enhanced by RA (Fig. 7Go).



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  		Figure 7. Modulation by RA and TGF-ß1 of PL activity: effects on limited proteolysis of IGFBP-2. Cells were cultured in serum-free medium for 24 h and, after removal of the medium, treated for 48 h either with 1 ng/ml TGF-ß1 or 2 µM RA alone or in association with 5 µg/ml PLG. A, Western ligand blotting: 1 ml eq medium per slot was submitted to electrophoresis and the IGFBPs revealed by their binding to radiolabeled IGFs. B, Immunoblotting: the same conditions were used for analysis of IGFBP-2 using specific polyclonal antibody.

 
t-PA and PAI-1 mRNAs were detected by Northern blotting in both cell lines, but u-PA and PAI-2 mRNAs were found in neither (Fig. 8Go). RA clearly increased the amounts of t-PA mRNA (11-fold ± 2 (SEM)), and TGF-ß1 strongly induced PAI-1 mRNA (87-fold ± 10 (SEM)) (Fig. 8Go).



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  		Figure 8. Northern blot analysis of IGFBP-2, t-PA, and PAI-1 mRNAs: effects of RA and TGF-ß1. Culture conditions were the same as those described in Fig. 7Go. Thirty micrograms of total RNA were separated by electrophoresis and transferred to nitrocellulose. Then the same mRNAs were successively hybridized to 32P-labeled IGFBP-2, t-PA, and PAI-1 cDNA probes. Ethidium bromide staining was used to visualize the 18S and 28S ribosomal RNA controls to confirm homogeneity of gel loading and RNA integrity.

 
Under the same experimental conditions, IGFBP-2 mRNA was marginally increased by TGF-ß1 (1.5-fold ± 0.2 (SEM)) and decreased by RA (1.5-fold ± 0.3 (SEM)).

The same results were obtained for T + 24 h and T + 72 h.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The PL system is known to play a role in regulating cell proliferation and migration during embryonal development and in tumoral invasion and metastasis (27, 28). It affects cell adhesion to the extracellular matrix and activation or release of growth factors (41, 42). Our findings confirm the implication of the PL system in proliferation in the case of neuroblastoma cells, which synthesize t-PA and PAI-1 (8): cell number increased in the presence of PLG and decreased in the presence of the serine protease inhibitor, Pc. They also demonstrate that the mitogenic effects of PL involve the IGFs, in this case IGF-II, which is the only form produced by these cells and whose effects on proliferation are well established (4, 5). PLG-induced mitogenesis was significantly depressed in cells cultured with either anti-IGF-II antibody and/or the {alpha}IR-3 antibody, which blocks the type 1 IGF receptor. A further indication from our observations is that IGF-II activity reflects its bioavailability, in large part, mediated by IGFBP-2, which is the major IGFBP produced by neuroblastoma cells and which has stronger affinity for IGF-II than the less abundant IGFBP-4. IGFBP-2 was found to be proteolysed by PL, resulting in a marked loss of affinity for IGF-II. Consequently, the bioavailability of IGF-II was enhanced in the presence of PLG, in view of the increased proportion of proteolysed IGFBP-2, and reduced in the presence of Pc, which inhibits the action of PL. Because t-PA binds to the cell surface (42) and t-PA activity was found to be low when tested in vitro, it can be concluded that IGFBP-2 proteolysis occurred essentially either on contact with the cells or in the pericellular environment. A similar mechanism has been described for IGFBP-3 secreted by the prostate adenocarcinoma cell line, PC-3 (26). However, this is the first report of IGFBP proteolysis (in this case, IGFBP-2) modulating cell growth in neuroblastoma cells.

Another aspect of our findings concerns the effects of RA and TGF-ß. RA is known to promote differentiation (43) but also has some mitogenic activity in neuroblastoma cells (18). TGF-ß is more commonly a growth inhibitor (44) and also is so in these cells. Our data indicate that the effects of these factors, at least partly, involve the IGFBPs. RA promoted PL activity in the cell environment by stimulating t-PA synthesis, whereas TGF-ß1 inhibited it via stimulation of PAI-1 synthesis. Such effects have been reported previously in other cell models (30, 31). At the same time, PLG-induced IGFBP-2 proteolysis was augmented by RA and diminished by TGF-ß1, indicating that changes in the PL system affect IGFBP-2 proteolysis. Furthermore, RA treatment decreased the amount of IGFBP-2 mRNA, whereas TGF-ß increased it. With RA, t-PA synthesis was stimulated, which in turn would promote IGFBP-2 proteolysis and increase IGF-II bioavailability. The effect would be enhanced by the fact that RA reduces the amount of intact IGFBP-2 available to sequester IGF-II and also stimulates IGF-II expression, the result being an enlarged pool of free IGF-II inducing proliferation (18). Conversely, in the presence of TGF-ß, which stimulates PAI-1 synthesis and IGFBP-2 expression, the amount of intact IGFBP-2 is increased. This means greater sequestration of IGF-II and reduced cell proliferation.

Our findings also point towards a secondary effect of RA. It is pertinent that IGFBP-6, whose affinity for IGF-II is the strongest among the IGFBPs (21) and whose expression is accompanied by an arrest of cell proliferation (18, Babajko et al., in press), seemed relatively insensitive to PL. Also, its synthesis is stimulated by RA (18). This secondary effect of RA on the expression of IGFBP-6, which is likely to sequester IGF-II, would then limit its primary mitogenic action mediated by IGF-II.

Our study therefore demonstrates that the interactions between RA and TGF-ß and the IGF system, including IGFBP proteolysis by PL, are important in the control of neuroblastoma cell proliferation.


   Acknowledgments
 
We are indebted to Brigitte de Gallé for her technical assistance.


   Footnotes
 
1 This work was supported by INSERM, the Association de la Recherche sur le Cancer, and the Ligue Nationale contre le Cancer. Back

Received July 5, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sara VR 1992 The role of insulin-like growth factors in the nervous system. In: Schoffield PN (ed) Insulin-Like Growth Factors. Structure and Biological Functions. Oxford University Press, pp 80–109
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