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Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways

RICCARDA GRANATA, LETIZIA TROVATO, GIOVANNI GARBARINO, MARINA TALIANO, RENATA PONTI^*, GIUSY SALA, RICCARDO GHIDONI and EZIO GHIGO¹

Division of Endocrinology and Metabolism, Department of Internal Medicine,
^* Section of Clinics and Oncological Dermatology, University of Turin, Turin, Italy; and
Laboratory of Biochemistry and Molecular Biology, San Paolo University Hospital, University of Milan, Milan, Italy

¹ Correspondence: Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, C.so Dogliotti 14-10126 Turin, Italy. E-Mail: ezio.ghigo{at}unito.it

SPECIFIC AIMS

Our aims were to 1) establish the role of IGFBP-3 on primary human umbilical vein endothelial cell (HUVEC) apoptosis and survival; 2) determine the role of sphingolipid signaling in mediating IGFBP-3 effects, providing mechanistic insight into IGFBP-3 function in HUVEC; 3) assess IGFBP-3 induction of IGF-I expression and IGF-IR signaling; and 4) evaluate IGFBP-3 influence on endothelial cell (EC) motility.

PRINCIPAL FINDINGS

1. IGFBP-3 enhances the apoptotic effect of doxorubicin and reduces apoptosis induced by serum starvation
To investigate the effect of IGFBP-3 on apoptosis induced by doxorubicin and serum starvation, HUVEC were preincubated for 1 h with 1000 ng/mL IGFBP-3 in serum-free medium and cultured with or without 0.5 µM doxorubicin for 24 h. In serum-deprived HUVEC, cell viability as measured by MTT was 2.7 fold decreased compared with cells grown in the presence of serum. Under serum deprivation, IGFBP-3 significantly increased cell viability. HUVEC survival was reduced by doxorubicin and IGFBP-3 significantly enhanced doxorubicin-induced cell death. IGFBP-3 significantly reduced the number of apoptotic cells in serum-free conditions (evaluated by FACS analysis and Hoechst 33258) and increased apoptosis after addition of doxorubicin. These results indicate that IGFBP-3 induces dual effects on apoptosis triggered by different stimuli.

2. IGFBP-3 potentiates the fumonisin B1 reversal of doxorubicin-induced cell death and apoptosis
Based on studies showing that doxorubicin induces apoptosis through de novo ceramide synthesis, we investigated whether IGFBP-3 may be involved in this pathway. IGFBP-3 effects on apoptosis induced by serum deprivation and doxorubicin were studied in the presence of fumonisin B1, a specific inhibitor of ceramide synthase. HUVEC were preincubated with IGFBP-3 (1000 ng/mL) for 1 h before addition of fumonisin B1 (100 µM) for 40 min and doxorubicin (0.5 µM). Viability and apoptosis were evaluated after 24 h. In serum-free conditions, fumonisin B1 had no significant effect on cell viability and apoptosis whereas IGFBP-3 increased cell survival and decreased apoptosis not only alone, but also in the presence of fumonisin B1. In doxorubicin-treated cells, fumonisin B1 significantly reduced cell death and apoptosis. IGFBP-3 with fumonisin B1 further reduced cell death and apoptosis but exerted a proapoptotic effect in the absence of fumonisin B1. These results show that fumonisin B1 acts as an antagonist of apoptosis induced by doxorubicin, likely inhibiting the production of ceramide. IGFBP-3 increases apoptosis in cells treated with doxorubicin but displays an opposite, antiapoptotic effect in the presence of fumonisin B1.

3. IGFBP-3 reduces ceramide in serum-starved conditions and increases doxorubicin-induced ceramide levels
Next we aimed to determine the effect of IGFBP-3 on ceramide levels. Cells were treated for 24 h in serum-free medium in the presence or absence of IGFBP-3 (1000 ng/mL), doxorubicin (0.5 µM), and fumonisin B1 (100 µM); ceramide was quantified using the diacylglycerol kinase method. IGFBP-3 attenuated ceramide in serum-free medium and further increased doxorubicin-induced ceramide levels. Fumonisin B1 decreasedthe production of ceramide in cells with or without doxorubicin; in the presence of fumonisin B1, IGFBP-3 further reduced ceramide levels. The results agree with experiments on apoptosis performed under the same conditions and suggest that IGFBP-3 may influence ceramide levels in ECs.

4. IGFBP-3 stimulates sphingosine kinase (SK) activity and induces SK1 mRNA
Sphingosine-1-phosphate (S1P), the metabolite of ceramide, has been implicated in cellular proliferation and survival; thus, we sought to determine IGFBP-3 effect on SK activation. IGFBP-3 caused a rapid increase in SK activity in HUVEC (Fig. 1 A), peaking at 30 min (230% compared with basal). SK1 mRNA, the SK isoform that stimulates cell growth and survival, was also analyzed. We found that IGFBP-3 significantly increased SK1 mRNA in a time-dependent manner, reaching 450% of control at 180 min (Fig. 1B ). The competitive inhibitor of SK, N,N-dimethylsphingosine (DMS) (0.5 µM), completely blocked the IGFBP-3 antiapoptotic effect in serum-deprived HUVEC after 24 h (Fig. 1C ), suggesting that IGFBP-3 reduces ceramide levels and exerts its protective effect by increasing S1P levels.

View larger version (17K): [in this window] [in a new window]   Figure 1. SK activation by IGFBP-3 and reversion of IGFBP-3 antiapoptotic effect by DMS. A) SK activity measured in HUVEC treated with IGFBP-3 (1000 ng/mL). S1P was visualized by autoradiography (upper panel). Results are the mean ± SE of 3 independent experiments (*P<0.05, **P<0.001). B) HUVEC were treated as for the SK activity assay, SK1 mRNA was determined by RT-PCR. Values from densitometric analysis were normalized to ß-actin and reported as % of the basal time point. Data are the mean ± SE from 3 separate experiments (*P<0.001 for all time points vs. the basal time point). C) Inhibition of the antiapoptotic effect of IGFBP-3 by DMS (0.5 µM). HUVEC were incubated 40 min in the presence of DMS before addition of IGFBP-3. Apoptosis was quantified after 24 h by Hoechst 33258; values are the mean ± SE of 500 cells counted in duplicate from 3 independent experiments (*P<0.05; **P<0.01).

5. IGFBP-3 up-regulates IGF-I expression
IGFBP-3 is known to modulate IGF actions, and IGFBP-3 inhibitory or mitogenic activities are either dependent or independent of binding to IGFs. Our next aim was to evaluate IGFBP-3 induction of IGF-I expression. HUVEC were treated in serum-free medium with or without IGFBP-3, fumonisin B1, and doxorubicin; secretion of IGF-I into cell-conditioned medium was evaluated by radioimmunoassay. Low IGF-I levels did not change in the absence of IGFBP-3, but in the presence of IGFBP-3, alone or with doxorubicin and fumonisin B1, IGF-I secretion was dramatically increased (6 fold). Even IGF-I mRNA was significantly increased after 24 h treatment with IGFBP-3 compared with unstimulated cells. The increase induced by IGFBP-3 suggests that IGF-I may act, in association with its major binding protein, as an antiapoptotic and survival factor in ECs.

6. IGFBP-3 induces phosphorylation of the IGF-IR and activates PI3K/Akt and ERK
Based on the reported increase in IGF-I expression, we aimed to determine whether IGFBP-3 protection in HUVEC was associated with stimulation of IGF-IR signaling. In serum-starved cells, tyrosine phosphorylation of the IGF-IR ß subunit increased at 10 min and peaked 30 min after IGFBP-3 stimulation. The IGFBP-3 antiapoptotic effect after 24 h was abolished by the inhibitor of IGF-IR phosphorylation, tyrphostin AG1024. IGFBP-3 increased Akt and ERK1/2 phosphorylation at 30 and 5 min, respectively. These results suggest that IGFBP-3 protection from apoptosis may depend on IGF-I increase via IGF-IR phosphorylation and activation of its downstream survival pathways PI3K/Akt and ERK.

7. IGFBP-3 enhances motility of endothelial cells
IGF-I and S1P are both known to enhance EC motility. We wanted to study whether IGFBP-3 could affect HUVEC migration. In serum-starved conditions, motility never exceeded 4 µm/h whereas IGFBP-3 induced an immediate increase that remained significantly higher (6 µm/h) throughout the observation period. Fumonisin B1 acted on cell motility (4 µm/h); with IGFBP-3, motility, initially similar to fumonisin B1 alone, significantly increased in a time-dependent manner (5 µm/h), being lower, however, than in cells without the ceramide synthase inhibitor. Compared with untreated cells or cells with fumonisin B1, doxorubicin decreased HUVEC motility (<3µm/h). IGFBP-3 showed no significant enhancement of motility when added to doxorubicin, but migratory speed values were higher at almost every time point (3 µm/h). Fumonisin B1 increased motility of doxorubicin-treated cells, with speed similar to that of cells without doxorubicin. IGFBP-3 significantly increased cell motility by fumonisin B1 over doxorubicin in a time-dependent manner, reaching speed values comparable to those in the absence of the drug (5 µm/h). These results indicate that IGFBP-3 affects EC motility, increasing migration either alone or with fumonisin B1 and doxorubicin.

CONCLUSIONS AND SIGNIFICANCE

This study shows that IGFBP-3 has dual effects on HUVEC, potentiating doxorubicin-induced apoptosis but enhancing survival in serum-starved conditions. Ceramide was increased by IGFBP-3 in the presence of doxorubicin and decreased when IGFBP-3 alone was added to cells cultured in serum-free medium. Protection exerted by the ceramide synthase inhibitor fumonisin B1 over doxorubicin-induced apoptosis was enhanced by IGFBP-3 with a concomitant reduction of ceramide levels (Fig. 2 ).

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic diagram.

IGFBP-3 has been shown to act as both a proliferative and proapoptotic factor depending on the cell type and experimental conditions. Our results in HUVEC show that ceramide levels and doxorubicin-induced apoptosis are enhanced by IGFBP-3, in agreement with previous findings on IGFBP-3 effects in the presence of cytotoxic drugs. However, it has to be explained why IGFBP-3, in the presence of doxorubicin with fumonisin B1 or in serum-free medium with or without the inhibitor, decreases ceramide levels and acts as an antiapoptotic factor. We suggest that IGFBP-3 is in fact a survival factor for ECs except when in the presence of doxorubicin, which likely activates different signaling mechanisms. The dual effects of IGFBP-3 on ceramide production and biologic activity in HUVEC may be due to the paradigm of the ceramide/S1P rheostat as a mechanism for regulating proliferation and apoptosis through sphingolipids. Ceramide is known to be proapoptotic whereas S1P is involved in cell survival and protection against apoptosis; in ECs it stimulates migration and angiogenesis. We show that IGFBP-3 not only reduced ceramide and apoptosis in serum-starved cells but did so even in the presence of doxorubicin with fumonisin B1, the inhibitor of ceramide synthase that may shift the balance from ceramide to its metabolite S1P. S1P is generated by activation of SK, an important signaling molecule stimulated by different mitogenic and survival factors. We found that IGFBP-3 potently and rapidly activated SK, inducing the expression of its antiapoptotic isoform SK1. Moreover, the SK inhibitor DMS completely blocked the IGFBP-3 antiapoptotic effect, suggesting that SK activation and S1P generation serve as signals for the IGFBP-3 survival effect in HUVEC. Indeed, we found that IGFBP-3 increased the expression of IGF-I, a strong survival factor that induces EC migration and angiogenesis. IGFBP-3 even stimulated IGF-IR tyrosine phosphorylation and activated PI3K/Akt and ERK. Inhibition of IGF-IR phosphorylation with tyrphostin AG1024 abrogated IGFBP-3 protection against serum starvation-induced apoptosis, suggesting this effect is mediated by the IGF-IR. Finally, we found that IGFBP-3 significantly increased EC motility (Fig. 2) .

Hence, IGFBP-3 has a novel role in regulating EC fate. Given that IGF-I and S1P both potently stimulate EC motility and based on our results and data from others, we suggest the existence of an interplay between sphingolipids and the IGF-I system in the regulation of endothelial biologic outcome. Moreover, the IGFBP-3-induced increase in cell migration may suggest a role for this binding protein in promoting angiogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1618fje; doi: 10.1096/fj.04-1618fje

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