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Apelin (65-77) activates p70 S6 kinase and is mitogenic for umbilical endothelial cells

Institut National de la Santé et de la Recherche Médicale INSERM U-589, C.H.U. Rangueil, Toulouse Cédex, France

¹ Correspondence: Institut National de la Santé et de la Recherche Médicale INSERM U-589, TSA 50032, C.H.U. Rangueil, Bât. L3, 1 avenue Jean-Poulhès, 31059-Toulouse Cédex 9, France. E-mail: yaudig{at}toulouse.inserm.fr

SPECIFIC AIMS

The present study was designed to reconstitute the transduction cascade induced by the apelin peptide and to characterize the function of apelin signaling in endothelial cells.

PRINCIPAL FINDINGS

The transduction cascade leading to activation of p70S6 kinase by apelin was first demonstrated in CHO cells, but the principal data are now reproduced in umbilical endothelial cells (HUVECs). We show that apelin is mitogenic for these cells.

1. Apelin (65-77) induces phosphorylation of p70 S6 kinase and Akt in CHO cells
Apelin (65-77) promoted a time-dependent phosphorylation on two residues (T421/S424) in the pseudosubstrate domain that peaked at 6 min. At the same concentration, apelin induced a rapid phosphorylation of p70S6K on T389, a residue critical for enzyme activation. Activation of p70S6K is often linked to upstream stimulation of a PI3 kinase and the Akt kinase and, indeed, apelin induced a time-dependent phosphorylation of Akt on residues T308 or S473. Consequently, these results demonstrate that apelin (65-77) promotes robust phosphorylation of Akt and p70S6K in key residues linked to their activation.

2. Apelin-mediated phosphorylation of p70 S6 kinase in CHO cells involves a rapamycin-sensitive pathway regulated by PI3K and Akt
To show that Akt was positioned between PI3K and p70S6K, we analyzed the effect of selective inhibitors of each enzyme on the phosphorylation of Akt and P70S6K. Wortmannin fully blocked the phosphorylation of p70S6K at residue T389 but partially decreased phosphorylation of p70S6K at residues T421/S424. SH6, a specific inhibitor of Akt, and rapamycin fully blocked the phosphorylation of p70S6K at T389 promoted by apelin, whereas the same concentrations of SH6 or rapamycin partially abolished apelin-mediated phosphorylation at T421/S424. On the other hand, and as expected, phosphorylation of T308 and S473 of Akt was abolished by SH6 but not modified by rapamycin. All these findings reveal that a component of the phosphorylation of p70S6K initiated by apelin via the stimulation of its receptor involves a rapamycin-sensitive pathway regulated by PI3K and Akt.

3. Apelin-mediated phosphorylation of p70 S6 kinase in CHO cells involves a PD098059-sensitive pathway regulated by ERK
The T421 and S424 residues of p70S6K are embedded in a canonical MAPK phosphorylation site. We previously showed that apelin activates extracellular signal-regulated kinases (ERKs). Accordingly, a specific inhibitor of MEK, PD098059, decreased the phosphorylation of T421 and S424 in a concentration-dependent manner. The same compound partially diminished phosphorylation of p70S6K at residue T389. Taken together, these data suggest a dual phosphorylation of p70S6K, which involves a wortmannin-sensitive pathway that targets T389 and a PD098059-sensitive pathway that targets T421/S424.

4. The apelin-mediated phosphorylation of p70S6K in CHO cells is blocked by pertussis toxin (PTX) and involves a member of the src family
To dissect the rapamycin-sensitive and rapamycin-insensitive transduction cascades, we decided to characterize the various intermediates between the apelin receptor and its downstream effectors. Pretreatment of the CHO cells with pertussis toxin (PTX) fully abrogated the apelin-mediated phosphorylation of p70S6K and Akt. Herbymicin A, and notably PP2, abolished the phosphorylation of Akt at residue 473 and of p70S6K at residues T421/S424 and T389. An identical effect was observed on ERK phosphorylation.

5. The apelin-mediated phosphorylation of p70S6K in CHO cells involves an atypical PKC
GF109203X, a general inhibitor of PKC isoforms, strongly inhibited not only phosphorylation of Akt at either T308 or S473, but also that of p70S6K at T389 and at T421/S424. In addition, a pseudosubstrate sequence of PKC zeta blocked the phosphorylation of p70S6K at residues T421/S424 and T389. As overnight preincubation with PMA did not alter its effect, these results suggest that an atypical PKC isoform is involved in the transduction cascade initiated by apelin that leads to the dual phosphorylation of p70S6K via ERK and Akt.

6. Regulation of p70S6K phosphorylation correlates with regulation of its enzymatic activity
As previously reported for other ligands of serpentine receptors, the activity of p70S6K was enhanced 5-fold by apelin (Fig. 1 ). This activation was abolished by PTX pretreatment of the cells as well as by incubation with rapamycin. Inhibition of the PI3K/Akt pathway by wortmannin was clearly more pronounced than the inhibition of the ERK pathway by PD098059. In addition, the inhibition was increased by the simultaneous addition of both compounds. Consonant with their effect on the dual phosphorylation of p70S6K, GFX and PP2 were very efficient antagonists of apelin-induced activation of p70S6K. To summarize, these findings all clearly confirm that the dual regulation of phosphorylation of p70S6K induced by apelin correlates accurately with that of kinase activity.

View larger version (17K): [in this window] [in a new window]   Figure 1. Regulation of p70S6 kinase activity in CHO cells. Serum-deprived CHO cells were pretreated for 1 h with various effectors (20 nM rapamycin, 50 µM PD98059, 100 nM wortmannin, 10 µM PP2, and 10 µM GF109203X) or overnight (25 ng/mL pertussis toxin), then stimulated with 1 µM apelin (65-77) for 7 min. Cell lysates (100 µg) were immunoprecipitated with anti-p70S6K antibodies, followed by precipitation with protein A-Sepharose beads. In vitro kinase assays were performed using a synthetic peptide substrate (AKRRRLSSLRA), and p70S6K activity was measured using a S6 kinase assay kit (Upstate Biotechnology Inc.) in the presence of [-32P] ATP according to the manufacturer’s instructions.

7. Apelin (65-77) also induces phosphorylation of p70 S6 kinase in umbilical endothelial cells via a dual pathway
Although the analysis of receptor transduction in CHO cells has a good predictive value, it was interesting to attempt to reproduce the most significant results in HUVECs. Apelin induced a time-dependent phosphorylation of p70S6K that was fully abrogated by PTX. In addition, the main inhibitors of the intermediate proteins characterized in CHO cells were efficient antagonists of the apelin-induced phosphorylation of p70S6K.

8. Apelin (65-77) is a mitogenic peptide for umbilical endothelial cells
To confirm that activation of p70S6K was linked to endothelial cell proliferation, we determined the mitogenic activity of apelin by analyzing the incorporation of thymidine into DNA. As shown in Fig. 2 , apelin (65-77) almost doubled thymidine incorporation and was as efficient as VEGF. The thymidine incorporation was partially sensitive to rapamycin. It can be concluded that activation of endothelial cell proliferation by apelin is linked to the stimulation of p70S6K.

View larger version (16K): [in this window] [in a new window]   Figure 2. Apelin (65-77) stimulates thymidine incorporation in endothelial cells. HUVECs (5x104 cells) at passage 3 were plated on 0.2% gelatin-coated 6-well plates in medium supplemented with 20% heat-inactivated FCS. The next day cells were washed twice with serum-free medium and incubated in a medium containing 0.5% fetal calf serum (FCS)/0.25% bovine serum albumin. Cells were then incubated for 24 h with [3H]thymidine (1 µCi/mL) in the presence or absence of 10–7 M apelin (65-77), 10 ng/mL VEGF, or 1 ng/mL FGF-2. In a second group, stimulation by apelin and growth factors was determined in the presence of 1 nM rapamycin. The cells were then treated with 10% trichloroacetic acid (TCA) for 30 min at 4°C, rinsed with 5% TCA, and lysed with 0.1M NaOH. After filtration of the lysates on glass fiber filters, [3H]thymidine incorporation was determined using a liquid scintillation counter. Data are shown for cells incubated in the absence (open bars) or presence (gray bars) of 1 nM rapamycin. The cpm/well was determined in sextuplicate for each condition and expressed as percentage of the basal thymidine incorporation. The results of 2 separate experiments were pooled.

CONCLUSIONS AND SIGNIFICANCE

We report here for the first time that the peptide ligand apelin (65-77) activates p70S6 kinase, S6K1, like other growth factors and peptides (Fig. 3 ). Although the various pathways and intermediates of the transduction cascade were initially characterized in CHO cells stably transfected with the apelin receptor, the principal findings have now been reproduced in umbilical endothelial cells. In addition, apelin (65-77) promotes proliferation of these endothelial cells.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram of apelin transduction in the endothelial cell.

In this paper, we have characterized intracellular proteins operating in this new signaling pathway such as src, PI3K, aPKC, Akt, and ERK. Moreover, we demonstrate that stimulation of p70S6K by apelin in HUVECs and CHO cells results from activation of two signaling cascades, the PI3K/Akt cascade and the ERK cascade (Fig. 3) . At the molecular level, apelin (65-77) promotes the phosphorylation of different subsets of residues in distinct domains of P70S6K through activation of a dual pathway. At the biochemical level, phosphorylation of p70S6K induced by apelin correlates accurately with that of kinase activity. Finally, at the cellular level apelin (65-77) behaves as a mitogenic peptide for the endothelial cell.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1930fje;

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