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Angiopoietin-1 activates both anti- and proapoptotic mitogen-activated protein kinases¹

RANIA HARFOUCHE^*, JEAN-PHILIPPE GRATTON, GEORGE D. YANCOPOULOS, MICHELA NOSEDA, ALY KARSAN and SABAH N. A. HUSSAIN^*,2

^* Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Québec, Canada;
Institut de Recherches Cliniques de Montréal (IRCM), Université de Montréal, Montréal, Québec, Canada;
Regeneron Pharmaceutical Inc., Tarrytown, New York, USA;
Department of Pathology and Laboratory Medicine, University of British Columbia, and Department of Medical Biophysics, British Columbia Cancer Agency, and
Department of Pathology and Laboratory Medicine, University of British Columbia, and Departments of Medical Biophysics and Pathology and Laboratory Medicine, British Columbia Cancer Agency, Canada

²Correspondence: Room L3.05, Critical Care Division, Royal Victoria Hospital, 687 Ave. West, Montreal, Quebec, Canada H3A 1A1. E-mail: sabah.hussain{at}muhc.mcgill.ca

SPECIFIC AIMS

Recent studies have documented that angiopoietin-1 (Ang-1) promotes endothelial cell survival through activation of Tie-2 receptors, which in turn activate the PI-3 kinase/AKT pathway. Whether other pathways are involved in the anti-apoptotic effects of Ang-1 remains unknown. We evaluated whether Ang-1 activates the ERK1/2, p38 and JNK/SAPK members of the mitogen-activated protein kinases (MAPKs) in human umbilical vein endothelial cells (HUVECs) and whether these kinases are involved in the anti-apoptotic effects of Ang-1. We also examined the nature of the interactions between PI-3 kinase, ERK1/2, and p38 MAP kinase in the absence and presence of Ang-1.

PRINCIPAL FINDINGS

1. Ang-1 elicits simultaneous activation of ERK1/2 and p38 MAPKs in serum-deprived endothelial cells
Treating serum-deprived HUVECs with Ang-1 (300 ng/mL) elicited a time-dependent increase in ERK1/2 phosphorylation that rose by 15-fold within 15 min and declined thereafter. Selective inhibitors of the ERK1/2 pathway (PD098059 or U0126) significantly attenuated Ang-1-induced ERK activation (Fig. 1) . Ang-1 also elicited phosphorylation of Elk-1 transcription factor and p90 kDa ribosomal S6 kinase (p90^RSK), two MAPK substrates. Ang-1 evoked a fivefold rise in p38 phosphorylation, which peaked within 30 min of Ang-1 and declined thereafter. Ang-1-induced p38 phosphorylation was inhibited by a p38 pharmacological inhibitor, SB203580.

View larger version (37K): [in this window] [in a new window]   Figure 1. A) Mean and SE values of the number of apoptotic ECs evaluated with PI DNA staining and FACS analysis (n=6 in each group). *P < 0.05 compared with control conditions (2% FBS). P < 0.05 compared with serum deprivation. #P < 0.05 compared with Ang-1. B: Effect of 24 h of serum deprivation (0.1% FBS) in the absence and presence of Ang-1 (with or without MEK1/2 and p38 inhibitors) on EC apoptosis (assessed with DNA fragmentation). DNA extracts were electrophoresed on agarose gel, stained with SYBR Green I, and visualized under UV epi-illumination. Also shown is DNA standard (right lane). Ang-1 attenuated the effect of serum deprivation on DNA fragmentation. When PD098059 was present along with Ang-1, DNA fragmentation increased, whereas incubation of Ang-1 along with SB203580 reduced DNA fragmentation. C) Activation of caspase-3 (assessed with selective cleaved caspase-3 antibody) after 24 h of serum deprivation (0.1% FBS) in the absence and presence of Ang-1. D) Mean and SE values of cleaved caspase-3 protein band (19 kDa) ODs. **P < 0.01 compared with the control (2% FBS). P < 0.05 compared with serum deprivation. #P < 0.05 compared with Ang-1. E) Mean values of caspase-3 activity measured under control conditions (2% FBS), 24 h of serum deprivation (0.1% FBS), and serum deprivation+Ang-1 (n=6 in each group). Symbols are identified above.

2. Ang-1-mediated ERK1/2 activation inhibits serum deprivation-induced apoptosis while p38 activation by Ang-1 promotes apoptosis
When growth supplements and fetal bovine serum (FBS) was removed for 12 or 24 h, the percentage of apoptotic HUVECs (evaluated by propidium iodide DNA profiling or DNA laddering) and caspase-9, -7, and -3 activation (measured with selective fluorescent substrates) and cleavage (evaluated with antibodies specific to the cleaved forms) rose significantly compared with cells grown with growth supplements and 2% FBS (Fig. 1 ). The presence of Ang-1 (300 ng/mL) in the culture medium significantly attenuated serum deprivation-induced apoptosis and reduced caspase-9, -7, and -3 activation and cleavage (Fig. 1) . However, the presence of PD098059 or U0126 along with Ang-1 completely abrogated the inhibitory effects of Ang-1 on apoptosis and enhanced caspase-9, -7, and -3 activities and cleavage. These results suggest that ERK1/2 activation by Ang-1 promotes HUVEC survival. In contrast, the inclusion of SB203580 along with Ang-1 resulted in a small but significant decline in the degree of apoptosis and significantly reduced caspase-7 and -3 activation, indicating that p38 MAPK activation by Ang-1 promotes HUVEC apoptosis (Fig. 1) .

3. Modulation of JNK/SAPK phosphorylation by Ang-1
Serum deprivation of HUVECs for 24 h resulted in by an 50% increase in the level of JNK/SAPK MAPK phosphorylation. The presence of Ang-1 (300 ng/mL) eliminated this rise in JNK/SAPK activation, suggesting that the anti-apoptotic effects of Ang-1 may be mediated in part through inhibition of JNK/SAPK activation. This conclusion was confirmed in HUVECs expressing a dominant negative mutant of JNK/SAPK. The effect of Ang-1 on serum deprivation-induced apoptosis in these cells was attenuated compared with control cells.

4. Interactions between MAPKs and the PI-3 kinase pathway in response to Ang-1
We next assessed whether Ang-1-induced ERK1/2 activation is mediated via the PI-3 kinase pathway. Ang-1-induced phosphorylation of Raf-1, MEK1/2, and ERK1/2 in serum-starved HUVECs was significantly reduced by selective pharmacological inhibitors of the PI-3 kinase pathway (wortmannin or LY294002) and by expressing a dominant negative mutant of the p85 subunit of PI-3 kinase that prevents the association of this subunit with the catalytic subunit of this enzyme. Activation of the ERK pathway by Ang-1 was not inhibited in the presence of a dominant negative mutant of AKT, suggesting that the PI-3 kinase per se rather than AKT promotes ERK activation in the presence of Ang-1. Unlike these effects of PI-3 kinase pathway on ERK activation, pharmacological inhibitors or dominant negative mutant of this kinase elicited substantial augmentation of Ang-1-induced phosphorylation of p38 MAPK. Ang-1-induced p38 phosphorylation was also augmented by a MEK1/2 inhibitor (PD098059). These results suggest that both the PI-3 kinase and the ERK pathways exert inhibitory influences on p38 phosphorylation in the presence of Ang-1 and that when these two pathways are inhibited, the full stimulatory effect of Ang-1 on p38 activation becomes unmasked.

CONCLUSIONS

We describe here for the first time that both ERK1/2 and p38 MAPKs, as well as their downstream targets Elk-1 and p90^RSK kinase, are activated in response to Ang-1 in serum-deprived HUVECs. Many reports have confirmed that activation of ERK1/2 MAPKs in response to various proangiogenesis factors such as VEGF, FGF, and PDGF mediate the proliferative effects of these factors on endothelial cells. There is recent evidence, however, that ERK1/2 kinases play an important role in the regulation of apoptosis in various cells. In nonendothelial cells, ERK1/2 kinases inhibit apoptosis induced by a wide range of stimuli including hyperoxia, reactive oxygen species, and growth factor withdrawal. In endothelial cells, ERK1/2 kinases have recently been shown to mediate the anti-apoptotic effects of VEGF. Our study indicates for the first time that the ERK pathway plays an important role in the anti-apoptotic effects of Ang-1 since inhibition of this pathway augmented the number of apoptotic cells. The mechanisms by which MAPKs modulate apoptosis remain under investigation. In the case of ERK1/2 kinases, several authors have attributed their anti-apoptotic effects to activation of cellular proliferation. Translocation of ERK1/2 kinases to the nucleus and activation of transcription form a well-described process by which ERKs influence cell cycle genes. Another possible target for the ERK pathway is caspase activation. Our previous observation that Ang-1 had no effect on cytochrome c release from the mitochondria and our current finding that MEK1/2 inhibitors reverse the inhibitory effects of Ang-1 on caspase-9, -3, and -7 activation strongly support the notion that the ERK pathway inhibits endothelial cell apoptosis by targeting caspase-9, -3, and -7. Several authors have reported that Ang-1 inhibits endothelial cell apoptosis by activating the prosurvival PI-3 kinase/AKT axis. We found in the current study that activation of ERK1/2 MAPKs in response to Ang-1 is mediated through the PI-3 kinase, since selective pharmacological inhibitors and dominant negative mutant of this kinase significantly reduced Ang-1-induced ERK1/2 activation. Many cytokines and growth factors such as insulin-like growth factor-I are known to inhibit apoptosis by simultaneously activating both the PI-3 kinase/AKT and ERK pathways. There is also evidence that the PI-3 kinase operates upstream from the ERKs in order to mediate several biological effects, including inhibition of apoptosis. The exact sites where the PI-3 kinase pathway activates ERK1/2 kinases in our study appear to be upstream from Raf-1 since pharmacological inhibitors and dominant negative mutant of PI-3 kinase inhibited Ang-1-mediated Raf-1 and MEK1/2 phosphorylation. One possible target of the interaction is Rac, which upon stimulation by the PI-3 kinase causes activation of a Raf-1/MEK/ERK pathway cascade. It is also possible that the PI-3 kinase stimulates ERK phosphorylation by indirectly activating Ras (see Fig. 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Schematic diagram.

We report here for the first time that Ang-1 elicits p38 MAPK activation in HUVECs and that inhibition of the and ß isoforms of p38 MAPKs by SB203580 potentiates the inhibitory effect of Ang-1 on apoptosis, suggesting that p38 MAPKs promote HUVEC apoptosis. The simultaneous activation of anti-apoptotic (PI-3 kinase and ERK1/2) and proapoptotic (p38) pathways by Ang-1 is not unique to Ang-1. Parallel activation of survival and death pathways has recently been documented in response to VEGF, FGF, PDGF, and cytokines such as TNF-. It is interesting that despite activation of p38 kinases, Ang-1 still inhibited serum deprivation-induced apoptosis, suggesting that the effects of anti-apoptotic pathways (PI-kinase/AKT and ERK1/2) predominate over the proapoptotic p38 MAPKs. A similar observation has recently been confirmed in endothelial cells in response to VEGF exposure. We did not evaluate the exact mechanisms by which PI-3 kinase inhibits p38 kinase activation, but a recent study of the effect of VEGF on endothelial cells indicates that inhibition of p38 activation by the PI-3 kinase pathway is mediated through phosphorylation and inhibition of MEKK3 (a p38 kinase) by AKT. That observation and our current study suggest that the negative influence of PI-3 kinase pathway on p38 MAPK signaling is a general phenomenon in ECs and is not limited to a specific mitogens such as VEGF or Ang-1.

In summary, our study indicates that Ang-1 activates simultaneously the anti-apoptotic ERK1/2 and proapoptotic p38 MAPK pathways in HUVECs and that both the PI-3 kinase and ERK1/2 pathways mask the anti-apoptotic properties of p38 pathway, resulting in overall attenuation of serum deprivation-induced apoptosis by Ang-1.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0698fje; doi: 10.1096/fj.02-0698fje

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