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Inhibition of endothelial cell proliferation by Notch1 signaling is mediated by repressing MAPK and PI3K/Akt pathways and requires MAML1

Zhao-Jun Liu^*, Min Xiao^*, Klara Balint^*, Akinobu Soma^*, Chelsea C. Pinnix^*, Anthony J. Capobianco^*, Omaida C. Velazquez and Meenhard Herlyn^*,1

^* Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania, USA; and

Department of Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA

¹Correspondence: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA. E-mail: herlynm{at}wistar.org

SPECIFIC AIMS

This study aims to elucidate the signaling mechanisms by which the activated Notch pathway inhibits endothelial cell proliferation. We show here that Notch-induced suppression is mediated by the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways.

PRINCIPAL FINDINGS
1. Activation of the Notch1 pathway suppresses endothelial cell proliferation
We have previously documented that enforced activation of Notch1 signaling by transient expression of the intracellular portion of the Notch transmembrane protein (N^IC) induces cell growth arrest of human iliac venous and arterial endothelial cells (HIAECs). To study whether stimulation of Notch1 receptors on the cell surface results in proliferation inhibition of HIAECs, we tested two soluble forms of the Notch1 ligands, sDll4 and sJag1, for their effects on cell proliferation. We constructed sDll4 as a chimeric fusion gene, in which the cDNA encoding the extracellular domain was fused to the Fc region of the human IgG1 heavy-chain gene. Expression of the sDll4 protein was confirmed by immunoprecipitation and immunoblotting assays. The biological effects of sDll4 and sJag1 on the activation of cell surface Notch1 were validated by detecting a cleaved form of Notch1 in cells. HIAECs treated with sDll4 or sJag1 displayed reduced proliferation rates (15–18%) compared to those treated or untreated with controls. This finding implicates suppressed activation of Notch1 signaling on HIAEC proliferation. To determine whether sustained activation of the Notch1 pathway achieves a similar effect, we constructed a lentiviral vector encoding the N^IC gene and transfected it into HMVECs and HIAECs. We found that proliferation rates of N^IC-transfected cells markedly decreased compared with the rates of either parental or GFP-transfected control cells. These results are consistent with sDll4-initiated Notch pathway activation, but enforced constitutive activation of the Notch1 pathway has a more profound inhibitory effect on cell proliferation. Taken together, our data demonstrate that activation of the Notch1 pathway inhibits human endothelial cell proliferation in vitro.

2. Activation of Notch1 signaling represses MAPK and PI3K/Akt pathway signaling in endothelial cells
To determine the intracellular signaling pathways that mediate Notch’s effect on proliferation inhibition, we tested the potential of activated Notch1 to regulate two proliferation-related signal transduction pathways—the MAPK and PI3K/Akt pathways. Both pathways are activated following vascular endothelial growth factor (VEGF) or serum stimulation in HIAEC and HMVEC. In endothelial cells expressing N^IC, phosphorylation of Erk1/2 (p44/42) 30 min after mitogen stimulation was significantly inhibited compared with that in control cells. Similarly, phosphorylation of Akt 1 h after mitogen stimulation was drastically suppressed (Fig. 1 A). The effects of Notch signaling on regulating MAPK and PI3K/Akt pathways were confirmed by stimulation of HMVECs with sJag1. Phosphorylation of Erk1/2 and Akt was suppressed (Fig. 1B ). Thus, activation of Notch1 signaling negatively regulates MAPK and Akt pathways in endothelial cells.

View larger version (42K): [in this window] [in a new window]   Figure 1. Phosphorylation of MAPK and Akt by Notch1 activation. A) Endothelial cells transfected with GFP/lenti or NIC-GFP/lenti were restimulated with 50 ng/ml of h-VEGF after starvation for 16 h. Cells were harvested at different time points (as indicated) and lysed for Western blot analyses. Membranes were blotted with antiphospho-Erk1/2 and -Akt Abs and reblotted with antitotal Erk1/2 and Akt Abs. X-ray films were scanned with a densitometer, and specific bands were quantitatively plotted. A representative result is shown. B) Starved HMVECs were treated with sJag1 or sPTPBR7 and restimulated with h-VEGF. Cells were harvested and lysed for Western blot analyses.

3. MAPK and PI3K/Akt pathways are responsible for mediating activated Notch1-induced cell proliferation inhibition
To investigate whether changes in the activity of the PI3K/Akt pathway account for Notch1-induced cell proliferation control, we introduced either an active mutant of Akt (Myr-Akt) or GFP (as a control) into HMVEC-N^IC cells. The Myr-Akt–transfected cells showed increased phosphorylation of Akt. Cell proliferation assays for HMVEC-N^IC demonstrated that their decreased proliferation rate was restored if the Akt activity was enhanced. Similarly, we tested the mediating effect of the MAPK pathway on Notch endothelial cell proliferation inhibition by transfecting an active mutant of H-Ras^V12 or hemagglutinin (HA)-tagged B-RAF^V600E into HMVEC-N^IC cells. Expression of H-Ras^V12 or B-RAF^V600E rescued the decreased proliferation rate of HMVEC-N^IC cells, implying that the MAPK pathway, like the PI3K/Akt pathway, is indeed responsible for mediating Notch1 signaling-induced inhibition of endothelial cell proliferation. Similar effects were observed in HIAEC cells. Taken together, our results strongly indicate that Notch1 pathway activation inhibits endothelial cell proliferation by concurrently regulating both the MAPK and PI3K/Akt signaling pathways.

4. The role of Notch1 signaling in cell proliferation control is transcription-dependent
To further elucidate the mechanism by which Notch pathway activation suppresses PI3K/Akt and MAPK pathway-mediated endothelial cell proliferation, we tested whether the process is transcription-dependent or -independent. We first tested the effect of HES1 on HMVEC growth. HES1 is a downstream target molecule of the Notch signaling cascade whose expression depends on N^IC/CSL/MAML complex-mediated gene transcription. When introduced into HMVEC, HES1 suppressed cell proliferation relative to control. This finding suggests a transcription-dependent mechanism in the execution of Notch signaling. Because MAML is required to transcribe downstream target genes of the Notch pathway, we decided to investigate the potential role of MAML1 in controlling endothelial cell proliferation by introducing a dominant-negative mutant of MAML1/pBabe into HMVEC-N^IC cells (DN-MAML1-HMVEC-N^IC). We used pBabe as a control (Control-HMVEC-N^IC). As expected, DN-MAML1 altered the rate of cell proliferation induced by activated Notch1. This result suggests that the effects of Notch signaling on cell proliferation control are mediated by the Notch/MAML-HES cascade and are transcription-dependent.

5. Effect of Notch signaling on controlling MAPK and PI3K/Akt pathways is MAML1-dependent
To address whether the effects of MAML1 on endothelial cell proliferation control are mediated through the MAPK and PI3K/Akt pathways, we examined whether DN-MAML1 could alter N^IC-induced MAPK and PI3K/Akt pathway activities. Compared with Control-HMVEC-N^IC cells, phosphorylation of Erk1/2 following mitogen stimulation was significantly re-established in DN-MAML1-HMVEC-N^IC cells. Similarly, phosphorylation of Akt following mitogen stimulation was also remarkably reversed. Very similar results were obtained in HIAECs. These data demonstrate that MAML1 is indeed responsible for mediating the effects of activated Notch signaling on cell proliferation control by regulating the MAPK and PI3K/Akt signaling pathways.

CONCLUSIONS AND SIGNIFICANCE
Endothelial cell proliferation is required for vascular spouting and elongation during the new blood vessel formation. When endothelial cells differentiate, which is required for remodeling and maturation of newly formed blood vessels, the cell cycle needs to be arrested. This process is known as differentiation-associated cell cycle arrest. In the matured vasculature, most of the endothelial cells remain in quiescent states. Notch signaling is known to be temporally and spatially involved in the remodeling of newly formed vessels. It has been suggested that Notch signaling is silent during the early stages of angiogenesis (when active endothelial cell proliferation is required) but activated during vessel maturation (when endothelial cells cease proliferation and likely undergo differentiation-associated cell cycle arrest). Notch pathway activation may also be involved in endothelial cell contact inhibition. If true, this would support the hypothesis that Notch signaling helps control vessel homeostasis by maintaining endothelial cell quiescence.

The current study suggests that the MAPK and PI3K/Akt pathways are under the control of Notch signaling and that both pathways are ultimately responsible for Notch signaling-induced cell proliferation inhibition (Fig. 2 ). Our work establishes a direct link between the pathways of Notch, MAPK, and PI3K/Akt and provides an explanation for the mechanisms of controlling cell proliferation by Notch signaling in endothelial cells. The signaling cascades delivered from both MAPK and PI3K/Akt pathways regulate cell cycle machinery. It has been demonstrated in endothelial cells that Notch signaling inhibits phosphorylation of Rb and mitogen-induced up-regulation of p21^Cip1. Our findings may well bridge the gap between Notch activation and the negative regulation of cell cycle machinery.

View larger version (37K): [in this window] [in a new window]   Figure 2. Schematic illustration of a suggested mechanism by which Notch signaling inhibits endothelial cell proliferation. Activation of Notch signaling suppresses MAPK and PI3K/Akt pathways. The Notch/MAML-HES cascade likely regulates MAPK and PI3K/Akt pathways indirectly through the induction/suppression of other targeting gene(s) (e.g., suppressing VEGF-R2). This induction or suppression, in turn, mediates Notch’s effect. The signaling cascades from both MAPK and PI3K/Akt pathways ultimately control components of cell cycle machinery, such as Rb and p21Cip1.

Classical Notch signaling is transmitted by means of the Notch-N^IC/CSL/MAML cascade. Recent evidence suggests that a transcription-independent mechanism exists for mediating multiple effects of Notch signaling. It is likely that Notch signaling regulates the MAPK and PI3K/Akt pathways at the post-transcriptional concentration because (a) HES1, a post-transcriptional target gene product, can represent the effect of Notch on endothelial cell proliferation control; and (b) MAML1 is an essential element for initiating transcription, DN-MAML1 can antagonize the effect of Notch on MAPK and PI3K/Akt pathway-mediated cellular proliferative or inhibitory signals. Consistently, our preliminary efforts in detecting a direct association between N^IC and MAPK or Akt by coimmunoprecipitation were unsuccessful (data not shown). This finding supports the notion that intracellular Notch itself does not directly regulate the MAPK and PI3K/Akt pathways. Because MAPK and PI3K/Akt pathways are simultaneously controlled by the Notch/MAML-HES cascade, and these two parallel pathways are initiated by receptor tyrosine kinases/phosphatases or adaptor-mediated kinases/phosphatases, it is more likely that the regulation of MAPK and PI3K/Akt pathways by the Notch/MAML-HES cascade is mediated by regulating common kinases/phosphatases. It has been reported that Notch activation down-regulates VEGF-R2 expression on endothelial cells. Likely, decreased expression of VEGF-R2 is responsible for initiating weak MAPK and PI3K/Akt pathway activation (considering that VEGF stimulation was used in our experiments). However, since the effects of complete M199 medium are similar to those of VEGF, VEGF-R2 is obviously not the only target molecule mediating Notch-induced suppression. Future studies to identify such Notch signaling-induced target molecules will help define the mechanism underlying the crosstalk between Notch signaling and the MAPK and PI3K/Akt pathways.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4880fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4880fjev1 20/7/1009    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liu, Z.-J. Articles by Herlyn, M. Search for Related Content PubMed PubMed Citation Articles by Liu, Z.-J. Articles by Herlyn, M.