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Transcriptional and post-translation regulation of the Tie1 receptor by fluid shear stress changes in vascular endothelial cells¹

LIMOR CHEN-KONAK^,2, YULIA GUETTA-SHUBIN^,2, HAVA YAHAV, AYELET SHAY-SALIT, MICHAL ZILBERMAN, OFER BINAH^¶ and NITZAN RESNICK^,3

The Interdepartmental Program in Biotechnology,
Department of Anatomy and Cell Biology,
^¶ Department of Physiology, The Rappaport Family Institute for Research in the Medical Sciences and the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel

³Correspondence: Department of Anatomy and Cell Biology, The Rappaport Family Institute for Research in the Medical Sciences and the Bruce Rappaport Faculty of Medicine, Efron St., P.O. Box 9697, Haifa, Israel 31096. E-mail: nitzanr{at}tx.technion.ac.il

SPECIFIC AIMS

Hemodynamic forces, and more specifically fluid shear stress, are suggested to play a role in blood vessel formation and maturation. Recent studies from our lab and others suggest that shear stress-induced blood vessel restructuring may be mediated by the regulation of angiogenic ligands and receptors in vascular endothelial cells. The present study tested the hypothesis that shear stress changes that activate endothelial cells regulate the levels of Tie1, thus promoting the instability of the endothelium that results in vascular restructuring.

PRINCIPAL FINDINGS

1. Tie1 expression in arterial endothelial cells exposed to shear stress and its dependence on VE-cadherin expression
Arterial endothelial cells were grown to confluence and exposed to physiological levels of laminar shear stress (10 dynes/cm²) for various intervals. Minutes after exposing the cells to shear stress, Tie1 levels declined beyond the static control (Fig. 1 A) and returned to static control level only 2 h after exposure to shear stress. In contrast, angiopoietin receptor Tie2 levels did not change in short intervals after the onset of flow.

View larger version (35K): [in this window] [in a new window]   Figure 1. Tie1 expression in endothelial cells exposed to shear stress. Bovine aortic endothelial cells were incubated under static conditions or exposed to short intervals of physiological shear stress (10 dynes/cm2). Cells were extracted and tested for Tie1 expression using Western blot analysis. Data quantification and statistical analysis (n=3) were carried out.

2. Tie1 expression in response to shear stress changes
The rapid and transient change in Tie1 levels suggested that the molecule is sensitive to shear stress changes rather than to the force per se. To test this, endothelial cells were subjected to 6 h of continuous shear stress on a magnitude of 10 dynes/cm², followed by 30 min shear stress of 15 dynes/cm². As a control, cells were exposed to 7 h of continuous shear stress of 10 or 15 dynes/cm². These experiments revealed that compared with the control static cultures, Tie1 levels did not change significantly in cells exposed to continuous shear stress (7 h) of 10 or 15 dynes/cm² (data not shown). In contrast, Tie1 levels decreased significantly in cells experiencing changes in shear stress. While Tie1 levels were suppressed by an increase (step-up) and a decrease (step-down) in shear stress,an increase in shear stress (step-up) had a more prominent effect on Tie1 levels. Shear stress changes (more commonly an increase/step-up in shear stress) mimic the hemodynamic environment to stimulate arteriogenesis.

3. Tie1 is cleaved in response to shear stress and binds to Tie2
Down-regulation of Tie1 has been documented in endothelial cells treated with TNF-, PMA, and VEGF. It was found that this down-regulation of the full-size Tie1 is the result of its cleavage into two proteins: the endodomain (45 kDa) and ectodomain (90 kDa). The rapid down-regulation of Tie1 in response to shear stress or shear stress changes raised the question of whether shear stress stimulates the cleavage of Tie1. Using antibodies targeted to the carboxyl terminus of Tie1, we found that as soon as 15 min after the onset of flow, Tie1 is cleaved and the 45 kDa fraction can be identified. Tie1 endodomain levels remain significantly high in endothelial cells exposed to 1 h of shear stress and decline thereafter. The appearance of the 45 kDa endodomain band correlates with the reduction in intensity of the whole 130 kDa molecule. A Tie1 antibody that recognizes Tie1 amino terminus was also able to detect the decrease in 130 kDa Tie1 levels (data not shown).

Brindle and co-workers demonstrated that Tie1 cleavage in response to PMA, TNF-, or VEGF is accompanied by binding of the Tie1 endodomain to the angiopoietin receptor Tie2. To test the interaction of Tie1 and Tie2 in response to shear stress, BAEC cultures were extracted and immunoprecipitated at various intervals after exposure to shear stress using Tie1 or Tie2 antibodies. Samples immunoprecipitated with the Tie1 antibodies revealed a decrease in Tie1 130 kDa fragment levels, an increase in Tie1 45 kDa fragment, and an increase in Tie2 levels, suggesting that the interaction occurred between the Tie2 and Tie1 endodomains. This binding increased with time and could still be observed 1 h after onset of flow, supporting previous observations that Tie1-Tie2 binding is stable. Immunoprecipitation with Tie2 antibodies revealed unchanged levels of Tie1 130 kDa and Tie2 in response to shear stress but an increase in the Tie1 45 kDa fragment bound to Tie2. Thus, both immunoprecipitation assays imply that shear stress induces the binding of Tie2 to Tie1 endodomain.

4. Shear stress regulates Tie1 transcription
Northern blot analysis of endothelial cells subjected to shear stress revealed that Tie1 is down-regulated by shear stress as soon as 30 min after the onset of flow (data not shown). To further characterize the transcriptional regulation of Tie1 by shear stress, BAEC cultures were transfected with the murine Tie1 promoter construct (Tie1-Luc) and exposed to shear stress. Luciferase expression regulated by the Tie1 promoter was down-regulated >threefold in endothelial cells exposed to 1 h of shear stress. The levels of luciferase expression were not affected by the flow in cultures transfected with the PGL3/basic backbone. We tested the responsiveness of the Tie1 promoter to additional stimuli. Endothelial cultures transfected with the Tie1 construct were exposed to laminar shear stress for 1 and 4 h, treated with TNF-, treated with PMA, or exposed to various intervals of hypoxia (1% oxygen). Down-regulation of the luciferase expression was observed in transfected cells exposed to shear stress or treated with TNF-, but not with PMA, which had no effect on the luciferase expression regulated by the Tie1 promoter. Exposure of endothelial cells to 1 h or 24 h of hypoxia leads to the induction of luciferase expression (Fig. 2) . These results are in accord with recent publications demonstrating the induction of Tie1 protein levels in endothelial cells exposed to hypoxia.

View larger version (21K): [in this window] [in a new window]   Figure 2. Tie1 is transcriptionally regulated by shear stress. Bovine aortic endothelial cells were transfected with the murine Tie1-Luc promoter and cotransfected with Renilla-Luc construct. The cultures were untreated, treated with TNF- (200 units/4 h), or exposed to shear stress (10 dynes/cm2 for 1 and 4 h) or hypoxia (1% O2 for 1 and 24 h). Luciferase expression is normalized to that of Renilla luciferase and expressed as the ratio between transfected treated/transfected untreated cultures (n=3).

5. A novel negative shear stress response element in the Tie1 promoter
Transcriptional induction or suppression of endothelial genes in response to shear stress is mediated by promoter elements termed SSREs. These elements bind transcription factors to mediate transcriptional activation or repression. So far, only one negative SSRE (NSSRE) has been defined in the promoter of VCAM-1. This SSRE is an AP1 binding site, but the nature of the transcription factor(s) that bind to it is yet to be defined. To define the NSSRE in the Tie1 promoter, deletion analysis was carried out. We found that a 250 bp fragment in the 3' end of the promoter retains shear stress responsiveness, as the level of repression of this deletion construct was identical to that of the full-size promoter. Although this fragment does not encode for the VCAM-1-negative SSRE, it has a single AP1-like site and seven AP4 sites.

CONCLUSIONS AND SIGNIFICANCE

Tie 1 is an orphan receptor important for vessel development and integrity. Tie1-deficient mice die after E13.5 due to severe hemorrhage and edema; studies using transgenic mice carrying a chimeric Tie1 suggested that the molecule is important for proliferation and migration of capillary endothelial cells in embryogenesis and adulthood. Yet little is known about the mechanism of action of Tie1 and its regulation in vivo and in vitro by physiological or pathological stimuli. Tie1 and Tie2 expression is dependent both in vitro and in vivo on cell confluence and is regulated at the level of transcription. The intracellular signaling pathways of Tie1 are not fully understood. The cytoplasmic domain has been shown to undergo autophosphorylation in transfected NIH3T3 cells, which leads to its binding to P85. But such autophosphorylation has not been shown in unstimulated or stimulated endothelial cells. Recently Tie1 was shown to undergo proteolytic cleavage in response to various stimuli such as PMA, TNF-, and VEGF-A. This proteolysis is mediated by yet to be identified zinc-dependent proteases, can be inhibited in some cases by metaloprotease inhibitors, and results in the appearance of a 45 kDa endodomain. We found that shear stress-mediated cleavage of Tie1 is only partially inhibited by Phenantroline and GM1489 (data not shown), suggesting that more than one protease type may be involved. These results are further supported by the finding that Tie1 cleavage in response to PMA treatment is inhibited by a PKC inhibitor that has no effect on Tie1 cleavage in response to VEGF. Cleavage of Tie1 may affect its ability to send intracellular signals through the remaining endodomain, which is very stable. Alternatively, the cleaved extracellular domain may compete with the binding of potential ligands to the uncleaved receptor.

The study of Tie1 signaling became more complicated when its endodomain was shown to bind to Tie2. This binding occurs in endothelial cells stimulated by PMA or VEGF and in endothelial cells exposed to shear stress (this study). The binding occurs rapidly after the cleavage of Tie1 and remains stable for several hours. Tie2 serves as a receptor for multiple angiopoietins. Crystal structure of Tie2 revealed a complex cytoplasmic domain structure, suggesting that Tie2 activation is distinct from other known tyrosine kinase receptors. Thus, the complex signaling via Tie2 and its broad consequences calls for a tight regulation of this receptor activity. One regulator of Tie2 signaling may indeed be Tie1 through its binding to Tie2. If so, shear stress regulation of Tie1 cleavage and its binding to Tie2 are an avenue through which biomechanical forces affect Tie2 activity without affecting its level of expression.

Human and mice Tie1 promoters have been isolated and partially characterized. In the present study, we have shown that the promoter of Tie1 is responsive to an array of stimuli, including TNF- and shear stress, that suppress its activity, and hypoxia, which increases it. Deletion analysis of the promoter led to the identification of a 250 bp region that retains the responsiveness of the promoter to shear stress. This stretch contains a single AP1 and seven AP4 binding sites. Characterization of the novel Tie1-NSSRE may shed light on the mechanisms by which shear stress regulates endothelial gene expression.

Of the known angiogenic receptors, Tie1 is the less studied mainly because of the lack of a putative ligand and the complexity of its intracellular signaling. However, there is no doubt that Tie1 plays a major role in blood vessel restructuring, maturation, and maintenance in the embryo and the adult. The finding that biomechanical forces modulate the level and activity of this receptor is significant in our attempt to understand how these forces play a role is blood vessel restructuring.

View larger version (62K): [in this window] [in a new window]   Figure 3. A) An excess flow to an arterial branch that results in the increase in shear stress magnitude occurs after partial or complete blockade of an adjacent branch. B) This increase in shear stress is transduced by the vascular endothelial cells into cellular signals, including changes in Tie1 level and activity. 1) Tie1 is rapidly cleaved, leaving behind a 45 kDa endodomain. 2) This endodomain binds to Tie2, regulating the activity and cellular signaling of Tie2. 3) Tie1 levels decrease as a result of transcriptional suppression, mediated by a novel NSSRE. C) Decrease in Tie1 activity may affect the interaction of the endothelium with the extracellular matrix (ECM) and neighboring cells. Loosening of these interactions may trigger shear stress-mediated formation of new blood vessels.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1151fje; doi: 10.1096/fj.02-1151fje

² These authors contributed equally to this work.

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