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TGF-β Signaling in Endothelial-to-Mesenchymal Transition: The Role of Shear Stress and Primary Cilia

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Science Signaling  21 Feb 2012:
Vol. 5, Issue 212, pp. pt2
DOI: 10.1126/scisignal.2002722
A Presentation from the Keystone Symposium on Epithelial Plasticity and Epithelial to Mesenchymal Transition, Vancouver, Canada, 21 to 26 January 2011.

Abstract

Endothelial-to-mesenchymal transition (EndoMT) is an instrumental step in the development of valves in the embryonic heart. This process is driven by activation of transforming growth factor–β (TGF-β) signaling and is characterized by the loss of endothelial and gain of mesenchymal phenotype, and by delamination of cells from the surface into the underlying endocardial cushion matrix. The endothelial cells (ECs) overlying the cushions are typically exposed to high blood flow and concomitant shear stress and do not have a primary cilium. Here, we show that shear stress activates TGF-β–Alk5 signaling in ECs, which is necessary for EndoMT in the cushions. Moreover, we show that the absence of a primary cilium is critically important for this transition process.

Presentation Notes

Slide 1: Science Signaling logo

The slideshow and notes for this presentation are provided by Science Signaling (http://www.sciencesignaling.org).

Slide 2: TGF-β signaling in endothelial-to-mesenchymal transition: The role of shear stress and primary cilia

This presentation focuses on the role of transforming growth factor–β (TGF-β), shear stress, and primary cilia in endothelial-to-mesenchymal transition (EndoMT).

Slide 3: Shear stress, primary cilia, and cardiac development

Endothelial cells (ECs) respond to blood flow and concomitant shear stress by changing their morphology, gene expression, and function (1). Abnormal patterns of shear stress or abnormal EC responses can lead to vascular diseases, such as atherosclerosis (2), and to congenital cardiovascular anomalies.

In the embryonic heart, the valves develop from the endocardial cushions, the purple areas in the illustration (3). These cushions consist of a thick layer of extracellular matrix that forms between the myocardium (the cardiac muscle) and the endocardium (the endothelium that lines the lumen of the heart). The cushions extend into the lumen of the heart, which causes narrowing of the lumen and an increase in local blood flow velocity and shear stress. Endothelial-to-mesenchymal transition (EndoMT) is responsible for populating the cushions with cells. In this process, ECs, which do not have primary cilia (3), lose their endothelial characteristics, acquire a mesenchymal phenotype, and delaminate from the luminal surface into the cushion matrix.

Slide 4: EndoMT in endocardial cushion formation

High shear stress induces the expression of many genes such as Krüppel-like factor-2 (Klf2), endothelial nitric oxide synthase (eNOS), and TGF-β and represses expression of Endothelin-1 (Et-1) and primary cilia assembly in the ECs. All these factors are instrumental for endothelial function. Activation of TGF-β signaling in the ECs induces EndoMT (4), which coincides with a loss of endothelial markers such as the cell-surface proteins VE-cadherin and CD31, and a gain of mesenchymal markers such as Smooth muscle actin, Vimentin, and the transcription factors Snail and Slug. Activation of TGF-β signaling through the type I receptor Alk5 (activin receptor–like kinase 5) is further reflected by an increase of Pai1 (plasminogen activator inhibitor 1) messenger RNA (mRNA) levels and by phosphorylation of the Smad2 and Smad3 transcription factors. Our research addressed the following questions: First, what is the role of the primary cilium in the endothelial response to shear stress? Second, is shear stress–induced EndoMT mediated by TGF-β signaling?

Slide 5: Primary cilia are mechanosensors for fluid flow

Each EC can bear a solitary, nonmotile primary cilium that has a microtubule bundle core. These cilia are typically 1 to 5 μm in length and 200 nm in diameter (5, 6). They are attached to the microtubules of the cytoskeleton and function in calcium signaling, nitric oxide production, and in the regulation of gene expression (7, 8).

Slide 6: Experimental model to examine the role of primary cilia in shear stress–induced EndoMT

To examine the role of the primary cilium on ECs, we used a ciliated cell line from wild-type (WT) mice and a nonciliated cell line from the Oak Ridge polycystic kidney disease (Tg737orpk/orpk) mouse model (8, 9). The latter carry a mutation in the gene that encodes intraflagellar transport protein 88 (Ift88, also called polaris), which is necessary for ciliary assembly and maintenance. The cells were exposed to 0.5 Pa (5 dyne/cm2) of shear stress for 24 hours in a recirculation parallel plate flow system (10).

Slide 7: Cilia and the response to shear stress

In the absence of fluid flow, both WT and Tg737orpk/orpk ECs grow to form a cobblestone monolayer. Under flow (shear stress), the WT ciliated cells retained this morphology, whereas the nonciliated cells underwent EndoMT and became spindle-shaped (9). This phenotype could be mimicked in both ciliated and nonciliated cells by exposure to TGF-β ligand. Flow-induced EndoMT was characterized by the loss of CD31 expression from the surface of the ECs and by the gain of the mesenchymal markers α-smooth muscle actin (αSMA) and N-cadherin (Ncad).

Slide 8: Nonciliated ECs: Shear-induced EndoMT in a TGF-β–Alk5 dependent manner

This slide shows the molecular response of the cells to shear stress. The graphs show mRNA abundance for CD31, αSMA, Pai1, and Snail1 by means of quantitative polymerase chain reaction (PCR), on extracts from WT (left bar) and Tg737orpk/orpk cells (3 bars on the right) exposed to 0.5 Pa shear stress (red boxes) compared with static controls (represented by the horizontal dashed line). The loss of CD31 and gain of αSMA in Tg737orpk/orpk cells, as shown in the previous slide, was confirmed at the mRNA level. This coincided with a strong induction of Pai1 and Snail1, expression of which is strongly associated with EndoMT in ECs. The flow-induced loss of CD31 and gain of αSMA and Snail1 in Tg737orpk/orpk cells could be prevented, and the induction of Pai1 strongly reduced, by applying shear stress in the presence of antibodies that block the binding of TGF-β to its receptors or of the Alk5 inhibitor SB431542 (designated as “SB” on the slide). These changes in gene expression were accompanied by retention of the cobblestone phenotype. The asterisks in the figures indicate significant changes (P < 0.05) as compared with the static conditions, and “ns” denotes nonsignificant changes. The hash sign represents significant changes between the assigned groups. These experiments were included in a paper recently published in Circulation Research (9).

Slide 9: Shear stress represses Klf4 expression in nonciliated ECs

In ECs, Klf4 is a functional homolog of Klf2, and expression of both these transcription factors is induced by shear stress in WT ciliated ECs (9). However, in nonciliated Tg737orpk/orpk ECs, Klf2 is not induced by shear stress, and Klf4 is dramatically down-regulated by shear stress at the mRNA level [red boxes in (A) and (B)] and at the protein level (C). This is a TGF-β–dependent process because induction of both transcription factors by shear stress is restored upon inhibition of TGF-β–Alk5 signaling by blocking antibodies or by SB.

Slide 10: Klf4 overexpression inhibits shear-induced EndoMT

To determine whether this down-regulation of Klf4 is the result of EndoMT or is required for this transition, we overexpressed Klf4 in Tg737orpk/orpk ECs before exposing the cells to shear stress. This resulted in a 10-fold increase in Klf4 transcription (A) and a twofold increase in the abundance of Klf4 protein (B). Klf4 overexpression also prevented flow-induced EndoMT, as illustrated by the maintenance of the cells’ cobblestone morphology (C), the retention of CD31 expression (D), and the lack of induction of αSMA, Pai1, Snail, and Ncad under shear stress (D). This shows that down-regulation of Klf4 in ECs is a prerequisite for EndoMT (9).

Slide 11: Primary cilia inhibit shear-induced EndoMT

To determine whether shear-induced EndoMT was caused by the loss of primary cilia and was not a consequence of some other attribute of this cell line, we rescued the orpk mutant phenotype by overexpressing an mCherry-tagged version of Ift88 (Ift88*). This restored expression of Ift88 at both the mRNA (A) and protein (B) levels and rescued the assembly of primary cilia, as shown with α-tubulin staining (C). When these cells were exposed to shear stress, the EndoMT transition phenotype was prevented, as illustrated by maintenance of the cobblestone morphology in (D). Quantitative PCR analysis showed a loss of repression of CD31 and Klf4, a loss of induction of Snail and Ncad, and restoration of Klf2 induction in the presence of flow (E). Flow induced αSMA and Pai1 in these rescued mutant cells in amounts comparable with those of WT cells under shear stress reflecting a “ciliary” response. This demonstrates that flow-induced EndoMT depends on the loss of the endothelial primary cilium (9).

Slide 12: In vivo implications of loss of primary cilia

In the hearts of Tg737orpk/orpk embryos at day 11.5 after conception (E11.5), ECs lining the ventricular septum and trabeculations (D and F) and the atrial wall (H and J) show increased abundance of αSMA and phosphorylated Smad2 (P*Smad2), indicating enhanced TGF-β–Alk5 activation, compared with WT (compare the brown immunostaining in the cells indicated by the arrows between WT and Tg737orpk/orpk). These ECs are ciliated in WT hearts but lack cilia in hearts from the mutant embryos (3). This indicates that the loss of endothelial primary cilia primes these cells for activation of shear stress–induced TGF-β–Alk5 signaling (9).

Slide 13: Lack of primary cilia primes shear-induced EndoMT

In conclusion, we have shown that the loss of primary cilia primes ECs for shear-induced EndoMT and is a necessary prerequisite for this transition. The left panel illustrates that ciliated ECs retain their cobblestone morphology and up-regulate Klf2 and Klf4 under fluid flow. Nonciliated cells, however, activate TGF-β–Alk5 signaling under shear stress and undergo EndoMT (right), which is accompanied by a loss of Klf2 induction and by repression of Klf4 expression (9).

Slide 14: Acknowledgments

Most of this work was performed by Anastasia Egorova from the Hierck and Poelmann lab at the Leiden University Medical Center (LUMC) in Leiden, The Netherlands, and involved collaborators from the ten Dijke (supported by the Leducq Foundation, the Centre for Biomedical Genetics and the Dutch Research Foundation NWO/MWO) and Goumans lab at the LUMC, the Nauli lab at the University of Toledo, and the Yoder lab at the University of Alabama at Birmingham.

Editor’s Note: This contribution is not intended to be equivalent to an original research paper. Note, in particular, that the text and associated slides have not been peer-reviewed.

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