Data on the regulation of moesin and merlin by the urokinase receptor (uPAR): Model explaining distal activation of integrins by uPAR

The data presented herein are connected to our research article (doi: 10.1016/j.biocel.2017.04.012) [1], in which we investigated the functional connections between the urokinase receptor (uPAR), and the ezrin/radixin/moesin (ERM) proteins, moesin and merlin [1]. Firstly, a model of action is proposed that enlightens how uPAR regulates distal integrins. In addition, data show the effects of expressing wild-type moesin or permanently active T558D mutant of moesin on angiogenesis and morphology of human aortic endothelial cells (HAEC). Additional data compare the effects of urokinase (uPA, the main ligand of uPAR) on the same cells. Lastly, we provide technical data demonstrating the effects of specific siRNA for moesin and merlin on moesin and merlin expression, respectively.


a b s t r a c t
The data presented herein are connected to our research article (doi: 10.1016/j.biocel.2017.04.012) [1], in which we investigated the functional connections between the urokinase receptor (uPAR), and the ezrin/radixin/moesin (ERM) proteins, moesin and merlin [1]. Firstly, a model of action is proposed that enlightens how uPAR regulates distal integrins. In addition, data show the effects of expressing wild-type moesin or permanently active T558D mutant of moesin on angiogenesis and morphology of human aortic endothelial cells (HAEC). Additional data compare the effects of urokinase (uPA, the main ligand of uPAR) on the same cells. Lastly, we provide technical data demonstrating the effects of specific siRNA for moesin and merlin on moesin and merlin expression, respectively.
& Data are useful for understanding the effects of expressing mutants of moesin on cell morphology and angiogenesis.
Show the effects of urokinase on angiogenesis and morphology of cells expressing mutants of moesin.
Provide a model of action explaining how the urokinase receptor activates distal integrins.

Data
uPAR is a membrane receptor involved in cell migration, adhesion and angiogenesis [2][3][4][5][6][7][8]. uPAR is a glycosyl-phosphatidyl-inositol receptor that is not connected to the intracellular compartment. Thereby, to induce intracellular signalling, uPAR interacts proximally with other membrane receptors such as the integrins [9][10][11]. Moesin and merlin belong to the family of ERM proteins. Moesin links membrane proteins to actin filaments permitting cell flexibility, and merlin regulates membrane receptors activity and signalling [12][13][14]. Fig. 1 represents a model built using experimental data from our research study [1], illustrating the functions of moesin and merlin. In line with this model, microscopy pictures show the effects of overexpressing wild-type moesin or permanently active mutant T558D on angiogenesis and morphology of human aortic endothelial cells (HAEC) (Fig. 2). The same HAEC transfected or not with wild-type moesin or mutant T558D, were also used to determine the effects of urokinase (uPA) on angiogenesis and cell morphology (Fig. 2). These qualitative data are completed by quantification of angiogenesis performed in our research paper [1]. Knocking down a protein using siRNA is a convenient method to investigate the function of that particular protein [15][16][17]. Here, we provide technical data for knocking down either moesin or merlin in HAEC using specific siRNA (Figs. 3 and 4). Fig. 3 displays the effects of moesin siRNA compared to parental HAEC. In addition, HAEC transfected with scrambled siRNA or without siRNA (mock) served as negative controls. The effects of increasing doses of merlin siRNA were compared to the higher dose of scrambled siRNA used as negative control (Fig. 4).  The binding of SRSRY sequence of uPAR to formyl peptide receptors such as FPRL1 (thick blue arrow) or the binding of D2A sequence located in domain 2 of uPAR to integrins (thick red arrow) initiates outside-in signalling converging towards phosphorylated moesin (P-moesin) and merlin (thin blue and thin red arrows), which results in the de-phosphorylation of moesin and phosphorylation of merlin (thick black arrow). This latter step initiates inside-out signalling (thick purple arrow) activating distal integrins (yellow arrow) that are involved in cell adhesion and migration (green arrow).

DNA transfection, Gene knockdown
According to manufacturer's instructions plasmid DNA was purified using the QIAGEN-tip HiSpeed kit. Nucleofector and a basic Nucleofector kit for endothelial cells (Lonza) were employed for transient transfection. 10 6 HAEC and 3 µg DNA were added in 100 µl basic solution, transferred into an amaxa cuvette, and electroporated. Knockdown of moesin or merlin gene was performed using specific small interfering RNA (siRNA) for moesin (s8984, Ambion), and merlin/NF2 (s194647, Ambion). Scrambled siRNA (4390825, Applied Biosystems) served as negative control. Transfection was performed with 10 6 HAEC and 4 µg of siRNA mixed in 250 µl of serum-free medium plus 7 μl of TransIT-siQUEST® reagent (Mirus), which were incubated for 20 min at room temperature, then seeded into 6-well plate containing 2 ml/well of fresh medium, and cultured for 2 days at 37°C. Alternatively, transfection of 500,000 HAEC with 1-100 nM siRNA was realized with Labtech microporation unit (1000 V, 30 ms pulse width and 3 pulse number).

Microscopy
8,000 HAEC were seeded onto thick layer of matrigel (Corning) in cell culture media plus 0.5% of FCS, and cultured for 12 hours in the presence or in the absence of 10 nM uPA. Then, low magnification photographs (4 lens) were taken under the microscope (Olympus DP-50). Photographs shown in Fig. 2 are representative of one out of six independent experiments performed in triplicate.

Statistical analysis
Student's t test for pairwise comparisons of treatments was performed with the GraphPad Prism software.