br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments This work is supported by national Portuguese funding through FCT, Fundação para a Ciência e a Tecnologia, project UID/BIM/04773/2013 CBMR, projects PEst-OE/EQB/LA0023/2013 and PTDC/SAU-ENB/111702/2009 to J.B., U01HL100408, NIH/NHLBI to I.D., and SFRH/BPD/74807/2010 to G.M.O. I.P.L. thanks the Biomedical Sciences PhD program and FCT for funding (SFRH/BD/62054/2009). J.M.A.S. is a PhD student of the ProRegeM - PhD Program in Mechanisms of Disease and Regenerative Medicine of the University of Algarve and New University of Lisbon financed by the FCT. We thank Shoumo Bhattacharya (University of Oxford) for CITED2 expressing vectors, Austin Smith (University of Cambridge) for E14/T RGDfK and pPyCAGIP vector, Gergana Dobreva (Max Planck Institute for Heart and Lung Research) for Mef2c-luc reporter, Claudia Florindo, head of the Microscopy Facility (University of Algarve), and José A. Belo (New University of Lisbon) for reagents.
Introduction G-protein-coupled receptors (GPCRs), such as PAR1 (Protease Activated Receptor 1, also referred to as CF2R, F2R, TR, and HTR), are transmembrane receptors that transmit extracellular signals into cells by coupling to specific heterotrimeric guanine nucleotide binding proteins (G proteins) and thus mediate an array of responses (Rosenbaum et al., 2009; Vassart and Costagliola, 2011). G-protein-activated pathways constitute the largest class of therapeutic targets (Ding et al., 2015; Thompson et al., 2005). The function ascribed to GPCRs is the result of agonist binding to the receptor, resulting in activation of specific G proteins such as stimulatory Gαs and inhibitory Gαi subunits, which selectively activate or inactivate effector pathways to mediate the desired responses (Kobilka, 2007; Wess, 1997). However, little is known about the role of GPCRs in mediating the differentiation of stem cells to terminally differentiated cells (Callihan et al., 2011; Kobayashi et al., 2010). To date, work has centered on pathways in adult stem cells such as signals emanating from specialized GPCRs (Frizzled proteins) of the WNT pathway and chemokine receptors such as CXCR4 expressed in stem cells (Holland et al., 2013; Van Camp et al., 2014). The role of GPCR signaling in mediating the differentiation of pluripotent embryonic stem cells (ESCs) into differentiated cells has not been widely explored. ESCs are critical for regenerative therapies because unlike adult stem cells they expand indefinitely and are ideal for generating mature cells to replace injured tissue. Studies showed that the transcriptional programs underlying ESC differentiation mirror those during embryonic development (James et al., 2005; Shiraki et al., 2014). One example is the differentiation of ESCs into regenerative vascular endothelial cells (ECs), which requires upregulation of the developmental transcription factors such as ER71 (Kohler et al., 2013) and which serves as a window for investigation of signaling pathways mediating vascular regeneration in ischemic tissue. Here we used a GPCR gene expression screen to identify GPCRs expressed in mouse ESCs (mESCs) undergoing differentiation to ECs. We observed that PAR1 was highly upregulated, and further that it was required for EC differentiation. PAR1 functions as a scaffold for the transforming growth factor β (TGF-β) receptor TGFβRII, which thereby dampens SMAD signaling and activates the transition of ESCs to ECs capable of forming new blood vessels.
Results
Discussion Studies in Par1 mouse embryos showed that PAR1 is a key regulator of vascular development; that is, ∼50% of Par1 mice died in utero because of defective vasculogenesis (Griffin et al., 2001). PAR1 utilizes multiple heterotrimeric G proteins, Gαi, Gαq, and Gα12/13, to transmit intracellular signals (Coughlin, 2000; Soh et al., 2010). Only EC-specific Gα13 embryos died at embryonic days 9.5–11.5 with a phenotype resembling the Par1 mice (Ruppel et al., 2005). Furthermore, embryos re-expressing Gα13 in ECs did not differ from their Gα13 littermates and also showed intracranial bleeding (Ruppel et al., 2005), pointing to a key function of PAR1 independent of its associated canonical heterotrimeric G-protein signaling.