Signaling pathways that control rho kinase activity maintain the embryonic epicardial progenitor state

Abstract

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.

Keywords: Cardiovascular; Epac; Epicardial Cells; Epithelial to Mesenchymal Transformation; Guanine Nucleotide Exchange Factor (GEF); Ras Homolog Gene Family, Member A (RhoA); Rnds; Signal Transduction; Transformation; p190RhoGAP.

FIGURE 1.

A, structure and characterization of the developing E9.5 mouse PE. The PE develops as a mesothelial outgrowth and arises from the septum transversum in the region of the atrioventricular junction in the developing looped heart. The scanning electron micrographs show the PE as a “grape-like” structure with villus projections (A, panels a and b) composed of cuboidal mesothelium. PE vesicles migrate to the primitive heart, and the epicardial mesothelial cells are shown attached and spread on the E10.5 cardiac myocytes of the heart (panel c)). These mesothelial cells are reported to undergo EMT and migrate into the heart differentiating into SMCs to form the walls of the coronary vessels (not shown). B, PE explants from mouse (E9.5) undergo EMT. At 24 h the PE was attached, and there was an outward migration of epicardial cells in all directions over time. C, SMA and SM myosin heavy chain mRNA expression increased over time. D, immunolabeling of the 48-h explants showed that cells toward the center (panels d, e, and f), but not those at the periphery (panels a, b, and c), developed vinculin-containing focal adhesions (panel d) and that subcortical actin (panel a) was now reorganized into stress fibers (panels e and f) consistent with epicardial mesenchymal cells expressing SM marker proteins.

FIGURE 2.

TGF-β1 treatment of EEC cells induced RhoA-mediated EMT. A, transcription levels of SMA and SM22 increased over a 24–72-h period in the presence of 250 pm TGF-β1. ROCK inhibitor Y-27632 Epac activator 007 and C3 treatment to inhibit RhoA activity partially suppressed or abolished this effect but were without effect on control cells (data not shown). GAPDH was used as a loading control. Data are presented as the means ± S.E., n = 3–24. Western blots show that SMA and SM22 protein expression also increased with TGF-β1 treatment (lower panel). B, TGF-β1 treatment of EECs for 72 h at 37 °C significantly increased RhoA activity as measured with the Rhotekin activity assay and with the phosphorylation of MYPT1 at the ROCK sites Thr-696 and Thr-853. The vertical lines in the Western blots indicate that an intervening band was deleted. C, distribution of E-cadherin, β-catenin, ZO1, and expression of actin (phalloidin) and SMA detected by immunolabeling of EECs under control conditions and with TGF-β1-induced EMT. Note that TGF-β1 treatment (panels b, d, and f) initiated separation of cell-cell contacts based on E-cadherin, β-catenin, and ZO1. The appearance of actin stress fibers (panels h and j) appeared only with TGF-β1 treatment.

FIGURE 3.

TGF-β1-induced EMT increased the expression and activity of p63RhoGEF and GEF-H1, but not VAV2 and EMT, and partial silencing of p63RhoGEF and GEF-H1 expression suppressed EMT as indicated by a decrease in SMA expression. A, protein expression, detected by Western blotting, of p63RhoGEF, GEF-H1, but not LARG, VAV2, or p115RhoGEF, significantly increased with TGF-β1 treatment, n = 12–16. Cells were stimulated with TGF-β1 (250 pm) for 72 h. The protein level of PDZRhoGEF (PRG) was decreased. n.s., not significant. B, GEF-H1, but not VAV2 activity, measured with a RhoG17A mutant to capture active RhoGEFs increased with TGF-β1-induced EMT; n = 3. A trend in increase of p63RhoGEF activity did not reach significance. RhoGEF protein levels were normalized to tubulin. Data are presented as the means ± S.E. C and D, lentiviral shRNAs to p63RhoGEF and GEF-H1 and empty vectors (controls) were introduced into mouse embryonic epicardial cells, and the extent of GEF suppression and SMA protein expression was measured by Western blotting 48 h after stimulation with TGF-β1, n = 3. Expression of SMA was significantly decreased with suppression of p63RhoGEF or GEF-H1.

FIGURE 4.

The cAMP target, Epac, suppresses EMT and cell migration in epicardial cell outgrowth from heart explants. A, Western blot showing that Epac1, p190RhoGAP, and Rap1 proteins were expressed in EMCs and the PE. B, the Epac activator, 007 (50 μm), increased active Rap1·GTP in EMCs. C, immunolabeling showed Rap1 localized to junctional membranes (arrows) and in the cytosol in 007 treated EMCs (panel a) stained with TRITC-labeled phalloidin (panel b) and co-localized with cortical actin (panel c). D, activation of Epac1 with 007 treatment suppressed EMT in epicardial cells grown out from E10.5 heart explants for 48 h in the presence of 50 ng of bFGF showing compact rounder cells outlined by cortical actin (panels c and d). bFGF treatment alone resulted in migratory cells with many processes (panel a) and actin stress fibers (panel b). E, migration of mesenchymal cells grown out from E10.5 heart explants into collagen gels in the presence of 50 ng of bFGF with/without 50 μm 007 for 6 days was retarded in the presence of 007. Counting of cells at different distances from the edge of the heart (distance = 0) is detailed under “Experimental Procedures.” F, Rap1 activity shown as a fraction of total Rap1 at 72 h decreased by 4-fold with TGF-β1-induced EMT in EECs consistent with a fall in Epac activity. Data are the means ± S.E., n = 3. G, epicardial cells grown out from WT and Epac1 null E10.5 heart explants for 48 h in the presence of 50 ng of bFGF, showing that the Epac1 null cells exhibit an increased migration indicated by the greater separation of the nuclei and the increased presence of lamellipodia; data for cell separation were from 6 WT and 9 Epac1 null hearts, number of cells counted were WT = 167 and Epac1 null = 73. The western blot shows absence of Epac1 protein in the Epac1 null cells.

FIGURE 5.

Activation of Epac with 007, partial silencing of Epac with shRNA, inhibition of cAMP phosphodiesterase with IBMX, or inhibition of RhoA by C3 toxin altered TGF-β1-induced EMT in EECs. The epicardial cells were treated with 250 pm TGF-β1 with/without 50 μm 007, 10 μm IBMX, or 3–5 μg/ml C3 toxin for 72 h followed by immunolabeling with antibodies for detection of SM22 and ZO1. TGF-β1 but not 007 treatment alone significantly increased the expression of SM22 protein (C versus B) and disrupted the cell-cell contacts as indicated with ZO-1 staining (G versus F), whereas these TGF-β1-induced changes were suppressed by treatment with either 007 (D and H), IBMX (L), or C3 toxin (P). Partial silencing of Epac1 by 45% ± 17 S.E., n = 8 (panel U), resulted in an increase in SM22 in both control and TGF-β1 treated EECs detected by immunohistochemistry (panels Q–T) consistent with Epac suppression of EMT (three independent experiments).

FIGURE 6.

Rnds 1 and 3 mRNA and protein were expressed in epicardial cells but were suppressed and their membrane/cytosol distribution changed in cells having undergone EMT. A, relative Rnd 1 and 3 mRNA expression in control and TGF-β1-treated EECs; n = 3. n.s., not significant. B, relative Rnd 1 and 3 and p190RhoGAP protein expression in control and TGF-β1-treated EECs n = 3. C, Rnds 1 and 3 were found in both the cytosolic (c) and the Triton-soluble fractions (m) in control EECs but decreased in the membrane fraction with TGF-β1 treatment for 72 h; n = 3. Data are the means ± S.E.

FIGURE 7.

Rnd3 associates with p190RhoGAP in epicardial cells in the epithelial state; overall signaling scheme. A, anti-p190RhoGAP antibody, but not a nonspecific mouse IgG antibody, coimmunoprecipitated (IP) endogenous p190RhoGAP and Rnd 3. Anti-Rnd3 antibody, but not a nonspecific IgG, coimmunoprecipitated endogenous Rnd3 and p190RhoGAP. B, PLA demonstrating a proximity of <40 nm of Rnd3 and p190RhoGAP in EMCs as evidenced by each bright dot. C, control for PLA illustrating that under the same conditions no signal was observed for p190RhoGAP antibody and ROCK antibody. The presence of cells is indicated by the nuclear stain DAP1. D and E, control panels for the PLA assay showing the immunofluorescence of p190 RhoGAP and Rnd3 antibodies in these cells. G, p190RhoGAP activity, as indicated by Tyr-1105 phosphorylation, significantly decreases in the presence of 250 pm TGF-β1 (36 h) in EECs; n = 8. H, scheme of signaling pathways contributing to the maintenance of the proepicardial progenitor phenotype. EMT initiated by growth factors leads to activation of p63RhoGEF and GEF-H1 to exchange GTP for GDP and activate RhoA to stimulate the transcription of SM marker proteins such as SMA, SM myosin, and SM22 and transformation of the proepicardial cells into coronary vascular SM cells. RhoA activity is suppressed and kept in check in the proepicardial cells by the increased activity of p190RhoGAP and Rnd proteins as well as through signaling from cAMP to Epac, the GTP exchange factor for the small GTPase, Rap1, to further down-regulate RhoA activity.