BMP-2 and TGFβ2 Shared Pathways Regulate Endocardial Cell Transformation

Abstract

Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-β receptor (TGFβR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFβR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFβ signaling pathways in TGFβR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFβR3-dependent EMT stimulated by TGFβ2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFβR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFβ2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFβR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFβR3-dependent endocardial cell EMT stimulated by either TGFβ2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.

Common pathways regulate Type III TGFβ receptor-dependent cell invasion in epicardial and endocardial cells

“…Targeting TGFβR3 in cardiac cushion explants revealed a requirement of the receptor for endocardial cell invasion in vitro [10] which is dependent on the 3 C-terminal amino acids of TGFβR3 and interaction with GIPC [20]. Experiments in both endocardial cells and epicardial cells have also revealed a role for the Par6/Smurf/Rho pathway and multiple ALKs in cell invasion [20, 28, 31, 37, 38]. Here, incubation of atrioventricular cushion explants, which include endocardial cells that express TGFβR3 that undergo EMT and cell invasion in response to TGFβ released from associated cardiac myocytes, with ALK-specific small molecule inhibitors (2µM DMH1; ALK2 & ALK3 or 2.5µµ SB431542; ALK5) resulted in a 60% decrease in endocardial cell invasion when compared to vehicle incubated explants (Fig.…”

“…We next asked directly if ALK2 and ALK3 were required downstream of the TGFβR3 by overexpressing GFP or GFP and TGFβR3-FL in ventricular explants, which include endocardial cells that lack TGFβR3 expression, and EMT was measured by scoring the percentage of total GFP-positive cells as epithelial, activated or transformed (Fig. 3D) as described previously [15, 20, 37, 38]. Ventricular endocardial cells overexpressing GFP alone show a small percentage of cells that undergo EMT as measured by the percentage of epithelial cells, activated cells (elongated cells on surface of gel), and transformed cells (cells that have entered the gel).…”

“…In endocardial cells, as in epicardial cells, TGFβR3-T841A stimulates ligand-independent cell invasion. We used a combination of small molecule inhibitors and siRNA to confirm that ALK2, ALK3, and ALK5, known mediators downstream of TGFβR3 [37], are required for ligand-independent invasion in endocardial cells. These results suggest that TGFβR3 activation results in a coordinated and concomitant stimulation of multiple ALKs to regulate cell invasion in both epicardial and endocardial cells.…”

BMP2 rescues deficient cell migration inTgfbr3−/−epicardial cells and requires Src kinase

“…[12][13][14] The role of BMPs in endocardial cell transformation have been heavily investigated revealing a requirement for TGFbR3 in valve development. [15][16][17] TGFb1, BMP7 and BMP2, directly bind to TGFbR3. 34 TGFbR3 is required for BMP2 activation of Smad1 (canonical signaling) and BMP2-dependent endocardial cell transformation.…”

Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development

“…For example, BMP2 and TGFβ2 can bind to Betaglycan to induce EMT in chick ventricular and AVC explant assays [174,175], which suggests this interaction plays an important role in AVC cushion formation. Loss of ALK2 in endothelium leads to reduced phosphorylation of TGFβ and BMP Smads [45] and indicates either that loss of BMP signalling reduces TGFβs expression (as mentioned earlier) or that BMP signalling can induce TGFβ Smads specifically via different combinations of receptor-ligand complexes or vice versa.…”