Transforming growth factor-beta (TGF-beta) signaling is mediated by a complex of type I (TBRI) and type II (TBRII) receptors. The type III receptor (TBRIII) lacks a recognizable signaling domain and has no clearly defined role in TGF-beta signaling. Cardiac endothelial cells that undergo epithelial-mesenchymal transformation express TBRIII, and here TBRIII-specific antisera were found to inhibit mesenchyme formation and migration in atrioventricular cushion explants. Misexpression of TBRIII in nontransforming ventricular endothelial cells conferred transformation in response to TGF-beta2. These results support a model where TBRIII localizes transformation in the heart and plays an essential, nonredundant role in TGF-beta signaling.

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-␤ (TGF-␤) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-␤ type III receptor (T␤RIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-␤ and inhibin receptor. Here we demonstrate that T␤RIII functions as a BMP cell surface receptor. T␤RIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-␤. T␤RIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous T␤RIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for T␤RIII function, we demonstrate that BMP-2 specifically stimulates T␤RIII-mediated epithelial to mesenchymal cell transformation. The ability of T␤RIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-␤ signaling suggests a broader role for T␤RIII in orchestrating TGF-␤ superfamily signaling.

Abstract-Transforming growth factor (TGF)␤ receptor III (TGF␤R3), or ␤-glycan, binds all 3 TGF␤ ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGF␤R1, TGF␤R2). TGF␤R3 facilitates signaling via TGF␤R1/TGF␤R2 but also has been suggested to play a unique and nonredundant role in TGF␤ signaling. Targeted deletion of Tgfbr3 revealed a requirement for Tgfbr3 during development of the coronary vessels. Coronary vasculogenesis is significantly impaired in null mice, with few vessels evident and numerous, persistent blood islands found throughout the epicardium. Tgfbr3-null mice die at embryonic day 14.5, the time when functional coronary vasculature is required for embryo viability. However, in null mice nascent coronary vessels attach to the aorta, form 2 coronary ostia, and initiate smooth muscle recruitment by embryonic day 14. Analysis of earlier developmental stages revealed defects in the epicardium. At embryonic day 13.5, these defects include an irregular and hypercellular epicardium with abundant subepicardial mesenchyme and a thin compact zone myocardium. Tgfbr3-null mice also displayed other defects in coronary development, including dysmorphic and distended vessels along the atrioventricular groove and subepicardial hemorrhage. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild-type littermates. These data demonstrate a requirement for Tgfbr3 during coronary vessel development that is essential for embryonic viability. Key Words: coronary vessels Ⅲ transforming growth factor ␤ receptor Ⅲ mice, null C oronary artery disease is responsible for 54% of all cardiovascular disease in the United States. 1 Coronary vessels have a unique derivation from mesothelial cells that form a transitory structure termed the proepicardium. Proepicardial cells are transferred to the heart, form the epicardium, and give rise to endothelial cells, smooth muscle cells, and cardiac fibroblasts (reviewed elsewhere 2,3 ). Endothelial cells derived from the epicardium form a vascular plexus by the process of vasculogenesis. This vascular network attaches to the aorta and recruits epicardially derived mesenchyme to become vascular smooth muscle. The identification of the molecular and cellular processes that regulate coronary vessel development may provide insight into coronary vessel disease and reveal novel therapeutic opportunities.The transforming growth factor (TGF)␤ family of growth factors regulates cell growth and differentiation in the cardiovascular system during both development and disease. 4 -6 Three ligands, TGF␤1, TGF␤2, and TGF␤3, 7-9 bind 4 cell surface proteins. These include two transmembrane serine/ threonine kinase receptors, the type I TGF␤ receptor (TGF␤R1) and the type II TGF␤ receptor (TGF␤R2). 10 -12 Several type I receptors, termed activin receptor-like kinases (ALKs), have been described. TGF␤R2 has a constitutively active cytoplasmic...

Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor ␤ (TGF␤) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGF␤1 or TGF␤2 lose epithelial character and undergo smooth muscle differentiation. To further study TGF␤ Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor 1 (WT1). TGF␤ induced the loss of zonula occludens-1 (ZO-1) and the appearance of SM22␣ and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGF␤ while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGF␤ induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation. Developmental Dynamics 237:366 -376, 2008.

Epithelial-mesenchymal transformation (EMT) occurs during both development and tumorigenesis. Transforming growth factor beta (TGFbeta) ligands signal EMT in the atrioventricular (AV) cushion of the developing heart, a critical step in valve formation. TGFbeta signals through a complex of type I and type II receptors. Several type I receptors exist although activin receptor-like kinase (ALK) 5 mediates the majority of TGFbeta signaling. Here, we demonstrate that ALK2 is sufficient to induce EMT in the heart. Both ALK2 and ALK5 are expressed throughout the heart with ALK2 expressed abundantly in endocardial cells of the outflow tract (OFT), ventricle, and AV cushion. Misexpression of constitutively active (ca) ALK2 in non-transforming ventricular endocardial cells induced EMT, while caALK5 did not, thus demonstrating that ALK2 activity alone is sufficient to stimulate EMT. Smad6, an inhibitor of Smad signaling downstream of ALK2, but not ALK5, inhibited EMT in AV cushion endocardial cells. These data suggest that ALK2 activation may stimulate EMT in the AV cushion and that Smad6 may act downstream of ALK2 to negatively regulate EMT.