Transforming growth factor-beta stimulates epithelial-mesenchymal transformation in the proepicardium

Abstract

The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE-derived cells undergoes epithelial-mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor-beta (TGFbeta) 1 and TGFbeta2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFbeta receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE-derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFbeta stimulates transformation in the PE and suggest that ALK2 partially mediates this effect.

Fig. 1

Effect of TGFβ and FGF on PE explants. A, Representative TGFβ2- and vehicle-incubated PE explants photographed at 24, 48 and 72 hours after harvest. Explants incubated with 200 pM TGFβ2 contain more elongate cells radiating from the explant (arrowhead) as compared to vehicle incubated explants. B, The number of transformed cells was increased significantly in explants incubated with 200 pM of either TGFβ1 or TGFβ2, *=p<0.05. C, EMT in PE explants incubated with 10 ng/ml FGF1 or FGF7 did not differ from vehicle incubated explants. All photomicrographs at 100x.

Fig. 2

TGFβ alters cytokeratin and ZO1 expression in PE cells. PEs were incubated with vehicle, TGFβ1 or TGFβ2 for 24 hours. After fixation, explants were processed to detect either cytokeratin or ZO1 expression by immunofluorescence. A, Epithelial cells in PEs incubated with vehicle express cytokeratin abundantly. B,C, Cells from explants incubated with 200 pM TGFβ1 (B) or TGFβ2 (C) separate from the epithelial sheet, become spindle shaped, and have decreased expression of cytokeratin. D, ZO1 immunoreactivity in vehicle incubated explants is located at the periphery of cells demarcating cell-cell contact points between adjacent epithelial cells. E,F, Cells from explants incubated with 200 pM TGFβ1 (E) or TGFβ2 (F) separated from the epithelial sheet and changed shape. ZO1 immunoreactivity was less prominent at the cell periphery, consistent with a loss of cell-cell contacts and the transition from an epithelial to a mesenchymal cell phenotype. All panels, 400x.

Fig. 3

Expression of TGFβ2, ALK2, ALK5, and Smad6 in PE. Representative sections are depicted that reflect the pattern of expression from stages 14–20. A,B, At stage 18, TGFβ2 mRNA is seen in cells within the body of the PE (asterisks) and in cells forming the primitive epicardium (arrowheads). C, D, ALK2 mRNA in the PE (asterisk) and endocardium (arrow) at stage 18. E, F, ALK5 and G, H, Smad6 are both expressed in the PE (asterisks), shown here at stages 18 and 19, respectively. I, RT-PCR was performed as described in “Experimental Procedures” to amplify specific regions of the mRNAs encoding TGFβ1, TGFβ2, ALK2, ALK5, Smad6 and GAPDH from PEs. GAPDH was not appreciably amplified when the reaction was performed without reverse transcriptase enzyme (-RT) or template (-RNA). All photomicrographs at 200x. avc, atrioventricular cushion; pe, proepicardium; sv, sinus venosus

Fig. 4

ALK2, but not ALK5, promotes epithelial cell activation in PE explants. Explants were infected with adenovirus coding for GFP alone or GFP and constitutively active ALK2 (caALK2) or ALK5 (caALK5). A, Cells expressing caALK2 were less likely to be epithelial and more likely to be activated than cells expressing only GFP (*=p<0.05). B, The distribution of GFP-positive cells in PEs infected with caALK5 virus was similar to the distribution of GFP-positive cells in explants infected with virus encoding only GFP. C, GFP-positive cells were scored as epithelial (arrowheads, left panel), activated (arrowhead, middle panel), or transformed (arrowhead, right panel) as defined in “Results.” Asterisks indicate transformed (left panel) or epithelial (right panel) cells in different focal planes of the same field.

Fig. 5

Smad6 inhibits epithelial cell activation in PE explants. Explants were infected with adenovirus coding for GFP alone or GFP and Smad6. GFP-positive cells were scored as epithelial, activated, or transformed as described in “Results.” Cells overexpressing Smad6 were more likely to be epithelial and less likely to be activated than cells expressing GFP alone (*=p<0.05).

Fig. 6

BMP7 does not increase epithelial cell activation. Explants were infected with adenovirus coding for GFP alone and incubated with 15 nM BMP7 or vehicle. After 72 hours in culture, GFP-positive cells were scored as epithelial, activated, or transformed as described in “Results.” The distribution of GFP-positive cells in PE explants incubated with BMP7 was similar to the distribution of GFP-positive cells in vehicle-incubated explants.

Fig. 7

Model of TGFβ signaling during PE cell EMT. While TGFβ stimulates PE explant cells to undergo EMT, caALK5 is insufficient to recapitulate this response. caALK2 drives PE cells to undergo activation, the first step in EMT, while Smad6 inhibits epithelial cell activation. BMP-7 does not stimulate epithelial cell activation, suggesting ALK2-mediated activation is independent of BMP-7 signaling via the Type II activin receptor (ActRII). These data are consistent with a model where TGFβ signals via ALK2, and possibly a second unidentified pathway, to elicit EMT in PE explants. Data from the endocardial cushion model of EMT suggest that ALK2 signaling may also require TBRIII.