Abstract
Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming growth factor-beta (TGFbeta) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Recently Par6c (Par6) has been shown to function downstream of TGFbeta to recruit Smurf1, an E3 ubiquitin ligase, which targets RhoA for degradation to control apical-basal polarity and tight junction dissolution. We tested the hypothesis that Par6 functions in a pathway that regulates endocardial cell EMT. Here we show that the Type I TGFbeta receptor ALK5 is required for endocardial cell EMT. Overexpression of dominant negative Par6 inhibits EMT in AVC endocardial cells, whereas overexpression of wild-type Par6 in normally non-transforming ventricular endocardial cells results in EMT. Overexpression of Smurf1 in ventricular endocardial cells induces EMT. Decreasing RhoA activity using dominant negative RhoA or small interfering RNA in ventricular endocardial cells also increases EMT, whereas overexpression of constitutively active RhoA in AVC endothelial cells blocks EMT. Manipulation of Rac1 or Cdc42 activity is without effect. These data demonstrate a functional role for Par6/Smurf1/RhoA in regulating EMT in endocardial cells.
Highlights
Congenital heart defects occur at a rate of almost 1 in 100 live births [1, 2] and are responsible for the largest proportion of deaths due to birth defects in the first year of life and remain a major cause of death during childhood [3]
Using an in vitro model of endocardial cell transformation we demonstrate that ALK5 activity is required for epithelial-mesenchymal transformation (EMT) in atrioventricular cushion (AVC) explants
Each inhibitor or vehicle was added to the collagen gel 12 h prior to the placement of AVC explants isolated from Stage 14 chick embryos
Summary
Construction of Adenoviral Constructs—Adenoviruses were generated using the pAdEasy system [33]. After 24 h, ventricular or AVC explants were excised from the infected hearts and placed in culture as described previously [35] (supplemental Fig. S1, A and B). The appropriate final concentration (for 300 l of total volume) of siRNA derived from 21-bp RNA (target sequence) was added to a final volume of 100 l. These two tubes were mixed and incubated for 15 min at room temperature to allow siRNA complexes to form. For control siRNA, a scrambled 21-oligonucleotide template containing the same number of the bases of the RhoA siRNA target that did not blast to any gene in the chicken genome, 5Ј-AGACTGTCGCGTGCTCTGTCC-3Ј was used as previously described [36].
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