The ubiquitin ligase Nedd4-1 is required for heart development and is a suppressor of thrombospondin-1

Abstract

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.

FIGURE 1.

Generation of the Nedd4-1 knock-out (Nedd4-1^−/−,trap) mice. A, schematic representation of Nedd4-1, highlighting the insertion locus of the trapping cassette (in an intron within the C2 domain region). B, PCR analysis performed on ES cells and embryonic tissues to genotype the wild type (+/+), Nedd4-1^+/−,trap, and Nedd4-1^−/−,trap mice. C, immunoblot analysis for Nedd4-1 of MEF cells and embryos from the wild type, Nedd4-1^+/−,trap, and Nedd4- 1^−/−,trap mice, demonstrating the loss of Nedd4-1 in the Nedd4-1^−/−,trap mice and MEFs. D, immunoblot analysis for Nedd4-1 and Nedd4-2, demonstrating the loss of Nedd4-1, but not Nedd4-2, in the hearts of Nedd4-1^−/−,trap mice. E, LacZ staining revealing the expression of knock-out cassette (which contains the β-gal gene) in the Nedd4-1^+/−,trap and Nedd4-1^−/−,trap E11.5 embryos (blue).

FIGURE 2.

Pattern of endogenous expression of Nedd4-1 in embryos and hearts of Nedd4-1^+/−,trap mice. A, sagittal sections showing the pattern of expression of Nedd4-1 as revealed from LacZ staining (blue) of E10.5 and E13.5 Nedd4-1^+/−,trap embryos. B, cross-section of the heart of E10.5 Nedd4-1^+/−,trap embryos, demonstrating Nedd4-1 (LacZ, blue) expression in the myocardium.

FIGURE 3.

Heart defects and vasculature abnormalities in the Nedd4-1^−/−,trap embryos. A, H&E staining of cross-sections from E13.5 WT ((+/+), upper panels) and Nedd4-1^−/−,trap ((−/−), lower panels) embryos, revealing DORV and ventricular septal defect in the Nedd4-1^−/−,trap embryos. B, heart defects in the Nedd4-1^exon9.10 embryos (−/−) relative to WT (+/+), including a common atrioventricular canal defect in some of the knock-out embryos. In A and B, RV, right ventricle; LV, left ventricle; A, atrium; Ao, aorta; PA, pulmonary artery; VSD, ventricular septal defect; CAVC, common atrioventricular canal. More detailed serial sections are shown in supplemental Fig. S1. C, PECAM-1 staining of the vasculature of E10.5 WT (+/+) and Nedd4-1^−/−,trap (−/−) embryos followed by OPT analysis. The head region is shown, demonstrating fewer and flatter veins in the cephalic plexus of the knock-out embryos (red circles). A full three-dimensional view of vasculature in the whole embryo is depicted in supplemental Movies S1 and S2.

FIGURE 4.

Levels of Tsp-1 are increased in the Nedd4-1^+/−,trap MEFs and embryos. A and B, immunoblot analysis of Tsp-1 (either the monomer or trimer (native) forms of the protein) in the WT (+/+), Nedd4-1^+/−,trap (+/−), and Nedd4-1^−/−,trap (−/−) MEFs (A) or E12.5 embryos (B). For the left panels in A and B, nonreducing conditions and gels were used to observe the native Tsp-1 trimers. Note the increase in Tsp-1 expression in the knock-out embryos and MEFs (in A and B) observed when probing for either the native/trimeric or monomeric Tsp-1. C, similar pattern expression of Nedd4-1 and Tsp-1 in the heart. Embryonic hearts (E13.5, +/−) were immunostained for Tsp-1 and β-gal (Nedd4-1) in parallel, using monoclonal anti Tsp-1 and polyclonal anti β-gal antibodies, respectively. The control panel shows no primary antibodies. Scale bar, 20 μm.

FIGURE 5.

Elevated transcription from the Tsp-1 promoter in the Nedd4-1^−/−,trap MEFs. A, luciferase activity reporting Tsp-1 promoter activity in WT (+/+) or Nedd4-1^−/−,trap (−/−) MEFs. The Tsp-1 promoter was expressed from the pGL3 vector in the presence/absence of the indicated amounts of c-Myc cDNA. Shown is a representative experiment, performed in duplicate. **, p ≤ 0.01, unpaired t test. In three separate experiments, each performed in duplicate, there was a 3.3-fold increase in Tsp-1 transcription in the Nedd4-1^−/−,trap relative to WT MEFs (p = 3 × 10⁻⁹, unpaired t test). B, semi-quantitative PCR (sqPCR) revealing increased mRNA levels in Nedd4-1^−/−,trap MEFs relative to WT. Quantification of mRNA levels, analyzed by quantitative PCR, revealed 3.98-fold increase in mRNA of Tsp-1 in the Nedd4-1^−/−,trap MEFs relative to WT MEFs.

FIGURE 6.

Aspirin reduces Tsp-1 levels but does not correct the heart defects. A, WT (+/+) and Nedd4- 1^−/−,trap (−/−) MEFs were treated with the indicated amounts of aspirin for 12 h, and the amount of Tsp-1 was analyzed by immunoblotting. The same results were obtained after aspirin treatment for 4 or 8 h (not shown). B, aspirin treatment of pregnant Nedd4-1^+/−,trap mothers causes reduction in the levels of Tsp-1 in the embryos. Pregnant mothers were treated with aspirin in the drinking water, embryos were harvested at E12.5, and their tissues were analyzed by immunoblotting for levels of Tsp-1. C, the heart defects in the Nedd4- 1^−/−,trap embryos persist after aspirin treatment. Aspirin-treated embryos (E18.5) were harvested, and tissue sections were analyzed by H&E for heart integrity. Shown are the persistent defects in the heart despite aspirin treatment. RA, right atrium; LA, left atrium; AVSD, atrioventricular septal defect; CAVC, common atrioventricular canal; Pap, papillary muscle.

FIGURE 7.

Bleeding in the Nedd4-1^−/−,trap embryos. A, whole Nedd4-1^−/−,trap embryo (−/−), E12.5, revealing bleeding, mainly in the head area. B, H&E staining of E13.5 embryos showing blood vessel rupture and bleeding in the (−/−) but not WT (+/+) embryos.