Differential regulation of transforming growth factor beta signaling pathways by Notch in human endothelial cells

Abstract

Notch and transforming growth factor beta (TGFbeta) play critical roles in endothelial-to-mesenchymal transition (EndMT), a process that is essential for heart development. Previously, we have shown that Notch and TGFbeta signaling synergistically induce Snail expression in endothelial cells, which is required for EndMT in cardiac cushion morphogenesis. Here, we report that Notch activation modulates TGFbeta signaling pathways in a receptor-activated Smad (R-Smad)-specific manner. Notch activation inhibits TGFbeta/Smad1 and TGFbeta/Smad2 signaling pathways by decreasing the expression of Smad1 and Smad2 and their target genes. In contrast, Notch increases SMAD3 mRNA expression and protein half-life and regulates the expression of TGFbeta/Smad3 target genes in a gene-specific manner. Inhibition of Notch in the cardiac cushion of mouse embryonic hearts reduces Smad3 expression. Notch and TGFbeta synergistically up-regulate a subset of genes by recruiting Smad3 to both Smad and CSL binding sites and cooperatively inducing histone H4 acetylation. This is the first evidence that Notch activation affects R-Smad expression and that cooperative induction of histone acetylation at specific promoters underlies the selective synergy between Notch and TGFbeta signaling pathways.

FIGURE 1.

NICD affects the expression and TGFβ-induced phosphorylation of R-Smads. HMEC were transduced with empty vector or NICD. A, protein expression of R-Smads in whole cell lysates was examined by immunoblotting. B, R-Smad band intensity was measured by densitometry and normalized to tubulin. The relative density of R-Smad proteins was expressed as the -fold changes relative to the vector control and shown as the mean ± S.E. of three or four independent experiments. *, p < 0.05. C, HMEC transduced with empty vector or NICD were left untreated (UT) or treated with 2.5 ng/ml TGFβ1 for 2 h. R-Smad mRNA level was examined by qRT-PCR and normalized to GAPDH. mRNA levels were expressed as -fold changes relative to the untreated vector samples and shown as mean ± S.E. of three independent experiments. *, p < 0.05. D, HMEC transduced with empty vector or NICD were left untreated or treated with 2.5 ng/ml TGFβ1 for 1 h. The amount of total and phosphorylated R-Smad proteins in whole cell lysates was examined by immunoblotting. Tubulin was used as a loading control. E, R-Smad protein level in cytosolic and nuclear fractions was examined by immunoblotting. Tubulin and poly(ADP-ribose) polymerase (PARP) were used as loading controls for cytosolic and nuclear fractions, respectively.

FIGURE 2.

NICD differentially affects TGFβ/Smad target gene expression. HMEC transduced with empty vector or NICD were left untreated (UT) or treated with 2.5 ng/ml of TGFβ1 for 2 h. The mRNA level of target genes of TGFβ/Smad1 (A), TGFβ/Smad2 (B), and TGFβ/Smad3 (C and D) was examined by qRT-PCR and normalized to GAPDH. mRNA levels were expressed as -fold changes relative to the untreated vector samples and shown as mean ± S.E. of three independent experiments. *, p < 0.05.

FIGURE 3.

Effects of Dll4-induced Notch activation on TGFβ signaling. HMEC cocultured with Dll4-expressing HMEC at a 1:1 ratio were left untreated (UT) or treated with 2.5 ng/ml TGFβ1 for 1 h (B and C) or 2 h (A, D, E, F, and G). A, the mRNA level of Smad1, Smad2, Smad3, and Smad5 was examined by qRT-PCR and normalized to GAPDH. mRNA levels were expressed as -fold changes relative to the untreated vector sample and shown as mean ± S.E. of three independent experiments. *, p < 0.05. B, the amount of total and phosphorylated R-Smad proteins in whole cell lysates was examined by immunoblotting. Tubulin was used as a loading control. C, the R-Smad proteins in cytosolic and nuclear fractions were examined by immunoblotting. Tubulin and poly(ADP-ribose) polymerase (PARP) were used as loading control for cytosolic and nuclear fractions, respectively. D–G, the mRNA level of target genes of TGFβ/Smad1 (D), TGFβ/Smad2 (E), and TGFβ/Smad3 (F and G) was examined by qRT-PCR and normalized to GAPDH. mRNA levels were expressed as -fold changes relative to the untreated vector samples and shown as mean ± S.E. of three independent experiments. *, p < 0.05.

FIGURE 4.

Inhibition of Notch activity reduces Smad3 expression in mouse embryonic hearts. Notch activity was inhibited by induction of dnMAML1 at E8.5 or E9.5, and cells in the cardiac cushion were stained by DAPI at E10.5 (A) and quantified using ImageJ software (B). A, atrium; V, ventricle; AVC, atrioventricular cushion. Bars, 50 μm. Ten sections of control hearts and six sections of dnMAML1 hearts at 24 h and three sections of control hearts and six sections of dnMAML1 hearts at 48 h were analyzed. Cardiac cushion cell number per section represents mean ± S.E. *, p < 0.05 compared with controls. C, Notch activity was inhibited by induction of dnMAML1 at E9.5, and Smad3 expression in cardiac cushion cells was examined at E10.5 by immunofluorescence staining using Smad3 antibody. Red, Smad3; green, CD31 (endocardium); blue, DAPI (nuclei). Bars, 50 μm. D, Smad3 expression in cardiac cushion cells from 13 sections of six control hearts and 15 sections of five dnMAML hearts were examined by immunofluorescence staining. The intensity of Smad3 staining was analyzed using ImageJ software, compared with the number of nuclei in the same area, and expressed as intensity per cell. Intensity per cell (arbitrary units) represents the means ± S.E. *, p < 0.05.

FIGURE 5.

NICD interacts with Smad3 and increases protein stability. A, HMEC transduced with empty vector or NICD were treated with 50 μg/ml cycloheximide for various times. Smad1, Smad2, and Smad3 protein level in whole cell lysates was examined by immunoblotting. The density of the Smad3 bands was measured by densitometry and normalized to tubulin. The relative density of Smad3 protein at each time point is shown as the mean ± S.E. of four independent experiments. *, p < 0.05. B, 293T cells were transiently transfected with FLAG-Smad3 (2 μg) with or without cotransfection of various amounts of NICD-HA (1, 2, or 3 μg). Empty vector was used to equalize total plasmid concentration for each transfection. FLAG-Smad3 level was examined using anti-Smad3 or anti-FLAG antibodies. NICD expression was detected using anti-HA antibody. Tubulin was used as a loading control. The density of the FLAG-Smad3 and NICD bands was measured by densitometry and normalized to tubulin. The relative density of FLAG-Smad3 and NICD is shown as the mean ± S.E. of three independent experiments. The density of FLAG-Smad3 in cells transfected with FLAG-Smad3 alone and that of NICD in cells transfected with the lowest amount of NICD were designated as 1 for FLAG-Smad3 and NICD, respectively. *, p < 0.05. C, 293T cells were transiently transfected with Smad3, NICD-HA, or both for 48 h. Physical interaction between Smad3 and NICD was examined in the whole cell lysates by co-immunoprecipitation using anti-Smad3 or anti-HA antibodies. IP, immunoprecipitation; IB, immunoblot.

FIGURE 6.

Synergistic effect between Dll4 and TGFβ signaling requires both ALK5 and Notch activation. HMEC with or without Dll4 coculture were pretreated with 10 μm SB431542 (ALK5 kinase inhibitor) or 10 μm DAPT (γ-secretase inhibitor) overnight and then left untreated (UT) or treated with 2.5 ng/ml TGFβ1 for 4 h in the presence of the inhibitors. ANKRD1, Snail, Smad3, and phospho-Smad3 expression was examined in whole cell lysates by immunoblotting. Tubulin was used as a loading control.

FIGURE 7.

Smad3 occupancy on SBE and CSL sites in the promoters of Smad3/CSL target genes. HMEC with or without Dll4 co-culture were left untreated or treated with TGFβ1 for 1 h. Smad3 occupancy on SBE and/or CSL sites in PAI1 (top), ANKRD1 (middle), and HEY1 promoter (bottom) was examined by Smad3 ChIP with IgG as a negative control. ChIP-qPCR was conducted using primers that amplify the SBE or CSL sites (see supplemental Tables 1 and 3 for SBE and CSL sites and primer sequences). Smad3 occupancy on these sites was normalized against the respective input DNA and expressed as a percentage of input DNA. Values were shown as mean ± S.E. of four independent experiments. *, p < 0.05.

FIGURE 8.

Effects of TGFβ and Dll4-induced Notch activation on histone modification. HMEC with or without Dll4 co-culture were left untreated or treated with TGFβ1 for 2 h. Histone H4 acetylation (AcH4) (A) and H3K4Me3 (B) were examined by ChIP followed by qPCR, using anti-acetyl-histone H4 and anti-histone H3 (trimethyl Lys⁴) antibodies, respectively, with IgG as a negative control. ChIP-qPCR was conducted using primers that amplify the proximal promoter regions and/or 5′-ends of the PAI1 (top), ANKRD1 (middle), and HEY1 genes (bottom) (see supplemental Table 4 for primer sequences). The enrichment of these regions was calculated as a percentage of the respective input DNA concentration and expressed as relative signal after normalization against the untreated vector samples (designated as 1). Values are shown as the mean ± S.E. of four independent experiments. *, p < 0.05.