The Delta intracellular domain mediates TGF-beta/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway

Abstract

Delta is a major transmembrane ligand for Notch receptor that mediates numerous cell fate decisions. The Notch signaling pathway has long been thought to be mono-directional, because ligands for Notch were generally believed to be unable to transmit signals into the cells expressing them. However, we showed here that Notch also supplies signals to neighboring mouse neural stem cells (NSCs). To investigate the Notch-Delta signaling pathway in a bi-directional manner, we analyzed functional roles of the intracellular domain of mouse Delta like protein 1 (Dll1IC). In developing mouse NSCs, Dll1IC, which is released from cell membrane by proteolysis, is present in the nucleus. Furthermore, we screened for transcription factors that bind to Dll1IC and demonstrated that Dll1IC binds specifically to transcription factors involved in TGF-beta/Activin signaling--Smad2, Smad3 and Smad4--and enhances Smad-dependent transcription. In addition, the results of the present study indicated that over-expression of Dll1IC in embryonic carcinoma P19 cells induced neurons, and this induction was blocked by SB431542, which is a specific inhibitor of TGF-beta/Activin signaling. These observations strongly suggested that Dll1IC mediates TGF-beta/Activin signaling through binding to Smads and plays an important role for bi-directional Notch-Delta signaling pathway.

Figure 1.

Alignment of amino acid sequences of Delta homologs from various vertebrate species and Drosophila was generated using DNASIS program (Hitachi Software, Japan). Amino acids that are identical to those of mouse Dll1 are shown in black. Amino acids that differ from those of mouse Dll1 are shown in red. Gaps that were introduced for the alignment are shown as lines. The transmembrane region is shaded gray. RKRP predicted nuclear localization signal is shaded yellow, ATEV PDZ-binding motif is shaded green. M-Dll1: mouse Delta1, C-Dll1: chicken Delta1, X-Dll1: Xenopus Delta1, Z-Dll1: zebra fish DeltaD, D-D: Drosophila Delta, M-Dll3: mouse Delta3.

Figure 2.

Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with pCDNA-transfected COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.***P < 0.001, Student's t-test (versus appropriate controls).

Figure 3.

Dll1 was cleaved and Dll1IC presented in the nucleus. (a) Aliquots of 20 μg of lysates from NSCs and aliquots of 30 μg of subcellular protein fractions were subjected to western blotting with anti-Dll1IC antibody. From the sizes of the bands, band 1 seemed to be the full-length mouse Delta (Dll1), while the others appeared to be cleavage products of Dll1. The band with the lowest molecular weight (band 2) seemed like Dll1IC (18 kDa) released from the cell membrane. S, lysates from NSCs; N, nuclear fraction; C, cytoplasm fraction; M, membrane fraction. (b) NSCs were stained with anti-Dll1IC antibody (1) and DAPI (2). Merged image (3) shows that immunoreactivity had accumulated in the nucleus. However, inhibitor of γ-secretase strongly blocked these accumulations; stained with anti-Dll1IC antibody (4), DAPI (5), merged image (6).

Figure 4.

Co-culture of Dll1-expressing cells with Notch1-expressing cells. Each expression vector for full-length Notch1, full-length Dll1 or vector alone was transfected into COS7 cells. Forty-eight hours after transfection, Dll1-expressing cells (Dll1/COS7) were mixed with Notch-expressing cells (Notch/COS7) or control cells (CTRL/COS7) at a ratio of 1:8 and cultured for indicated times. DAPT was added to the culture medium as an inhibitor of γ-secretase. Dll1 processing was detected by western blotting with anti-Dll1IC antibody. From the size of the band, the 90 kDa protein (top band) seemed to be full-length Dll1, while the other appeared to be cleaved products of Dll1. Cleavage products of Dll1 were remarkably increased by co-culture with Notch1-expressing COS7 cells and these increases were strongly inhibited by DAPT. The same filter was also reacted with anti-G3PDH antibody as an internal control, showing that almost the same amount of cell lysates were processed for western blotting (bottom of the figure).

Figure 5.

Dll1IC bound to Smad2, Smad3 and Smad4. (a) Result of screening for transcription factors that bind to Dll1IC. Note that Smad binding sequences showed strong signals (boxed). (b) Signal from Smad binding sequences (boxed) were enhanced by the addition of recombinant Dll1IC protein to the nuclear extract before immunoprecipitation. Signals from Pax-5 binding sequence and mineral corticoid response element (MRE) were also enhanced by the addition of recombinant Dll1IC protein. On the other hand, signals from NF-κB binding site and NF-E2 binding sequence were disappeared by the addition of recombinant Dll1IC protein. Spots along the right and bottom side of arrays are markers for alignment. (c) COS7 cells were transiently co-transfected with each expression vector for 8 Smads and Dll1IC expression vector with or without expression vectors for constitutive activated receptor (caALK5-HA or caALK6-HA). Forty-eight hours after transfection, lysates from co-transfected COS7 cells were subjected to immunoprecipitation (IP) with the indicated antibodies, followed by western blotting (WB). αDll1IC, rabbit anti-Dll1IC antibody; αFlag, mouse monoclonal anti-FLAG M2 antibody. Levels of expression of Smads and Dll1IC were determined by western blotting and are shown in the bottom two panels.

Figure 6.

Dll1IC enhanced Smad-dependent transcription. (a) HepG2 cells were transiently transfected with the indicated amounts of Dll1IC expression vector and 9 × CAGA-Luc promoter–reporter plasmid, which responds specifically to Smad3. Cells were stimulated by the addition of TGF-β to the medium. Luciferase activities were normalized using the pRL-CMV vector that was always co-transfected as an internal control. Values are shown as the means ± S.D. of four independent experiments. (b) 9 × CAGA-Luc promoter–reporter plasmid was transfected into P19 cells stably over-expressing Dll1IC (Dll1IC/P19). P19 cells, which carry the vector alone (pCDNA/P19), were used as control. After transfection, cells were stimulated with TGF-β at various concentrations. Luciferase activities were normalized using the pRL-CMV vector that was always co-transfected as an internal control. Values are shown as the means ± S.D. of four independent experiments.

Figure 7.

Over-expression of Dll1IC in P19 cells induced neurons. (a) Dll1IC-expressing P19 cells (Dll1IC/P19) and those carrying vector alone (pCDNA/P19) were cultured without RA. For inhibition experiments, Dll1IC/P19 cells were cultured with 1 μM SB431542. (1)–(3) are phase-contrast micrographs, and (4)–(6) show the results of staining with Tuj1, a marker of neurons (red) and DAPI (blue). (b) Cells were cultured as described and lysates were prepared from subcultured cells (1), aggregated cells (2) and re-plated cells (3). Aliquots of 10 μg of lysates were subjected to western blotting with Tuj1.