RBP-Jκ/CBF-1 and GC-rich palindromic sites are needed for the activation of the Herp2 promoter by Notch and BMP receptor signaling pathways. (A) Dominant-negative RBP-Jκ/CBF-1 interferes with synergy between Notch and BMP signaling pathways. −1221Herp2-luc was cotransfected with N1ICD, RBP-Jκ and/or RBP(R218H). (B) Cartoon of the luciferase constructs used in Figure 3. t+i: adenovirus major late promoter; luc: luciferase. Closed symbols: wild-type motif; open symbols: mutated motif. Small letters indicate mutated nucleotides. (C) RBP-Jκ/CBF-1 site in Herp2 promoter is important for activation by Notch and BMP signaling pathways. MEECs were transfected with indicated reporters in the absence or presence of N1ICD. (D) MEECs were transfected with indicated reporters in the absence or presence of N1ICD. (E) CBF-1 binding and GC-rich palindromic sites mimic synergy by BMP and Notch signaling pathways. MEECs were transfected with indicated reporters in the absence or presence of N1ICD. (F) The activity of (CBF-1)₄-luc in the presence of N1ICD can not be enhanced by BMP-6 in MEECs. MEECs were transfected with (CBF-1)₄-luc in the absence or presence of N1ICD.

Notch and BMP signaling pathways synergize to induce Herp2 mRNA expression. (A) Northern blot analysis of Herp2 mRNA expression in MEECs challenged with LacZ, caALK5/HA and caALK6/HA in the absence or presence of N1ICD adenoviruses (upper panel). The DNA fragment encoding the C-terminal part of mouse Herp2, which has less identity among other Herp family, was used as a probe. Equal loadings of RNA samples are shown by expression of GAPDH mRNA (middle panel) and ethidium bromide staining pattern of 28S and 18S ribosomal RNAs before Northern blotting (lower panel). Relative intensity of bands corresponding to Herp2 mRNA was normalized using the intensity of bands for GAPDH mRNA. Each value is shown at the bottom. (B) Expression of ectopically expressed HA-tagged caALKs (upper panel) and N1ICD (lower panel). As a loading control, the same blot was stained with anti-actin antibody (lower panel). (C) Herp2 is a direct target gene for BMP. Northern blot analysis of Herp2 mRNA in MEECs stimulated with 100 ng/ml BMP-6 for the indicated times in the absence or presence of 5 μg/ml CHx (upper panel). Equal loadings of RNA samples are shown by expression of GAPDH mRNA (middle panel) and ethidium bromide staining pattern of 28S and 18S ribosomal RNAs before Northern blotting (lower panel).

Herp2 accelerates degradation of Id1 in MEECs. (A) MEECs infected with Flag-Herp2 and/or Flag-Id1 adenoviruses in the absence and/or presence of 10 μM MG-132 were subject to be labeled with Trans[³⁵S-Label]. The cells were lysed at the indicated times. (B) The intensity of bands corresponding to Id1 in (A) without MG-132 was measured using the phosphoimager. (C) The intensity of bands corresponding to Id1 in (A) with MG-132 was measured using the phosphoimager.

Schematic model of the functional role of Herp2 and Id1 in the Notch and BMP receptor signaling pathways in ECs. (i) BMP signaling alone induces Id1 expression, which activates EC migration. (ii) Notch signaling alone induces Herp2 expression. Subsequently, Herp2 inhibits EC migration. (iii) When ECs simultaneously receive both BMP and Notch signaling, Herp2 expression is synergistically induced. Then, Herp2 is able to promote the degradation of Id1. As a consequence, EC migration is efficiently blocked.

The Herp2 promoter is synergistically induced upon activation of the Notch and BMP receptor pathways. (A) C2C12 cells or (C) MEECs were transfected with each reporter construct in the absence or presence of N1ICD. (B) Detection of Notch receptors and their ligands in MEECs. The corresponding cDNA fragment is a main band in each lane. (D) MEECs were transfected with −1221Herp2-luc in the absence or presence of N1ICD or N4ICD. (E) −1221Herp2-luc was cotransfected with Delta1, Jagged1 or Jagged2 in MEECs. (F) −1221Herp2-luc was cotransfected with N1ICD and/or Smad1 in MEECs. (G) MDA-MB468 cells were transfected with −1221Herp2-luc in the presence of N1ICD and/or Smad4.

Notch and BMP signaling pathways cooperate in transcriptional responses via protein–protein interactions that also involve p/CAF. (A) Notch pathway affects BMP-6-induced BRE-luc activity. MEECs were transfected with N1ICD and BRE-luc. (B) BMP/Smad pathway potentiates activated Notch/RBP-Jκ/CBF-1-driven transcriptional response. MEECs were transfected with N1ICD, Smad1 and pGa981-6. (C) N1ICD potentiates transcriptional activity of Smad1 independent of direct binding of Smad1 to DNA. HepG2 cells were transfected with combinations of N1ICD, Gal4, Gal4-Smad1 and Gal4-M1-Luc. (D) p/CAF potentiates the synergy between Notch and BMP signaling pathways. MEECs were transfected with N1ICD, Smad1, p/CAF and −1221Herp2-luc. (E) Activated BMP receptor and p/CAF enhance the interaction between Smad1 and N1ICD. 6xMyc-Smad1, Flag-CBF-1, Flag-p/CAF, caALK6/HA and N1ICD-V5 were transfected in COS7 cells. The cell lysates were immunoprecipitated with anti-V5 antibody. The immunoprecipitates were subjected to Western blotting with anti-myc antibody (upper panel). Using total lysates, the expressions of 6xmyc-Smad1 (second panel), N1ICD-V5 (third panel), caALK6/HA (fourth panel), Flag-p/CAF (fifth panel) and Flag-CBF-1 (lower panel) are shown. (F) BMP-6 and/or p/CAF enhance the interaction between Smad5 and N1ICD. 6xMyc-N1ICD and Flag-p/CAF were transfected in MEECs. The cells were stimulated with 100 ng/ml BMP-6 2 h before lysis. The cell lysates were immunoprecipitated with anti-Smad5 antibody. The immunoprecipitates were subjected to Western blotting with anti-myc antibody (upper panel). Using total lysates, the expressions of Flag-p/CAF (second panel), 6xMyc-N1ICD (third panel) and endogenous Smad5 (lower panel) are shown. (G) N1ICD potentiates the ability of Smad5 to bind to SBE. 6xMyc-N1ICD was transfected in MEECs. The cells were stimulated with 100 ng/ml BMP-6 2 h before lysis. After DNAP, Western blotting was performed with anti-Smad5 (upper panel) or anti-myc antibody (second panel). Using total lysates, the expressions of endogenous Smad5 (fourth panel), 6xMyc-N1ICD (fifth panel), phospho-Smad1/5/8 (sixth panel), endogenous Smad3 (seventh panel) and phospho-Smad2 (lower panel) are shown. As a positive control, the lysates from MEECs stimulated with 5 ng/ml TGF-β for 2 h were used. Then, association of Smad3 with SBE was observed using anti-Smad3 antibody (third panel).

Activation of Notch and BMP receptors and their downstream targets regulate EC migration. (A) Effect of N1ICD in the absence or presence of caALK6 or caALK5 on the migration of MEECs. MEECs infected with the indicated adenoviruses were used. The cells were infected with the same amounts of adenoviruses (a moi of 500). The value for the migration in MEECs infected with both GFP and LacZ was set as 100% (upper panel). Using total lysates, the expressions of N1ICD (middle panel) and caALKs (lower panel) are shown. (B) Effect of Herp2 on the migration of MEECs. MEECs infected with adenoviruses expressing LacZ and/or Flag-Herp2 were used. The cells were infected with the same amounts of adenoviruses (a moi of 400). Using adenovirus expressing LacZ, total amounts of adenoviruses were adjusted. The value for the migration in MEECs infected with LacZ alone was represented as 100% (upper panel). Using total lysates, the expressions of Herp2 (middle panel) and actin (lower panel) are shown. (C) Herp2 inhibits Id1-induced cell migration. MEECs infected with adenoviruses expressing LacZ, Flag-Herp2 and Flag-Id1 were used in a migration assay. The cells were infected with the same amounts of adenoviruses (a moi of 800). Using adenovirus expressing LacZ, total amounts of adenoviruses were adjusted. The number of cells migrated in MEECs infected with LacZ alone was set at 100% (upper panel). Cell lysates of MEECs infected with adenoviruses expressing Flag-Herp2 (second panel) and Flag-Id1 (third panel) were subjected to Western blotting with anti-Flag M5 antibody. To show equal loading of each sample, anti-actin antibody (lower panel) was used. (D) Overexpression of Herp2 decreases caALK6-induced Id1 expression. MEECs infected with adenoviruses expressing caALK6/HA and Flag-Herp2 were used. The cells were infected with the same amounts of adenoviruses (a moi of 800). Using adenovirus expressing LacZ, total amounts of adenoviruses were adjusted. Cell lysates of MEECs infected with adenoviruses were subjected to Western blotting with anti-Id1 (upper panel), anti-Herp2 (middle panel) and anti-HA (lower panel) antibodies, respectively. (E) Effect of Herp2 on Id1 expression in MEECs or C2C12 cells. MEECs (left panel) or C2C12 cells (right panel) were transfected with Flag-Id1 and different amounts of Myc-Herp2. Total cell lysates were used for the detection of Myc-Herp2 (upper panel), Flag-Id1 (middle panel) and actin (lower panel). (F) Interaction of Herp2 with Id1 in COS7 cells. 6xMyc-Herp2 and Id1 were transfected in COS7 cells, and the cell lysates were immunoprecipitated with anti-Id1 antibody. The immunoprecipitates were subjected to Western blotting with anti-myc antibody (upper panel). Using total lysates, the expressions of 6xMyc-Herp2 (second panel) and Id1 (lower panel) are shown.

Herp2 acts as a critical switch gene for ECs downstream of Notch and BMP signaling pathways. (A) Effect of Herp2-specific siRNA on the migration of MEECs challenged with adenoviruses expressing N1ICD and/or caALK6/HA. MEECs infected with adenoviruses expressing LacZ, N1ICD and caALK6/HA were used in a migration assay. The cells were infected with 200 moi of caALK6 and/or 400 moi of N1ICD. Using adenovirus expressing LacZ, total amounts of adenoviruses were adjusted. The value for the migration in MEECs infected with LacZ alone when treated with the control siRNA was represented as 100%. (B) The expression of N1ICD and caALK6 in MEECs. Anti-N1ICD (upper panel) and anti-HA antibodies (lower panel) were used to detect the expression of N1ICD and caALK6, respectively. (C) Detection of Herp2 mRNA from the cells treated with Herp2-specific siRNAs. RT–PCR was performed with primers specific for Herp2 and actin.