br COMP leads to activation of
COMP leads to activation of Notch3 113
Fig. 3. Notch3 is activated due to COMP expression in breast cancer cells. (A) Volcano plot of quantitative real time PCR Array of Notch pathway related genes in cDNA produced from MDA-MB-231 Necrosulfonamide expressing COMP compared with the control cells. (B) Luciferase reporter assay showed increased activity of the Notch pathway activity in MDA-MB-231 and BT-20 cells that express COMP compared to control cells. (C) Western blot analysis of total cell lysates of MDA-MB-231 cells immunodetecting Notch1, Notch2, Notch3, with β-tubulin used as a loading control. (D) Expression of mRNA coding for Notch3 is activated by the expression of COMP protein in MDA-MB-231 cells. Two-way ANOVA Bonferroni's multiple comparisons test was used (*b0.05, **b0.01, ***b0.001, ****b0.0001). NICD: Notch Intracellular Domain, NEXT: Notch Extracellular Truncated, FL: Full-length protein.
COMP increases the interaction between Notch3 and Jagged1
In cartilage, pentameric COMP acts as an adaptor protein bringing together different collagen fibers. Therefore, we hypothesized that COMP may also mediate the interaction between Notch3 and its ligand, Jagged1. Indeed, a proximity ligation assay (PLA) indicated that COMP interacts with both Notch3 (Figs. 4A, B, S3A) and Jagged1 (Figs. 4A, C, S3A) in the MDA-MB-231 and BT-20 cells. These observations were confirmed using ELISAs detecting Notch3-COMP (Fig. 4D) and Jagged1-COMP (Fig. 4E) interactions. In both cases, interactions were enhanced in the COMP-expressing cells compared with the control cells. Furthermore, COMP was co-immunoprecipitated with Notch3 and Jagged1 from COMP-expressing MDA-MB-231 and BT-20 cells, which were transiently transfected with Myc-tagged Jagged1 or N-terminally
FLAG-tagged Notch3, confirming the interactions of Jagged1, Notch3, and COMP (Fig. 4F). Finally, the interaction between Notch3 and Jagged1 was enhanced in COMP-expressing cells compared to control cells (Figs. 4G, S3A). Interestingly, supplemen-tation of control cells with recombinant COMP signifi-cantly increased the interaction between Notch3 and Jagged1 in both MDA-MB-231 and BT-20 cell lines (Figs. 4G, S3A).
Notch3 activation leads to cross-talk with other molecular pathways
COMP may affect cells both during intracellular processing and after secretion and rebinding to the cell membrane . During 48 h of culture, MDA-MB-231 and BT-20 cells secrete approximately 6 μg/ml and 2.3 μg/ml COMP, respectively, to the supernatant (Fig. S2C). In order to confirm that surface-bound
114 COMP leads to activation of Notch3
Fig. 4. COMP acts as an adaptor protein facilitating the interaction between Notch3 and Jagged1. (A) Confocal z-stacked maximum projection representative images of proximity ligation assay (PLA) of MDA-MB-231 and BT-20 cells. (B) Results of PLA showing that COMP interacts with Notch3 and (C) with Jagged1 in two different cell lines. In an ELISA assay an anti-Notch3 antibody was used to capture the Notch3 protein complexes (D) and an anti-Jagged1 antibody was used to capture the Jagged1 protein complexes (E), lysed under non-denatured conditions and an anti-COMP antibody applied to detect the COMP interactions with Notch3 and Jagged1. (F) MDA-MB-231 or BT-20 cells expressing COMP and their respective control cells were transiently transfected with constructs that express Notch3 protein tagged with FLAG and Jagged1 tagged with c-Myc. Cell lysates were immunoprecipitated using antibodies against the respective tags followed by immunodetection of COMP protein. (G) Interaction between Notch3 and Jagged1 was enhanced in the COMP-expressing cells and also by the supplementation of cell culture medium of Mock cells with 80 μg/ml of recombinant COMP protein. All experiments were repeated at least 3 times. Two-way ANOVA Bonferroni's multiple comparisons test was used (*b0.05, **b0.01, ***b0.001, ****b0.0001).
COMP leads to activation of Notch3 115
COMP activates Notch3, we estimated the binding affinity of COMP to the MDA-MB-231 cells to KD = 96.76 μmol (Fig. S2D) and cultured MDA-MB-231 cells supplemented with 40 μg/ml and 80 μg/ml of recombi-nant COMP for 24 h. Recombinant COMP induced 3-times more Notch3 activation than observed in control cells incubated with BSA (Fig. 5A, B), as measured by western blot detection of the Notch3 intracellular domain. Interestingly, the amount of Jagged1 was reduced (Fig. 5C, D), which is likely due to the fact that upon Notch3 activation, Jagged1 is internalized into the signaling cell together with the extracellular domain of Notch3, followed by ubiquitin-mediated degradation.
A distinctive feature of the Notch pathway is the ability to cross-talk with other molecular pathways, which modulates the Notch signaling output. Based on the aforementioned results of the qPCR array (Fig. 3A), we examined the status of the β-catenin pathway, in the presence and absence of COMP. We first utilized a Luciferase reporter assay and detected increased β-catenin activation in the presence of COMP expres-sion, which was further enhanced by blocking Notch activation with DAPT (Fig. 5E). Moreover, total β-catenin protein expression, as well as the activated form of β-catenin, was higher in COMP-expressing cells compared to control cells and further elevated when Notch activation was blocked in COMP-expressing cells using DAPT (Fig. 5F). Additionally, there was more total β-catenin both in the cytoplasmic and nuclear fraction of COMP-expressing cells com-pared to control cells (Fig. 5G), a hallmark of β-catenin activation.