Abstract 1004: Disruption of Notch Receptor Signaling Promotes Smooth Muscle Cell Differentiation
The objective of this study was to examine the role of Notch receptor signaling during VSMC lineage commitment in our multipotent P19 model system. To address this objective, we utilized a ‘gain’ or ‘loss of function’ approach to modulate Notch signaling during VSMC differentiation. Notch1 (pN1-IC) and Notch3 (pN3-IC) expression vectors were employed to examine Notch signaling under constitutive activation. P19 cells transfected with pN3-IC exhibit a significant reduction in Octamer3A/4 (Oct4) expression by RT-PCR (−12.46 fold; p<0.005; n=6) and immunoblot when compared to pN1-IC transfected cells (1.56 fold; n=6) or cells transfected with empty vector. To disrupt global Notch signaling we utilized the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). Interestingly, P19 cells which were pretreated with 1μM DAPT for 24 hours resulted in a significant reduction in Oct4 expression (−16.23 fold; p<0.001; n=5) and immunofluorescence, with an increase in alpha-smooth muscle actin (αSMA) expression (+6.91 fold; p<0.05; n=5) when compared to vehicle treated cells. To assess the effects of Notch specific receptor expression on VSMC differentiation, we utilized Notch1, or Notch3-specific siRNAs. Oct4 expression was significantly reduced during transient knockdown of both Notch1 (−24.93 fold; p<0.001; n=3) and Notch3 (−13.77 fold; p<0.005; n=3) when compared to a scrambled control. Although a marked induction of VSMC marker αSMA was observed in siNotch3 treated cells (+2.10 fold; p=ns; n=3), a robust elevation in αSMA transcription was observed in siNotch1 treated (+27.29 fold; p<0.001; n=3) cells. Taken together, these data suggests that repression of Notch signaling is essential for VSMC differentiation.