Notch-mediated CBF-1/RBP-J{kappa}-dependent regulation of human vascular smooth muscle cell phenotype in vitro.
Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of alpha-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-Jkappa (RBP-Jkappa) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of alpha-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-Jkappa-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jkappa-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-Jkappa-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation.
Morrow
David
D
Vascular Health Research Centre, Faculty of Science and Health, Dublin City University, Dublin 9, Ireland.
Scheller
Agnieszka
A
Birney
Yvonne A
YA
Sweeney
Catherine
C
Guha
Shaunta
S
Cummins
Philip M
PM
Murphy
Ronan
R
Walls
Dermot
D
Redmond
Eileen M
EM
Cahill
Paul A
PA
D000199:Actins / Q000096:biosynthesis; D002454:Cell Differentiation / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism*; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051880:Receptors, Notch; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D000199:Actins; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C497761:NOTCH1 protein, human; C106075:NOTCH4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D014157:Transcription Factors