Abstract
Notch signaling is essential for vascular patterning and response of the vasculature to injury and growth factor stimulation. Despite these findings, the molecular basis of Notch signaling in the vasculature is poorly understood. Here we report that activation of Notch signaling mediated through members of the HRT family of basic helix-loop-helix transcription factors represses smooth muscle cell (SMC) differentiation and expression of genes encoding smooth muscle cell contractile markers. Activation of Notch receptors by Jagged1 or forced expression of the constitutively active Notch1 intracellular domain in C3H10T1/2 fibroblasts inhibited myocardin-dependent transcription of SMC-restricted genes and activity of multiple SMC-restricted transcriptional regulatory elements. Consistent with these findings, forced expression of HRT2 inhibited myocardin-induced expression of SMC-restricted genes and activity of SMC-restricted transcriptional regulatory elements. Moreover, forced expression of HRT2 repressed transcription of multiple SMC-restricted transcriptional regulatory elements in A10 SMCs. The repressive function of HRT2 was not mediated via the capacity of HRT2 to bind SMC CArG elements or by disruption of myocardin-SRF protein complexes. Structure-function analyses of HRT2 indicated that repression required the basic DNA binding domain and additional C-terminal sequence. Taken together, these results demonstrate that Notch signaling represses myocardin-dependent SMC transcription. These data are consistent with a model wherein Notch signaling represses SMC differentiation and maintenance of the contractile SMC phenotype.
Highlights
Vascular smooth muscle cells (VSMCs)1 express a unique set of genes encoding SMC-restricted contractile and cytoskeletal proteins required for the modulation of arterial tone and vas
Jagged1 can activate Notch signaling whether it is on a neighboring cell or immobilized on a tissue culture plate [46]. 10T1/2 cells plated on either Jagged1-coated or control tissue culture dishes were transfected with the pcDNA3-myocardin expression plasmid or the control plasmid pcDNA3. 48 h post-transfection, the cells were harvested, and SM␣-actin, SM-MyHC, and SM22␣ mRNA were quantified by real time RT-PCR
We have shown that Notch signaling, mediated by Hairy-related transcription (HRT) family members, represses myocardin-dependent smooth muscle differentiation of SMCs from mesodermal precursors and antagonizes transcription of endogenous contractile genes in SMCs
Summary
Plasmids—The expression plasmid (pcDNA3-ICN1) encoding the mouse Notch intracellular domain (ICN1) was prepared by subcloning the PCR-amplified ICN1 cDNA into pcDNA3 (Invitrogen). Expression plasmids for mouse HRT1 (pHRT1-V5) and HRT3 (pHRT3-V5) were prepared by subcloning PCR-amplified HRT1 and HRT3 cDNA, respectively, into pcDNA3.1 with a C-terminal V5 epitope tag. Primer sequences for real time PCR of cDNAs for glyceraldehyde-3-phosphate dehydrogenase, SM␣-actin, SM-MyHC, SM22␣, SM-calponin-h1, myocardin, HRT1, HRT2, and HRT3 were described previously [15, 25] with the exception of the reverse primer for HRT3, which was 5Ј-TAGCTGACTGCTCAGGGAAGGCAA-3Ј. Luciferase Reporter Assays—10T1/2 or A10 cells were seeded at 20,000 cells/well in a 24-well tissue culture plate and transfected with 25 ng of luciferase reporter construct, 25 ng of myocardin expression plasmid, and the indicated amounts of ICN1 or HRT expression plasmids with additional pcDNA3 vector added to normalize total DNA concentration in each transfection. Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs were performed with in vitro transcribed and translated HA-SRF, FLAG-myocardin, and HRT2-V5 and the radiolabeled SME-4 CArG-box containing
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