Vascular smooth muscle cells (SMC) undergo significant phenotype modulation during embryonic differentiation and following vascular injury. The differentiated SMC (the contractile phenotype) is associated with high expression of several specific contractile proteins including smooth muscle alpha-actin (SMA), smooth muscle myosin heavy chain (SM-MHC), SM22α, and calponin1. The dedifferentiated SMC (the synthetic phenotype) is characterized by low expression of contractile proteins. The phenotype transition of SMC from contractile to synthetic phenotype plays a pivotal role in pathological processes such as atherosclerosis, hypertension, and restenosis. The investigation of SMC phenotype transition may contribute to the understanding and therapies of cardiovascular diseases.
The expression of Notch ligands and receptors is dysregulated after arterial injury and in human vascular diseases. Mutations of Notch pathway components also were found in human vascular diseases. To investigate the role of Notch signaling in SMC differentiation, we activated Notch signaling using Notch receptor intercellular domains (NotchICDs) or immobilized Jagged1-Fc in human primary aortic SMC. Jagged1-activated Notch signaling or constitutive activation of Notch signaling after expression of Notch1ICD, Notch2ICD, Notch3ICD, or Notch4ICD were able to induce SMC differentiation. Differentiation was monitored by the up-regulation of the SMC specific gene products SMA, SM22a, calponin1, and SM-MHC at both mRNA and protein levels. Expression of Notch downstream effectors hairy related transcription factors (HRTs) did not mimic Notch function. On the contrary, HRTs repress endogenous and NotchICD-induced SMC differentiation. Further study indicated that HRTs did not interfere with the formation of NotchICD/CBF1 complex, but they repressed the complex binding to SMA promoter. This is the first reported evidence that HRTs exert a negative feedback in Notch-meditated SMC differentiation.
In addition to Notch signaling, I confirmed that TGFβ is another important signaling pathway regulating SMC differentiation. This study demonstrated that TGFβ1-dependent SMC differentiation is time and dosage-dependent. TGFβ1 treatment of SMC leads to the phosphorylation of Smad2/3 and Smad1/5/8. TGFβ1-induced phosphorylation of Smad1/5/8 is BMP-independent. I also observed that ALK5 is required for the TGFβ1-mediated SMC differentiation and Smads phosphorylation events, and that the MAPK signaling pathway is required for the TGFβ1-regulated SMC differentiation.
Since Notch and TGFβ1 both induce a differentiated SMC phenotype, my studies tested the hypothesis that they cooperatively activate the expression of SMC markers through parallel signaling axes. Notch signaling enhanced SMC response to TGFβ1 by promoting pSmad2/3 binding to SMC specific promoters. In addition, HRTs expression inhibited TGFβ1-modulated SMC differentiation, suggesting HRTs function as a common negative regulator of SMC differentiation.
In summary, both Notch and TGFβ are important inducers of SMC differentiation. These studies identify a mechanism by which activated Notch signaling enhances TGFβ1 responsiveness in SMC. Further, my data demonstrate the activity of HRTs transcription factors as common negative regulators of Notch and TGFβ1-induced SMC differentiation.
|School:||The University of Maine|
|School Location:||United States -- Maine|
|Source:||DAI-B 72/10, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Developmental biology|
|Keywords:||Cell differentiation, Notch signaling, Smooth muscle cells, TGF-beta|
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