Yuefeng Tang

Date of Award

5-2011

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Lucy Liaw

Second Committee Member

Jeong Yoon

Third Committee Member

Douglas Spicer

Abstract

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 calponin 1. 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 (NotchlCDs) or immobilized Jagged 1-Fc in human primary aortic SMC. Jagged1-activated Notch signaling or constitutive activation of Notch signaling after expression of Notch 1ICD, 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, calponin 1, 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 NotchlCD-induced SMC differentiation. Further study indicated that HRTs did not interfere with the formation of NotchlCD/CBF 1 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 TGFß is another important signaling pathway regulating SMC differentiation. This study demonstrated that TGFß1-dependent SMC differentiation is time and dosage-dependent. TGFß1 treatment of SMC leads to the phosphorylation of Smad2/3 and Smad 1/5/8. TGFß1-induced phosphorylation of Smad 1/5/8 is BMP-independent. I also observed that ALK5 is required for the TGFß1-mediated SMC differentiation and Smads phosphorylation events, and that the MAPK signaling pathway is required for the TGFß 1-regulated SMC differentiation. Since Notch and TGFß1 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 TGFß1 by promoting pSmad2/3 binding to SMC specific promoters. In addition, HRTs expression inhibited TGFß1-modulated SMC differentiation, suggesting HRTs function as a common negative regulator of SMC differentiation. In summary, both Notch and TGF(3 are important inducers of SMC differentiation. These studies identify a mechanism by which activated Notch signaling enhances TGFß1 responsiveness in SMC. Further, my data demonstrate the activity of HRTs transcription factors as common negative regulators of Notch and TGFß1-induced SMC differentiation.

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