Abstract
Proliferation of SMC after vascular injury accounts for clinical
conditions in transplant vasculopathy, in-stent restenosis and vein
bypass graft failure. Vascular injury upregulates the expression of
many transcription factors, two important ones are Yin yang-1 (YY-1)
and early growth response-1 (Egr-1). The aims of this thesis were (1)
to examine a possible mechanism by which a transcriptional
repressor YY-1 inhibits SMC proliferation and (2) the development of
a phospho-specifc antibody to a transcription activator, Egr-1 found
to be a positive regulator of SMC proliferation. The results show that
YY-1 inhibits p21WAF1/Cip1 transcription that perturbs the formation of
p21WAF1/Cip1/cdk4/cyclin D1 complex thus blocking the downstream
pRBSer249/Thr252 phosphorylation and expression of PCNA and TK-1.
This inhibition was observed only in SMCs and not in ECs.
Moreover, inhibition of endogenous YY-1 was performed to show the
gain- and loss-of function of this transcription factor. YY-1 binds with
Sp1 and prevents its occupancy of a Sp1 binding element in the
p21WAF1/Cip1 promoter without YY-1 itself binding to the promoter. YY-
1 suppression of p21WAF1/Cip1 also involves p53 ubiquitination and
proteasomal degradation. Further studies showed that
overexpression of the first two-zinc finger region of YY-1 can inhibit
smooth muscle cell proliferation and not ECs. This cell-type specific
effect of YY-1 could be a potential tool in controlling SMC
proliferation in drug eluting stent.
The second part of this dissertation is the generation of phosphospecific
antibody to Egr-1. Egr-1 controls a variety of genes
implicated to SMC proliferation. Phosphorylation of Egr-1 can induce
or repress the expression of its target gene depending on what type
of kinase is involved. Preliminary data show that a phospho-specific
antibody to Egr-1, pS26, can detect the Egr-1 phosphorylated protein
from cell extract. Specificity of pS26 was determined also using slot
blots of synthetic peptides and recombinant proteins, peptide
blocking and phosphatase treatment. Further validation is needed to
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fully confirm the specificity of this new phospho-specific antibody to
Egr-1. A phospho-specific antibody to Egr-1 will serve as a tool to
dissect mechanism by which this immediate early gene product
exerts it control on fibroproliferative vasculopathies.
The results generated by this thesis have added a new layer on the
understanding of mechanism on inhibition of SMC proliferation and a
generation of potential tool to dissect the mechanism of
phosphorylation of a transcription factor implicated to SMC
proliferative vasculopathy.