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(2011-08) Snyder, Russell Odell; Thekkumkara, Thomas; Abbruscato, Thomas J.; Karamyan, Vardan; Stoll, James
Transcriptional repression through cis and trans acting factors enabling an
alternate approach to control of angiotensin type 1 receptor (ATlR) expression has not
been studied. In prior investigation treatment with retinoic acid was associated with
enhanced insulin sensitivity. In our previous study, expression of ATlR has been
inversely correlated with intracellular glucose concentrations. Therefore we hypothesized
that 13-cis retinoic acid (13cRA), an antioxidant, enhances insulin sensitive glucose
mediated down-regulation of the ATlR. Our study shows that cells exposed to 13cRA
specifically down-regulated the ATlR protein in a dose and time dependent manner.
Down-regulation of the ATlR expression leads to reduced Angil mediated intracellular
calcium release, a hallmark of receptor mediated intracellular signaling. Consistently with
receptor down-regulation, we observed significant reduction in ATlR mRNA; however,
the AT 1 R down-regulation was independent of insulin sensitive glucose uptake.
Treatment with 13cRA results in phosphorylation of p42/p44 MAP Kinases in these cells.
Subsequent studies using MEK inhibitor PD98059 prevented 13cRA mediated ATlR
down-regulation as well as restored Angil mediated intracellular calcium response.
Furthermore, serially deleted promoter constructs of the ATlR indicated that a region
between - 2541 and -1836 bp upstream of the mRNA start site has an Spl binding site
(5' -GGGGCGGGGC-3'), which was involved in 13cRA's ability to down-regulate
ATlR gene expression. Mobility shift assay using the Spl binding site displayed a
blockade of Spl binding activity upon treatment with 13cRA. Under similar conditions,
we observed a significant increase in early growth response protein 1 (Egr-1) expression.
Blocking MAP Kinase phosphorylation results in no increase in Egr-1 protein expression and no down-regulation of ATlR mRNA and protein. Treatment with cycloheximide
indicates that ATlR down-regulation requires de novo protein synthesis of Egr-1 . The
essential role and interplay of Egr-1 and Sp 1 in the regulation of ATlR transcription were
confirmed by siRNA mediated effective silencing of these proteins and their actions on
their respective targets. By protein-protein interaction with Egr-1, Sp 1 loses its ability to
bind cis acting elements in the ATlR promoter. Our data indicates that 13cRA, through
MAP Kinase, activates Egr-1 leading to a direct interaction of Egr-1 and Sp I. This
interaction suppresses Spl 's ability to promote transcription of the ATIR gene. In
summary, our study demonstrates for the first time that 13cRA has a glucose independent
mechanism for transcriptional inhibition of ATlR. The observed effects by 13cRA
suggest its therapeutic potential in insulin sensitive and insensitive tissues in which ATIR
expression is up-regulated.