Galley, Joseph
(2021)
Mechanisms of Guanylyl Cyclase Gene Regulation by FoxO Transcription Factors.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
Nitric oxide (NO) is a known vasodilator molecule produced in the vascular endothelium which freely diffuses to the smooth muscle cells (SMC) due to its small, non-polar nature. Once in the SMC, NO binds its cognate receptor, soluble guanylyl cyclase (sGC) to catalyze formation of cGMP to cause dilation. This process maintains healthy blood pressure and hemodynamic function. Therapeutic strategies have sought to target this pathway by elevating available NO treatment, stimulation of NO-sensitive sGC, or activation of NO-insensitive sGC. Despite successes of several sGC modulator clinical trials, little study had been devoted to transcription mechanisms responsible for sGC gene expression in SMC. We thus sought to identify transcriptional regulators of sGC in SMC tissue.
We identified several predicted Forkhead box subclass O (FoxO) protein binding sites on sGC promoters. We then inhibited the FoxO proteins in aortic SMC and observed significant loss of sGC gene and protein expression as well as cGMP production. Treated ex vivo murine aortas showed loss of sGC expression and loss of NO-dependent vasodilatory function. We next showed that a murine 2-kidney-1-clip (2K1C) model of hypertension causes increased vasodilatory function and expression of sGC in the contralateral renal arteries. Because angiotensin II (Ang II) causes many of the blood pressure effects in renovascular hypertension, we treated cultured renal SMC with Ang II and observed increased sGC expression through Ang II type 1 receptor (AT1R)-dependent agonism. This increase in sGC expression and downstream function was dependent upon functional FoxO transcriptional activity. We knocked down each FoxO protein in aortic SMC and showed that loss of FoxO1 and FoxO3 increase sGC expression and downstream function, while FoxO4 loss decreased sGC expression and function. We then used sGCβ promoter-luciferase vectors to show which regions are necessary for transcriptional function and again show that expression requires FoxO transcription. Finally, we show that FoxO4 binds several predicted locations using chromatin immunoprecipitation of the sGCβ promoter. Combined, we are the first to identify the FoxO family as transcriptional regulators of sGC in SMC, opening a new avenue for therapeutic innovation in basic and clinical vascular research.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
20 September 2021 |
| Date Type: |
Publication |
| Defense Date: |
23 June 2021 |
| Approval Date: |
20 September 2021 |
| Submission Date: |
6 August 2021 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
156 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
N/A |
| Related URLs: |
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| Date Deposited: |
20 Sep 2021 18:54 |
| Last Modified: |
20 Sep 2021 18:54 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/41797 |
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