Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Nelson, Mark

Abstract

The goals of this dissertation were to explore the role of the Ca2+-sensitive transcription factor NFAT in the regulation of smooth muscle contractility. We have identified a conserved NFAT binding site that overlaps an intronic SRF-binding CArG element that has previously been demonstrated to be essential for expression of smooth muscle α-actin. Transfection of a reporter construct containing the composite CArG/NFAT element, designated SNAP, into SMCs resulted in robust basal reporter activity that was sensitive to the calcineurin/NFAT pathways inhibitors FK506 and CsA. Mutations to either the NFAT or adjacent SRF binding site essentially abolished reporter activity, indicating that both were required. Co-immunoprecipitation assays revealed that NFATc3 and SRF formed a complex in solution, and that the formation of this complex was facilitated by the presence of the SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased α-actin expression in cultured SMCs, suggesting that NFAT plays a role in the expression of smooth muscle contractile proteins such as α-actin. To determine if NFAT transcriptional activity is involved in modulating urinary bladder smooth muscle contractility, we compared the contractile and electrophysiological properties of NFATc3-null mice to wild-type mice. UBSM strips taken from NFATc3-null mice displayed an elevated contractile response to EFS compared to strips from wild-type mice. This increased contractility was due to a decrease in IBTX-sensitive BK current and was supported at the molecular level by reduced expression of mRNA for the pore-forming α-subunit of the BK channel. Single-channel recordings revealed that the β-subunit of the BK channel, which modulates the sensitivity of the BK channel to voltage and Ca2+, was not altered. Interestingly, TEA-sensitive KV currents, and expression of the pore-forming KV2.1 subunit, were increased in the NFATc3-null myocytes. However, the increased contractile response of UBSM strips from NFATc3-null mice indicates that, at least in response to electrical field stimulation, the downregulation of BK current plays a more significant role than does the increase in KV current. Presumably, this is due to the prominent role that BK channels play in shaping the UBSM action potential. Thus, this dissertation provides evidence that NFAT plays a role in modulating smooth muscle contractility via its role in regulating the expression of contractile proteins and ion channels and, furthermore, lays the foundation for future investigations into the specific role of NFAT in the pathological response of the urinary bladder to outlet obstruction.

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