Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Russell (Chip) Norris

Second Advisor

Robin C. Muise-Helmericks

Third Advisor

Jeffrey Jones

Fourth Advisor

Antonis Kourtidis

Fifth Advisor

Joshua Lipschutz

Abstract

Non-syndromic Mitral Valve Prolapse (MVP) is a common disease with associated morbidities and mortality. Affecting 2-3% of the global population, MVP has become a significant health burden in developed countries. We recently identified mutations in the cilia gene, DZIP1 in multiple families with MVP. To initially identify the function of DZIP1 in valve biology, we performed proteomics-based approaches with the goal of identifying unique binding partners for DZIP1. These studies revealed a direct interaction between DZIP1 and the β-catenin antagonist, CBY1. We hypothesized that DZIP1 suppresses the Wnt/ β-catenin pathway during mitral valve development through CBY1. Immunofluorescence staining revealed overlap between DZIP1 and CBY1 protein at the basal body of the primary cilium. Increase of activated β-catenin was observed in the Dzip1S14R/+ valves. Co-immunoprecipitation confirmed an interaction between DZIP1, CBY1 and β-catenin. Ensuing immunofluorescence staining suggested overlap between β-catenin and the basal body. DZIP1 truncation mutants identified a minimal CBY1 interaction motif within the C-terminus of DZIP1. A membrane permeant mimetic peptide against this motif was synthesized and confirmed as being able to interact with CBY1 and β-catenin. Treatment of chicken valve interstitial cells with the mimetic peptide resulted in significant decrease in activated nuclear β-catenin. To test whether this pathway was relevant in the context of the DZIP1 mutation, we assayed nuclear vs. cytoplasmic β-catenin expression in Dzip1S14R/+ MEFs. Western blot analysis showed a significant increase in nuclear β-catenin from the mutant cells. An additional family was identified with a rare DZIP1 variant within the DZIP1-CBY1 interaction motif. The mutation resulted in reduced DZIP1 and CBY1 protein stability and a peptide synthesized with the mutation resulted in an enhanced interaction between DZIP1 and β-catenin and an inhibitory effect on β-catenin signaling. Through analysis of nuclear β-catenin expression profiles during cardiac valve development, we conclude that Wnt/β-catenin signaling is temporally and spatially regulated. It is down regulated after E13.5 and undetectable in the adult. However, β-catenin signaling is significantly upregulated in human myxomatous valves and thus may be a major contributor to disease phenotype. In conclusion, DZIP1 suppresses Wnt activity to direct mitral valve development through interacting with and stabilizing CBY1. This study characterizes the β-catenin expression profile during murine cardiac valve development and reveals a molecular mechanism, by which mutations in DZIP1 alter valve development leading to increased β-catenin signaling. Altered β-catenin signaling may be an early initiating signal in the pathogenesis of MVP.

Rights

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