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YB-1 Stress-Response Protein Conformation Implicated in Post-transcriptional Control of Myofibroblast Differentiation

Willis, William L
http://rave.ohiolink.edu/etdc/view?acc_num=osu1376593223

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program.
Vascular smooth muscle alpha-actin (SMA) is an indicator of myofibroblast differentiation, as well as one of several fetal contractile protein isoforms re-expressed in adult cardiomyocytes in response to mechanical stress-injury. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMA mRNA and regulates its translational activity. Our central hypothesis is that YB-1 drives maladaptive SMA expression in injury-activated myofibroblasts by modulating the packaging, delivery, and translational efficiency of its cognate mRNA. In a mouse model for cardiac fibrosis, we observed that accumulation of fetal SMA protein in cardiac sarcomeres was associated with accumulation of punctate YB-1 deposits which localized to perinuclear regions as well as polyribosome-enriched cytosol proximal to cardiac intercalated discs. Samples from both fibrotic mouse hearts as well as SMA positive endomyocardial biopsies from human heart transplant patients were enriched with high molecular weight, heat-denaturing resistant YB-1 oligomers migrating in the range of 100-250 kDa during reducing SDS-PAGE. Based on these intriguing observations, which suggested that YB-1 oligomer formation may be associated with the packaging and translation control of SMA mRNA, we examined the regulatory aspects of YB-1 oligomerization using a model system based on isolated human pulmonary fibroblasts (hPFBs). Activation of SMA gene expression in hPFBs by TGFbeta1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a SMA exon-3 translation-silencer sequence. We discovered that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent gamma-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers distributed during SDS-PAGE over a 75 kDa to 250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMA mRNA. YB-1 crosslinking was reversible as a function of free-calcium concentration and TG2 enzyme availability. Metabolic stress incurred during tissue injury may also promote conversion of fibroblasts to the myofibroblast phenotype, as temporary loss of oxygen perfusion promotes a hypoxic, energy-deficient pro-oxidative cellular microenvironment. Stimulation of AMP-activated kinase (AMPK) activity with 5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) activated TG2 transamidation and induced the formation of high molecular weight YB-1 oligomers with enhanced affinity for the SMA mRNA exon-3 translation-silencer sequence. AMPK and peroxide administration differentially regulated phosphorylation of the YB-1 cold-shock domain (CSD), which modulates YB-1 subcellular localization and SMA mRNA binding efficiency. AICAR suppressed YB-1 phosphorylation, which prevented nuclear translocation and activated SMA mRNA binding. In contrast, peroxide stimulation activated Erk/MAPK dependent phosphorylation of the YB-1 CSD and dispersed YB-1: SMA mRNA complexes. Thus, we propose that coordinated AMPK activation and ROS production during myofibroblast differentiation regulates SMA expression at the post-transcriptional level, by coordinating the respective packaging and deployment/translation activation of SMA mRNA from YB-1 ribonucleoprotein complexes. In summary, intracellular calcium accumulation and increased ROS levels incurred during metabolic and biomechanical stress may govern SMA mRNA translational activity during wound healing and cardiopulmonary stress responses via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.
Arthur Strauch, PhD (Advisor)
Denis Guttridge, PhD (Committee Member)
Lai-Chu Wu, PhD (Committee Member)
Mark Ziolo, PhD (Committee Member)
196 p.
Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology
YB-1; transglutaminase 2; translational control; RNA-binding protein; myofibroblast; AMPK; smooth muscle alpha-actin; cardiac remodeling; cardiac intercalated disc; metabolic stress; fibrosis; ROS; TGFbeta1

Recommended Citations

Citations

  • Willis, W. L. (2013). YB-1 Stress-Response Protein Conformation Implicated in Post-transcriptional Control of Myofibroblast Differentiation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376593223

    APA Style (7th edition)

  • Willis, William. YB-1 Stress-Response Protein Conformation Implicated in Post-transcriptional Control of Myofibroblast Differentiation. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1376593223.

    MLA Style (8th edition)

  • Willis, William. "YB-1 Stress-Response Protein Conformation Implicated in Post-transcriptional Control of Myofibroblast Differentiation." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376593223

    Chicago Manual of Style (17th edition)

osu1376593223
882
© 2013, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.