Novel Mechanisms of Type I Collagen Regulation in Liver Fibrosis

• Other Authors: Fritz, Dillon (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Medical sciences
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-4395

The process of normal wound repair after tissue injury follows a closely regulated sequence involving inflammation, the recruitment, activation and proliferation of fibroblasts and the secretion of extracellular matrix, which culminates in healing and termination of the proliferative and secretory processes. In pathological fibrosis, the normal healing and termination stages are disregulated and fibroblast activity continues unabated. This persistent "activated" state of the fibroblasts is the cause of the excessive accumulation of ECM, predominantly fibrillar collagens type I and III, which results in the disruption of the normal tissue function . In cirrhosis, the end stage of liver fibrosis, type I collagen represents up to 50% of liver proteins. Hepatic stellate cells (HSCs) are the major cell type responsible for collagen synthesis in the liver. In normal liver, quiescent HSCs store vitamin A, but only express trace amounts of type I collagen. Upon a fibrogenic stimulus, HSCs become activated, a process in which they lose vitamin A, proliferate, change morphologically into myofibroblasts, and increase their synthesis of extracellular matrix proteins. In order to understand the pathophysiology of cirrhosis, as well as other fibroproliferative disorders which can effect the heart, lungs, and skin, it is critical to elucidate the molecular mechanisms which regulate the expression and synthesis of type I collagen. In the first aim of this dissertation, we examined the role of the RNA-binding protein RBMS3 in regulating type I collagen expression. RBMS3 expression increases upon activation of HSCs, and is also increased in liver fibrosis. Through our research, we showed that RBMS3 specifically interacts with a conserved 60 nucleotide sequence in the 3' UTR of the homeobox transcription factor Prx1. Prx1, which is also upregulated in activated HSCs and liver fibrosis, transactivates the collagen á1(I) promoter and stimulates transcription of the gene. The binding of RBMS3 to the 3' UTR of the Prx1 mRNA results in the stabilization of the mRNA and increased protein synthesis. Since Prx1 is a transcription factor which increases collagen gene transcription, this mechanism may promote the profibrotic phenotype of HSCs. In the second aim of the dissertation, we focused on the posttranscriptional regulation of type I collagen expression. In the 5' UTR of á1(I), á2(I), and á1(III) collagen mRNA there is a stem-loop structure that encompasses the translation initiation codon. This 5' stem-loop is strongly conserved in evolution differing by only two nucleotides between fish and human collagen mRNAs. In order to study the role of the 5' stem-loop, our collaborators designed a transgenic mouse in which the 5' stem-loop structure of the collagen á1(I) gene was abolished. The collagen á1(I) mRNA stability and protein synthesis in fibroblasts from these transgenic mice was significantly decreased. Since it was clear that the 5' stem-loop was essential for proper type I collagen synthesis, we examined the role that 5' stem-loop binding proteins have in regulating this mechanism. Through the use of 2-D gel SDS-PAGE and MALDI-TOF MS, we identified several 5' stem-loop associated proteins which included LARP6, non-muscle myosin IIb, nucleolin, and vimentin. We discovered that LARP6, through direct binding of the collagen 5' stem-loop, enables the aggregation of type I collagen mRNAs into large complexes. Additional proteins identified in these RNA-protein complexes are components of stress granules, which regulate RNA metabolism. This type I collagen mRNA stress granule formation, along with non-muscle myosin IIb, may facilitate the mechanism by which the coordinated translation of á1(I) and á2(I) collagen mRNAs can occur. Overall, my dissertation research has accomplished two major findings. The first finding is how the transcription of the collagen á1(I) gene is regulated through the binding of RBMS3 to the mRNA encoding transcription factor Prx1. The second finding is how the translation of type I collagen is regulated by the LARP6 mediated aggregation of collagen mRNAs and their association with non-muscle myosin. Elucidation of these mechanisms may help in the development of antifibrotic drugs. === A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Fall Semester, 2008. === August 7, 2008. === RNA, Liver Fibrosis, Biomedical Science === Includes bibliographical references. === Branko Stefanovic, Professor Directing Dissertation; Ken Roux, Outside Committee Member; Yanchang Wang, Committee Member; James Olcese, Committee Member.