Characterization of Mechanisms Controlling Translational Regulation of Type I Collagen in Liver Fibrosis as Target for Novel Anti-Fibrotics and the Regulation of Neural Tube Closure during Embryonic Development

Pathological hepatic fibrosis represents a worldwide medical problem with substantial mortality and the number of cases is increasing. Despite all medical advances, effective therapy to treat progressive liver fibrosis is still unavailable and is urgently needed, as the disorder results in organ death. The pathogenesis and the molecular mechanism underlying fibrosis are similar in most organs in human body, therefore, my work, which focused on liver fibrosis as the primary disease model, has broader implications. Liver fibrosis is categorized as an abnormal wound-healing response (excessive scarring), predominantly caused by increased production of type I collagen protein. Activation of a specialized cell type in the liver; hepatic stellate cells (HSC), is responsible for excessive collagen synthesis and pathogenesis of liver fibrosis. The dramatic up-regulation of collagen expression is primarily controlled at the level of initiation of protein production (post-transcriptional and translational level of regulation). The long-term goal of my work is to elucidate factors involved in translational regulation of type I collagen mRNAs and to target these factors as a potential anti-fibrotic therapy. This would lead to the first development of collagen specific drugs. mRNAs encoding for type I collagen have a unique structure, the 5' stem loop (5'SL), which regulates collagen synthesis. To understand this regulation, our lab has recently characterized a novel protein La ribonucleoprotein domain family member 6 (LARP6), as the key protein regulating translation of type I collagen mRNAs. LARP6 directly binds the 5'SL of collagen mRNAs and acts as a crucial adapter protein that recruits other factors required for collagen synthesis and progression of scarring. My dissertation has three independent projects. The two projects of my proposal are to identify and characterize the role of novel factors that are recruited by LARP6 to enhance collagen synthesis in fibrosis; these factors are RNA Helicase A (RHA) and FK506 Binding Protein 3 (FKBP3). Detailed understanding of the mechanism by which these factors stimulate collagen synthesis was needed, as they may be potential targets for development of the specific antifibrotic treatment. In addition, in collaboration with Dr. Kato's group I identified additional functions of LARP6 in neural tube development during embryogenesis. This complemented the work on the role of LARP6 in fibrosis. Studies on RHA have indicated that RHA is tethered to the 5'SL of collagen mRNAs by interaction with LARP6. Knock down of RHA prevented formation of polysomes on collagen mRNAs and dramatically reduced synthesis of collagen protein, without affecting the level of collagen mRNAs. During activation of quiescent HSCs into collagen producing HSCs expression of RHA is highly upregulated. We postulated that RHA is recruited to the 5'UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of type I collagen in fibrosis. The second project on the role of FKBP3 has discovered that FKBP3 controls collagen synthesis prior to formation of polysomes, probably at the level of translation initiation. We determined that the FKBP3 regulation is in the cytoplasm where it] controls the amount of free LARP6 and thus affecting the next cycle of translation initiation on collagen mRNAs. I further extended these studies to animal model of liver fibrosis. I have used an alcoholic liver fibrosis model in rats and administered Tacrolimus (FK506), an immunosuppressive drug that binds FKBP3, in order to disrupt the LARP6/collagen mRNA complex. Our study showed that FK506 inhibited secretion of type I collagen by HSCs in vitro. In vivo, administration of FK506 completely prevented development of liver fibrosis and activation of HSCs. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis in vivo and in defective secretion of type I collagen in vitro. This is the first report of such activity of FK506 and may renew the interest in using FK506 to alleviate hepatic fibrosis. This work also validated my approach to discovery of novel antifibrotic drugs. The third project, which focused on the functions of LARP6 other than collagen synthesis, discovered its role in neural tube development in the embryo. Neural tube defects are among the most common human congenital malformations, but very little is known about the factors that regulate the neural tube formation. Our results showed that LARP6 controls the formation of cilia, the structures that are required for proper neural tube development during embryogenesis. LARP6 knockdown prevented cilia formation and as a result the embryos failed to develop proper central nervous system. Understanding this mechanism and targets of LARP6 in this process may be used as in prenatal diagnosis and therapies for neural tube defects. In conclusion, my dissertation work has characterized novel factors involved in synthesis of type I collagen and development of hepatic fibrosis. In addition, a mechanism of neural tube formation has been discovered. Overall, these findings will contribute to development of antifibrotic drugs and better understanding of congenital malformations of the nerve system.