Liver fibrosis is the 9th leading cause of death in the United States and represents a precancerous state for developing of hepatocellular carcinoma. There is no cure for liver fibrosis, so one of the goals is to identify potential molecular targets for development of antifibrotic drugs. To this goal, mechanisms regulating collagen synthesis by hepatic stellate cells (HSCs) have to be elucidated at the molecular level. Posttranscriptional regulation of expression of type I collagen is the main regulatory mechanism in HSCs. This regulation is executed through a unique element of collagen mRNAs, the 5'stem-loop, which tethers collagen alpha 1(I) and alpha 2(I) mRNAs to myosin filaments. This is required for coordinated translation of the peptides forming type I collagen. Myosin dependent collagen synthesis operates in cells which synthesize large amount of type I collagen, like activated HSCs. Disruption of myosin filaments almost completely inhibits collagen synthesis by HSCs. 5'stem-loop interacting proteins, RBM14, nucleolin, vimentin and RNA helicase A, have been identified by proteomics approach. These proteins tether collagen mRNAs to the nonmuscle myosin for coordinated translation. This proposal is to test the above hypothesis.
Specific aim 1 in this proposal will characterize the effects of binding of RBM14 to the 5'stem-loop on collagen synthesis by HSCs. This will be achieved by: 1 adenoviral delivery of RBM14 to quiescent HSCs, 2. inhibition of RBM14 expression by adenovirus delivered siRNA into quiescent HSCs. Binding affinity of RBM14 to 5'stem-loop RNA will also be measured, as a prerequisite for development of specific inhibitors.
Specific aim 2 will address the role of RNA helicase A in unwinding the 5'stem-loop and facilitating translation by using a combination of reporter gene assays and loss of function studies in HSCs. The model of myosin dependent collagen synthesis by HSCs will be tested in specific aim 3. Formation of myosin filaments will be prevented in HSCs by using specific inhibitors of myosin polymerization and the effects on collagen synthesis will be analyzed. The roles of nucleolin and vimentin will be addressed by inhibiting these proteins in HSCs with either siRNA or dominant negative mutants. The results will elucidate this unique mechanism of collagen synthesis in HSCs and identify the regulatory roles of the factors involved, as potential targets for development of antifibrotic drugs.
Liver fibrosis is the 9th leading cause of death in the USA and the number of cases is increasing. There is no effective cure for liver fibrosis and development of therapeutics must await elucidation of the molecular mechanisms of collagen synthesis by hepatic stellate cells. This proposal will characterize the newly discovered mechanism of collagen synthesis, which is dependent on structural protein myosin.
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