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.
|Wang, Hao; Stefanovic, Branko (2014) Role of LARP6 and nonmuscle myosin in partitioning of collagen mRNAs to the ER membrane. PLoS One 9:e108870|
|Manojlovic, Zarko; Earwood, Ryan; Kato, Akiko et al. (2014) RFX7 is required for the formation of cilia in the neural tube. Mech Dev 132:28-37|
|Vukmirovic, Milica; Manojlovic, Zarko; Stefanovic, Branko (2013) Serine-threonine kinase receptor-associated protein (STRAP) regulates translation of type I collagen mRNAs. Mol Cell Biol 33:3893-906|
|Stefanovic, Branko (2013) RNA protein interactions governing expression of the most abundant protein in human body, type I collagen. Wiley Interdiscip Rev RNA 4:535-45|
|Manojlovic, Zarko; Stefanovic, Branko (2012) A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs. RNA 18:321-34|
|Fritz, D; Cai, L; Stefanovic, L et al. (2011) Progress towards discovery of antifibrotic drugs targeting synthesis of type I collagen. Curr Med Chem 18:3410-6|
|Challa, Azariyas A; Stefanovic, Branko (2011) A novel role of vimentin filaments: binding and stabilization of collagen mRNAs. Mol Cell Biol 31:3773-89|
|Cai, Le; Fritz, Dillon; Stefanovic, Lela et al. (2010) Nonmuscle myosin-dependent synthesis of type I collagen. J Mol Biol 401:564-78|
|Cai, Le; Fritz, Dillon; Stefanovic, Lela et al. (2010) Binding of LARP6 to the conserved 5' stem-loop regulates translation of mRNAs encoding type I collagen. J Mol Biol 395:309-26|
|Jiang, Feng; Stefanovic, Branko (2008) Homeobox gene Prx1 is expressed in activated hepatic stellate cells and transactivates collagen alpha1(I) promoter. Exp Biol Med (Maywood) 233:286-96|
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