Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis

Stefanovic B (2013) Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis. PLoS ONE 8(6): e65897. doi:10.1371/journal.pone.0065897 Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis Zarko Manojlovic 0 John Blackmon 0 Branko Stefanovic 0 Arthur Veis, Northwestern University, United States of America 0 Department of Biomedical Sciences, College of Medicine, Florida State University , Tallahassee, Florida , United States of America Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen a2(I) mRNA was at normal levels. Collagen a1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into a1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. 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. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis. - Funding: Funding: R01 National Institutes of Health 5R01DK059466-08 to BS, F31 National Institute on Alcohol Abuse and Alcoholism, National Research Service Award AA019845-01A1 to ZM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Fibroprolifirative disorders can target any organ system, ultimately leading to organ failure, and are a major contributor to significant morbidity and mortality worldwide [1,2]. Despite all the medical advances in the last several years, there are only supportive therapies, with no approved, effective and specific antifibrotic treatments available [3]. Normal wound healing is initiated by tissue injury and inflammation with release of fibrogenic cytokines, resulting in proliferative activation of fibroblasts and deposition of extracellular matrix (ECM) proteins [4]. Liver fibrosis is an out of control wound healing response that is usually irreversible [5]. During liver injury, quiescent hepatic stellate cells (HSC), which normally store vitamin A, activate and differentiate into myofibroblast-like cells [6]. Activated HSC undergo proliferation and are the major cell type responsible for hepatic fibrogenesis [7]. Pathogenesis of alcoholic liver disease is mediated by free radicals and suppression of innate immunity [8,9]. Alcohol metabolism in hepatocytes results in production of reactive oxygen species and acetaldehyde that can directly activate HSCs. In addition, alcohol results in increased uptake of lipopolysaccharides (LPS) from the gut flora. Increased levels of LPS trigger Kupffer cells to release pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-a), interleukins (IL-6 and IL-1), as well as profibrotic cytokines, such as TGF-b [10,11]. Although several collagen types are secreted by activated HSC, type I collagen is the most abundant and responsible for clinical manifestations of liver fibrosis, as well as manifestations of other fibroprolifirative disorders [4,12]. The excessive collagen deposition in hepatic fibrosis is primarily due to the dramatic up-regulation of type I collagen synthesis at the post-transcriptional level [13]. This includes stabilization and more efficient translation of collagen mRNAs [1316]. Type I collagen is a heterotrimer composed of two a1(I) and one a2(I) polypeptides and is the most abundant protein in the human body [17]. mRNAs encoding for type I collagen have an unique 59 stem loop structure (59SL) in their 59 untranslated regions that contains the start codon [16]. Our lab has cloned and characterized La ribonucleoprotein domain family member 6 (LARP6) as the protein which binds 59SL and regulates translation of type I collagen mRNAs [18]. LARP6 binds the 59SL of collagen mRNAs with high affinity and specificity and is the central component of a ribonucleoprotein complex that assembles on the 59SL [18]. LARP6 associates collagen mRNAs with two types of cytoskeletal filaments: with intermediate filaments composed of vimentin that prolonged the half-life of collagen mRNAs and with nonmuscle myosin filaments required for synthesis of natural heterotrimer of type I collagen [19,20]. Disulfide bonding and post-translational modifications of collagen polypeptides take place during the translational elongation phase and before the heterotrimer is released into the lumen of the endoplasmic reticulum (ER) [21]. We postulated that nonmuscle myosin filaments facilitate translation of collagen a1(I) and a2(I) mRNAs within the subcompartments of the ER to allow coordinated synthesis and folding of the heterotrimeric type I collagen [19]. In the past years great efforts have been made to understand the molecular mechanism of collagen synthesis [1820,22]. However, there has been no report on involvement of the FK506 binding proteins (FKBPs) in this process. FKBPs represent a superfamily of proteins that are implicated in T-Cell activation, ribosome biogenesis, tumor suppression, and transcription regulation [23 26]. All FKBPs have cis-trans prolyl isomerase (PPIase) activity and bind multiple immunosuppressant drugs, like rapamycin, FK506 (tacrolimus) and cyclosporin A [24,27]. Tacrolimus (FK506), a macrolide antibiotic with potent immunosuppressive effects was isolated from Streptomyces tsukubaensis and has been previously used to prevent allograft and for treatment of autoimmune disorders in humans [2830]. Animal studies have indicated that FK506 can inhibit neutrophil infiltration, reduce free radicals, decrease generation of reactive oxygen species and suppress pro-inflammatory cascade [3135], thus, potentially affecting the factors which mediate alcoholic liver injury. A study on pulmonary fibrosis in mice suggested that FK506 can be a potent antifibrotic agent [36]. However, studies on liver fibrosis induced by bile duct ligation o (...truncated)