The Specific Regulation of Type I Collagen Synthesis in Fibrosis
Fibrosis is characterized by excessive synthesis of type I collagen which impedes the normal function of an affected organ. Type I collagen is the most abundant protein in the human body, produced by the folding of two a1(I) polypeptides and one a2(I) polypeptides into the triple helix. Expression of collagen 1a(I) gene is predominantly regulated at the level of mRNA stability and translation. A conserved stem'Cloop structure is found in the 5' untranslated region of collagen mRNAs. The 5'stem-loop structure which is critical for the coordinated translation encompasses the start codon in collagen mRNAs and regulates collagen synthesis by the binding of RNA binding proteins. Assembly of collagen heterotrimer occurs on the membrane of endoplasmic reticulum (ER) while a1(I) and a2(I) mRNAs are associated with polysomes. I have found that the binding of 5'stem-loop binding proteins LARP6 and nonmuscle myosin II to the 5'stem-loop regulates coordinated translation of collagen mRNAs and is required for collagen triple helix formation. My work describes that the coordinated translation of collagen mRNAs increases local concentration of the chains necessary for productive folding. Chapter 1 presents the identification of 5'stem- loop binding protein and its function in regulating collagen synthesis. To identify 5'stem-loop binding proteins we performed expressional cloning and cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein which binds to both 5'stem- loop of collagen a1(1) and a2(1) mRNAs a sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain (amino acids 32-45 and 218-300) which is not found in other members of the La super family. The RNA binding domain of.LARP6 interacts with the two single-stranded regions of the 5'stem-loop. The Kd for binding of LARP6 to the 5'stem-loop is 1.4 nM. The binding is to the single stranded regions of the bulge of 5'stem-loop RNA, in both the nucleus and the cytoplasm. Recombinant LARP6 has a similar binding affinity and specificity to the 5'stem-loop as the endogenous LARP6. The C-terminal region of LARP6 has a nuclear localization signal, which allows LARP6 to accumulate in the nucleus. Combination of gain of function by adenoviral delivery and loss of function by using either dominant negative forms of LARP6 or siRNA directly against the RNA sequence of LARP6 were employed to determine the function of LARP6. Over expression of LARP6 decreased synthesis of collagen protein, however it did not change collagen mRNA steady state level. Also, collagen mRNAs were redistributed from polysomal fractions to the fractions representing the free polysomes in LARP6 overexpressed cells. This suggests that overexpression of LARP6 blocked ribosomal loading on collagen mRNAs and inhibited collagen mRNA translation. Endogenous LARP6 and overexpressed LARP6 were not associated with polysomes. These results suggested that LARP6 prevents premature translation of collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased steady state level of collagen polypeptide in the cell as well as the section rate of the protein. However, collagen mRNA stability was not affected, nor was the degradation of collagen protein by proteasome. Therefore, it is likely that collagen mRNA translation had been inhibited by the depletion of LARP6. We describe that collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using a collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but it was observed only when the reporter was encoded by mRNA with the 5'stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulated that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer. In Chapter 2, we describe that nonmuscle myosin IIB plays an important role in regulating collagen synthesis. First, we identified nonmuscle myosin II B as LARP6 binding protein by tobramycin affinity purification and confirmed the binding specificity of myosin II B to LARP6 and to collagen mRNAs. We showed that the C terminus of LARP6 was required for the binding to nonmuscle myosin IIB, and that this binding was not RNA dependent. Secondly, we identify the role of nonmuscle myosin IIB in regulating type I collagen synthesis. Nonmuscle myosin II filaments are required for the secretion of collagen a2(I) peptide and the colocalization of a1(I) and a2(I) peptides in the cell. The motor activity of myosin II as well as the integrity of the filaments is involved in this process. We also discovered that the effect of myosin II on type I collagen synthesis was mediated by 5'stem-loop of LARP6 and through the protein-protein interaction with LARP6. At the end, we determined that nonmuscle myosin IIB filaments are involved in directing collagen mRNAs to polysomes for translation. Therefore, we concluded that nonmuscle myosin II interacts with LARP6 and collagen 5'stem-loop to regulates coordinate translation of collagen mRNAs. In Chapter 3, we explain the role of LARP6 and nonmuscle myosin II in collagen synthesis stimulated by cytokines and growth factors TGF-beta and ouabain. Cytokines and growth factors including TGF-beta and ouabain are stimulators of fibrosis and they increase collagen synthesis in scleroderma skin fibroblasts, hepatic stellate cells, kidney fibroblasts and cardiac fibroblasts. When we down regulated LARP6 or disrupted nonmuscle myosin II filaments, the stimulation of type I collagen secretion by TGF-beta 1 and ouabain was diminished. Therefore, we concluded that LARP6 and nonmuscle myosin II regulate inducible collagen synthesis in fibrosis. This dissertation describes the specific regulation of type I collagen synthesis in fibrosis. The first finding was that collagen protein synthesis took place of discrete regions on the ER membrane through by the binding of LARP6 to 5'stem-loop. The second finding was that coordinated synthesis of type I collagen polypeptide requires nonmuscle myosin II. The third finding discovered that profibrotic cytokines like TGF-beta and ouabain induced collagen synthesis through LARP6 and nonmuscle myosin II mechanism in human scleroderma skin fibroblasts and rat cardiac fibroblasts. In conclusion, we have discovered that LARP6 and nonmuscle myosin II regulated collagen synthesis. This pathway may contribute to excess collagen deposition in fibrosis. This information will help to find future anti-fibrotic therapy.
March 15, 2010.
A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Branko Stefanovic, Professor Directing Dissertation; Hengli Tang, University Representative; Myra M. Hurt, Committee Member; Yanchang Wang, Committee Member.
Florida State University
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