Characterization of Sequence-Specific Binding of LARP6 to the 5' Stem-Loop of Type I Collagen mRNAs and Implications for Rational Design of Antifibrotic Drugs.

Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5' stem-loop (5'SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5'SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5'SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the ß-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5'SL. The major site of crosslinking of LARP6 to 5'SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5'SL. Presence of RRM increases the stability of complex between La domain and 5'SL RNA and RRM domain does not make extensive contacts with 5'SL RNA. We propose a model in which the initial recognition of 5'SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.

Imaging of type I procollagen biosynthesis in cells reveals biogenesis in highly organized bodies; Collagenosomes.

Mechanistic aspects of type I procollagen biosynthesis in cells are poorly understood. To provide more insight into this process we designed a system to directly image type I procollagen biogenesis by co-expression of fluorescently labeled full size procollagen α1(I) and one α2(I) polypeptides. High resolution images show that collagen α1(I) and α2(I) polypeptides are produced in coordination in discrete structures on the ER membrane, which we termed the collagenosomes. Collagenosomes are disk shaped bodies, 0.5-1 µM in diameter and 200-400 nm thick, in the core of which folding of procollagen takes place. Collagenosomes are intimately associated with the ER membrane and their formation requires intact translational machinery, suggesting that they are the sites of nascent procollagen biogenesis. Collagenosomes show little co-localization with the COPII transport vesicles, which export type I procollagen from the ER, suggesting that these two structures are distinct. LARP6 is the protein which regulates translation of type I collagen mRNAs. The characteristic organization of collagenosomes depends on binding of LARP6 to collagen mRNAs. Without LARP6 regulation, collagenosomes are poorly organized and the folding of α1(I) and α2(I) polypeptides into procollagen in their cores is diminished. This indicates that formation of collagenosomes is dependent on regulated translation of collagen mRNAs. In live cells the size, number and shape of collagenosomes show little change within several hours, suggesting that they are stable structures of type I procollagen biogenesis. This is the first report of structural organization of type I collagen biogenesis in collagenosomes, while the fluorescent reporter system based on simultaneous imaging of both type I collagen polypeptides will enable the detailed elucidation of their structure and function.

Technology for Discovery of Antifibrotic Drugs: Phenotypic Screening for LARP6 Inhibitors Using Inverted Yeast Three Hybrid System.

Fibrosis is defined by excessive production of type I collagen in various organs. Excessive type I collagen production in fibrosis is stimulated by binding of RNA protein LARP6 to the structural element of collagen mRNAs, the 5' stem loop (5'SL). The LARP6-dependent regulation is specific for type I collagen and critical for fibrosis development. Inhibitors of LARP6 binding have potential to be specific antifibrotic drugs, as evidenced by the discovery of one such inhibitor. To create technology for phenotypic screening of additional compounds we developed an inverted yeast three hybrid system. The system is based on expression of human LARP6 and a short RNA containing the 5'SL of human collagen α1(I) mRNA in Saccharomyces cerevisiae cells. The cells were engineered in such a way that when LARP6 is bound to 5'SL RNA they fail to grow in a specific synthetic medium. Dissociation of LARP6 from 5'SL RNA permits the cell growth, allowing identification of the inhibitors of LARP6 binding. The assay simply involves measuring optical density of cells growing in multiwall plates and is pertinent for high throughput applications. We describe the specificity of the system and its characteristics for high throughput screening. As a proof of principle, the result of one screen using collection of FDA approved drugs is also presented. This screen demonstrates that using this technology discovery of novel LARP6 inhibitors is possible.

Discovery and evaluation of inhibitor of LARP6 as specific antifibrotic compound.

Fibrosis is characterized by excessive production of type I collagen. Biosynthesis of type I collagen in fibrosis is augmented by binding of protein LARP6 to the 5' stem-loop structure (5'SL), which is found exclusively in type I collagen mRNAs. A high throughput screen was performed to discover inhibitors of LARP6 binding to 5'SL, as potential antifibrotic drugs. The screen yielded one compound (C9) which was able to dissociate LARP6 from 5' SL RNA in vitro and to inactivate the binding of endogenous LARP6 in cells. Treatment of hepatic stellate cells (liver cells responsible for fibrosis) with nM concentrations of C9 reduced secretion of type I collagen. In precision cut liver slices, as an ex vivo model of hepatic fibrosis, C9 attenuated the profibrotic response at 1 µM. In prophylactic and therapeutic animal models of hepatic fibrosis C9 prevented development of fibrosis or hindered the progression of ongoing fibrosis when administered at 1 mg/kg. Toxicogenetics analysis revealed that only 42 liver genes changed expression after administration of C9 for 4 weeks, suggesting minimal off target effects. Based on these results, C9 represents the first LARP6 inhibitor with significant antifibrotic activity.

Screening for antifibrotic compounds using high throughput system based on fluorescence polarization.

Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I) mRNA and α2(I) mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL). Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis in vivo. Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.

Characterization of binding of LARP6 to the 5' stem-loop of collagen mRNAs: implications for synthesis of type I collagen.

Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5' UTRs, the 5'SL. LARP6 binds the 5'SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5'SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5'SL of collagen α2(I) mRNA is more stable than the binding to 5'SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5'SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5'SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5'SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis.

Role of cytokine receptor-like factor 1 in hepatic stellate cells and fibrosis.

AIM: To elucidate the role of cytokine receptor-like factor 1 (CRLF1) in hepatic stellate cells and liver fibrosis. METHODS: Rat hepatic stellate cells (HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro. Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays. Injections of carbon tetrachloride (CCl(4)) for 4 wk were employed to induce liver fibrosis. The degree of fibrosis was assessed by Sirius red staining. Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver. The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro. Expression of CRLF1, CLCF1 and ciliary neurotrophic factor receptor (CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Expression of profibrotic cytokines and collagens was analyzed by the same method. RESULTS: CRLF1 is a secreted cytokine with unknown function. Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin (IL)-6. Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs. Likewise, expression of CRLF1 was very low in normal livers, but was highly upregulated in fibrotic livers, where its expression correlated with the degree of fibrosis. A cofactor of CLRF1, cardiotrophin-like cytokine factor 1 (CLCF1), and the receptor which binds CRLF1/CLCF1 dimer, the CNTFR, were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers, indicating a constitutive expression. Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines, suggesting that the factor itself is not pro-inflammatory. Ectopic expression in quiescent HSCs, however, retarded their activation into myofibroblasts and specifically decreased expression of type III collagen. Inhibition of type III collagen expression by CRLF1 was also seen in the whole liver. Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type III collagen in fibrosis. CONCLUSION: By regulating activation of HSCs and expression of type III collagen, CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.

Nonmuscle myosin-dependent synthesis of type I collagen.

Type I collagen, synthesized in all tissues as the heterotrimer of two alpha1(I) polypeptides and one alpha2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5' stem-loop in collagen alpha1(I) and alpha2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen alpha1(I) homotrimer, diminished intracellular colocalization of collagen alpha1(I) and alpha2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen alpha1(I) and alpha2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.

Binding of LARP6 to the conserved 5' stem-loop regulates translation of mRNAs encoding type I collagen.

Type I collagen is the most abundant protein in the human body, produced by folding of two alpha1(I) polypeptides and one alpha2(I) polypeptide into the triple helix. A conserved stem-loop structure is found in the 5' untranslated region of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein that binds the collagen 5' stem-loop in a sequence-specific manner. LARP6 has a distinctive bipartite RNA binding domain not found in other members of the La superfamily. LARP6 interacts with the two single-stranded regions of the 5' stem-loop. The K(d) for binding of LARP6 to the 5' stem-loop is 1.4 nM. LARP6 binds the 5' stem-loop in both the nucleus and the cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes; however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but 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 postulate that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.

Sequence-dependent effect of interruptions on microsatellite mutation rate in mismatch repair-deficient human cells.

Although microsatellite mutation rates generally increase with increasing length of the repeat tract, interruptions in a microsatellite may stabilize it. We have performed a direct analysis of the effect of microsatellite interruptions on mutation rate and spectrum in cultured mammalian cells. Two mononucleotide sequences (G(17) and A(17)) and a dinucleotide [(CA)(17)] were compared with interrupted repeats of the same size and with sequences of 8 repeat units. MMR-deficient (MMR(-)) cells were used for these studies to eliminate effects of this repair process. Mutation rates were determined by fluctuation analysis on cells containing a microsatellite sequence at the 5' end of an antibiotic-resistance gene; the vector carrying this sequence was integrated in the genome of the cells. In general, interrupted sequences had lower mutation rates than perfect ones of the same size, but the magnitude of the difference was dependent upon the sequence of the interrupting base(s). Some interrupted repeats had mutation rates that were lower than those of perfect sequences of the same length but similar to those of half the length. This suggests that interrupting bases effectively divide microsatellites into smaller repeat runs with mutational characteristics different from those of the corresponding full-length microsatellite. We conclude that interruptions decrease microsatellite mutation rate and influence the spectrum of frameshift mutations. The sequence of the interrupting base(s) determines the magnitude of the effect on mutation rate.

Mechanism of direct hepatotoxic effect of KC chemokine: sequential activation of gene expression and progression from inflammation to necrosis.

This work aimed to show that an important, yet unrecognized, role of KC chemokine in the liver is regulation of gene expression. KC expression in the liver stimulated three classes of genes in this temporal order: immediate-early genes, proinflammatory genes, and profibrotic genes. Transcription factors E2F5 and early growth response 1 (EGR1), Ca(2+) signaling molecules S100A8 and S100A9, and two oxidative stress-induced genes were identified as immediate-early genes of KC. Expression of these genes was stimulated at 3-5-fold increased KC concentrations. Expression of proinflammatory genes was activated 6 h after the immediateearly genes, and they included interleukin-1alpha (IL-1alpha) and IL-1beta. KC receptor gene CXCR2 was also upregulated, suggesting that KC may act through a positive feedback loop. Stimulation of expression of profibrotic genes, including type I collagen, was seen only after the proinflammatory genes were highly expressed for 12 h. KC is a potent regulator of gene expression that proceeds in a sequential manner. Immediate-early genes of KC stimulation were identified. The positive feedback regulation and an increased oxidative stress induced by KC may explain the poor prognosis in liver patients with elevated levels of CXC chemokines.

Direct hepatotoxic effect of KC chemokine in the liver without infiltration of neutrophils.

KC is a mouse homolog of human chemokine gro-alpha (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection of CCl(4), followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/ gro-alpha in liver pathology.

Variation in efficiency of DNA mismatch repair at different sites in the yeast genome.

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.

Inhibitory effect of dicationic diphenylfurans on production of type I collagen by human fibroblasts and activated hepatic stellate cells.

Excessive production of extracellular matrix is responsible for clinical manifestations of fibroproliferative disorders and drugs which can inhibit excessive synthesis of type I collagen are needed for the therapy. Several dicationic diphenylfurans were synthesized and were found to bind RNA. Two of these type compounds were able to reduce synthesis of type I collagen by human fibroblasts and human activated hepatic stellate cells (HSCs). Activated HSCs are responsible for collagen production in liver fibrosis. When added at 40 microM compound 588 reduced intracellular level and secretion of procollagen alpha1(I) by 50%, while compound 654 reduced these parameters by more than 80% at 20 microM. 654 also significantly reduced secretion of fibronectin. Toxic effects were observed at 80 microM for 588 and 40 microM for 654. 654 reduced expression of a reporter gene with collagen signal peptide, while expression of the same gene without signal peptide was unaffected. Also, expression of intracellular proteins tubulin and calnexin was unchanged. 654 accumulated inside the cell in the cytoplasm and did not change the steady-state level of collagen mRNAs. Treatment of cells with proteosome inhibitor MG132 did not change the inhibitory effect of 654, suggesting that 654 acts as suppressor of translation of proteins containing a signal peptide. Most secreted proteins of fibroblasts and activated HSCs are components of extracellular matrix. Therefore inhibition of their production, as shown here for procollagen alpha1(I) and fibronectin, may be a useful property of some of diphenylfurans, making these compounds a basis for development of antifibrotic drugs.

TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis.

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding.