The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease.

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

Treatment of keloids with a single dose of low-energy superficial X-ray radiation to prevent recurrence after surgical excision: An in vitro and in vivo study.

BACKGROUND: Although keloids have been empirically treated with steroids and radiation, evidence-based radiation parameters for keloid therapy are lacking. OBJECTIVE: To determine evidence-based radiation parameters for blocking keloid fibroblast proliferation in vitro and apply them to patients. METHODS: The effects of various radiation parameters and steroids on cell proliferation, cell death, and collagen production in keloid explants and fibroblasts were evaluated with standard assays. Effective radiation parameters were then tested on patients. RESULTS: No differences were observed between the effects of 50 and 320 kV radiation or between single and fractionated radiation doses on keloid fibroblasts. A 3 Gy, 50 kV dose inhibited keloid fibroblast proliferation in culture, whereas 9 Gy completely blocked their outgrowth from explants by inducing multiple cell death pathways and reducing collagen levels. Thirteen of 14 keloids treated with a single 8 Gy, 50 kV dose of radiation did not recur, although 4 patients with 6 keloids were lost to follow-up. LIMITATIONS: Seventy-five percent of patients received steroids for pruritus, whereas approximately 25% of patients were lost to follow-up. CONCLUSIONS: A single 8 Gy dose of superficial 50 kV radiation delivered an average of 34 days after keloid excision maybe sufficient to minimize recurrence, including in individuals resistant to steroids. Higher radiation energies, doses, or fractions may be unnecessary for keloid therapy.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. RESULTS: We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. CONCLUSIONS: Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression.

Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60-70 days). However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6), is specifically involved in type I collagen regulation. In the 5'untranslated region (5'UTR) of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL) structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5'SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP), 25 kD FK506 binding protein (FKBP25) and RNA helicase A (RHA), contribute to this process.

Insulin-like growth factor-1 increases synthesis of collagen type I via induction of the mRNA-binding protein LARP6 expression and binding to the 5' stem-loop of COL1a1 and COL1a2 mRNA.

Collagen content in atherosclerotic plaque is a hallmark of plaque stability. Our earlier studies showed that insulin-like growth factor-1 (IGF-1) increases collagen content in atherosclerotic plaques of Apoe(-/-) mice. To identify mechanisms we investigated the effect of IGF-1 on the la ribonucleoprotein domain family member 6 (LARP6). LARP6 binds a stem-loop motif in the 5'-UTR of the mRNAs encoding the collagen type I α-subunits (α1(I) and α2(I)), and coordinates their translation into the heterotrimeric collagen type I molecule. In human aortic smooth muscle cells (SMCs), IGF-1 rapidly increased LARP6 expression and the rate of collagen synthesis and extracellular accumulation. IGF-1 increased both LARP6 and collagen type I expression via a post-transcriptional and translation-dependent mechanism involving PI3K/Akt/p70S6k-signaling. Immunoprecipitation of LARP6, followed by qPCR indicated that IGF-1 increased the level of COL1a1 and COL1a2 mRNA bound to LARP6. Mutation of the 5' stem-loop of Col1a1 mRNA, which inhibits binding of LARP6, abolished the ability of IGF-1 to increase synthesis of collagen type I. Furthermore, overexpression of a 5' stem-loop RNA molecular decoy that sequesters LARP6, prevented the ability of IGF-1 to increase pro-α1(I) and mature α1(I) expression in cultured medium. IGF-1 infusion in Apoe(-/-) mice increased expression of LARP6 and pro-α1(I) in aortic lysates, and SMC-specific IGF-1-overexpression robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, we identify LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, which may play an important role in promoting atherosclerotic plaque stability.

A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs.

Type I collagen is composed of two α1(I) polypeptides and one α2(I) polypeptide and is the most abundant protein in the human body. Expression of type I collagen is primarily controlled at the level of mRNA stability and translation. Coordinated translation of α(I) and α2(I) mRNAs is necessary for efficient folding of the corresponding peptides into the collagen heterotrimer. In the 5' untranslated region (5' UTR), collagen mRNAs have a unique 5' stem-loop structure (5' SL). La ribonucleoprotein domain family member 6 (LARP6) is the protein that binds 5' SL with high affinity and specificity and coordinates their translation. Here we show that RNA helicase A (RHA) is tethered to the 5' SL of collagen mRNAs by interaction with the C-terminal domain of LARP6. In vivo, collagen mRNAs immunoprecipitate with RHA in an LARP6-dependent manner. Knockdown of RHA prevents formation of polysomes on collagen mRNAs and dramatically reduces synthesis of collagen protein, without affecting the level of the mRNAs. A reporter mRNA with collagen 5' SL is translated three times more efficiently in the presence of RHA than the same reporter without the 5' SL, indicating that the 5' SL is the cis-acting element conferring the regulation. During activation of quiescent cells into collagen-producing cells, expression of RHA is highly up-regulated. We postulate that RHA is recruited to the 5' UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of the most abundant protein in the human body.

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.

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

[This corrects the article DOI: 10.1371/journal.pone.0065897.].

Predictive analysis of the scoliotic curve using a subject's 3D model.

A predictive analysis of the conservative scoliosis treatment is necessary, in which a 3D model of an optimal treatment algorithm is a basic part in the design of a prosthetic corset. Since CAD technology has proven to be very useful in the field of prosthetics and orthotics, we used an open-source software to plan the correction of the scoliotic curve on a virtual model of the subject's torso. The shape of the scoliosis was simplified by means of a directional polygon, which was drawn in a reverse manner depending on the directional arcs of the scoliotic curve. The resulting scoliosis correction, simulated in a predictive analysis, was defined by changing the Cobb angle, eccentricity, and torso height. With the proposed low-cost method of predictive analysis, it is possible to help CPOs to a more accurate and effective design of orthoses and corrective aids and to comprehensively determine the entire treatment procedure.

Mutation of the 5'-untranslated region stem-loop structure inhibits α1(I) collagen expression in vivo.

Type I collagen is a heterotrimeric extracellular matrix protein consisting of two α1(I) chains and one α2(I) chain. During liver fibrosis, activated hepatic stellate cells (HSCs) are the major source of the type I collagen that accumulates in the damaged tissue. Expression of α1(I) and α2(I) collagen mRNA is increased 60-fold compared with quiescent stellate cells and is due predominantly to post-transcriptional message regulation. Specifically, a stem-loop structure in the 5'-untranslated region of α1(I) collagen mRNA may regulate mRNA expression in activated HSCs through its interaction with stem-loop binding proteins. The stem-loop may also be necessary for efficient production and folding of the type I collagen heterotrimer. To assess the role of the stem-loop in type I collagen expression in vivo, we generated a knock-in mouse harboring a mutation that abolished the stem-loop structure. Heterozygous and homozygous knock-in mice exhibited a normal phenotype. However, steady-state levels of α1(I) collagen mRNA decreased significantly in homozygous mutant MEFs as well as HSCs; intracellular and secreted type I collagen protein levels also decreased. Homozygous mutant mice developed less liver fibrosis. These results confirm an important role of the 5' stem-loop in regulating type I collagen mRNA and protein expression and provide a mouse model for further study of collagen-associated diseases.

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.

Fibrotic Scar in CNS Injuries: From the Cellular Origins of Fibroblasts to the Molecular Processes of Fibrotic Scar Formation.

Central nervous system (CNS) trauma activates a persistent repair response that leads to fibrotic scar formation within the lesion. This scarring is similar to other organ fibrosis in many ways; however, the unique features of the CNS differentiate it from other organs. In this review, we discuss fibrotic scar formation in CNS trauma, including the cellular origins of fibroblasts, the mechanism of fibrotic scar formation following an injury, as well as the implication of the fibrotic scar in CNS tissue remodeling and regeneration. While discussing the shared features of CNS fibrotic scar and fibrosis outside the CNS, we highlight their differences and discuss therapeutic targets that may enhance regeneration in the CNS.

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.

Discovery of a Lead Compound for Specific Inhibition of Type I Collagen Production in Fibrosis.

Fibrosis is a major medical problem caused by excessive synthesis of the extracellular matrix, composed predominantly of type I collagen, in various tissues. There are no approved antifibrotic drugs, and the major obstacle in finding clinically relevant compounds is the lack of specificity of current experimental drugs for type I collagen. Here we describe the discovery of a lead compound that specifically inhibited secretion of type I collagen by fibroblasts in culture at IC50 = 4.5 µM. The inhibition was specific for type I collagen, because secretion of fibronectin was not affected. In vitro, the compound inhibited binding of LARP6, the master regulator of translation of type I collagen mRNAs, to the 5' stem-loop sequence element which regulates their translation. Because binding of LARP6 to collagen mRNAs is crucial for the development of fibrosis, this inhibitor represents a promising lead for optimization into specific antifibrotic drugs.

Innovative approaches to designing and manufacturing a prosthetic thumb.

CASE DESCRIPTION: Conventional methods for producing custom prosthetic fingers are time-consuming, can be uncomfortable for the patient, and require a skilled prosthetist. The subject was a 40-year-old male with congenital absence of the thumb and related metacarpal bone on the right non-dominant hand, anomaly of the lengths of individual upper limb segments, and contracture of the elbow joint. This hand presentation made it impossible for him to perform thumb opposition, which is a very important function for common daily activities. OBJECTIVE: The goal was to design an individual passive thumb prosthesis using free open-source software, 3D scanning technology, and additive manufacturing methods (i.e., fused filament fabrication). STUDY DESIGN: Case report. TREATMENT: Artificial thumb prostheses with two types of bases and fastening interfaces were designed and manufactured. One combination was chosen as the best alternative. OUTCOMES: The shape, positioning, firmness, and fastening of the prosthesis were compliant enough for the patient to be able to hold objects with his healthy fingers and artificial thumb. This innovative approach to fabrication of a custom thumb prosthesis provided considerable advantages in terms of custom sizing, manufacturing time, rapid production, iteration, comfort, and costs when compared to conventional methods of manufacturing a hand prosthesis. CONCLUSION: The methodology of designing and manufacturing a prosthetic thumb using 3D scanning and additive manufacturing technologies have been demonstrated to be adequate from a practical point of view. These technologies show potential for use in the practice of prosthetics.

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.

RNA-binding protein RBMS3 is expressed in activated hepatic stellate cells and liver fibrosis and increases expression of transcription factor Prx1.

Hepatic stellate cells (HSCs) are mesenchymal cells of the liver, activation of which is responsible for excessive synthesis of extracellular matrix, including type I collagen, and development of liver fibrosis. The activation of HSCs is driven by transcription factors and pair-related homeobox transcription factor Prx1 was identified as one of the transcription factors involved in this process, because transcription of collagen alpha1(I) gene is stimulated by Prx1 in HSCs and in the liver. Here, we show that expression of the RNA-binding protein RBMS3 is upregulated in the activation of HSCs and fibrotic livers. Immunoprecipitation followed by differential display identified Prx1 mRNA as one of the mRNAs interacting with RBMS3. The RBMS3 sequence-specific binding site was mapped to 60 nt located 1946 nt 3' of the stop codon of Prx1 mRNA. Ectopic expression of RBMS3 in quiescent HSCs, which express trace amounts of type I collagen, increased expression of Prx1 mRNA and collagen alpha1(I) mRNA. Expression of reporter Prx1 mRNA containing the RBMS3 binding site was higher than the mRNA lacking this site. Over-expression of RBMS3 further increased the steady-state level of the reporter mRNA-containing RBMS3 binding site, but had no effect on the mRNA lacking this site. Binding of RBMS3 to the Prx1 3' UTR increased the half-life of this mRNA, resulting in increased protein synthesis. These results suggest that RBMS3, by binding Prx1 mRNA in a sequence-specific manner, controls Prx1 expression and indirectly collagen synthesis. This is the first description of the function of RBMS3, as a key regulator of profibrotic potential of HSCs, representing a novel mechanism by which activated HSCs contribute to liver fibrosis.

Homeobox gene Prx1 is expressed in activated hepatic stellate cells and transactivates collagen alpha1(I) promoter.

Hepatic stellate cells (HSCs) are mesenchymal cells of the liver, which are normally in quiescent state and synthesize tracing amounts of extracellular matrix proteins. Upon fibrogenic stimulus, HSCs become activated and increase synthesis of type I collagen 50-100 fold. Prx1 and Prx2 are two homeobox transcription factors which are required for mesenchymal tissue formation during embryogenesis. The present study shows that Prx1 mRNA is expressed in in vivo and in vitro activated HSCs, but not in quiescent HSCs. Prx1 is also expressed in fibrotic livers, while it is undetectable in normal livers. Overexpression of Prx1a in quiescent HSCs cultured in vitro induced collagen alpha1(I) mRNA and TGFbeta3 mRNA expression. Prx1 transactivated TGFbeta3 promoter 3 fold in transient transfection experiments. In the whole liver, Prx1a induced expression of collagen alpha1(I), alpha2(I), alpha1(III) and alpha-smooth muscle mRNAs, which are the markers of activation of HSCs. Prx1 also increased expression of collagen alpha1(I) mRNA after acute liver injury. This suggests that Prx1a promotes activation of HSCs and expression of type I collagen. Several regions in the collagen alpha1(I) promoter were identified which mediate transcriptional induction by Prx1. The regions are scattered throughout the promoter and individually have modest effects; however, the cumulative effect of all sequences is >50 fold. This is the first description of the effects of Prx1 in HSCs and in the liver, and identification of the two Prx1 target genes, which play a pivotal role in development of liver fibrosis, is a novel finding for liver pathophysiology.