Preproendothelin-1 expression is negatively regulated by IFNγ during hepatic stellate cell activation

Endothelin-1 (ET-1), a powerful vasoconstrictor peptide, is produced by activated hepatic stellate cells (HSC) and promotes cell proliferation, fibrogenesis, and contraction, the latter of which has been thought to be mechanistically linked to portal hypertension in cirrhosis. Interferon-γ (IFNγ), a Th1 cytokine produced by T cells, inhibits stellate cell proliferation, fibrogenesis, and muscle-specific gene expression. Whether IFNγ-induced inhibitory effects are linked to regulation of ET-1 expression in activated stellate cells remains unknown. Here we examined IFNγ's effects on preproET-1 mRNA expression and the signaling pathways underlying this process. We demonstrated that preproET-1 mRNA expression in HSCs was prominently increased during cell culture-induced activation; IFNγ significantly inhibited both preproET-1 mRNA expression and ET-1 peptide production. Similar results were found in an in vivo model of liver injury and intraperitoneal administration of IFNγ. PreproET-1 promoter analysis revealed that IFNγ-induced inhibition of preproET-1 mRNA expression was closely linked to the AP-1 and Smad3 signaling pathways. Furthermore, IFNγ reduced JNK phosphorylation, which tightly was associated with decreased phosphorylation of downstream factors c-Jun and Smad3 and decreased binding activity of c-Jun and Smad3 in the preprpET-1 promoter. Importantly, IFNγ reduced both c-Jun mRNA and protein levels. Given the important role of ET-1 in wound healing, our results suggest a novel negative signaling network by which IFNγ inhibits preproET-1 expression, highlighting one potential molecular mechanism for IFNγ-induced host immunomodulation of liver fibrogenesis.     

Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes.

During the wound healing process lysis of basement membranes precedes keratinocyte migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming growth factor-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming growth factor-beta 1 was found to increase the expression of both 92- and 72-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal keratinocytes. The 92-kDa enzyme predominated in both unstimulated and stimulated cultures. The 92-kDa form was stimulated over 5-fold, and the other form by a factor of 2-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 92-kDa enzyme was similar in culture medium containing either 0.15 or 1.2 mM calcium chloride. Rat mucosal keratinocytes secreted only 92-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.2-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human keratinocytes were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 92-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human keratinocytes, offering a possible explanation of how keratinocytes detach from basement membranes prior to the migration over the wound bed.     

Suppression of type I collagen production by microRNA-29b in cultured human stellate cells

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through imperfect base pairing with the 3′ untranslated region (3′UTR) of target mRNA. We studied the regulation of alpha 1 (I) collagen (Col1A1) expression by miRNAs in human stellate cells, which are involved in liver fibrogenesis. Among miR-29b, -143, and -218, whose expressions were altered in response to transforming growth factor-β1 or interferon-α stimulation, miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 3′UTR. miR-29b also had an effect on SP1 expression. These results suggested that miR-29b is involved in the regulation of type I collagen expression by interferon-α in hepatic stellate cells. It is anticipated that miR-29b will be used for the regulation of stellate cell activation and lead to antifibrotic therapy.     

Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl₄ administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl₄-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12?h after acute CCl₄-intoxication. Tissue levels of ET-1, ET_A- and ET_B receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl₄-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.     

Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene

Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.     

Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.     

The role of integrin-linked kinase in liver wound healing

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle alpha-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle alpha-actin, transforming growth factor-beta, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n = 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n = 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha.

Changes in endothelial nitric oxide synthase (eNOS) expression may be involved in the endothelium-dependent vasorelaxation dysfunction associated with several vascular diseases. In the present work, we demonstrate that eNOS mRNA contains a previously undescribed cis element in the 3' untranslated region (3' UTR). A U+C-rich segment in the 3' UTR is critical in complex formation with bovine aortic endothelial cell cytosolic proteins. Tumor necrosis factor alpha (TNF-alpha), which destabilizes eNOS mRNA, increased the binding activity of the cytosolic proteins in a time-dependent manner. These data suggest that endothelial cytosolic proteins bind to the 3' UTR of eNOS mRNA. These proteins may play a role in TNF-alpha-induced eNOS mRNA destabilization.     

Binding of a phosphoprotein to the 3'' untranslated region of the mouse protamine 2 mRNA temporally represses its translation.

The synthesis of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated during germ cell development by mRNA storage for about 7 days in the cytoplasm of differentiating spermatids. Two highly conserved sequences, the Y and H elements present in the 3' untranslated regions (UTRs) of all known mammalian protamine mRNAs, form RNA-protein complexes and specifically bind a protein of 18 kDa. Here, we show that translation of fusion mRNAs was markedly repressed in reticulocyte lysates supplemented with a mouse testis extract enriched for the 18-kDa protein when the mRNAs contained the 3' UTR of mouse protamine 2 (mP2) or the Y and H elements of mP2. No significant decrease was seen when the fusion mRNAs contained the 3' UTR of human growth hormone. The 18-kDa protein is developmentally regulated in male germ cells, requires phosphorylation for RNA binding, and is found in the ribonucleoprotein particle fractions of a testicular postmitochondrial supernatant. We propose that a phosphorylated 18-kDa protein plays a primary role in repressing translation of mP2 mRNA by interaction with the highly conserved Y and H elements. At a later stage of male gamete differentiation, the 18-kDa protein no longer binds to the mRNA, likely as a result of dephosphorylation, enabling the protamine mRNA to be translated.     

Endothelin-1 and endothelin-converting enzyme-1 in human granulomatous pathology of eyelid: an immunohistochemical and in situ hybridization study in chalazia

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in several functions of eye pathophysiology, such as regulation of intraocular tension and retinal reactive vasoconstriction. As ET-1 pro-inflammatory and fibrosing activity is emerging in different fields of pathology, we investigated the expression of ET-1 and endothelin-converting enzyme-1 (ECE-1) in chalazia, granulomatous lesions of the eyelid. ET-1 and ECE-1 were analyzed by immunohistochemistry (IHC) in twenty surgically removed chalazia, with regard to expression in eyelid structures and inflammatory infiltrate. Phenotype of ET-1 expressing inflammatory cells was established by double immunofluorescence. The cellular localization of prepro-ET-1 (pp-ET-1) mRNA and ECE-1 mRNA was studied by nonisotopic in situ hybridization (ISH). Neutrophils (PMNs), macrophages and T-lymphocytes were scattered in stroma, around alveoli and grouped in lipogranulomas. PMNs, macrophages, basal epithelium of meibomian adenomers and central ducts immunostained for ET-1. ECE-1 protein was found in meibomian adenomers, conjunctival epithelium, tarsal mucous glands and in inflammatory cells. Hybridization signals for pp-ET-1 mRNA and ECE-1 mRNA were recognized in healthy and degenerating meibomian ducts, adenomers, inflammatory cells, as well as in vessel walls. ECE-1 mRNA was also present in conjunctival epithelium and Henle's crypts. Our findings suggest that the multifunctional peptide ET-1 may have a role in molecular genesis of tissue damage in chalazia. ET-1 cytokine activity is likely to support the migration of inflammatory cells and the setting of lipogranulomas; ET-1 stimulation might contribute to proliferation of fibroblasts and collagen synthesis. ET-1 upregulation on meibomian adenomers and ducts may further enhance granulomas formation by stimulating lipid release.     

Knockout of Endothelial Cell-Derived Endothelin-1 Attenuates Skin Fibrosis but Accelerates Cutaneous Wound Healing

Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.