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This study was designed to develop an animal model of alcoholic pancreatitis and to test the hypothesis that the dose of ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in corn oil (unsaturated fat), with or without a standard or high dose of ethanol, and medium-chain triglycerides (saturated fat) with a high dose of ethanol for 8 wk enterally. The dose of ethanol was increased as tolerance developed, which allowed approximately twice as much alcohol to be delivered in the high-dose group. Serum pancreatic enzymes and histology were normal after 4 wk of diets rich in unsaturated fat, with or without the standard dose of ethanol. In contrast, enzyme levels were elevated significantly by the high ethanol dose. Increases were blunted significantly by dietary saturated fat. Fibrosis and collagen alpha1(I) expression in the pancreas were not detectable after 4 wk of enteral ethanol feeding; however, they were enhanced significantly by the high dose after 8 wk. Furthermore, radical adducts detected by electron spin resonance were minimal with the standard dose; however, the high dose increased carbon-centered radical adducts as well as 4-hydroxynonenal, an index of lipid peroxidation, significantly. Radical adducts were also blunted by approximately 70% by dietary saturated fat. The animal model presented here is the first to demonstrate chronic alcohol-induced pancreatitis in a reproducible manner. The key factors responsible for pathology are the amount of ethanol administered and the type of dietary fat.

Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis.

The regulation of collagen alpha1(I) expression in hepatic stellate cells (HSCs) occurs in a complex fashion that is just beginning to be determined. The presence of regulatory sequences in both the 5' and 3' regions of the mRNA appear to be critical to its regulation in HSCs and are involved in the increased expression of collagen in activated HSCs. The 3' UTR contains a C-rich site that binds alphaCP, a known RNA-binding protein that is responsible for the increased stability of the mRNA in activated HSCs. Given that alphaCP is present in both activated and quiescent HSCs, there must be a mechanism for modifying alpha(CP to bind to the RNA in activated but not quiescent HSCs. The 5' region contains an evolutionary conserved stem-loop region that encompasses the translation initiation codon. This stem-loop can bind protein(s) in activated HSCs in an RNA cap-dependent manner. Such binding, together with the binding of alphaCP to the 3' UTR, can facilitate translation of collagen alpha1(I) mRNA, resulting in increased mRNA steady-state levels and collagen synthesis. A role of alphaCP in activating translation initiation has also been demonstrated. These two mechanisms work together to upregulate collagen alpha1(I) production in activated but not quiescent HSCs.

Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene.

Background/aims: The activation of hepatic stellate cells is a key initiating event in hepatic fibrogenesis. Although TGFbeta1 is a potent inducer of collagen alpha1(I) expression in vitro and elevated levels of TGFbeta1 are found in patients and experimental animals with hepatic fibrosis and cirrhosis, the role of increased TGFbeta1 in the initiation of hepatic stellate cell activation in vivo is unknown. We used two experimental approaches to study this relationship: 1) Induction of an acute liver injury with carbon tetrachloride (CCl4) in normal and TGFbeta1-knockout (ko) mice, and 2) overexpression of TGFbeta1 in the liver of wild-type mice using a recombinant replication-deficient adenovirus encoding human TGFbeta1 (Ad-TGFbeta1)., Methods: TGFbeta1-ko mice (n=6) and normal mice (n=6) were injected once intraperitoneally (i.p.) with CCl4 (1 microl/g BW) or mineral oil. Wild-type mice (n=3) were injected intravenously with Ad-TGFbeta1 (10(10) pfu) or a control virus expressing beta-galactosidase (Ad-LacZ, 10(10) pfu). Animals were sacrificed after 3 days and total liver RNA was prepared. The expression of collagen alpha1(I) mRNA normalized to GAPDH mRNA was measured by RNase protection assay, asmooth muscle actin (alpha-sma) protein expression was analyzed by Western blotting. The expression of TGFbeta1, TGFbeta2, and TGFbeta3 mRNAs were determined semi-quantitatively with RT-PCR., Results: The collagen alpha1(I) mRNA was increased 10-fold in CCl4-treated wild-type mice compared to the controls. This increase was reduced about 80% in the TGFbeta1-ko mice. The TGFbeta1 mRNA levels in the wild-type mice were proportional to the collagen alpha1(I) mRNA levels. a-sma, a marker of hepatic stellate cell activation, was expressed earlier and at a higher level in wild-type mice than TGFbeta-ko mice after CCl4 treatment. The Ad-TGFbeta1 infected mice had 14-fold higher hepatic TGFbeta protein levels and 15-fold higher collagen alpha1(I) mRNA levels than the Ad-LacZ-infected control mice. Collagen alpha1(I) mRNA levels were proportional to the transgenic TGFbeta1 mRNA levels, while the endogenous TGFbeta1 was only slightly higher than in the controls. TGFbeta2 and TGFbeta3 mRNA levels were elevated in CCl4-treated wild-type and TGFbeta1-ko mice and in Ad-TGFbeta1-infected mice compared to the controls., Conclusions: Absence of TGFbeta1 inhibits hepatic collagen alpha1(I) mRNA and alpha-sma protein expression by the toxic stimulus CCl4, and targeted TGFbeta1 overexpression increases collagen alpha1(I) mRNA and alpha-sma protein levels in the liver in vivo. Other TGFbeta family members do not compensate for the TGFbeta1 deficiency. This indicates that TGFbeta1 accelerates, but is not absolutely required, for the activation of hepatic stellate cells.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.

Posttranscriptional events may play an important role in regulating collagen alpha 1(I) mRNA in hepatic stellate cells. Quiescent stellate cells contain a very low level of this mRNA, but treatment of the cells with Actinomycin D or cycloheximide increases the mRNA accumulation 5 fold in 3 hours. The same treatment has no effect on activated stellate cells, which produce a large amount of the collagen alpha 1(I) mRNA. This suggests that there is a labile degrading activity which normally prevents accumulation of the mRNA in quiescent cells. Half life of the mRNA in activated cells is about 48 hours. We detected a sequence specific RNA binding activity present in these cells which binds to the C-rich sequence in the 3' UTR of collagen mRNA. Such binding can not be detected in quiescent cells and may be responsible for the stabilization of the mRNA in activated stellate cells. Thus, collagen alpha 1(I) mRNA levels in stellate cells may be modulated by negative regulation in quiescent cells and by positive regulation in activated cells.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.

There are two gene sets for U2 snRNA in the sea urchin L. variegatus, the U2E gene, which is expressed in oogenesis and early embryogenesis and then silenced, and the U2L gene, which is expressed constitutively. There are four major promoter elements found in both U2 snRNA genes; an essential TATA box at -25 to -30, a proximal element (PSE) at -55 required for expression, an element at -100 necessary for maximal expression, and an upstream activating sequence (UAS) necessary for maximal expression. The elements of the U2E and U2L promoters were interchanged and the effect on the temporal pattern of expression determined after microinjection of the genes into sea urchin zygotes. When the U2E PSE element was introduced into the U2L gene, the temporal pattern of expression of the gene was changed to that of the U2E gene. Converting the U2L gene proximal element into the early U2 gene proximal element by altering 14 nucleotides in the promoter also changed the temporal pattern of expression of the U2L gene. Factors that interact with the U2E PSE, detected by a gel mobility shift assay and DNaseI footprinting, were present in blastula but not late gastrula embryos. In contrast, interchanging the -100 element did not greatly affect the temporal pattern of expression, and factors that interact with the U2E gene -100 box were present in both late gastrula and blastula embryos.

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The -100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene.

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.