Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor beta 1 stimulation of alpha 2(I) collagen gene expression.

Transforming growth factor beta 1 (TGF-beta 1) exerts a positive effect on the transcription of genes coding for several extracellular matrix-related products, including collagen I. We have previously identified a strong TGF-beta 1-responsive element (TbRE) in the upstream promoter sequence of the alpha 2(I) collagen (COL1A2) gene. Our experiments have shown that TGF-beta 1 stimulates COL1A2 transcription by increasing binding of an Sp1-containing complex (TbRC) to the TbRE. They have also suggested that the change occurs via posttranslational modification of a protein(s) directly or indirectly interacting with Sp1. Here, we provide evidence showing that tyrosine dephosphorylation of nuclear proteins mimics the stimulation of COL1A2 transcription by the TGF-beta 1-activated signaling pathway. Preincubation of nuclear extracts with protein tyrosine phosphatase (PTPase) but not with protein phosphatase type 2A (PP2A), a serine/threonine phosphatase, enhanced binding of the TbRC to the same degree as culturing cells in TGF-beta 1. Consistent with these in vitro findings, genistein, a tyrosine kinase inhibitor, led to markedly increased COL1A2 gene expression, whereas sodium orthovanadate, a tyrosine phosphatase inhibitor, decreased it substantially. These results were supported by transfection experiments showing that genistein and sodium orthovanadate have opposite effects on TbRE-mediated transcription. Moreover, nuclear proteins isolated from genistein-treated cells were found to interact with the TbRE significantly more than those from untreated cells. Furthermore, pretreatment of cells with sodium orthovanadate virtually abrogated nuclear protein binding to the TbRE, but not to a neighboring cis-acting element unresponsive to TGF-beta 1. The results of this study, therefore, provide the first correlation between tyrosine dephosphorylation, increased binding of a transcriptional complex, and TGF-beta 1 stimulation of gene expression.

Tumor necrosis factor alpha inhibits type I collagen synthesis through repressive CCAAT/enhancer-binding proteins.

Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits ECM accumulation by stimulating the expression of matrix proteolytic enzymes and by downregulating the deposition of structural macromolecules such as type I collagen. Stimulation of ECM degradation has been linked to prolonged activation of jun gene expression by the cytokine. Here we demonstrate that TNF-alpha inhibits transcription of the gene coding for the alpha2 chain of type I collagen [alpha2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-alpha-responsive element. This conclusion was based on the concomitant identification of C/EBPbeta and C/EBPdelta as TNF-alpha-induced factors by biochemical purification and expression library screening. It was further supported by the ability of the C/EBP-specific dominant-negative (DN) protein to block TNF-alpha inhibition of alpha2(I) collagen but not TNF-alpha stimulation of the MMP-13 protease. The DN protein also blocked TNF-alpha downregulation of the gene coding for the alpha1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-alpha-induced signaling pathway that controls ECM formation and remodeling.

Accelerated development of liver fibrosis in CCl4-treated rats by the weekly induction of acute phase response episodes: upregulation of alpha1(I) procollagen and tissue inhibitor of metalloproteinase-1 mRNAs.

Patients with alcoholic hepatitis have several manifestations of the acute phase response (APR) and have elevated blood levels of interleukin-1, interleukin-6 and tumor necrosis factor-alpha. We have previously shown that liver stellate cells express interleukin-6 mRNA and protein and respond to this cytokine with increased expression of alpha1(I) procollagen mRNA. We further showed that the production of an APR episode stimulates a transient expression of alpha1(I) procollagen mRNA in the liver. In this communication we demonstrate that the concomitant induction of a weekly APR episode in rats with a schedule of CCl4 to produce cirrhosis, accelerates the development of liver fibrosis. We show that the enhancement of liver fibrosis is due, in part, to further upregulation in the expression of alpha1(I) procollagen and tissue inhibitor of metalloproteinases-1 mRNAs above values observed in control rats receiving only CCl4. The effect of the APR appears to have specificity since not all the mRNAs measured were equally affected. Altogether, these results suggest that increased blood or liver levels of APR cytokines, whether induced by APR episodes, endotoxin or other unrelated causes, may contribute to the development of liver fibrosis by enhancing the expression of type I collagen and of tissue inhibitor of metalloproteinases-1 mRNAs.

Ethanol induces the expression of alpha 1(I) procollagen mRNA in a co-culture system containing a liver stellate cell-line and freshly isolated hepatocytes.

To study the fibrogenic action of ethanol in vitro we used a co-culture system of freshly isolated hepatocytes and a liver stellate cell line (CFSC-2G) developed in our laboratory. Our results show that in this co-culture system ethanol induces the expression of alpha 1(I) procollagen mRNA in a dose- and time-dependent manner. This effect of ethanol was due to its metabolism by alcohol dehydrogenase since 4-methylpyrazole prevented the ethanol-mediated increase in alpha 1(I) procollagen mRNA. Ethanol was more efficient than acetaldehyde in inducing alpha 1(I) procollagen mRNA expression and its effect was protein synthesis-independent. Transfection of either hepatocytes or liver stellate cells with a reporter gene, chloramphenicol acetyl transferase (CAT), driven by 3700 bp of the mouse alpha 1(I) procollagen promoter demonstrated that only LSC expressed significant CAT activity and that this activity was enhanced by ethanol. Overall, our results suggest that this co-culture system is a useful model to study alcohol-induced fibrogenesis in vitro and that mechanisms other than acetaldehyde formation may also play an important role in alcohol-induced fibrogenesis.

Acetaldehyde-mediated collagen regulation in hepatic stellate cells.

Alcohol-induced liver cirrhosis is one of the major causes of death worldwide. Strong evidence has established that ethanol's first metabolite, acetaldehyde, is highly fibrogenic and enhances the deposition of many extracellular matrix components by hepatic stellate cells. This article reviews our current knowledge of the molecular mechanisms whereby acetaldehyde induces these activities, with particular emphasis on those related to the upregulation of type I collagen.

Fibroblast-stimulating factor 1, a novel lymphokine produced in schistosomal egg granulomas, stimulates liver fat-storing cells in vitro.

We report that the novel lymphokine fibroblast-stimulating factor 1, produced within hepatic schistosomal egg granulomas, stimulates liver fat-storing cells (FSC) in vitro to proliferate and express fibronectin genes. Because FSC are critical in hepatic fibrogenesis generally, we propose that in vivo stimulation of FSC by fibroblast-stimulating factor 1 may be an important step in schistosomal liver fibrosis.

Kupffer cells from CC1(4)-treated rat livers induce skin fibroblast and liver fat-storing cell proliferation in culture.

Conditioned media of Kupffer cells from normal rat liver produce in culture two factors that inhibit fibroblast proliferation. The inhibitory factors have molecular masses of approximately 25 and 5 kDa. In contrasts to these results, the conditioned media of Kupffer and mononuclear macrophagic cells, obtained 48 hours after CC1(4) administration to rats, contains a 17 kDa factor that stimulates fibroblast proliferation (FSF). FSF also stimulates [3H]-thymidine incorporation into DNA of cultured rat liver fat-storing cells. Two peaks with FSF activity were demonstrated after isoelectrofocusing; one with a pI of 6.1 and a second with a pI of 7.5. The fraction containing FSF is devoid of interleukin-1 (IL-1) activity and no inhibitory activity is detected in this conditioned media. Production of FSF is inhibitable by colchicine but not by indomethacin, it is thermolabile and trypsin-sensitive. In animals treated chronically with CC1(4) to produce cirrhosis, FSF activity is demonstrable from the first to the 8th week of treatment. However, the activity is lower at 8 weeks post-CC1(4) as compared with 2 weeks. The results suggest that homeostasis of cells in the liver is controlled by factors produced locally, that act by autocrine and paracrine mechanisms. When homeostasis is altered, fibroblast proliferation occurs, and excess collagen deposition leads to fibrosis. We propose that the antifibrogenic activity of colchicine is associated, in part, with its capacity to inhibit the release of FSF by Kupffer cells and liver mononuclear macrophage cells.

Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription.

Transforming growth factor-beta1 (TGFbeta) is a strong activator of extracellular matrix accumulation. TGFbeta stimulates the gene coding for human alpha2(I)-collagen (COL1A2) by inducing binding of an Sp1-containing complex to an upstream promoter element (TGFbeta responsive element or TbRE) that contains a CAGA box. Here we report that the CAGA box of the TbRE is the binding site of the Smad3/Smad4 complex, and that the binding of the complex is required for TGFbeta-induced COL1A2 up-regulation. Recombinant Smad3 and Smad4 bind in vitro to the CAGA box of COL1A2; TGFbeta treatment of cultured fibroblasts induces Smad3/Smad4 binding to the TbRE; transient overexpression of Smad3 and Smad4 in fibroblasts transactivates TbRE-driven transcription; and COL1A2 gene up-regulation by TGFbeta is abolished in cells stably transfected with plasmids that express dominant negative forms of Smad3 or Smad4. In Sp1-deficient Drosophila Schneider cells, there was cooperative synergy between Smad3/Smad4 and Sp1 at the TbRE site. The analysis also emphasized the requirement of both Sp1- and Smad-binding sites for optimal promoter transactivation. In cells stably transfected with a plasmid expressing a dominant negative form of Sp1, the synergy was shown to be promoter-specific and dependent on the binding of Sp1 to the TbRE. Interestingly, overexpression of dominant negative Sp1 was found to block the antagonistic signal of tumor necrosis factor-alpha on COL1A2 transcription, as well. These results provide the first linkage between the Smad3 and Smad4 proteins and TGFbeta stimulation of type I collagen biosynthesis.

Structural and functional analysis of the promoter of the human alpha 1(XI) collagen gene.

In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.

Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43.

BACKGROUND: Immunocytochemical analysis of liver has revealed that fat-storing cells (FSC) are heterogeneous with regard to vitamin A content, staining for cytokeratins, desmin, and vimentin and the cytoskeletal protein alpha-smooth muscle actin. Since fat-storing cells play an important role in collagen deposition in normal and cirrhotic liver, we considered it important to study whether fat-storing cells were heterogeneous with regard to cell proliferation, expression of mRNAs coding for cytokines interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta), and extracellular matrix components alpha 1(I), alpha 1(III), alpha 1(IV) procollagens, laminin B1 chain and fibronectin. EXPERIMENTAL DESIGN: We used a FSC line (CFSC) that was developed in our laboratory after spontaneous immortalization of a primary culture of fat-storing cells that were obtained from the liver of a CCl4-cirrhotic rat (Lab. Invest. 65:644-653, 1991). The cells were cloned by limiting dilution and have been maintained in culture for over 3 years without appreciable changes in the parameters investigated. RESULTS: In this communication we report the characterization of 4 of the clones obtained. We show that they are heterogeneous with regard to proliferation index, expression of alpha 1(I), alpha 1(III) and alpha 1(IV) procollagen, IL-6 and TGF-beta mRNAs. The clones also differ in their response to IL-6. We also showed that clones are coupled through functional gap junctions but that they are heterogeneous with regard to the expression of the gap junction protein connexin 43. CONCLUSIONS: We suggest that clonal heterogeneity of FSC may occur in vivo. Since each of the clones expresses a unique phenotype, these FSC clones could be excellent models to study the role of defined extracellular matrices on the expression of liver specific genes by cultured hepatocytes.

Regulation of the alpha 2(I) collagen gene transcription in fat-storing cells derived from a cirrhotic liver.

Fat-storing cells (FSC) are the main producers of type I collagen in both normal and fibrotic livers. In order to elucidate the molecular mechanisms controlling collagen expression in FSC, we examined the transcription of the alpha 2(I) collagen gene (COL1A2) in two distinct FSC clones, CFSC-2G and CFSC-5H, derived from a single CCl4-induced cirrhotic liver. The phenotype of CFSC-2G resembles freshly isolated FSC, whereas that of CFSC-5H mimics activated myofibroblasts. Cell transfection experiments showed that the upstream sequence between nucleotides -378 and -183 is essential for COL1A2 transcription in both FSC clones. This is the same promoter region that is transcriptionally active and contains the binding site of a multimeric protein complex that mediates TGF-beta stimulation of COL1A2 expression in dermal fibroblasts. We therefore examined the relative levels of endogenous and transfected COL1A2 transcription and their response to TGF-beta treatment in the two FSC clones. The results showed that CFSC-5H expresses a significantly higher level of the COL1A2 mRNA than CFSC-2G. They also showed that TGF-beta treatment increases both endogenous and transfected COL1A2 transcription in CFSC-2G but not in CFSC-5H. Interestingly, nuclear proteins from both FSC clones bind to the TGF-beta-responsive element more strongly than those from dermal fibroblasts. Altogether, the data suggest that collagen production in CFSC-5H cells has been already activated by the autocrine stimulation of TGF-beta. In contrast, CFSC-2G cells are only partially activated but can be easily recruited to produce collagen when stimulated by exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1.

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.

Hydrogen peroxide: a link between acetaldehyde-elicited alpha1(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells.

Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic actions of acetaldehyde and the induction of an oxidative stress response. However, the mechanisms responsible for these activities, and the possible connections between oxidative stress and acetaldehyde-induced fibrosis are not well understood. In this communication we investigated the molecular mechanisms whereby acetaldehyde induces mouse alpha1(I) procollagen (col1a1) gene expression in cultured hepatic stellate cells. Transfection assays using reporter plasmids driven by different segments of the col1a1 promoter localized an acetaldehyde-responsive element (AcRE) between nucleotides -370 and -345. We also show that acetaldehyde enhances binding of a CCAAT/enhancer binding protein-beta (C/EBPbeta)-containing complex to this element, and that this effect is due, at least in part, to an increase in the concentration of nuclear p35C/EBPbeta protein. Although this element overlaps to a previously described transforming growth factor beta1 (TGF-beta1)-responsive element, the stimulatory effect of acetaldehyde is not mediated through this cytokine, because addition of neutralizing anti-TGF-beta1 antibodies does not prevent acetaldehyde-elicited col1a1 up-regulation. On the other hand, this effect is blocked by the addition of catalase, an H(2)O(2) scavenger. Moreover, this ethanol metabolite stimulates production of H(2)O(2) in stellate cells. Thus, these results suggest that acetaldehyde-induced col1a1 up-regulation is mediated, at least in part, through H(2)O(2). Altogether, these data suggest that the -370 to -344 region of the col1a1 gene is a point of convergence of the action of numerous extracellular stimuli that ultimately leads to col1a1 up-regulation. In addition, we have established a direct connection between oxidative stress and enhanced col1a1 expression induced by acetaldehyde.

CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells.

Hepatic stellate cells (HSCs) are a major source of extracellular matrix, which, during fibrogenesis, undergo a process of "activation" characterized by increased proliferation and collagen synthesis. Oxidative stress can stimulate HSC proliferation and collagen synthesis in vitro. Cytochrome P4502E1 (CYP2E1) is an effective producer of reactive oxygen species. To study how intracellular oxidative stress modulates alpha 2 collagen type I (COL1A2) gene induction, a rat HSC line (HSC-T6) was transfected with human CYP2E1 complementary DNA in the sense and antisense orientation and with empty vector, and stable cell lines were generated. The cells expressing CYP2E1 displayed elevated production of reactive oxygen species and showed a 4-fold increase in COL1A2 messenger RNA (mRNA) levels; expression of this mRNA among different clones appeared to correlate with the level of CYP2E1. COL1A2 expression was decreased by vitamin E treatment or transfection with manganese superoxide dismutase, and was further increased after treatment with L-buthionine sulfoximine (BSO) to lower GSH levels. Thus, CYP2E1-dependent oxidative stress plays a major role in the elevation of COL1A2 mRNA levels in this system. Nuclear run-on assay showed a 3-and-a-half-fold increase in COL1A2 transcription in the cells expressing CYP2E1; stabilization of COL1A2 mRNA was also observed. These results indicate that under oxidative stress conditions, COL1A2 mRNA expression is regulated both transcriptionally and through mRNA stabilization. The CYP2E1-expressing HSC appear to be a valuable model for the sustained generation of reactive oxygen species and may allow the elucidation of signaling pathways responsible for oxidant stress-mediated collagen gene induction.

Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers. Differences in the production of interleukin-6.

Liver fat-storing cells (FSC) play an important role in collagen deposition. During the induction of liver cirrhosis, FSC lose their fat droplets, acquire an actin-rich cytoskeleton and transform into myofibroblasts. Myofibroblasts have been associated with increased collagen production in cirrhotic livers. Cultured FSC resemble myofibroblasts. However, it is not known whether regulation of collagen gene expression is similar in FSC obtained from normal or cirrhotic livers. In this communication, we describe the characterization of two fat-storing cell lines, one from normal (NFSC) and one from CCl4-cirrhotic liver (CFSC), obtained after spontaneous immortalization in culture. We studied the effect of serum and various growth factors on cell proliferation. We determined the production of collagen and fibronectin and we analyzed the presence of mRNA transcripts of collagens type I, III, and IV, fibronectin laminin, transforming growth factor-beta and interleukin-6. We found that CFSC have a greater serum-dependency than NFSC. NFSC grow with a mixture of insulin and epidermal growth factor, whereas CFSC proliferate only with platelet-derived growth factor. Although we did not find significant differences in the expression of mRNAs for collagen type I, fibronectin and transforming growth factor-beta, collagen and fibronectin synthesis was increased 2- and 1.5-fold respectively. NFSC contained 1.6- and 2.0-fold more type III collagen and laminin mRNAs, respectively, than CFSC. Neither cell line expressed type IV collagen mRNA. NFSC but not CFSC produced interleukin-6. These results suggest that, except for the lack of transcripts of collagen type IV, both cell lines resemble primary cultures of FSC. However, significant differences in cell proliferation and interleukin-6 production between the two cell lines were found. We suggest that these cell lines could be useful tools to study possible differences in regulation of matrix production by FSC.

Interaction between GC box binding factors and Smad proteins modulates cell lineage-specific alpha 2(I) collagen gene transcription.

Type I collagen is produced predominantly in mesenchymal cells, but molecular mechanisms responsible for cell type-specific expression are virtually unknown. During fibrogenic process in the liver, activated hepatic stellate cells (HSC) are the main producers of type I collagen, whereas parenchymal hepatocytes produce little, if any, of this protein. We have previously reported that Sp1 and an interacting unknown factor(s) bind to the -313 to -255 sequence of the alpha2(I) collagen gene (COL1A2) and play essential roles for basal and TGF-beta-stimulated transcription in skin fibroblasts and HSC. Recently, Smad3 has been shown to bind to this region, and its interaction with Sp1 has been implicated in TGF-beta-elicited COL1A2 stimulation. The present study demonstrates predominant binding of Sp3 rather than Sp1 to this regulatory element in parenchymal hepatocytes. In these cells, this region did not exhibit strong enhancer activity or mediate the effect of TGF-beta. Transfection of HSC with an Sp3 expression plasmid abolished the COL1A2 response to TGF-beta, whereas overexpression of Sp1 in hepatocytes increased basal COL1A2 transcription and conferred TGF-beta responsiveness. Functional and physical interactions between Sp1 and Smad3, but not between Sp3 and Smad3, were demonstrated using the bacterial GAL4 system and immunoprecipitation-Western blot analyses. These results indicate that cell lineage-specific interactions between GC box binding factors and Smad protein(s) may account, at least in part, for differential COL1A2 transcription and TGF-beta responsiveness in HSC and parenchymal hepatocytes.

A transcription activator with restricted tissue distribution regulates cell-specific expression of alpha1(XI) collagen.

Different regulatory programs are likely to control expression of the alpha1(XI) collagen (COL11A1) gene in cartilaginous and non-cartilaginous tissues and in coordination with different collagen genes. Here, we report the identification of a cis-acting element that is required for constitutive and tissue-specific activity of the proximal COL11A1 promoter. The element binds an apparently novel activator whose expression is restricted mostly, but not exclusively, to cells of mesenchymal origin. Transient transfection experiments using wild-type and mutant constructs demonstrated the critical contribution of a 45-base pair upstream element (FP9) to promoter activity. The same functional tests and DNA binding assays narrowed down the critical portion of FP9 to a 20-base pair sequence, which consists of an imperfect palindrome with strong homology to the GATA consensus motif. Despite being able to bind GATA proteins in vitro, FP9 is actually recognized by a distinct approximately 100-kDa polypeptide (FP9C) probably belonging to the zinc-finger family of transcription factors. FP9C binding was mostly identified in nuclei from cells of mesenchymal origin, including those actively engaged in COL11A1 transcription. A positive correlation was also established between the level of FP9C binding and the degree of cell differentiation in vitro. Thus, FP9C represents an unusual example of tissue-specific and differentiation-related transcription factor with overlapping expression in hard and soft connective tissues.

Role of hydrogen peroxide and oxidative stress in healing responses.

Oxidative stress is a host defense mechanism whose involvement in maintaining homeostasis and/or inducing disease has been widely investigated over the past decade. Various reactive oxygen species (ROS) have been defined and the enzymes involved in generating and/or eliminating them have been widely studied. In this review we briefly discuss general mechanisms of oxidative stress and the oxidative stress response of the host. We focus primarily on hydrogen peroxide and summarize the systems involved in its formation and elimination. We describe mechanisms whereby hydrogen peroxide and other ROS can modify protein conformation and, thus, alter protein function, and describe a group of transcription factors whose biological activity is modulated by the redox state of cells. These basic aspects of oxidative stress are followed by a discussion of mechanisms whereby hydrogen peroxide and other ROS can modulate some physiological and pathological processes, with special emphasis on wound healing and scarring of the liver.

Rat liver fat-storing cell lines express sarcomeric myosin heavy chain mRNA and protein.

Fat-storing cells (FSC, lipocytes, or Ito cells) of liver store vitamin A and are the main producers of extracellular matrix in normal and cirrhotic liver. During liver injury, FSC undergo an activation process characterized by a decrease in vitamin A storage and an increase in cell proliferation and extracellular matrix deposition. This activation process also occurs upon culturing FSC from normal liver. In contrast to most cells of nonmuscle origin, activated FSC express two cytoskeletal proteins normally found in muscle, desmin, and smooth muscle alpha-actin. Based on their strategic perisinusoidal location, it has been hypothesized that FSC play a role in regulating blood flow. However, the nature of the contractile elements involved in this process remains to be determined. In this communication we demonstrate the presence of a sarcomeric myosin in proteins solubilized from liver biomatrix. In addition we demonstrate the expression of sarcomeric myosin heavy chain (MHC) mRNA and protein in two FSC clones derived from a CCl4-cirrhotic rat liver (CFSC). Through cloning the cDNA corresponding to the MHC gene expressed in these cells we demonstrate that it encodes fast IId skeletal MHC and thus represents a marker normally seen in adult muscle. The unexpected expression of an adult stage skeletal muscle molecular motor in FSC from cirrhotic liver is consistent with the proposed specialized contractile capacity of these cells.

Identification of connective tissue gene transcripts in freshly isolated parenchymal, endothelial, Kupffer and fat-storing cells by northern hybridization analysis.

The aim of the present study was to identify the cell types that express collagen alpha 1(I), alpha 1(III) and alpha 1(IV), fibronectin and laminin B1 genes in normal rat liver. Parenchymal, sinusoidal endothelial, Kupffer and fat-storing (Ito) cells were isolated and purified. Total RNA of the freshly isolated cells was subjected to Northern hybridization analysis. We also compared the steady state levels of specific mRNAs in freshly isolated fat-storing cells to the levels in myofibroblast-like cells obtained from purified fat-storing cells cultured for two passages. The average purity of each cell preparation, and the percentage of contaminating cells, were determined by transmission electron microscopy and by examining the presence of vitamin A-autofluorescent cells. Fibronectin and collagen alpha 1(III) mRNAs were detected in total RNA of purified parenchymal cells. In poly(A)+ enriched RNA, small amounts of collagen alpha 1(I) mRNA were also present. In total RNA of freshly isolated fat-storing cells, collagen alpha 1(III), alpha 1(IV), and laminin B1 transcripts were found, whereas collagen alpha 1(I) and fibronectin mRNAs were not detected. Cultured fat-storing cells, however, did contain high levels of collagen alpha 1(I) and fibronectin mRNAs. The molecular size of the latter transcript was larger than the fibronectin transcript found in parenchymal cells and the whole liver. Endothelial cells contained small amounts of alpha 1(IV) mRNA. Kupffer cells did not contain the investigated transcripts. We conclude that normal parenchymal, fat-storing and endothelial cells each express a typical pattern of connective tissue molecules. When fat-storing cells are allowed to differentiate into myofibroblast-like cells, they express high levels of collagen alpha 1(I) and fibronectin mRNAs, in addition to collagen alpha 1(III) and alpha 1(IV), and laminin B1 chain mRNAs.