p38 MAPK mediates the regulation of alpha1(I) procollagen mRNA levels by TNF-alpha and TGF-beta in a cell line of rat hepatic stellate cells(1).

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

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 hepatic stellate cells contribute to the acute-phase response with increased expression of alpha1(I) and alpha1(IV) collagens, tissue inhibitor of metalloproteinase-1, and matrix-metalloproteinase-2 messenger RNAs.

The acute-phase response (APR) represents a systemic reaction of the organism to multiple nonspecific inflammatory stimuli. In general, it is protective for the host, and hepatocytes are the main cells responding with alterations in the expression of a set of liver-specific proteins named the acute-phase proteins. We have previously shown that although a turpentine-induced APR is not fibrogenic per se, it enhances collagen deposition in rats treated with CCl(4) and up-regulates expression of hepatic alpha1(I) collagen and tissue inhibitor of metalloproteinases 1 (TIMP-1) messenger RNAs (mRNAs). In this report we extended our studies and showed that turpentine induced, in a time-dependent manner, expression of alpha1(I) and alpha1(IV) collagens, TIMP-1, and matrix-metalloproteinase 2 (MMP-2) mRNAs. We further showed that expression of these mRNAs occurs in hepatic stellate cells, but not in hepatocytes obtained 6 hours after the induction of an APR episode. These changes were accompanied by increased blood levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) without noticeable immediate changes in the expression of their respective mRNAs in the liver. In contrast to CCl(4)-induced liver damage, turpentine alone, whether administered as a single dose or as a weekly dose for 3 weeks did not up-regulate expression of transforming growth factor beta1 (TGF-beta1) mRNA and did not result in excess collagen deposition. Overall, these findings suggest that collagen deposition in the livers of rats with repeated APR episodes may be enhanced only when given together with a fibrogenic stimulus that activates hepatic stellate cells (HSCs) and/or up-regulates TGF-beta1 mRNA expression.

Lysophosphatidic acid enhances collagen gel contraction by hepatic stellate cells: association with rho-kinase.

We studied the effect of lysophosphatidic acid (LPA) on collagen gel contraction by cultured rat hepatic stellate cells (HSCs) in association with the function of Rho-kinase, one of the target molecules of small GTPase Rho. Binding studies showed a single class-binding site of LPA on HSCs. LPA enhanced the contraction of a collagen lattice seeded with HSCs. LPA increased the number of HSCs with polygonal morphology that contained actin stress fibers, and enhanced the phosphorylation of myosin light chain and the assembly of focal adhesion kinase and RhoA around fibronectin-coated beads seeded on HSCs. The electric cell-substrate impedance sensor system showed that LPA enhanced adhesion of HSC to extracellular substrate. All the effects of LPA were suppressed by Y-27632, Rho-kinase inhibitor. These data support the notion that LPA is involved in modulating HSC morphology, its attachment to surrounding extracellular matrix and its contraction by a mechanism involving Rho-kinase.

Tumor necrosis factor alpha down-regulates expression of the alpha1(I) collagen gene in rat hepatic stellate cells through a p20C/EBPbeta- and C/EBPdelta-dependent mechanism.

Tumor necrosis factor alpha (TNF-alpha) is one of the key cytokines of the acute phase response and of many inflammatory processes. This cytokine has several antifibrogenic actions and down-regulates the expression of the type I collagen genes and induces the expression of metalloproteinases. Because TNF-alpha directly antagonizes some fibrogenic actions of transforming growth factor beta(1) (TGF-beta(1)), we considered it important to map the cis-acting regulatory element of the alpha1(I) collagen (col1a1) promoter involved in TNF-alpha responsiveness in hepatic stellate cells (HSC), to investigate the transcription factors that bind to it, and to establish possible mechanisms by which TNF-alpha down-regulates its expression. In this article, we show the presence of a functional TNF-alpha-responsive element (TaRE) in the -378 to -345 region of the col1a1 promoter. This element colocalizes with a previously reported TGF-beta(1)-responsive element. We further demonstrate that TNF-alpha induces nuclear translocation and binding of transcriptional complexes containing p20C/EBPbeta, p35C/EBPbeta, and C/EBPdelta to this sequence of the promoter. Transient overexpression of C/EBPdelta or p20C/EBPbeta, the natural dominant negative form of C/EBPbeta in HSC, down-regulated activity of a CAT reporter vector driven by -412 to +110 of the col1a1 promoter. Taken together, these data suggest that the -378 to -340 region of the col1a1 promoter is the site of convergence of different stimuli that ultimately modulate col1a1 gene transcription.

H(2)O(2) is an important mediator of physiological and pathological healing responses.

BACKGROUND: TGF-beta1 is a pleiotropic cytokine that plays a key role in wound healing and organ fibrosis. We have recently demonstrated that, in part, some fibrogenic actions of TGF-beta1 are mediated via formation of H(2)O(2). We have also demonstrated that TGF-beta1 plays a key role in the accelerated healing response induced by a peptidoglycan derived from some strains of Staphylococcus aureus (SaPG). METHODS: To investigate further the role of H(2)O(2) in healing responses, we implemented and improved a method to measure this reactive oxygen species. Using this method, we quantified the production of H(2)O(2) by cultured hepatic stellate cells-the main cells involved in type I collagen production in the liver-and by saline- and SaPG-inoculated polyvinyl alcohol sponges that had been surgically subcutaneously implanted in the dorsum of rats. RESULTS: We show that cultured hepatic stellate cells produce significant amounts of H(2)O(2). We show also that H(2)O(2) formation by saline- and SAPG-inoculated sponges is more intense during the early inflammatory phase of the healing response and precedes collagen deposition. Moreover, the production of H(2)O(2) is much higher in SaPG-inoculated sponges than in those inoculated with saline solution. CONCLUSIONS: Based on these findings, and on the fact that H(2)O(2) is produced during TGF-beta-induced upregulation of the alpha1(I) procollagen gene, we conclude that H(2)O(2) is one of the mediators of healing responses.

A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA.

A number of pathogenic and proinflammatory stimuli, and the transforming growth factor-beta (TGF-beta) exert opposing activities in cellular and immune responses. Here we show that the RelA subunit of nuclear factor kappaB (NF-kappaB/RelA) is necessary for the inhibition of TGF-beta-induced phosphorylation, nuclear translocation, and DNA binding of SMAD signaling complexes by tumor necrosis factor-alpha (TNF-alpha). The antagonism is mediated through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-beta receptors and inhibitory Smad7. Down-regulation of endogenous Smad7 by expression of antisense mRNA releases TGF-beta/SMAD-induced transcriptional responses from suppression by cytokine-activated NF-kappaB/RelA. Following stimulation with bacterial lipopolysaccharide (LPS), or the proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta, NF-kappaB/RelA induces Smad7 synthesis through activation of Smad7 gene transcription. These results suggest a mechanism of suppression of TGF-beta/SMAD signaling by opposing stimuli mediated through the activation of inhibitory Smad7 by NF-kappaB/RelA.

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.

Role of metalloproteinases in liver fibrosis.

Collagen deposition in the cirrhotic liver is the result of an imbalance between the amount of collagen produced and that, which is degraded. Although several groups have actively investigated the mechanisms that regulate collagen gene expression in the liver, little is known regarding those involved in the regulation of interstitial collagenases. In this study, we shall express our personal ideas regarding the role of metalloproteinases in collagen degradation in the cirrhotic liver, with special emphasis on the interstitial collagenases and some factors that may limit collagen degradation in vivo.

Transforming growth factor beta1 induces the expression of alpha1(I) procollagen mRNA by a hydrogen peroxide-C/EBPbeta-dependent mechanism in rat hepatic stellate cells.

Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up-regulation of transforming growth factor beta (TGF-beta), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-beta induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-beta-elicited alpha1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-beta induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of alpha1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-beta-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (-370 to -344) essential for TGF-beta responsiveness. We further showed that TGF-beta induced the activation and binding of a C/EBPbeta-containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF-beta-mediated accumulation of H2O2 and the up-regulation of col1a1 gene in HSC.

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis.

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression.

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.

Wound fluids from saline solution- and Staphylococcus aureus peptidoglycan-inoculated sponges induce expression of matrix metalloproteinase 13 messenger ribonucleic acid by cultured rat fibroblasts.

Polyvinyl alcohol sponges inoculated with Staphylococcus aureus peptidoglycan induce an accelerated wound healing response when implanted subcutaneously in rats. S. aureus peptidoglycan leads to a marked increase (50%) in reparative tissue collagen (as measured by hydroxyproline) by 4 days. However, this effect drops by 7 days and by 14 days; hydroxyproline levels are similar in sponges inoculated with S. aureus peptidoglycan or saline solution. These data suggest a very active early remodeling process in S. aureus peptidoglycan sponge reparative tissue. Consistent with this observation, we had found that steady-state levels of matrix metalloproteinase-13 mRNA were higher and persisted longer in S. aureus peptidoglycan sponge reparative tissue than in controls. We hypothesized that S. aureus peptidoglycan might induce a change in reparative tissue fibroblast phenotype or modify the character of the wound fluid. Fibroblasts obtained from saline solution- and S. aureus peptidoglycan-inoculated sponges 4 days after subcutaneous implantation and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum were similar with respect to morphologic features, proliferation, and expression of pro alpha1 (I) and alpha1 (III) collagens and tissue inhibitor of metalloproteinase-1 mRNA by Northern blot analysis. Neither cell type expressed matrix metalloproteinase-13 mRNA. No changes in the above parameters were detected when such fibroblasts were cultured for 24 hours in the presence of 0.5 mg of S. aureus peptidoglycan per 10 ml of medium or with fluid obtained from control sponges cultured for 12 hours with phosphate-buffered saline solution. Wound fluids extracted with Eagle's minimal essential medium by homogenization of saline solution- and S. aureus peptidoglycan-inoculated sponges implanted subcutaneously for 12 hours did not affect the proliferation of the fibroblasts. However, the extracts had a profound effect on the cellular expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-13, and pro alpha1 (I) collagen mRNA. Specifically, expression of matrix metalloproteinase-13 mRNA was induced, expression of pro alpha1 (I) collagen mRNA was reduced by 70%, and expression of tissue inhibitor of metalloproteinase-1 mRNA was increased by 150%. These changes were the same irrespective of whether the wound fluid was obtained from saline solution- or S. aureus peptidoglycan-inoculated sponges. Fluid obtained from S. aureus peptidoglycan-inoculated sponges, which contain a greater inflammatory exudate than saline solution-inoculated sponges do, is enriched in matrix metalloproteinase-13 mRNA-inducing activity. The nature of the factor(s) that induces matrix metalloproteinase-13 mRNA expression is not known. However, preliminary data suggest that the matrix metalloproteinase-13-inducing factor(s) is heterogeneous with regard to size and is temperature sensitive and trypsin resistant.

Accelerating effects of nonviable Staphylococcus aureus, its cell wall, and cell wall peptidoglycan.

We have previously reported that local application of viable Staphylococcus aureus dramatically accelerates wound healing, but viable Staphylococcus epidermidis does not. Because the S. aureus effect occurred in the absence of infection and because the cell walls of the two bacterial species differ, we hypothesized that nonviable S. aureus, its cell wall, and its cell wall component(s) would accelerate healing. Nonviable S. aureus was prepared by chemical and physical means, and its cell wall and peptidoglycan was prepared from heat-killed cultures. In a large number of experiments, nonviable S. aureus (independent of the strain's protein A content), its cell wall, and peptidoglycan when instilled locally at the time of wounding each significantly increased the breaking strength of rat skin incisions (tested both in the fresh state and after formalin fixation). These agents also enhanced subcutaneous polyvinyl alcohol sponge reparative tissue collagen accumulation, generally by a factor of two. Histologic features of treated and control incisions were similar. In contrast, the reparative tissue of treated sponges contained more neutrophils, macrophages, capillaries, and collagen. These experimental data thus confirm our previous studies, as well as our hypothesis, and extend these observations of enhanced wound healing to specific fractions of the bacterial cell wall.

Molecular mechanisms underlying wound healing acceleration by Staphylococcus aureus peptidoglycan.

Molecular mechanisms involved in wound healing acceleration by Staphylococcus aureus peptidoglycan were investigated with the use of polyvinyl alcohol sponges implanted under the dorsal skin of rats. Total collagen and RNA content and messenger RNA levels of alpha1(I) and alpha1(III) procollagen, transforming growth factor-beta1, and matrix metalloproteinase-1 were analyzed in saline solution- and S. aureus peptidoglycan-inoculated sponges at 4, 7, 14, and 21 days after implantation. S. aureus peptidoglycan-inoculated sponges on the fourth and seventh post-operative day were surrounded and penetrated by a thick capsule of reparative connective tissue. They were considerably heavier and contained more collagen and total RNA than saline solution-inoculated sponges. Histologically, the S. aureus peptidoglycan-inoculated sponges early on contained a denser infiltrate of polymorphonuclear cells than saline solution-inoculated sponges, and later fibroblasts, macrophages, collagen, and newly formed blood vessels were more abundant in the S. aureus peptidoglycan sponges. Matrix metalloproteinase-1 messenger RNA expression was elevated at 4 days in both sponge types. However, although matrix metalloproteinase-1 mRNA levels decreased to undetectable levels by 14 days in saline solution-inoculated sponges, they remained elevated throughout the 21-day study period in S. aureus peptidoglycan-inoculated sponges. No other significant differences in the parameters analyzed were detected. These results suggest that S. aureus peptidoglycan induces an accelerated but normal wound healing process in which the markedly increased early deposition of connective tissue is rapidly remodeled likely because of a sustained expression of matrix metalloproteinase-1.

Regulation of monocyte chemoattractant protein 1 by cytokines and oxygen free radicals in rat hepatic fat-storing cells.

BACKGROUND & AIMS: Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte/macrophage chemoattractant expressed by fat-storing cells (FSCs) in rat models of liver injury. This study investigated the mechanism of this activation of hepatic MCP-1 expression. METHODS: The regulation of MCP-1 messenger RNA (mRNA) expression and protein synthesis was examined in FSC lines derived from CCl4-induced cirrhotic rat liver (cirrhotic FSCs) and normal rat liver (normal FSCs). RESULTS: Northern blot hybridization analysis revealed low levels of MCP-1 mRNA in cultured cirrhotic FSCs that increased markedly after treatment with tumor necrosis factor alpha, interleukin 1 alpha, or transforming growth factor beta 1. All three cytokines increased the synthesis and secretion of MCP-1 protein. Oxygen free radical production also increased MCP-1 mRNA levels. These increases in MCP-1 mRNA were blocked by dexamethasone. In normal FSCs, levels of MCP-1 mRNA and secreted protein were increased in response to cytokines or oxygen free radical production, but the magnitude and duration of this increase was less than in cirrhotic FSCs. CONCLUSIONS: In liver injury, monocyte/macrophage recruitment and activation from FSC production of MCP-1 may be stimulated by cytokines and oxygen free radicals. During chronic liver injury leading to cirrhosis, FSCs may become hypersensitive to these stimuli, further fueling the inflammatory response.

Matrix metalloproteinase activity in human intrahepatic biliary epithelial cell lines from patients with autosomal dominant polycystic kidney disease.

Hepatic cysts derived from intrahepatic bile ducts are the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD). Cyst enlargement involves cell proliferation, fluid secretion into cysts, and alterations in extracellular matrix. To study hepatic cyst formation, continuous cell lines from human normal intrahepatic biliary epithelium (IBE) and ADPKD liver cyst-derived epithelium (LCDE) were developed. Because matrix degradation and remodeling are important for cyst formation and growth, we investigated matrix modifying enzymes expressed in IBE and LCDE cell lines. Gelatin substrate zymography showed that two matrix degrading activities with characteristics of matrix metalloproteinases are secreted from these cell lines. Western immunoblotting suggests that these activities correspond to the 72 kDa (Gelatinase A) and 92 kDa (Gelatinase B) type IV collagenases. Although the level of Gelatinase A activity is comparable in both IBE and LCDE cell lines, Gelatinase B activity is substantially increased in LCDE lines.

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)

Rat hepatocytes attach to laminin present in liver biomatrix proteins by an Mg(++)-dependent mechanism.

Laminin belongs to a family of proteins that contains at least seven variants. Together with fibronectin, it is the most important cell-adhesion protein. Recent data from various laboratories have suggested that liver sinusoidal laminins differ from Engelbert-Holmes-Swarm tumor laminin (laminin 1), because the former contain alpha 2 instead of alpha 1 chains. Therefore, we compared the adhesion of hepatocytes to laminin 1 and a matrix extracted with dilute acetic acid from liver biomatrix (LBP). We show that LBP contains laminin and that this extracellular matrix protein is the main adhesion protein. Close to 70% of the hepatocytes attach to LBP after 15 minutes of incubation at 37 degrees C. Cell adhesion was Mg++ and Mn(++)-dependent and Ca(++)- and insulin-independent. Ethylenediaminetetraacetic acid prevented cell adhesion in the presence of divalent cations. We show that synthetic cell-adhesion peptide sequences present in laminin 1 (RGD and YIGSR) or an antibody to the cell-binding domain (SIKVAV) of the alpha 1 chain do not prevent hepatocyte adhesion to LBP. We also show that the LBP has cell specificity; hepatocytes adhere to it preferentially when compared with other epithelial and mesenchymal cell lines. We suggest that because of the differences in chain composition of laminin 1 and liver sinusoidal laminins as well as the described differences in cell adhesion to the two substrata, further studies are needed to determine the actual composition of liver laminin and establish the chains and domains to which hepatocytes adhere.