Patricia Greenwel

Synergistic Cooperation between Sp1 and Smad3/Smad4 Mediates Transforming Growth Factor β1 Stimulation of α2(I)-Collagen (COL1A2) Transcription

Transforming growth factor-β1 (TGFβ) is a strong activator of extracellular matrix accumulation. TGFβ stimulates the gene coding for human α2(I)-collagen (COL1A2) by inducing binding of an Sp1-containing complex to an upstream promoter element (TGFβ 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 TGFβ-induced COL1A2 up-regulation. Recombinant Smad3 and Smad4 bind in vitro to the CAGA box of COL1A2; TGFβ 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 TGFβ is abolished in cells stably transfected with plasmids that express dominant negative forms of Smad3 or Smad4. In Sp1-deficientDrosophila 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-α on COL1A2 transcription, as well. These results provide the first linkage between the Smad3 and Smad4 proteins and TGFβ stimulation of type I collagen biosynthesis.

Role of hydrogen peroxide and oxidative stress in healing responses

Abstract. 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.

Sp1 Is Required for the Early Response of α2(I) Collagen to Transforming Growth Factor-β1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor β1 (TGF-β) stimulation of the human α2(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-β and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of α2(I) collagen mRNA accumulation by TGF-β, 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-β-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 COL1A2transactivation 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.

Tumor Necrosis Factor Alpha Inhibits Type I Collagen Synthesis through Repressive CCAAT/Enhancer-Binding Proteins

ABSTRACT Extracellular matrix (ECM) formation and remodeling are critical processes for proper morphogenesis, organogenesis, and tissue repair. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) 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-α inhibits transcription of the gene coding for the α2 chain of type I collagen [α2(I) collagen] in cultured fibroblasts by stimulating the synthesis and binding of repressive CCAAT/enhancer proteins (C/EBPs) to a previously identified TNF-α-responsive element. This conclusion was based on the concomitant identification of C/EBPβ and C/EBPδ as TNF-α-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-α inhibition of α2(I) collagen but not TNF-α stimulation of the MMP-13 protease. The DN protein also blocked TNF-α downregulation of the gene coding for the α1 chain of type I collagen. The study therefore implicates repressive C/EBPs in the TNF-α-induced signaling pathway that controls ECM formation and remodeling.

Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells

Hepatic stellate cells (HSC) are the main producers of type I collagen in fibrotic liver, and transforming growth factor‐β (TGF‐β) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of α2(I) collagen gene (COL1A2) that is essential for its basal and TGF‐β‐stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF‐β‐responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF‐β signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor‐1 (PAI‐1) genes and low response to TGF‐β in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated from the cytoplasm to the nucleus upon TGF‐β treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation. Increased COL1A2 and PAI‐1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether, the results correlate abnormality in TGF‐β/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC.

A synthetic peptide from transforming growth factor β type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury

Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine, which displays potent profibrogenic effects and is highly expressed in fibrotic livers. For this reason, development of TGF-B1 inhibitors might be of great importance to control liver fibrogenesis as well as other undesired side effects due to this cytokine. Potential peptide inhibitors of TGF-beta1 (derived from TGF-beta1 and from its type III receptor) were tested in vitro and in vivo using different assays. Peptides P11 and P12, derived from TGF-beta1, and P54 and P144, derived from its type III receptor, prevented TGF-beta1-dependent inhibition of MV1Lu proliferation in vitro and markedly reduced binding of TGF-beta1 to its receptors. P144 blocked TGF-beta1-dependent stimulation of a reporter gene under the control of human alpha2(I) collagen promoter. Intraperitoneal administration of P144 also showed potent antifibrogenic activity in vivo in the liver of rats receiving CCl4. These rats also showed a significant decrease in the number of activated hepatic stellate cells as compared with those treated with saline only. These results suggest that short synthetic peptides derived from TGF-beta1 type III receptor may be of value in reducing liver fibrosis in chronic liver injury.

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.

Reciprocal Modulation of Matrix Metalloproteinase-13 and Type I Collagen Genes in Rat Hepatic Stellate Cells

Collagen degradation by matrix metalloproteinases is the limiting step in reversing liver fibrosis. Although collagen production in cirrhotic livers is increased, the expression and/or activity of matrix metalloproteinases could be normal, increased in early fibrosis, or decreased during advanced liver cirrhosis. Hepatic stellate cells are the main producers of collagens and matrix metalloproteinases in the liver. Therefore, we sought to investigate whether they simultaneously produce alpha1(I) collagen and matrix metalloproteinase-13 mRNAs. In this communication we show that expression of matrix metalloproteinase-13 mRNA is reciprocally modulated by tumor necrosis factor-alpha and transforming growth factor-beta1. When hepatic stellate cells are co-cultured with hepatocytes, matrix metalloproteinase-13 mRNA is up-regulated and alpha1(I) collagen is down-regulated. Injuring hepatocytes with galactosamine further increased matrix metalloproteinase-13 mRNA production. Confocal microscopy and differential centrifugation of co-cultured cells revealed that matrix metalloproteinase-13 is localized mainly within hepatic stellate cells. Studies performed with various hepatic stellate cell lines revealed that they are heterogeneous regarding expression of matrix metalloproteinase-13. Those with myofibroblastic phenotypes produce more type I collagen whereas those resembling freshly isolated hepatic stellate cells express matrix metalloproteinase-13. Overall, these findings strongly support the notion that alpha1(I) collagen and matrix metalloproteinase-13 mRNAs are reciprocally modulated.

Ethanol induces the expression of α1(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.

Kupffer Cells from CC14-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.