- Email: [email protected]
Activation of hepatic stellate cells is mediated by oxidative stress through C-MYB expression
HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
745
AASLD
RECEPTOR TYROSINE KINASE EXPRESSION, A CENTRAL COMPONENT OF RAT HEPATIC STELLATE CELL ACTIVATION CHARACTERIZATION BY HOMOLOGY PCR. V Ankoma-Seyr KB Chans~ RS Warren, SL Friedman. UCSE Liver Center & Department of Surgery, Umversity of California, San Francisco, CA Receptor tyrosine kinases (RTKs) are a superfamily of transmembrane growth factor receptors which transduce signals critical to cell growth and development. In liver injury, proliferation of hepatic stellate cells (HSCs) (lipocytes) results from induction of a well known RTK, the []PDGF receptor, during cellular activation. The aims of this study were to determine whether other RTKs are also induced during HSC activation and to explore their potential role in hepatic fibrogenesis. Methods: 'Homology PCR' of RTKs was performed using degenerate oligonucleotide primers defining a highly conserved 200 bp region within the kinase domain (Oncogene 6: 753): the template was a subtracted activation-specific cDNA library from HSC isolated 3 hrs after CC14 adminstration (Hepatology 18: 107A). PCR products were cloned into pGEM and sequenced. Induction of candidate RTK mRNAs was confirmed by RNAse protection assay using purified stellate cells from normal or CC14-treated rats. Results: Partial cDNAs for several members of the RTK superfamily were identified. These included receptors for vascular endothelial growth factor (VEGF): Fit-l, Flk-1; for HGF: c-met and for PDGF: PDGFR. RNase protection demonstrated reciprocal expression of the two VEGF receptors in HSCs during injury: Elk1 mRNA was induced 4 fold at 3 hr after CCI4 whereas Flt-1 mRNA was down-regulated. Endothelial cell contamination of HSC isolates was excluded by demonstrating specific high affinity saturable binding of VEGF in culture-activated pure HSCs with IQ = 7.6 x 10[° M and 37,525 sites/cell. In addition to the known RTK's, a novel RTK, clone 'RTK40', was recovered whose tyrosine kinase sequence assigns it to a new subfamily which have a lectin-like domain in their extracellular regions (other members include Tyro 10, DDR). Ligands for these receptors have not yet been identified. For clone 'RTK40', six fold induction of its mRNA was documented during HSCs activation 3 hr after CC14. Screening of an unsubtracted library from HSCs activated in vivo with the original 200 bp 'RTK40' probe yielded 800 bp of additional sequence, confirming the novelty of this eDNA. COnclusions: Homology PCR has successfully identified a large number of RTKs expressed during HSC activation. The identification of VEGF receptors suggests that HSCs, in addition to endothelial cells, may participate in angiogenesis associated with liver injury. Further characterization of RTK 40 and identification of its ligand could establish an important role for this RTK in liver injury.
747 z~, A
NOVEL ZINC FINGER GENE I N D U C E I ) DURING HEPATIC STELLATE CELL ACTIVATION BINDS 'GC BOX' DNA AND IS EXP~ED IN HUMANS. S Jen.sen*, "~ Ratziu, A Lalazar, L Wong, SL Friedman. UCSF Liver Center & Department of Medicine, University of California, San Francisco, CA Hepatic stellate cell (lipoeyte) activation is characterized by a diverse and coordinated increase in gene expression, but little is known about its regulation. We previously identified a rat eDNA, called Zt'9, which is an immediate-early gene induced rapidly after liver injury in vivo (Hepatotogy 20:769A). Zf9's 3' region is highly homologous to other C2H 2 zinc f'mger transcriptional regulatory proteins; these proteins contain thi'e6C-terminus zinc fingers which bind specific DNA sequences within gene promoters and enhancers. In contrast, the 5' putative activation domain of Zt'9 is novel, suggesting unique interactions with transcriptional co-activating factors. Aims: a) To characterize the DNA binding properties of recombinant Zf9 peptides and b) to determine Zf9's possible relevance to clinical disease by cloning its human homologue. Results: Several Zf9 recombinant peptides were expressed as GST fusion proteins in E. coli, containing portions of the 5' activation domain, the 3' zinc finger domain, or both. Each recombinant peptide was incubated with a V32P-DNA probe and binding analyzed by gel shift assay. Peptides centaining the zinc finger domain bound to a 21 bp 'GC box' motif in a concentration dependent manner, whereas peptides lacking this domain did not bind DNA. For human Zf9 cloning, homology RT-PCR was employed using eDNA from activated human steUate cells as template with degenerate primers based on the rat amino acid sequence. A 201 bp human cDNA corresponding to a portion of the novel 5' domain was 82% homologous to rat at the nucleotide and amino acid levels. This sequence was used to clone an additional 720 bp by anchored PCR encoding the zinc finger region. Sequencing confirmed 81% homology between these species. Condualons: i) Zf9 binds to a 'GC box' motif via its zinc finger domain. Since 'GC boxes' are present in genes induced during steUate eeU activation (e.g. collagen type I, TGFI~I), this finding suggests a functional role of Zf9 in modulating gene expression during hepatic injury, ii) Expression of Zf9 by activated human steUate cells and its conservation between species underscore its potential importance in human liver disease. (*A Howard Hughes Medical Institute Medical Student Research Training Fellow).
ABSTRACTS
746
293A
STIMULATION OF c-JUN KINASE BY FIBRONECTIN AND INTERLEUKIN-la IS ASSOCIATED WITH INCREASED GENE EXPRESSION OF STROMELYSIN IN LIPOCYTES. JE Poulos. AM~Di Bisce~lie. JM Bellezzo. JD Weber. RS Britton. BR Bacon. and dJ Baldassare. Divisions of Gastroenterology/Hepatology and Nephrology, Saint Louis University Health Sciences Center, St. Louis, MO. Activated lipoeytes play a key role in hepatic fibrogenesis and their activation state may be influenced by extraeellularmatrix components and cytokines, e-Jan kinase (JNK) is an important intermediate in a signal transduction pathway that is initiated by activation of some cell surface receptors. JNK phosphorylates c-Jun and has not been studied in lipocytes, c-Jan homodimers or c-Jurdc-Fos heterodimers can activate gene expression through AP-I promoter sites. Aims: The aims of this study were to investigate if fibronectin (FN) or interleukin- 1,Y(IL- 1~x)activate JNK in lipocytes, and to determine whether such activation induces AP-1 activity and increases the gene expression of the matrix metalloproteinase stromelysin. Methods: Cultured rat lipocytes were serum-starved before the experiments. JNK was measured in cell extracts using a phosphorylation assay, and AP-I binding activity was measured in nuclear extracts using a gel shift assay. Stromelysin mRNA levels were assessed with an RNAse protection assay. Results: Exposure of lipoeytes to fibronectin (10-100 ttg/ml) increased JNK activity by 1.5- to 2.4-fold. Adhesion of lipocytus to plates coated with an gGDS-peptide increased JNK activity by 3.0-fold, compared to cells maintained in suspension on Teflon plates. IL-I~ (15-150 ng/ml) also increased JNK activity in lipocytss by 2.1- to 2.6-fold. AP-1 binding activity was markedly increased by both FN and IL-la, and stromelysin mRNA levels were increased 2.3-fold by FN and 3.2-fold by IL-1 ~. Condu~ions: FN and IL-1 tx increase the activity of JNK in lipocytes, stimulate the activation of AP-1, and increase the gene expression of stromelysin. The response to FN may be mediated by binding to integrins on the lipoeyte surface, since its effects are mimicked by an RGDS peptide. Signal transduetion pathways involving JNK may play an important role in regulating lipocyte function and fibrogenesis. (Supported by NIH DK-41gI6 and HL-40901)
748
ACTIVATION OF HEPATIC STELLATE CELLS IS MEDIATED BY OXIDATIVE STRESS THROUGH C-MYB EXPRESSION. KS Lee, M Buck, K Houglum, and M Chojkier. Dept. of Medicine, VAMC, and Center for Molecular Genetics, University of California, San Diego CA 92161. Stellate cell activation is a critical step in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and s-smooth muscle actin (~-SMA) expression. Here we show that quiescent rat stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix, plastic and TGFc~ was blocked by antioxidants, such as d-c~-tocopherol and butylated hydroxytoluene. Moreover, these stimuli markedly increased stellate cell entry into S-phase, which was abolished by antioxidants. Activation of stellate cells was associated with nuclear translocation and activation of NFkB, as detected by immunofluorescence and gel shift analysis, which was abolished by antioxidants. Because c-myb is an important inducer of proliferation in smooth muscle cells, we tested whether c-myb expression was essential for steIIate cell proliferation and/or activation. We found that the nuclear expression of c-myb was enhanced in activated stellate cells, irrespective of the method of induction, and that this effect was inhibited by antioxidants. A c-myb antisense, but not a c-myb sense, oligonucleotide suppressed the nuclear expression of c-myb during stellate cell activation, and prevented almost completely TGFc~-induced activation and proliferation of these cells. Nuclear extracts from activated, but not from quiescent, stellate cells formed a high affinity protein-DNA complex with the critical promoter E box of the ~-SMA gene, which was disrupted by c-myb and NFkB6s antibodies, and competed by c-myb and NFkB cognate cis-acting elements. These results strongly suggest a role for c-myb (and presumably NFkB) on c~-SMA gene expression. In this context, malondialdehyde-protein adducts and c-myb expression were also stimulated in activated stellate cells in the liver of CCl4-treated rats. This study suggests that oxidative stress plays an essential role on stellate cell activation, through the induction of c-myb by probably targeting the conserved Cys redox sensor within the DNA binding domain.
745
AASLD
RECEPTOR TYROSINE KINASE EXPRESSION, A CENTRAL COMPONENT OF RAT HEPATIC STELLATE CELL ACTIVATION CHARACTERIZATION BY HOMOLOGY PCR. V Ankoma-Seyr KB Chans~ RS Warren, SL Friedman. UCSE Liver Center & Department of Surgery, Umversity of California, San Francisco, CA Receptor tyrosine kinases (RTKs) are a superfamily of transmembrane growth factor receptors which transduce signals critical to cell growth and development. In liver injury, proliferation of hepatic stellate cells (HSCs) (lipocytes) results from induction of a well known RTK, the []PDGF receptor, during cellular activation. The aims of this study were to determine whether other RTKs are also induced during HSC activation and to explore their potential role in hepatic fibrogenesis. Methods: 'Homology PCR' of RTKs was performed using degenerate oligonucleotide primers defining a highly conserved 200 bp region within the kinase domain (Oncogene 6: 753): the template was a subtracted activation-specific cDNA library from HSC isolated 3 hrs after CC14 adminstration (Hepatology 18: 107A). PCR products were cloned into pGEM and sequenced. Induction of candidate RTK mRNAs was confirmed by RNAse protection assay using purified stellate cells from normal or CC14-treated rats. Results: Partial cDNAs for several members of the RTK superfamily were identified. These included receptors for vascular endothelial growth factor (VEGF): Fit-l, Flk-1; for HGF: c-met and for PDGF: PDGFR. RNase protection demonstrated reciprocal expression of the two VEGF receptors in HSCs during injury: Elk1 mRNA was induced 4 fold at 3 hr after CCI4 whereas Flt-1 mRNA was down-regulated. Endothelial cell contamination of HSC isolates was excluded by demonstrating specific high affinity saturable binding of VEGF in culture-activated pure HSCs with IQ = 7.6 x 10[° M and 37,525 sites/cell. In addition to the known RTK's, a novel RTK, clone 'RTK40', was recovered whose tyrosine kinase sequence assigns it to a new subfamily which have a lectin-like domain in their extracellular regions (other members include Tyro 10, DDR). Ligands for these receptors have not yet been identified. For clone 'RTK40', six fold induction of its mRNA was documented during HSCs activation 3 hr after CC14. Screening of an unsubtracted library from HSCs activated in vivo with the original 200 bp 'RTK40' probe yielded 800 bp of additional sequence, confirming the novelty of this eDNA. COnclusions: Homology PCR has successfully identified a large number of RTKs expressed during HSC activation. The identification of VEGF receptors suggests that HSCs, in addition to endothelial cells, may participate in angiogenesis associated with liver injury. Further characterization of RTK 40 and identification of its ligand could establish an important role for this RTK in liver injury.
747 z~, A
NOVEL ZINC FINGER GENE I N D U C E I ) DURING HEPATIC STELLATE CELL ACTIVATION BINDS 'GC BOX' DNA AND IS EXP~ED IN HUMANS. S Jen.sen*, "~ Ratziu, A Lalazar, L Wong, SL Friedman. UCSF Liver Center & Department of Medicine, University of California, San Francisco, CA Hepatic stellate cell (lipoeyte) activation is characterized by a diverse and coordinated increase in gene expression, but little is known about its regulation. We previously identified a rat eDNA, called Zt'9, which is an immediate-early gene induced rapidly after liver injury in vivo (Hepatotogy 20:769A). Zf9's 3' region is highly homologous to other C2H 2 zinc f'mger transcriptional regulatory proteins; these proteins contain thi'e6C-terminus zinc fingers which bind specific DNA sequences within gene promoters and enhancers. In contrast, the 5' putative activation domain of Zt'9 is novel, suggesting unique interactions with transcriptional co-activating factors. Aims: a) To characterize the DNA binding properties of recombinant Zf9 peptides and b) to determine Zf9's possible relevance to clinical disease by cloning its human homologue. Results: Several Zf9 recombinant peptides were expressed as GST fusion proteins in E. coli, containing portions of the 5' activation domain, the 3' zinc finger domain, or both. Each recombinant peptide was incubated with a V32P-DNA probe and binding analyzed by gel shift assay. Peptides centaining the zinc finger domain bound to a 21 bp 'GC box' motif in a concentration dependent manner, whereas peptides lacking this domain did not bind DNA. For human Zf9 cloning, homology RT-PCR was employed using eDNA from activated human steUate cells as template with degenerate primers based on the rat amino acid sequence. A 201 bp human cDNA corresponding to a portion of the novel 5' domain was 82% homologous to rat at the nucleotide and amino acid levels. This sequence was used to clone an additional 720 bp by anchored PCR encoding the zinc finger region. Sequencing confirmed 81% homology between these species. Condualons: i) Zf9 binds to a 'GC box' motif via its zinc finger domain. Since 'GC boxes' are present in genes induced during steUate eeU activation (e.g. collagen type I, TGFI~I), this finding suggests a functional role of Zf9 in modulating gene expression during hepatic injury, ii) Expression of Zf9 by activated human steUate cells and its conservation between species underscore its potential importance in human liver disease. (*A Howard Hughes Medical Institute Medical Student Research Training Fellow).
ABSTRACTS
746
293A
STIMULATION OF c-JUN KINASE BY FIBRONECTIN AND INTERLEUKIN-la IS ASSOCIATED WITH INCREASED GENE EXPRESSION OF STROMELYSIN IN LIPOCYTES. JE Poulos. AM~Di Bisce~lie. JM Bellezzo. JD Weber. RS Britton. BR Bacon. and dJ Baldassare. Divisions of Gastroenterology/Hepatology and Nephrology, Saint Louis University Health Sciences Center, St. Louis, MO. Activated lipoeytes play a key role in hepatic fibrogenesis and their activation state may be influenced by extraeellularmatrix components and cytokines, e-Jan kinase (JNK) is an important intermediate in a signal transduction pathway that is initiated by activation of some cell surface receptors. JNK phosphorylates c-Jun and has not been studied in lipocytes, c-Jan homodimers or c-Jurdc-Fos heterodimers can activate gene expression through AP-I promoter sites. Aims: The aims of this study were to investigate if fibronectin (FN) or interleukin- 1,Y(IL- 1~x)activate JNK in lipocytes, and to determine whether such activation induces AP-1 activity and increases the gene expression of the matrix metalloproteinase stromelysin. Methods: Cultured rat lipocytes were serum-starved before the experiments. JNK was measured in cell extracts using a phosphorylation assay, and AP-I binding activity was measured in nuclear extracts using a gel shift assay. Stromelysin mRNA levels were assessed with an RNAse protection assay. Results: Exposure of lipoeytes to fibronectin (10-100 ttg/ml) increased JNK activity by 1.5- to 2.4-fold. Adhesion of lipocytus to plates coated with an gGDS-peptide increased JNK activity by 3.0-fold, compared to cells maintained in suspension on Teflon plates. IL-I~ (15-150 ng/ml) also increased JNK activity in lipocytss by 2.1- to 2.6-fold. AP-1 binding activity was markedly increased by both FN and IL-la, and stromelysin mRNA levels were increased 2.3-fold by FN and 3.2-fold by IL-1 ~. Condu~ions: FN and IL-1 tx increase the activity of JNK in lipocytes, stimulate the activation of AP-1, and increase the gene expression of stromelysin. The response to FN may be mediated by binding to integrins on the lipoeyte surface, since its effects are mimicked by an RGDS peptide. Signal transduetion pathways involving JNK may play an important role in regulating lipocyte function and fibrogenesis. (Supported by NIH DK-41gI6 and HL-40901)
748
ACTIVATION OF HEPATIC STELLATE CELLS IS MEDIATED BY OXIDATIVE STRESS THROUGH C-MYB EXPRESSION. KS Lee, M Buck, K Houglum, and M Chojkier. Dept. of Medicine, VAMC, and Center for Molecular Genetics, University of California, San Diego CA 92161. Stellate cell activation is a critical step in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and s-smooth muscle actin (~-SMA) expression. Here we show that quiescent rat stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix, plastic and TGFc~ was blocked by antioxidants, such as d-c~-tocopherol and butylated hydroxytoluene. Moreover, these stimuli markedly increased stellate cell entry into S-phase, which was abolished by antioxidants. Activation of stellate cells was associated with nuclear translocation and activation of NFkB, as detected by immunofluorescence and gel shift analysis, which was abolished by antioxidants. Because c-myb is an important inducer of proliferation in smooth muscle cells, we tested whether c-myb expression was essential for steIIate cell proliferation and/or activation. We found that the nuclear expression of c-myb was enhanced in activated stellate cells, irrespective of the method of induction, and that this effect was inhibited by antioxidants. A c-myb antisense, but not a c-myb sense, oligonucleotide suppressed the nuclear expression of c-myb during stellate cell activation, and prevented almost completely TGFc~-induced activation and proliferation of these cells. Nuclear extracts from activated, but not from quiescent, stellate cells formed a high affinity protein-DNA complex with the critical promoter E box of the ~-SMA gene, which was disrupted by c-myb and NFkB6s antibodies, and competed by c-myb and NFkB cognate cis-acting elements. These results strongly suggest a role for c-myb (and presumably NFkB) on c~-SMA gene expression. In this context, malondialdehyde-protein adducts and c-myb expression were also stimulated in activated stellate cells in the liver of CCl4-treated rats. This study suggests that oxidative stress plays an essential role on stellate cell activation, through the induction of c-myb by probably targeting the conserved Cys redox sensor within the DNA binding domain.