- Email: [email protected]
“Deactivation” of activated ITO cells cultured in and on collagen type I
282A
701
AASLD
T N F - a S T I M U L A T E S cxl(I) P R O C O L L A G E N G E N E EXPRESSION I N L I V E R S T E L L A T E C E L L S BY E N H A N C I N G BINDI N G O F N F k B T O ITS A C U T E P H A S E R E S P O N S I V E E L E M E N T (APPLE). MJ" Iraburu. E Ruiz-Garcfa-Treviiano and M Roikind. Marion Bessin Liver Research Center, and Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, N.Y.
ABSTRACTS
702
"DEACTIVATION" OF ACTIVATED ITO CELLS CULTURED IN AND ON COLLAGEN TYPE I. ~ Cunningham, G Celetta. G Gabbiani. A Haden.que. Division of Gastroenterology and Department of Pathology, Hfpital Cantonal Universitaire. 1211 Geneva 14, Switzerland. n the intial stages of nepatic fibrosis, Ilo cells undergo a phenotypic transformation, commonly termed 'activation', in which they lose their vitamin ,~ rich lipid droplets, acquire a prominent cytoske]eton, express the smooth muscle isofrom of a actin (o.-SM), proliferate rapidly and secrete abundant ouanlites of collagen type I A similar transformation occurs spontaneously when freshly isolated to cells are cultured orl plastic or on a collagen I matrix. However, these 2-dimensional culture systems are nonphysiologic. We therefore decided to investigate the activation of It cells when cultured in a 3-dimensional collagen lattice, Ito cells were isolated from Wislar rats by collagenasepronase digestion followed by centrifugation over a Nycodenz gradient. These cells were activated by CUlture on plastic Cells at passage 2-4 (>95% positive for a-SIV by immunofluorescence [IF]) were then seeded or 0[astic dishes (control) or on to dishes coated with collagen I (gpl) or mixed with a collagen i solution which was then allowed to polymerize by culture at 37°C. Medium was DMEM with 10% FCS throughout. After 9 days, the cells were released by either lrypsinization or digestion with collagenase an(] counted in a hemocytometer, al(I)procollagen gene expression was studied by Northerrl Blotting and c~-S~ protein expression by Western plotting. The proportion of a-SMpositive cells was studied by double iF for total actin and ~-SM. Cell number increased after 5 and 9 days 2.09 and 3.53 fold (control), 1.9 and 2.6-fold Igpl) whereas there was a slight decrease in gp2 cells, 0.23-fold, at day 5 with no change in cell number through days to 9. By double IF more than 95% of control cells were ~ositive for a-SM compared to 48.5% those of gpl and <5% those of gp2. On Western blotting a-SM in gpl and gp2 cells decreased [o 48,5% and 3.3% respectively compared to the control cells, al(I) procollagen mRNA of gp2 cells decreased to 53.8% of the control cells.In conclusion, there is a profound inhibition of the three principal features of Ito cell activation, proliferation, a-SM and collagen expression wnen culture-activated Ito cells are cultured in a 3-dimensional collagen I lattice. This is apparently due both to the nature of the interaction of the cells with collagen I and to the three dimensional nature of the lattice. This system may be useful to study the factors leading to Ilo cells activation.
TISSUE TRANSGLUTAMINASE GENRE EXPRESSION IN HEPATIC FIBROGENESIS AND ITS REGULATION BY NF-tcB. A. Mi~a. E. Frizell. S-L Liu. J-L Zhu. P. Norton. and MA Zern. Department of Medicine, Iefferson Medical College, Philadelphia, PA 19107 Tissue Transgintaminase (tTG) has been found in association with apoptosis and cell necrosis. Its ability to catalyze stable cross-linking and its binding to collagen and fibronectin suggest .that tTG may participate in extracellular matrix remodeling. The present study was undertaken to delineate a possible role of tTO in hepatic fibmgenesis, and to explore whether NF-w.B may regulate its expression. To achieve our goals, rats were distributed into 9 groups. Animals in the control group remained m a t e d . The others were treated with carbon tetrachlorlde (CC14) solution (50% in mineral oil) at I mL/kg, intraperltoneally, twice per week. The rats treated with CC14 were sacrificed at different time-points of liver injury and fibmgenesis (6 hours, 24 hours, 4 days, and at 1, 2, 4, 6, and 8 weeks after the first CC14 injection). 20 Ixg of total RNA from the livers of treated and untreated animals were used to assess tTG gene expression by Nurthem blot. The results were evaluated by densitometry in a phosphor-imager apparatus, and standardized against 28S ribosomal R/flA. tTG mRNA levels were increased by 5-fold as early as 6 hours after the first injection. The steady state levels of the gene peaked at 24 hours and 4 days (10-12 fold), and remained increased during the later time-points. The activity of tTG -assessed by a cross-linking assay -- was increased in the rata treated with CC14, in a fashion that paralleled the Notrhem blot results. In an attempt to delineate factors that may regulate f i g gene expression during liver injury, we found an NF-r,B consensus sequence in the tTG promoter region. NF-~B was evaluated because increased binding of this transcriptional factor is associated with many forms of cell injury. Using an oligomer containing the NF-r,.B sequence in a gel retardation assay, we found increased binding to NF-r,.B in the CC14 treated samples, which also paralleled the increase in tTG gene expression. These data demonstrate an increase in tTG gene expression during the early injury phase of hepatic fibrosis, suggesting a role for this enzyme in forming cross-links to stabilize the fibrotie bands during hepatic fibregenesis. Moreover, increased NF-~cB binding to the tTO promoter may represent a mechanism by which ceil injury induces tTG transcription and thus potentiates the process of fibmgenesis.
B A C K G R O U N D : We have previously shown that: 1) liver ~ l ( I ) procollagen (COL-l) mRNA is stimul/~ted during the induction of an acute phase response and 2) the two acute phase cytokines, TNF-o~ and I L - 6 i n d u c e COL-1 m R N A e x p r e s s i o n in l i v e r s t e l l a t e cells (LSC). Because rat COL-1 promoter contains a putative APRE, we investigated whether TNF-c~ induces the formation of APRE/nuclearprotein complexes. M E T H O D S : Nuclear extracts (NE) were prepared from cultured LSC (CFSC-2G) treated with TNF-e for 2 hourS. Nuclear-protein/DNA complexes with A P R E and NFkB consensus sequences were monitored by gel mobility shift assays. COL-1 and IL-6 mRNAs were quantified by northern blot analysis. RESULTS: T N F - e induced IL-6 and COL-1 mRNA levels in CFSC-2G. NE from control cells did not form DNA/protein complexes with APRE. However, APRE formed a complex with N E from TNF-cc-treated cells. Supershifts performed with antibodies against several nuclear transcription factors inducible by TNF-~x showed that the A P R E complex contained NFkB. Moreover, binding to the APRE was abolished by a cold NFkB probe. Pretreatment of LSC with pyrrolidinedithiocarbamate (PD'IC) prevented formation of protein complexes with both APRE and NFkB probes, and inhibited TNF-c~-induced expression of IL-6 and COL-1 mRNAs. Cycloheximide induced the expression of IL-6 mRNA as well as A P R E / a n d NFkB/protein complexes. C O N C L U S I O N : We s h o w e d that T N F - a i n d u c e s f o r m a t i o n APRE/NFkB complexes in vitro and that up-regulation of IL-6 and COL-1 mRNAs by TNF-~ or their inhibition by PDTC in LSC are accompanied by parallel changes in the formation of APRE/NFkB complexes. We conclude that binding of NFkB to APRE in the COL1 promoter may be involved in COL-1 transcription induced by TNFc~. Supported in part by Grant AA10541 from NIAAA.
703
HEPATOLOGY O c t o b e r 1995
704
REVERSAL OF ACTIVATED LIPOCYTE PHENOTYPE BY CULTURE ON BASEMENT MEMBRANE-LIKE EXTRACELLULAR MATRIX. NA l ~ m . AM Soweid. R O'Neill. L Li. RS Britton. and KE Brown. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO. Activated lipocytes have a myofibroblastic phenotype, characterized by expression of g-smooth muscle actin (SMA) and increased cellular proliferation. These cells play a key role in hepatic fibroganesis, but little is known about factors that can reverse the myofibroblastic phenotype. The composition of the extraoellular matrix may be important in maintaining lipocytes in the activated, myofibroblastic state. Aim: To investigate whether the phenotype of activated lipocytes (myofibroblasts) can be reversed by culture on a basement membraue-like matrix. Methods: Lipocytes were isolated from normal rat liver by pronase-collagenase digestion, and were activated by culture on uncoated plastic through 7 passes. These activated cells were trypsinized, and plated at a density of IIY/60 mm dish on either plastic dishes, or plastic dishes Coated with a basement membrane-like matrix (Matrigel; Collaborative Biomedical). After 10 days of culture in M199 medium containing 10% serum, the ceils were harvested for determination of cell number and the content of the activation marker, SMA. Cell morphology was assessed using phase-contrast microscopy. Results: Activated lipocytes cultured for 10 days on plastic retained their proliferative myofibroblastic phenotype, with abundant expression of SMA and a 2.4-fold increase in cell number. Morphologically, these cells were spread and had numerous cellular extensions. In contrast, activated lipocytes plated on Matrigel essentially lost their rnyofibroblastic phenotype, as evidenced by only a trace amount of SMA expression, andno increase in cell number. These cells exhibited less cell spreading and had many fewer cellular extensions than the cells cultured on plastic. ~ : This is the first demonstration that activated lipocytes lose their myofibroblasticphenotype when plated on a basement membrane-like matrix. These results suggest that the myofibroblastic phenotype of activated lipocytes is perpetuated by adherence to abnormal matrix, and can be reversed by attachment to an extracellular matrix which mimics that found within the normal liver. (Supported by NIH DK-03907, DK-41816 and Glaxo Basic Research Award)
701
AASLD
T N F - a S T I M U L A T E S cxl(I) P R O C O L L A G E N G E N E EXPRESSION I N L I V E R S T E L L A T E C E L L S BY E N H A N C I N G BINDI N G O F N F k B T O ITS A C U T E P H A S E R E S P O N S I V E E L E M E N T (APPLE). MJ" Iraburu. E Ruiz-Garcfa-Treviiano and M Roikind. Marion Bessin Liver Research Center, and Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, N.Y.
ABSTRACTS
702
"DEACTIVATION" OF ACTIVATED ITO CELLS CULTURED IN AND ON COLLAGEN TYPE I. ~ Cunningham, G Celetta. G Gabbiani. A Haden.que. Division of Gastroenterology and Department of Pathology, Hfpital Cantonal Universitaire. 1211 Geneva 14, Switzerland. n the intial stages of nepatic fibrosis, Ilo cells undergo a phenotypic transformation, commonly termed 'activation', in which they lose their vitamin ,~ rich lipid droplets, acquire a prominent cytoske]eton, express the smooth muscle isofrom of a actin (o.-SM), proliferate rapidly and secrete abundant ouanlites of collagen type I A similar transformation occurs spontaneously when freshly isolated to cells are cultured orl plastic or on a collagen I matrix. However, these 2-dimensional culture systems are nonphysiologic. We therefore decided to investigate the activation of It cells when cultured in a 3-dimensional collagen lattice, Ito cells were isolated from Wislar rats by collagenasepronase digestion followed by centrifugation over a Nycodenz gradient. These cells were activated by CUlture on plastic Cells at passage 2-4 (>95% positive for a-SIV by immunofluorescence [IF]) were then seeded or 0[astic dishes (control) or on to dishes coated with collagen I (gpl) or mixed with a collagen i solution which was then allowed to polymerize by culture at 37°C. Medium was DMEM with 10% FCS throughout. After 9 days, the cells were released by either lrypsinization or digestion with collagenase an(] counted in a hemocytometer, al(I)procollagen gene expression was studied by Northerrl Blotting and c~-S~ protein expression by Western plotting. The proportion of a-SMpositive cells was studied by double iF for total actin and ~-SM. Cell number increased after 5 and 9 days 2.09 and 3.53 fold (control), 1.9 and 2.6-fold Igpl) whereas there was a slight decrease in gp2 cells, 0.23-fold, at day 5 with no change in cell number through days to 9. By double IF more than 95% of control cells were ~ositive for a-SM compared to 48.5% those of gpl and <5% those of gp2. On Western blotting a-SM in gpl and gp2 cells decreased [o 48,5% and 3.3% respectively compared to the control cells, al(I) procollagen mRNA of gp2 cells decreased to 53.8% of the control cells.In conclusion, there is a profound inhibition of the three principal features of Ito cell activation, proliferation, a-SM and collagen expression wnen culture-activated Ito cells are cultured in a 3-dimensional collagen I lattice. This is apparently due both to the nature of the interaction of the cells with collagen I and to the three dimensional nature of the lattice. This system may be useful to study the factors leading to Ilo cells activation.
TISSUE TRANSGLUTAMINASE GENRE EXPRESSION IN HEPATIC FIBROGENESIS AND ITS REGULATION BY NF-tcB. A. Mi~a. E. Frizell. S-L Liu. J-L Zhu. P. Norton. and MA Zern. Department of Medicine, Iefferson Medical College, Philadelphia, PA 19107 Tissue Transgintaminase (tTG) has been found in association with apoptosis and cell necrosis. Its ability to catalyze stable cross-linking and its binding to collagen and fibronectin suggest .that tTG may participate in extracellular matrix remodeling. The present study was undertaken to delineate a possible role of tTO in hepatic fibmgenesis, and to explore whether NF-w.B may regulate its expression. To achieve our goals, rats were distributed into 9 groups. Animals in the control group remained m a t e d . The others were treated with carbon tetrachlorlde (CC14) solution (50% in mineral oil) at I mL/kg, intraperltoneally, twice per week. The rats treated with CC14 were sacrificed at different time-points of liver injury and fibmgenesis (6 hours, 24 hours, 4 days, and at 1, 2, 4, 6, and 8 weeks after the first CC14 injection). 20 Ixg of total RNA from the livers of treated and untreated animals were used to assess tTG gene expression by Nurthem blot. The results were evaluated by densitometry in a phosphor-imager apparatus, and standardized against 28S ribosomal R/flA. tTG mRNA levels were increased by 5-fold as early as 6 hours after the first injection. The steady state levels of the gene peaked at 24 hours and 4 days (10-12 fold), and remained increased during the later time-points. The activity of tTG -assessed by a cross-linking assay -- was increased in the rata treated with CC14, in a fashion that paralleled the Notrhem blot results. In an attempt to delineate factors that may regulate f i g gene expression during liver injury, we found an NF-r,B consensus sequence in the tTG promoter region. NF-~B was evaluated because increased binding of this transcriptional factor is associated with many forms of cell injury. Using an oligomer containing the NF-r,.B sequence in a gel retardation assay, we found increased binding to NF-r,.B in the CC14 treated samples, which also paralleled the increase in tTG gene expression. These data demonstrate an increase in tTG gene expression during the early injury phase of hepatic fibrosis, suggesting a role for this enzyme in forming cross-links to stabilize the fibrotie bands during hepatic fibregenesis. Moreover, increased NF-~cB binding to the tTO promoter may represent a mechanism by which ceil injury induces tTG transcription and thus potentiates the process of fibmgenesis.
B A C K G R O U N D : We have previously shown that: 1) liver ~ l ( I ) procollagen (COL-l) mRNA is stimul/~ted during the induction of an acute phase response and 2) the two acute phase cytokines, TNF-o~ and I L - 6 i n d u c e COL-1 m R N A e x p r e s s i o n in l i v e r s t e l l a t e cells (LSC). Because rat COL-1 promoter contains a putative APRE, we investigated whether TNF-c~ induces the formation of APRE/nuclearprotein complexes. M E T H O D S : Nuclear extracts (NE) were prepared from cultured LSC (CFSC-2G) treated with TNF-e for 2 hourS. Nuclear-protein/DNA complexes with A P R E and NFkB consensus sequences were monitored by gel mobility shift assays. COL-1 and IL-6 mRNAs were quantified by northern blot analysis. RESULTS: T N F - e induced IL-6 and COL-1 mRNA levels in CFSC-2G. NE from control cells did not form DNA/protein complexes with APRE. However, APRE formed a complex with N E from TNF-cc-treated cells. Supershifts performed with antibodies against several nuclear transcription factors inducible by TNF-~x showed that the A P R E complex contained NFkB. Moreover, binding to the APRE was abolished by a cold NFkB probe. Pretreatment of LSC with pyrrolidinedithiocarbamate (PD'IC) prevented formation of protein complexes with both APRE and NFkB probes, and inhibited TNF-c~-induced expression of IL-6 and COL-1 mRNAs. Cycloheximide induced the expression of IL-6 mRNA as well as A P R E / a n d NFkB/protein complexes. C O N C L U S I O N : We s h o w e d that T N F - a i n d u c e s f o r m a t i o n APRE/NFkB complexes in vitro and that up-regulation of IL-6 and COL-1 mRNAs by TNF-~ or their inhibition by PDTC in LSC are accompanied by parallel changes in the formation of APRE/NFkB complexes. We conclude that binding of NFkB to APRE in the COL1 promoter may be involved in COL-1 transcription induced by TNFc~. Supported in part by Grant AA10541 from NIAAA.
703
HEPATOLOGY O c t o b e r 1995
704
REVERSAL OF ACTIVATED LIPOCYTE PHENOTYPE BY CULTURE ON BASEMENT MEMBRANE-LIKE EXTRACELLULAR MATRIX. NA l ~ m . AM Soweid. R O'Neill. L Li. RS Britton. and KE Brown. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO. Activated lipocytes have a myofibroblastic phenotype, characterized by expression of g-smooth muscle actin (SMA) and increased cellular proliferation. These cells play a key role in hepatic fibroganesis, but little is known about factors that can reverse the myofibroblastic phenotype. The composition of the extraoellular matrix may be important in maintaining lipocytes in the activated, myofibroblastic state. Aim: To investigate whether the phenotype of activated lipocytes (myofibroblasts) can be reversed by culture on a basement membraue-like matrix. Methods: Lipocytes were isolated from normal rat liver by pronase-collagenase digestion, and were activated by culture on uncoated plastic through 7 passes. These activated cells were trypsinized, and plated at a density of IIY/60 mm dish on either plastic dishes, or plastic dishes Coated with a basement membrane-like matrix (Matrigel; Collaborative Biomedical). After 10 days of culture in M199 medium containing 10% serum, the ceils were harvested for determination of cell number and the content of the activation marker, SMA. Cell morphology was assessed using phase-contrast microscopy. Results: Activated lipocytes cultured for 10 days on plastic retained their proliferative myofibroblastic phenotype, with abundant expression of SMA and a 2.4-fold increase in cell number. Morphologically, these cells were spread and had numerous cellular extensions. In contrast, activated lipocytes plated on Matrigel essentially lost their rnyofibroblastic phenotype, as evidenced by only a trace amount of SMA expression, andno increase in cell number. These cells exhibited less cell spreading and had many fewer cellular extensions than the cells cultured on plastic. ~ : This is the first demonstration that activated lipocytes lose their myofibroblasticphenotype when plated on a basement membrane-like matrix. These results suggest that the myofibroblastic phenotype of activated lipocytes is perpetuated by adherence to abnormal matrix, and can be reversed by attachment to an extracellular matrix which mimics that found within the normal liver. (Supported by NIH DK-03907, DK-41816 and Glaxo Basic Research Award)