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Posttranscriptional regulation of collagen α I(I) mRNA in hepatic stellate cells
292A
AASLD ABSTRACTS
741 S E R U M L E V E L S OF I L - 1 0 A N D s - I C A M - 1 IN P A T I E N T S WITH CHRONIC HEPATITIS C TREATED WITH INTERFERON-alpha. N Kuzushita. N Havashi. K Katavama. M Oshita. T Kanto, Y Kawanishi, A Kasahara, H Fusamoto, and T Kamada. First Dept. of Med., OSaka Univ. School of Medicine. Osaka, Japan
HCV RNA quantity and HCV genotype have been suggested to be important factors influencing the efficacy for interferon (IFN) therapy. However, it is unclear whether the host immune responses influence the efficacy for IFN therapy or not. ICAM-1 is required for induction of the cellular immune responses. In contrast, IL-10 can suppress the function of effector cells, such as macrophages and T cells. The aim of this study is to clarify whether the efficacy for IFN therapy might be affected by serum levels of s-ICAM-1 and those of IL-10. M e t h o d s Serum levels of IL-10 and s-ICAM-1 were determined collectively in the sera of 102 chronic hepatitis C patients before IFNalpha treatment using ELISA methods. Sustained responders (SR) were defined as those who showed persistent ALT normalization. Transient responders (TR) displayed ALT normalization during therapy and relapsed within 24 weeks after withdrawal. No responders (NR) continuously exhibited ALT abnormalities. HCV serotypes were determined using Kohara's method (Hepatology 1994 ;19:1347-1353). R e s u l t s [Serotype I (n:76) J [ SR(n=12) TR(n=43) I NR(n=21) I iL-10 (pg/ml) 8.24+3.94 a 9.66+5.82 b 13.16+7.23 I s-ICAM-I (n~/ml) I 543~_113 572'~198 628+_~21 [Serotype I1 (n=26) ] SR (n=19) TR (n=7) I IL-IO (pg/mi) 11.66+4.61 I s-ICAM-1 (ng/ml) [ 530+_130 622+126 I a: SR vs. NR p
1o.11+_3.96I
743 RAF & MAP KINASE DIFFERENTIALLY MODULATE STELLATE CELL TYPE I COLLAGEN GENE EXPRESSION. BH Davis,A Chen, and DWA Beno. Gastroenterology Section, Dept. of Medicine, Univ. of Chicago, Chicago, Illinois. The cytoplasmic signalling proteins which transmit extraeellular stimuli to the nucleus and lead to increased hepatic stellate cell (hsc) collagen gene transcription are unknown. An elucidation of these pathways should play a key role in fully understanding stellate cell activation and in ultimately designing selective inhibitors of hepatic fibrogenesis. We therefore evaluated the well-described ras-raf-MAP cytoplasmic kinase cascade as it plays a major role in most mitogenic pathways, including the stellate cell (JBC 270:3642;1995). Since MAP kinase can induce a large array of transcription factors, we reasoned it might affect collagen gene expression. Cultured rat hscs were co-transfected (co-t0 with a rat alpha I(I) collagen gene reporter (IntcolCAT: 3.6kb of 5' promoter and 1.6kb of the 1st intron linked to the CAT reporter gene) and dominant negative (d.n.) plasmids which specifically block ras, raf, or MAP kinase. Co-tf with d.n. ras or raf caused a marked concentration-dependent 3-6 fold increase in IntcolCAT. In contrast, d.n. MAP caused a 3-fold reduction of IntcolCAT and antagonized d.n. raf stimulation of IntcolCAT in a concentration-dependent fashion. To further explore the nuclear mediators of these effects, co-tf were performed with the d.n. plasmids and AP- 1 and Sp-1 reporter plasmids as well as with a series of IntcolCAT reporters which contain progressive truncations, internal deletions, and Sp-1 site mutations involving the 5' promoter and the 1st intron. D.n. raf enhanced AP-1 CAT while d.n. MAP suppressed AP-1 CAT. Both d.n. caused a modest suppression of Sp-1 CAT. While the d.n. raf effect may involve AP-1 transcription, the critical DNA domain resides largely in the -1.7- 1.3kb region of the 5' promoter. In contrast, the d.n. MAP effect requires the most proximal regions of the 5' promoter and the 1st intron. In conclusion these studies demonstrate that hsc type I collagen gene expression results from a complex balance between an inhibitory_ras-raf cascade independent of MAP and a stimulator3, - MAP cascade that each interact with distinct DNA domains of the 5' promoter and the 1st intron. Precise delineation of these domains and their associated DNA binding proteins should identify major regulators ofhsc type I collagen gene expression.
742
HEPATOLOGY October 1995
RESISTANCE TO INTERFERON TREATMENT IN CHRONIC HEPATITIS C PREDICTED BY PRETREATMENT iNTERFERON LEVELS. M Pidsi C Fabds. PL Toniutto. E Falleti, SG Tisminetzkv*, M Gerotto*. G Soardo. M Oel Fomo. D Vitulli. M CadattL F Baralle*, E Badoli. Dept. of Clin. and Exp. Pathology and Medicine, University of Udine; *ICGEB Tdeste, Italy. Since the rate of long-term response to interferon (IFN) treatment in patients with chronic hepatitis C vires (HCV) infection is low, strategies for identifying those patients most likely to respond are actively seeked. Previous studies have suggested that in chronic hepatitis the therapeutic efficacy of exogenous IFN might be related to its ability to replace a defective endogenous production; our aim was to vedfy the value of measuring pretreatment serum IFN levels in predicting the outcome of therapy in chronic HCV-related liver disease. Sixty patients (39 male, 21 female, age 46.1+12.2 years, mean+SD, range 14-70) positive to a 2rid generation screening test to anti HCV antibodies were studied: all had biopsy-proven liver disease (chronic hepatitis in 51, chronic hepatitis+cirrhosis in 9) and underwent treatment with IFN alpha, 3 Mega Units t.i.w, subcutaneously for up to 1 year. Serum IFN was measured by enzyme immunoassay (Cytoscreen T M human IFN-a, Biosource International, USA). HCV RNA genotyping was performed by dotblot assay of PCR amplified products of the 5' untranslated region as already described (Tisminetzky et al., Int Hepatol Commun 1994; 2:105-12). Fortythree patients (71.6%) had measurable pretreatment serum IFN; 13/60 patients (21.6%) had serum IFN >100 pg/ml. Forty-two patients normalized alanine aminotransferase (ALl') levels during IFN treatment; however, after its withdrawal 22 patients relapsed. Thus, 6 months after the end of treatment, only 20/60 patients (33.3%) maintained ALT levels consistently within the normal range and were considered to have a sustained response. Only 1/20 patients with sustained response had pretreatment serum IFN >100 pg/ml in comparison to 12/40 patients who did not respond or relapsed (Pearson X2 4.91, p<0.05), Stepwise multivariate analysis demonstrated that the predictive value of serum IFN was independent of age, sex, presence of cirrhosis, infection by genotype lb (improvemeit Z2 5.91, p<0.05). Our results suggest that, in patients with chronic HCV-rolated liver disease, measurement of serum IFN at baseline might be useful for the selection of patients with higher probability to achieve a long-term response.
744 PosTrRANSCRIPTIONAL REGULATIONOF COLLAGEN a 10) mRNA IN HEPATIC STELLATE CELLS. B Stcfanovic. C Hellcrbrand and DA Brenner. Dcpts. of Medicine, Biochcrnistry and Biophysics, University of North Carolina, Chapel Hill,NC. Liver fibrosis is characterized by excessive production of extraealhilar proteins by activated hepatic stellate cells. This study assessedthe contribution of posttmnseriptional regulation of collagen a 10) mRNA in these cells. We first confirmedthat quiesc~t stelinto cells have a very low level of collagen a 10) mRNA, and upon activation by culturing the cells on plastic for 14 days, the RNA accumulates to about a 15-fold higher level/elative to the GAPDH mRNA. Incubation of freshly isolated (nonactivated) stellato cells with ActinomycinD or cycloheximide increases the steady state level of collagen a 10) mRNA about 5-fold within 3h. No effect of Actinomycin D or cycloheximid¢ on the a 10) mRNAhas been observedin activated stellate cells. This finding suggests that the collagen mRNA is negatively regulated in nonactivated stellate cells. One possibilityis that here is a labile activity which degrades collagen m R N A in nonactivated stellato cells;disappearance of this activityrapidly leads to accumulation of the m R N A . Alternatively,a represser of transcriptionmay be involved.W e have identifiedan R N A binding proteinof 42 kD, which binds to the C-rich sequcoco located 20 nt downstream of the stop colon in the vt 10) mRNA. This blndlnZ activity is present in the cytoplasmic extracts of activated, but not quiescent stellate cells. Competition experiments showed that the protein hinds RNA in a sequence specific manner, although it can be competed with an excess of poly-C homopolymer.This RNA binding protein is clearly different from a protein of similar size that has been impfiented in maintainingthe high stability of a globin mRNA. ColLagena 10) mRNA is highly stable in activated stellatecells with minimal decay after 24h and probably with a half-lif~ of about 48h. It is possible that the protein bindingto the 3' region stabilizes the collagen c~ 1 0) mRNA in activated stellale cells.In summary, posRrsnseriptioanlregulation plays an important role in control of collagen a 1 0) mRNA levels in stellate cells. This regulation may be attained in two ways; by negatively regulating the mRNA in quiescent cells and by stabilizingthe m R N A in activatedcells.
AASLD ABSTRACTS
741 S E R U M L E V E L S OF I L - 1 0 A N D s - I C A M - 1 IN P A T I E N T S WITH CHRONIC HEPATITIS C TREATED WITH INTERFERON-alpha. N Kuzushita. N Havashi. K Katavama. M Oshita. T Kanto, Y Kawanishi, A Kasahara, H Fusamoto, and T Kamada. First Dept. of Med., OSaka Univ. School of Medicine. Osaka, Japan
HCV RNA quantity and HCV genotype have been suggested to be important factors influencing the efficacy for interferon (IFN) therapy. However, it is unclear whether the host immune responses influence the efficacy for IFN therapy or not. ICAM-1 is required for induction of the cellular immune responses. In contrast, IL-10 can suppress the function of effector cells, such as macrophages and T cells. The aim of this study is to clarify whether the efficacy for IFN therapy might be affected by serum levels of s-ICAM-1 and those of IL-10. M e t h o d s Serum levels of IL-10 and s-ICAM-1 were determined collectively in the sera of 102 chronic hepatitis C patients before IFNalpha treatment using ELISA methods. Sustained responders (SR) were defined as those who showed persistent ALT normalization. Transient responders (TR) displayed ALT normalization during therapy and relapsed within 24 weeks after withdrawal. No responders (NR) continuously exhibited ALT abnormalities. HCV serotypes were determined using Kohara's method (Hepatology 1994 ;19:1347-1353). R e s u l t s [Serotype I (n:76) J [ SR(n=12) TR(n=43) I NR(n=21) I iL-10 (pg/ml) 8.24+3.94 a 9.66+5.82 b 13.16+7.23 I s-ICAM-I (n~/ml) I 543~_113 572'~198 628+_~21 [Serotype I1 (n=26) ] SR (n=19) TR (n=7) I IL-IO (pg/mi) 11.66+4.61 I s-ICAM-1 (ng/ml) [ 530+_130 622+126 I a: SR vs. NR p
1o.11+_3.96I
743 RAF & MAP KINASE DIFFERENTIALLY MODULATE STELLATE CELL TYPE I COLLAGEN GENE EXPRESSION. BH Davis,A Chen, and DWA Beno. Gastroenterology Section, Dept. of Medicine, Univ. of Chicago, Chicago, Illinois. The cytoplasmic signalling proteins which transmit extraeellular stimuli to the nucleus and lead to increased hepatic stellate cell (hsc) collagen gene transcription are unknown. An elucidation of these pathways should play a key role in fully understanding stellate cell activation and in ultimately designing selective inhibitors of hepatic fibrogenesis. We therefore evaluated the well-described ras-raf-MAP cytoplasmic kinase cascade as it plays a major role in most mitogenic pathways, including the stellate cell (JBC 270:3642;1995). Since MAP kinase can induce a large array of transcription factors, we reasoned it might affect collagen gene expression. Cultured rat hscs were co-transfected (co-t0 with a rat alpha I(I) collagen gene reporter (IntcolCAT: 3.6kb of 5' promoter and 1.6kb of the 1st intron linked to the CAT reporter gene) and dominant negative (d.n.) plasmids which specifically block ras, raf, or MAP kinase. Co-tf with d.n. ras or raf caused a marked concentration-dependent 3-6 fold increase in IntcolCAT. In contrast, d.n. MAP caused a 3-fold reduction of IntcolCAT and antagonized d.n. raf stimulation of IntcolCAT in a concentration-dependent fashion. To further explore the nuclear mediators of these effects, co-tf were performed with the d.n. plasmids and AP- 1 and Sp-1 reporter plasmids as well as with a series of IntcolCAT reporters which contain progressive truncations, internal deletions, and Sp-1 site mutations involving the 5' promoter and the 1st intron. D.n. raf enhanced AP-1 CAT while d.n. MAP suppressed AP-1 CAT. Both d.n. caused a modest suppression of Sp-1 CAT. While the d.n. raf effect may involve AP-1 transcription, the critical DNA domain resides largely in the -1.7- 1.3kb region of the 5' promoter. In contrast, the d.n. MAP effect requires the most proximal regions of the 5' promoter and the 1st intron. In conclusion these studies demonstrate that hsc type I collagen gene expression results from a complex balance between an inhibitory_ras-raf cascade independent of MAP and a stimulator3, - MAP cascade that each interact with distinct DNA domains of the 5' promoter and the 1st intron. Precise delineation of these domains and their associated DNA binding proteins should identify major regulators ofhsc type I collagen gene expression.
742
HEPATOLOGY October 1995
RESISTANCE TO INTERFERON TREATMENT IN CHRONIC HEPATITIS C PREDICTED BY PRETREATMENT iNTERFERON LEVELS. M Pidsi C Fabds. PL Toniutto. E Falleti, SG Tisminetzkv*, M Gerotto*. G Soardo. M Oel Fomo. D Vitulli. M CadattL F Baralle*, E Badoli. Dept. of Clin. and Exp. Pathology and Medicine, University of Udine; *ICGEB Tdeste, Italy. Since the rate of long-term response to interferon (IFN) treatment in patients with chronic hepatitis C vires (HCV) infection is low, strategies for identifying those patients most likely to respond are actively seeked. Previous studies have suggested that in chronic hepatitis the therapeutic efficacy of exogenous IFN might be related to its ability to replace a defective endogenous production; our aim was to vedfy the value of measuring pretreatment serum IFN levels in predicting the outcome of therapy in chronic HCV-related liver disease. Sixty patients (39 male, 21 female, age 46.1+12.2 years, mean+SD, range 14-70) positive to a 2rid generation screening test to anti HCV antibodies were studied: all had biopsy-proven liver disease (chronic hepatitis in 51, chronic hepatitis+cirrhosis in 9) and underwent treatment with IFN alpha, 3 Mega Units t.i.w, subcutaneously for up to 1 year. Serum IFN was measured by enzyme immunoassay (Cytoscreen T M human IFN-a, Biosource International, USA). HCV RNA genotyping was performed by dotblot assay of PCR amplified products of the 5' untranslated region as already described (Tisminetzky et al., Int Hepatol Commun 1994; 2:105-12). Fortythree patients (71.6%) had measurable pretreatment serum IFN; 13/60 patients (21.6%) had serum IFN >100 pg/ml. Forty-two patients normalized alanine aminotransferase (ALl') levels during IFN treatment; however, after its withdrawal 22 patients relapsed. Thus, 6 months after the end of treatment, only 20/60 patients (33.3%) maintained ALT levels consistently within the normal range and were considered to have a sustained response. Only 1/20 patients with sustained response had pretreatment serum IFN >100 pg/ml in comparison to 12/40 patients who did not respond or relapsed (Pearson X2 4.91, p<0.05), Stepwise multivariate analysis demonstrated that the predictive value of serum IFN was independent of age, sex, presence of cirrhosis, infection by genotype lb (improvemeit Z2 5.91, p<0.05). Our results suggest that, in patients with chronic HCV-rolated liver disease, measurement of serum IFN at baseline might be useful for the selection of patients with higher probability to achieve a long-term response.
744 PosTrRANSCRIPTIONAL REGULATIONOF COLLAGEN a 10) mRNA IN HEPATIC STELLATE CELLS. B Stcfanovic. C Hellcrbrand and DA Brenner. Dcpts. of Medicine, Biochcrnistry and Biophysics, University of North Carolina, Chapel Hill,NC. Liver fibrosis is characterized by excessive production of extraealhilar proteins by activated hepatic stellate cells. This study assessedthe contribution of posttmnseriptional regulation of collagen a 10) mRNA in these cells. We first confirmedthat quiesc~t stelinto cells have a very low level of collagen a 10) mRNA, and upon activation by culturing the cells on plastic for 14 days, the RNA accumulates to about a 15-fold higher level/elative to the GAPDH mRNA. Incubation of freshly isolated (nonactivated) stellato cells with ActinomycinD or cycloheximide increases the steady state level of collagen a 10) mRNA about 5-fold within 3h. No effect of Actinomycin D or cycloheximid¢ on the a 10) mRNAhas been observedin activated stellate cells. This finding suggests that the collagen mRNA is negatively regulated in nonactivated stellate cells. One possibilityis that here is a labile activity which degrades collagen m R N A in nonactivated stellato cells;disappearance of this activityrapidly leads to accumulation of the m R N A . Alternatively,a represser of transcriptionmay be involved.W e have identifiedan R N A binding proteinof 42 kD, which binds to the C-rich sequcoco located 20 nt downstream of the stop colon in the vt 10) mRNA. This blndlnZ activity is present in the cytoplasmic extracts of activated, but not quiescent stellate cells. Competition experiments showed that the protein hinds RNA in a sequence specific manner, although it can be competed with an excess of poly-C homopolymer.This RNA binding protein is clearly different from a protein of similar size that has been impfiented in maintainingthe high stability of a globin mRNA. ColLagena 10) mRNA is highly stable in activated stellatecells with minimal decay after 24h and probably with a half-lif~ of about 48h. It is possible that the protein bindingto the 3' region stabilizes the collagen c~ 1 0) mRNA in activated stellale cells.In summary, posRrsnseriptioanlregulation plays an important role in control of collagen a 1 0) mRNA levels in stellate cells. This regulation may be attained in two ways; by negatively regulating the mRNA in quiescent cells and by stabilizingthe m R N A in activatedcells.