- Email: [email protected]
281 The contribution of the bone marrow to liver regeneration depends upon the endogenous hepatocytes replication potential
HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003
AASLD ABSTRACTS
280 A NOVEL LIVER SPECIFIC DEVELOPMENTALLY REGULATED SERINE PROTEASE/CONVERTASE. Krassimira V
Hadjiolova, Petko M Petkov, ; Mariana D Dabeva, Albert~ instein College of Medicine, Bronx, NY Using the SSH technology, we have identified previously 643 induced clones in 13 day fetal compared to adult liver; 202 of these clones were i n d e p e n d e n t transcripts, 64 of them represented ESTs and4 appeared to be new sequences (Petkov et al., Genomics 68: 197-209, 2000). Applying the rapid amplification of 5' and 3' cDNA ends to subtracted genes (GeneRace Kit, Invitrogen), we have obtained the full-length cDNAs for two of the subtracted clones. One of t h e m (clone 2G8) encodes a novel putative serine protease/ subtilase highly expressed in fetal liver and comparatively low in adult. The expression of 2G8 mRNA was analyzed by Northern blots with RNA isolated from different rat tissues: fetal and adult liver, isolated 14 day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. On Northern blot with total RNA, a strong signal was detected with fetal liver/ hepatoblasts RNA and a weak signal with adult liver RNA, which showed that the expression of this mRNA is developmentally regulated. To confirm the liver specific expression of 2G8, quantitative RT-PCR was carried out. The results of this analysis confirmed our previous results and revealed also a very low expression of 2G8 mRNA in the colon. The 2G8 cDNA is 3345 nucleotides in length and codes for a novel protein of 691 amino acids. We were able to identify in the data bases the sequences of a rat contig which contains the complete gene for 2G8. The gene coding for 2G8 comprises 21,920 bp of the rat genome and includes 12 exons and a long 3'-UTR segment of 1,000 nucleotides. The initiation of the translation begins with an AUG codon, 188 nucleotides downstream from the 5'-end of mRNA. Using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the PCR4 TOPO vector (Invitrogen). These two upstream and downstream clones were ligated to the 2G8 cDNA upstream and downstream sequences, respectively, so that the whole gene with the 5' and 3' regulatory sequences was obtained in the PCR4-TOPO vector. Analysis of the structure of 2G8 showed that this protein resembles 8 others mammalian subtilases, n a m e d convertases described during the last decade: furin, PC1/3, PC2, PC4, PACE4, PC5/6 and PC7/LPC/PC8 and SKI-I/SIP. These proteases are synthesized as proproteins and secreted from the cell. 2G8 contains a putative signal sequence for release from the ER located between amino acid 30 and 31 (alanine $ glutamine). However, it does not share the consensus cleavage sequences characteristic for the other convertases: (K,R)-(X)n-(K,R) for processing, release and secretion of the active enzyme form. At present, we do not know the processing site of the proprotein. In situ hybridization experiments showed that this serine protease is expressed in fetal liver hep atoblasts. Convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. They show tissue specificity and some are implicated in development and differentiation. 2G8 is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factors/cytokines or extracellular matrix proteins implicated in morphogenesis. Further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. Supported by NIH Grant R01 DK59321. Disclosures: Mariana D Dabeva - No relationships to disclose Krassimira V. Hadjiolova, Petko M. Petkov - No relationships to disclose
291A
281
THE CONTRIBUTION OF THE BONE MARROW TO LIVER REGENERATION DEPENDS UPON THE ENDOGENOUS HEPATOCYTES REPLICATION POTENTIAL. Pamela Vig,
Howard C Thomas, Malcolm R Alison, Stuart J Forbes, Imperial College, London, UK INTRODUCTION/AIMS: Bone marrow ceils (BMCs) can contribute to regeneration of the chronically damaged liver but in h u m a n studies and animal models the magnitude of this axis is highly variable. In a murine model of hepatitis B we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. METHODS: 2 month old female mice transgenic for Hepatitis B surface antigen (HBs-tg) received lethal irradiation and were then transplanted with C57BI/6J male BMCs by tail vein injection. 6 weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. Mice were sacrificed at 3 and 6 months following retrorsine injections. Y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins 8/18, cytochrome P450 and glycogen, negative for CD45). RESULTS: In the control mice with chronic liver damage there was an increase in Y chromosome positive hepatocytes over time, but the proportion remained <5% of the total number of hepatocytes. However, 19.3% (corrected count) of Y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. CONCLUSIONS: BMCs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. However, w h e n chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. Disclosures: Malcolm R Alison - No relationships to disclose Stuart J Forbes - No relationships to disclose Howard C Thomas - No relationships to disclose Pamela Vig - No relationships to disclose
AASLD ABSTRACTS
280 A NOVEL LIVER SPECIFIC DEVELOPMENTALLY REGULATED SERINE PROTEASE/CONVERTASE. Krassimira V
Hadjiolova, Petko M Petkov, ; Mariana D Dabeva, Albert~ instein College of Medicine, Bronx, NY Using the SSH technology, we have identified previously 643 induced clones in 13 day fetal compared to adult liver; 202 of these clones were i n d e p e n d e n t transcripts, 64 of them represented ESTs and4 appeared to be new sequences (Petkov et al., Genomics 68: 197-209, 2000). Applying the rapid amplification of 5' and 3' cDNA ends to subtracted genes (GeneRace Kit, Invitrogen), we have obtained the full-length cDNAs for two of the subtracted clones. One of t h e m (clone 2G8) encodes a novel putative serine protease/ subtilase highly expressed in fetal liver and comparatively low in adult. The expression of 2G8 mRNA was analyzed by Northern blots with RNA isolated from different rat tissues: fetal and adult liver, isolated 14 day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. On Northern blot with total RNA, a strong signal was detected with fetal liver/ hepatoblasts RNA and a weak signal with adult liver RNA, which showed that the expression of this mRNA is developmentally regulated. To confirm the liver specific expression of 2G8, quantitative RT-PCR was carried out. The results of this analysis confirmed our previous results and revealed also a very low expression of 2G8 mRNA in the colon. The 2G8 cDNA is 3345 nucleotides in length and codes for a novel protein of 691 amino acids. We were able to identify in the data bases the sequences of a rat contig which contains the complete gene for 2G8. The gene coding for 2G8 comprises 21,920 bp of the rat genome and includes 12 exons and a long 3'-UTR segment of 1,000 nucleotides. The initiation of the translation begins with an AUG codon, 188 nucleotides downstream from the 5'-end of mRNA. Using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the PCR4 TOPO vector (Invitrogen). These two upstream and downstream clones were ligated to the 2G8 cDNA upstream and downstream sequences, respectively, so that the whole gene with the 5' and 3' regulatory sequences was obtained in the PCR4-TOPO vector. Analysis of the structure of 2G8 showed that this protein resembles 8 others mammalian subtilases, n a m e d convertases described during the last decade: furin, PC1/3, PC2, PC4, PACE4, PC5/6 and PC7/LPC/PC8 and SKI-I/SIP. These proteases are synthesized as proproteins and secreted from the cell. 2G8 contains a putative signal sequence for release from the ER located between amino acid 30 and 31 (alanine $ glutamine). However, it does not share the consensus cleavage sequences characteristic for the other convertases: (K,R)-(X)n-(K,R) for processing, release and secretion of the active enzyme form. At present, we do not know the processing site of the proprotein. In situ hybridization experiments showed that this serine protease is expressed in fetal liver hep atoblasts. Convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. They show tissue specificity and some are implicated in development and differentiation. 2G8 is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factors/cytokines or extracellular matrix proteins implicated in morphogenesis. Further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. Supported by NIH Grant R01 DK59321. Disclosures: Mariana D Dabeva - No relationships to disclose Krassimira V. Hadjiolova, Petko M. Petkov - No relationships to disclose
291A
281
THE CONTRIBUTION OF THE BONE MARROW TO LIVER REGENERATION DEPENDS UPON THE ENDOGENOUS HEPATOCYTES REPLICATION POTENTIAL. Pamela Vig,
Howard C Thomas, Malcolm R Alison, Stuart J Forbes, Imperial College, London, UK INTRODUCTION/AIMS: Bone marrow ceils (BMCs) can contribute to regeneration of the chronically damaged liver but in h u m a n studies and animal models the magnitude of this axis is highly variable. In a murine model of hepatitis B we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. METHODS: 2 month old female mice transgenic for Hepatitis B surface antigen (HBs-tg) received lethal irradiation and were then transplanted with C57BI/6J male BMCs by tail vein injection. 6 weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. Mice were sacrificed at 3 and 6 months following retrorsine injections. Y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins 8/18, cytochrome P450 and glycogen, negative for CD45). RESULTS: In the control mice with chronic liver damage there was an increase in Y chromosome positive hepatocytes over time, but the proportion remained <5% of the total number of hepatocytes. However, 19.3% (corrected count) of Y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. CONCLUSIONS: BMCs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. However, w h e n chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. Disclosures: Malcolm R Alison - No relationships to disclose Stuart J Forbes - No relationships to disclose Howard C Thomas - No relationships to disclose Pamela Vig - No relationships to disclose