Specific Expression and Regulation of Hepassocin in the Liver and Down-regulation of the Correlation of HNF1α with Decreased Levels of Hepassocin in Human Hepatocellular Carcinoma

Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1α (hepatocyte nuclear factor-1α) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1α. An electrophoretic mobility shift assay demonstrated that HNF1α interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1α interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1α expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1α in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1α expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1α are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1α causes, at least in part, the transcriptional down-regulation of HPS in HCC. Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1α (hepatocyte nuclear factor-1α) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1α. An electrophoretic mobility shift assay demonstrated that HNF1α interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1α interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1α expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1α in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1α expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1α are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1α causes, at least in part, the transcriptional down-regulation of HPS in HCC. Hepassocin (HPS), 3The abbreviations used are: HPS, hepassocin; HCC, heptocellular carcinoma; HGF, hepatocyte growth factor; EGF, epidermal growth factor; TNF, tumor necrosis factor; BSA, bovine serum albumin; RACE, rapid amplification of cDNA ends; siRNA, small interfering RNA; PHx, partial hepatectomy; TNF, tumor necrosis factor; BSA, bovine serum albumin; IL, interleukin; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 3The abbreviations used are: HPS, hepassocin; HCC, heptocellular carcinoma; HGF, hepatocyte growth factor; EGF, epidermal growth factor; TNF, tumor necrosis factor; BSA, bovine serum albumin; RACE, rapid amplification of cDNA ends; siRNA, small interfering RNA; PHx, partial hepatectomy; TNF, tumor necrosis factor; BSA, bovine serum albumin; IL, interleukin; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. also called HFREP-1 (hepatocyte-derived fibrinogen-related protein) and fibrinogen-like 1, is a liver-specific gene and belongs to the fibrinogen superfamily, the members of which share a common fibrinogen-like domain at their carboxyl termini (1Hara H. Uchida S. Yoshimura H. Aoki M. Toyoda Y. Sakai Y. Morimoto S. Fukamachi H. Shiokawa K. Hanada K. Biochim. Biophys. Acta. 2000; 1492: 31-44Crossref PubMed Scopus (39) Google Scholar). HPS is translated into a mature 312-amino acid protein after cleavage of a hydrophobic secretion signal. The position within the human genome is mapped to chromosome 8p22–21.3. It has been reported that the expression of HPS mRNA was detected mainly in murine livers. In situ hybridization studies revealed its presence in parenchymal hepatocytes but not in endothelial cells (1Hara H. Uchida S. Yoshimura H. Aoki M. Toyoda Y. Sakai Y. Morimoto S. Fukamachi H. Shiokawa K. Hanada K. Biochim. Biophys. Acta. 2000; 1492: 31-44Crossref PubMed Scopus (39) Google Scholar). Subsequent studies of human adult tissues demonstrated that HPS mRNA was strongly expressed in adult livers, fairly strongly in fetal livers, and weakly in pancreases but not in other tissues (2Hara H. Yoshimura H. Uchida S. Toyoda Y. Aoki M. Sakai Y. Morimoto S. Shiokawa K. Biochim. Biophys. Acta. 2001; 1520: 45-53Crossref PubMed Scopus (45) Google Scholar). HPS was induced 2 h after a 70% hepatectomy of mouse livers, and the second peak arrived 24 h later. The expression of HPS remained high until 72 h later and declined to the basal level thereafter (3Yan J. Ying H. Gu F. He J. Li Y.L. Liu H.M. Xu Y.H. Cell Res. 2002; 12: 353-361Crossref PubMed Scopus (19) Google Scholar), suggesting that HPS may function as a regulator of cell growth in liver regeneration. Functionally, HPS was initially described as generating mitogenic activity on isolated hepatocytes, whereas it did not promote DNA synthesis in non-liver cell lines in vitro. Further studies revealed expression patterns inconsistent with a tumor suppressor (4Yan J. Yu Y. Wang N. Chang Y. Ying H. Liu W. He J. Li S. Jiang W. Li Y. Liu H. Wang H. Xu Y. Oncogene. 2004; 23: 1939-1949Crossref PubMed Scopus (44) Google Scholar). Expression of the HPS/LFIRE-1 (liver fibrinogen-related gene-1) was frequently down-regulated or lost in HCC at both mRNA and protein levels, compared with their adjacent normal liver tissues, and the expression level was found to be strongly associated with the tumors' differentiation statuses (4Yan J. Yu Y. Wang N. Chang Y. Ying H. Liu W. He J. Li S. Jiang W. Li Y. Liu H. Wang H. Xu Y. Oncogene. 2004; 23: 1939-1949Crossref PubMed Scopus (44) Google Scholar). Exogenous HPS expression in human HCC cells inhibited their anchorage-dependent or -independent growth in vitro, and down-regulation of HPS by an antisense approach enhances cancer cell proliferation and colony formation in soft agar (4Yan J. Yu Y. Wang N. Chang Y. Ying H. Liu W. He J. Li S. Jiang W. Li Y. Liu H. Wang H. Xu Y. Oncogene. 2004; 23: 1939-1949Crossref PubMed Scopus (44) Google Scholar). Taken together, these results suggest that HPS plays an important role in the liver's development and physiological function and is associated with the progression of liver tumors. It was recently suggested that HPS in plasma almost completely binds to the fibrin matrix during clot formation and is strongly associated with fibrin and possibly fibrinogen (5Rijken D.C. Dirkx S.P. Luider T.M. Leebeek F.W. Biochem. Biophys. Res. Commun. 2006; 350: 191-194Crossref PubMed Scopus (30) Google Scholar). Additionally, IL-6 could increase HPS expression in HepG2 hepatoma cells in a dose-dependent manner, indicating that HPS may be an acute phase Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). of gene expression in HCC a of studies loss of HPS/LFIRE-1 on chromosome in of of HCC the loss of (4Yan J. Yu Y. Wang N. Chang Y. Ying H. Liu W. He J. Li S. Jiang W. Li Y. Liu H. Wang H. Xu Y. Oncogene. 2004; 23: 1939-1949Crossref PubMed Scopus (44) Google Scholar). expression of is on the presence of cis-acting in their promoter and that with nuclear transcription that or transcriptional we that the down-regulation of HPS in HCC may at the transcriptional reduction in HPS gene transcription could be by in the or function of nuclear transcription In this study, we described and of the that the liver-specific regulation of the HPS results showed that HNF1α (hepatocyte nuclear factor-1α) was for expression of this and down-regulation or loss of transcription HNF1α at least in part, the transcriptional down-regulation of HPS in HCC. In we that HPS promoter activity was induced by of cells with a of cell HepG2 and human cell were hepatocyte cell and cell lines and were the of the of These were at in in and with and fetal bovine serum and at were with and in HCC were The tumor were at until RNA tumor and adjacent tissues were at and were by was using human liver cDNA and in the of and were as 2 of at by of for for of for for of for for and a at 72 for The second was with to of HPS and The were as 2 of at by at for for 72 for and a at 72 for were into and The on the was used to for the within HPS The was at The to to the transcription site of HPS was by using and The was with and and into a luciferase in The with of the promoter were by using as the and the in were and The the of the to the transcription used to of in a with a in the site to the transcription and sites and to the transcription in HPS were using the as described by the The and were used to the HNF1 The STAT3 of both was by using the following The DNA of were by The transcriptional HNF1α was by using and antisense the human liver cDNA The were with and and into the In in luciferase cells were into and with as 24 h cells were and in of were with of using the luciferase assay in a were expressed as induction to The results the of at least in luciferase activity was used as an for In in vivo luciferase were a normal with to and were into the mouse liver using the In by mouse to the In were with in of for at The was to of and for and the were to the at a for The were 24 h after luciferase were and and in of at the was for at of was with of luciferase assay and the was in a luciferase activity was used as an for The results the of at least were in and of the sites in the were were HepG2 and mouse as described M. Wang J. Li W. Li Xu Res. PubMed Scopus (30) Google Scholar). The the of which to HNF1 in the promoter of HPS in the and the which compared with the were with at the were using a of which was to the to the of the was using an The electrophoretic mobility shift assay was used to electrophoretic mobility shift assays. In the was to the and were on a in at and to a with and with the The was to HepG2 cells were with cell on for at for at in nuclear to and at for at The was as the cell were with HNF1α HMGB1 or of cell were with in of with protein as an were with of and in DNA were isolated and using was using and the following of and of were on The of HPS expression in human HCC tissues was using RNA in situ hybridization and a human HCC The was by at the and the sites by are in were with for and with for at were with in for at in and were at for h and with at for was by in of by in were with and using RNA was with and transcription was using a cDNA synthesis to the The cDNA was using to the the In was using the and The were as for for at for at and for at 72 was after the of a was by the to to was in The of HPS and HNF1α mRNA was using the and to that of The following were HNF1 HNF1α were as and HepG2 cells were in to and HNF1α were into HepG2 cells at with to the The RNA were used in were after h of and and were to cells were with of and and at a of of protein was the by were and with at the following HNF1α HPS and was using to the were by the of were by the of the of to the in of on for the and of of the were partial hepatectomy were to with 70% liver to and 12: Scholar). The of were the was and a was the the and the was F. M. J. PubMed Scopus Google Scholar). a small was in the of the The was the and were with and the was M. J. 2006; PubMed Scopus Google H. N. J. 2002; PubMed Scopus Google Scholar). the was and were at the after cells were with the following IL-6 endothelial growth or for 24 the of luciferase activity was as described and IL-6 cells were to and the was with of IL-6 with or 24 h luciferase activity was to HPS promoter was to the endogenous HPS mRNA with as the and was to the endogenous HPS protein level with as the were using and results are expressed as of was was used to The was used to the were used to the and RNA in situ hybridization HNF1α in of HPS for human HPS of by The site is within which is the transcription was using an in to RNA human liver and of these revealed DNA 2 and were in of of the HPS The into this site as was the cDNA HPS in the the the of of the transcriptional 1, and a of 1, were into a were with the human hepatocyte cell cells and human hepatoma cell HepG2 and this promoter showed transcriptional activity in both cells Furthermore, promoter were following of in by into The of this promoter activity in vivo F. Liu PubMed Scopus Google 2006; PubMed Scopus Google M. J. 2004; PubMed Scopus Google Scholar). 24 h after the promoter activity of in the liver was about for with significant was detected in other the and we the promoter by a of and a of 1, into of the luciferase in and showed significant promoter to about of the to resulted in an in promoter and of an or as found in led to an about reduction in promoter suggested that was a regulator of HPS expression within the to of the HPS promoter. for within this promoter and in the and this revealed binding sites for transcription and HNF1α to the and which the and HNF1α binding were and activity was in vivo as in in to was significant in Deletion of in to to the basal level and to indicating that this high level of was also with the promoter the to However, of a in to of activity compared with indicating that the promoter to possibly the was critical to the regulation of HPS promoter activity in vivo. of the HPS gene promoter revealed that the HNF1 element was and and to the of the HNF1 binding sites to HPS promoter or of the and were into mouse livers. In a the promoter activity of was to the basal of HNF1α with led to a of to the basal level and to the level with However, of the basal level of promoter activity the of expressed HNF1α showed a significant reduction of the HNF1 binding site Moreover, of the HNF1 binding site completely abolished the induction by of HNF1α. These strongly the that HNF1α was important to the expression of HPS in vivo. Additionally, we also the HNF1α of the promoter in cell in HNF1α promoter activity to the basal level and to the level with in the of cells with the HNF1α expression did not in a induction of the activity of the HPS promoter. may be by the that other M. F. J. 2001; PubMed Scopus Google with HNF1 were also in the regulation of human HPS expression. the binding of HNF1α to this element by an electrophoretic mobility shift assay using nuclear the human cell HepG2 of a were with nuclear HepG2 cells with the but not with the HNF1 The nuclear binding was to HPS, been by an of but not by an of The HepG2 nuclear with the was identified by with HNF1α binding of HNF1α to the in nuclear HepG2 cells with HNF1α in to the of cells with HNF1α into HepG2 cells the of HNF1α mRNA by and of HNF1α protein by The formation of nuclear cells and as These results that the HNF1α binding activity at the HNF1 site in the HPS promoter was to the down-regulation of HNF1α. nuclear the mouse liver a with the binding was by for binding by an of was to the of HNF1α in the In vivo of expressed HNF1α with the HPS promoter was by of in HepG2 in amplification of the the HNF1 site at using HNF1α as the a of the all which the of HNF1α with its in vivo. Moreover, a amplification was to that as M. F. J. 2001; PubMed Scopus Google and HMGB1 M. Wang J. Li W. Li Xu Res. PubMed Scopus (30) Google Scholar), interacted with HNF1α and were in liver-specific gene expression we using the or using or as both the HNF1 and suggested that and HMGB1 to be recruited by HNF1α and in HPS transcription by with HNF1α. was to the protein by HNF1α HMGB1 or HPS in HCC HPS mRNA expression in a of HCC using of HPS mRNA expression was in HCC were and with a of The and RNA in situ hybridization results of the HCC tumor are in the HCC the of HPS was the with or cells was with or and showed or However, the of HPS in HCC was not significantly correlated with and in HCC of in to at in a HNF1α Expression in HCC of we the regulation of HPS transcription by we to HNF1α was in the for the down-regulation of HPS expression in human HCC HNF1α and HPS mRNA showed expression patterns in HCC cell lines and the expression of HPS and HNF1α were by in of HNF1α mRNA expression were found in all HCC cell lines compared with human hepatocytes. of HNF1α mRNA expression in the tumor of HCC revealed a significant reduction of HNF1α expression in as compared with liver Moreover, HPS was found down-regulated in all HCC cell lines and HCC and reduction of HNF1α expression correlated significantly with the down-regulation of HPS expression in HCC tissues of HNF1α expression the transcriptional activity of the HPS of HNF1α were with a to the HPS promoter of the HNF1α expression with resulted in an about increase of promoter activity the basal level and in a the level with Further of the HNF1α expression led to a dose-dependent increase of the HPS promoter activity compared with the that HNF1α may as an of HPS gene and RNA were HepG2 cells were with and compared with cells with of HNF1α of HPS mRNA expression endogenous HNF1α expression was by siRNA, HPS mRNA was and protein was In with only the level of HNF1α protein of and on of HPS studies described HPS may function as a regulator of cell growth in liver (3Yan J. Ying H. Gu F. He J. Li Y.L. Liu H.M. Xu Y.H. Cell Res. 2002; 12: 353-361Crossref PubMed Scopus (19) Google Scholar). the of of expression in liver promoter were into mouse acute liver was induced by 70% liver and luciferase were 24 h later. in at 24 h after a 70% was found in whereas the promoter activity of and about the to about the basal In significant increase was in the compared with the normal However, did not increase the promoter activity of HPS promoter compared with the indicating that the promoter and was critical for the of HPS promoter activity in liver regeneration. In did not the promoter activity of with the suggesting that the site was in HPS by Moreover, we identified the of HNF1α expression following which may be in of HPS expression in liver regeneration. the of on HPS and were and the promoter activity of in liver was did not after PHx, the promoter activity of by about the and by the basal whereas and led to in promoter activity the normal and the suggested that the of HPS expression in may be an important role in liver and after by on cell proliferation and J. PubMed Scopus Google Scholar). the of HGF, EGF, endothelial growth and known to be during liver and HPS following serum were with the and with or for 24 and activity was in HPS promoter activity induced to in cells by of HGF, EGF, and The induction by was was detected with the that IL-6 induced a promoter activity increase of and arrived at about activity the basal the induction of HPS expression by endogenous HPS mRNA and protein in cells were in HPS mRNA expression was in a dose-dependent with with IL-6 resulted in a increase in HPS mRNA the were at IL-6 induced HPS mRNA expression that was the basal the of HPS transcription led to protein level with IL-6 as in we the of the IL-6 in HPS expression. could the of the hepatocyte IL-6 it is that the of IL-6 Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). in during with IL-6 the promoter activity of was induced compared with the cells with BSA, to the basal Moreover, using both IL-6 and a increase in HPS promoter activity compared with to whereas with led to a increase of HPS promoter activity not These results that the induction of HPS IL-6 also correlated with IL-6 the HPS gene promoter that was for promoter of the promoter were used in in cells in the or presence of IL-6 The did not to IL-6 The promoter activity of and to about in cells with IL-6 the basal level and to the cells with BSA, and the IL-6 in Moreover, of the HNF1 binding site resulted in the promoter not only the IL-6 but also the promoter These results suggested that to of the HPS promoter were for promoter activity of this gene and that the site an important role in the basal and promoter activity of this sites were in the and of the HPS promoter. studies revealed that STAT3 of 2001; PubMed Scopus Google Scholar). we the of sites the promoter activity of the HPS gene and which site was critical in the to IL-6 with sites in cells and promoter activity in the presence or of of these showed that the promoter with of the the of the second and of these showed significant in promoter However, the site did not the transcription activity of the The results of this suggested that sites a role in promoter activity of In this study, the promoter of the human HPS gene was and were and used to important in the DNA that could the liver-specific expression of this identified a binding site for HNF1 at position to of the transcription site of human HPS and demonstrated that the HNF1 binding site was for HPS promoter activity in vivo. Additionally, we the binding of endogenous HNF1α to the HPS promoter in by an electrophoretic mobility shift assay and in vivo by strongly the that HNF1α was important to the in vivo expression of of HNF1α in mouse could activity of the HPS and HPS promoter activity was by expression of HNF1α in human cell endogenous HNF1α expression by resulted in significant of HPS expression. These results to the that HNF1 binding sites in the HPS promoter expression of HNF1α protein is the regulator of liver-specific gene expression. binding sites been found in of as and the protein S. J. Scopus Google S. M. M. PubMed Scopus Google PubMed Scopus Google PubMed Scopus Google K. K. K. PubMed Scopus Google Wang PubMed Scopus Google Scholar). these sites are in promoter and with binding sites of other transcription However, it also has been that a liver-specific promoter be with only an HNF1 site M. M. M. H. PubMed Google Scholar). 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In we to the binding of to the HPS promoter in vivo. also that protein, was recruited to the HPS promoter by HNF1α. HNF1α HPS promoter of the of activity to its revealed that was a element in the HPS promoter at position to the transcription site of human binding sites for transcription were identified in this as and The of these transcription binding sites in HPS transcription are not studies found that HPS was down-regulated or lost in HCC (4Yan J. Yu Y. Wang N. Chang Y. Ying H. Liu W. He J. Li S. Jiang W. Li Y. Liu H. Wang H. Xu Y. Oncogene. 2004; 23: 1939-1949Crossref PubMed Scopus (44) Google Scholar). of HPS expression in HCC cell lines and tissues was in with and we also demonstrated that was a the expression of HPS and the expression of HNF1α. The critical role of HNF1α and the HNF1 binding site in HPS gene expression to that HPS gene transcription in HCC was HNF1α. of HPS gene is by that HCC the and binding activity of HNF1α. may the expression of other in HCC. the tumor suppressor activity and are in HCC, which with expression of HNF1α M. J. J. PubMed Scopus Google H. H. Res. Google S. S. PubMed Scopus Google Scholar). significant of HNF1α in hepatoma lines Y. Wang W. H. K. H. J. Scopus Google Scholar). and of gene expression is by a in HNF1α expression H.M. F. J. 2002; Scopus Google Scholar). Moreover, a tumor suppressor function for HNF1α is by the in mouse M. J. M. M. PubMed Scopus Google Scholar). Furthermore, the growth suppressor activity of HPS in HCC also has been a the in which HNF1α expression in HCC HPS liver-specific which are known to be by and and and protein, been reported to transcriptional by IL-6 during the acute phase is to and activity and Biochem. Biophys. Res. Commun. PubMed Scopus Google 2001; PubMed Scopus Google Scholar). In of we found that partial hepatectomy and IL-6 significantly induced HPS promoter and the induction was on IL-6 for the on the by IL-6 with of the HPS promoter revealed results showed that both of these sites were in induced promoter activity of HPS by IL-6 as was in the promoter with the of the HNF1 binding site in the presence or of It is known that a a transcription and DNA binding element are in the promoter of liver-specific may with induced transcription in to 2001; PubMed Scopus Google Scholar). we that HNF1α may with the to transcriptional of the HPS promoter to during of growth and the and 2001; PubMed Scopus Google Scholar). In we that the transcription HNF1α plays an important role in HPS promoter activity in vivo and demonstrate that down-regulation or loss of HNF1α causes, at least in part, the transcriptional down-regulation of HPS in HCC. liver induction of HPS mRNA expression at the transcriptional to of critical of HPS gene expression as