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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

2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês

10.1074/jbc.m806393200

1083-351X

Haitao Yu, Miao Yu, Chang‐Yan Li, Yi‐Qun Zhan, Wang‐Xiang Xu, Yonghui Li, Wei Li, Zhidong Wang, Chang‐Hui Ge, Xiaoming Yang,

Ion Transport and Channel Regulation

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 reactant (6Liu Z. Ukomadu C. Biochem. Biophys. Res. Commun. 2008; 365: 729-734Crossref PubMed Scopus (54) Google Scholar). Down-regulation of gene expression in HCC can occur via a variety of mechanisms. Previous studies have implicated allelic loss of HPS/LFIRE-1 on chromosome 8p22 in 57.1% (24 of 42) of HCC specimens through the loss of heterozygosity analysis (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). Because tissue-specific expression of genes is based on the presence of cis-acting sequences in their promoter and enhancer regions that interact with sequence-specific nuclear transcription factors that potentate or depress transcriptional initiation, we hypothesized that the down-regulation of HPS levels in HCC may occur at the transcriptional level. This reduction in HPS gene transcription could be caused by alterations in the quantity or function of regulatory nuclear transcription factors. In this study, we described identification and characterization of the regulatory elements that provided the liver-specific regulation of the HPS gene. Our results showed that liver-enriched HNF1α (hepatocyte nuclear factor-1α) was needed for appropriate expression of this gene, and down-regulation or loss of transcription factor HNF1α caused, at least in part, the transcriptional down-regulation of HPS in HCC. In addition, we have determined that HPS promoter activity was induced by pretreatment of cells with a variety of cellular mediators. Human hepatocarcinoma cell line HepG2 and human kidney cell line HEK293 were purchased from American Type Culture Collection (Manassas, VA). Human hepatocyte cell line L02, and hepatocarcinoma cell lines HL7702, SMMC-7721, and BEL-7402 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These were grown at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) and supplemented with penicillin (100 units/ml), streptomycin (10 μg/ml), and 10% fetal bovine serum (MDgenics Inc) and split 1:2 at confluences. Cells were detached via incubation with 0.05% trypsin and 0.04% EDTA in phosphate-buffered saline. All HCC specimens were obtained from patients who underwent surgical resection. The primary tumor specimens were immediately frozen at –80 °C until RNA extraction. Both tumor and adjacent nontumor tissues were sampled at 1 cm3, respectively, and were confirmed by pathological examination. 5′-RACE was performed using human liver Marathon-ready cDNA (BD Biosciences Clontech) and gene-specific primers anchored in the 3′-untranslated region of HPS: 5′-TGCATATCTGCTGTACTGAAAGC-3′ and adapter-specific primers. Touchdown PCR cycling conditions were as follows: 2 min of initial denaturation at 95 °C PCR followed by five cycles of 95 °C for 30 s, 70 °C for 2.5 min, 5 cycles of 95 °C for 30 s, 70 °C for 2.5 min, 25 cycles of 95 °C for 30 s, 68 °C for 2.5 min, and a final extension at 72 °C for 10 min. The second round PCR was carried out with nested primers corresponding to positions 625–647 bp of HPS cDNA: 5′-GCAAGGGAATCTCCAGCTGTTCC-3′ and adapter-specific primers. The cycling conditions were as follows: 2 min of initial denaturation at 95 °C PCR followed by 25 cycles at 95 °C for 30 s, 65 °C for 30s, 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. 5′-RACE products were cloned into pMD-18T simple vectors (TaKaRa Biotechnology Co., Ltd.) and sequenced. The TRANSFAC data base (available on the World Wide Web) was used to search for the potential cis-regulatory elements within HPS promoters. The threshold was set at 80.0. The –2304 to +135 bp region relative to the transcription start site of HPS (accession number NM_004467) was amplified by PCR using primers P-2304 (5′-CCGCTCGAGGATCTGGAATTAAACCTTCCC-3′) and P+135 (5′-CCCAAGCTTAGTTTTGGTTACTATCATACC-3′). The PCR product was digested with XhoI and HindIII and inserted into a promoterless luciferase reporter vector pGL3/Basic, resulting in plasmid pGL3/–2304. The deletion mutants with various lengths of the promoter regions were prepared by PCR using P+135 as the 3′-end primer and the 5′-primers shown in Table 1. They were named pGL3/–1654, pGL3/–1058, pGL3/–784, pGL3/–614, pGL3/–554, pGL3/–454, pGL3/–64, pGL3/+57, and pGL3/+117, respectively. The numbers behind sprit represent the boundaries of the inserts relative to the transcription start sites.TABLE 1Primers used to synthesize 5′-truncated constructs of HPSNameForward (5′-3′)P-16545′-CCGCTCGAGCCTGGCCATAATTTTGACCTT-3′P-10585′-CCGCTCGAGCAGTAGATAACAAAGAACAG-3′P-7845′-CCGCTCGAGGAATAATCCCAATATTTTTGCT-3′P-6145′-CCGCTCGAGTAATGGAGCTAACAAGTGCC-3′P-5545′-CCGCTCGAGAAGGAACAACTTGTCCAGAG-3′P-4545′-CCGCTCGAGCCTTGTTTTTTATAACTAGGA-3′P-645′-CCGCTCGAGTATTTCTAATCAAATAATGGA-3′P+575′-CCGCTCGAGAAGTTATTTAATGTCTCTGCA-3′P+1175′-GTATGATAGTAACCAAAACT-3′ Open table in a new tab Vectors with a deletion in the HNF1-binding site (–463/–457 bp relative to the transcription start site) and STAT3-binding sites (–665/–657 and –750/–743 bp relative to the transcription start site) in HPS promoters were introduced using the Takara MutanBest kit (Takara Biotechnology Co., Ltd.) as described by the manufacturer. The primers pGL3/–1058-HNF1-Del-F (5′-TGCCTTGTTTTTTATAACTAGG-3′) and pGL3/–1058-HNF1-Del-R (5′-AATTGCCAAGGTGAAATGGTC-3′) were used to amplify the HNF1 deletion mutant from pGL3/–1058. The STAT3 deletion mutant (deletion of both STAT3-binding sites) was amplified by PCR using the following primers: pGL3/-1058-STAT3-Del(–750/–743)-F, 5′-TCTGAGTGAATAAATAGGGA-3′; pGL3/–1058-STAT3-Del(–750/–743)-R, 5′-TACCAAAGTTTTGTTTTCTT-3′; pGL3/–1058-STAT3-Del(–665/–657)-F, 5′-AGAAACTGTTACACAATACG-3′; pGL3/–1058-STAT3-Del(–665/–657)-R, 5′-TCTGGGTTCATCATTTCTTC-3′. The DNA sequences of mutated vectors were confirmed by sequence analyses. The transcriptional factor HNF1α was generated by PCR using sense primer 5′-CCCAAGCTTATGGTTTCTAAACTGAGCCA-3′ and antisense primer 5′-GCTCTAGATACTGGGAGGAAGAGGCCA-3′ from the human liver cDNA library (BD Biosciences Clontech). The PCR products were digested with HindIII and XbaI and inserted into the pcDNA3.1/Myc-HisB vector (Invitrogen). In Vitro Luciferase Assays—For in vitro luciferase assays, cells were seeded into 24-well plates and transiently transfected with different reporter plasmids as indicators. 24 h later, cells were collected and lysed in 100 μl of 1× passive lysis buffer. Luciferase assays were carried out with 50 μl of lysate using the dual luciferase reporter assay system in a chemiluminescence analyzer (FB12 luminometer; Berthold Detection Systems). Luciferase activities were expressed as -fold induction relative to values obtained from control cells. The results represent the mean of at least three independent transfection experiments, each carried out in duplicate. Renilla luciferase activity was used as an internal control for transfection efficiency. In Vivo Luciferase Assays—For in vivo luciferase assays, mice were fed a normal diet with free access to food and water. Plasmids were transfected into the mouse liver using the TransIT In Vivo Gene Delivery System (Mirus, Madison, WI) by mouse tail vein injections according to the manufacturer's directions. In brief, 5.1-μl polymer solutions were incubated with 5–25 μgof plasmids in 200 μl of sterile water for 5 min at room temperature. The mixture was added to 1.9 ml of 1× delivery solution and incubated for 10–15 min, and the contents were delivered via syringe to the tail vein at a constant rate for 4–7 s. The mice were killed 24 h after injection. For luciferase determination, livers were removed and weighed (100 ± 3 mg) and homogenized in 1 ml of 1× passive lysis buffer at 4 °C; the lysate was centrifuged for 15 min at 13,000 rpm; 40 μl of supernatant was mixed with 40 μl of luciferase assay buffer (Promega, Madison, WI); and the chemiluminescence produced was measured in a luminometer (FB12 luminometer; Berthold Detection Systems). Renilla luciferase activity was used as an internal control for transfection efficiency. The results represent the mean of at least three independent transfection experiments; two individual mice were treated in every experiment, and three lobes of the liver, representing three different sites in the liver, were taken from each mouse. Nuclear extracts were prepared from HepG2 and mouse liver, as previously described (7Yu M. Wang J. Li W. Yuan Y.Z. Li C.Y. Qian X.H. Xu W.X. Zhan Y.Q. Yang X.M. Nucleic Acids Res. 2008; 36: 1209-1219Crossref PubMed Scopus (30) Google Scholar). The wild-type probe, 5′-CCTTGGCAATTATTAACCTGCC-3′, the underlined part of which corresponded to HNF1 recognition consensus sequenced in the promoter of HPS in the region –470/–457 bp, and the mutant probe, 5′-TCACCTTGGCAATTTGCCTT-3′, which deleted AA CC compared with the wild-type probe, were labeled with biotin at the 5′-end. Competition experiments were executed using a 100-fold excess of various unlabeled double-stranded DNA, which was added to the reaction mixture prior to the addition of the labeled probe. Supershifting was carried out using an anti-HNF1α antibody (catalog number sc-6547X; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The LightShift chemiluminescent electrophoretic mobility shift assay kit (catalog number 20148; Pierce) was used to perform electrophoretic mobility shift assays. In brief, the reaction system was first prepared according to the manufacturer's protocol, and then DNA-protein complexes were electrophoresed on a 4% polyacrylamide gel in 0.5× Tris borate/EDTA electrophoresis buffer at 100 V and then transferred to a positive nylon membrane, UV-cross-linked, probed with streptavidin-horseradish peroxidase conjugate, and incubated with the substrate. The membrane was then exposed to x-ray film. 107 HepG2 cells were lysed with cell lysis buffer (10 mm Tris-HCl, pH 8.0, 10 mm NaCl, 0.2% Nonidet P-40) on ice for 10 min, centrifuged at 2500 rpm for 5 min at 4 °C, resuspended in nuclear lysis buffer (50 mm Tris-HCl, pH 8.0, 10 mm EDTA, 1% SDS), vortexed three times, sonicated six times to shear chromatin, and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected as the whole cell extract. Protein-DNA immunocomplexes were immunoprecipitated with rabbit polyclonal antibodies against HNF1α (catalog number sc-8986; Santa Cruz Biotechnology), CBP (catalog number sc-22; Santa Cruz Biotechnology), HMGB1 (catalog number BC 003378; Protein Tech Group), or rabbit IgG (catalog number sc-2027; Santa Cruz Biotechnology). 100 μl of whole cell extract were incubated with antibodies in 900 μl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.0, 167 mm NaCl), with protein A/G-agrose beads as an adsorbent. Resins were washed with three kinds of buffers containing various detergents and NaCl in different concentrations. Following elution, DNA fragments were isolated and purified using phenol/chloroform. PCR was conducted using primers HPS-F(–503) (5′-CTTAGAGAGCAAATAAACTGACCAT-3′) and HPS-R(–309) (5′-TGAAGATTAAGTAAAAACGAAGTCC-3′) under the following conditions: 28 cycles, 52 °C, 5 units of Taq polymerase, and 25 pmol of each primer. Amplified products were analyzed on 1.5% Tris borate/EDTA-agrose gels. The detection of HPS expression in human HCC tissues was performed using RNA in situ hybridization and a tissue microarray containing 142 formalin-fixed, paraffin-embedded human HCC tissues. The probe was 5′-TGTCTGT(T)TTAGCCGTGTAGGGGCC(G)CTGTAGTATACACCATTC(A)GGTTTGC-3′, labeled by digoxigenin at the 3′-end, and the sites modified by L-leucyl-β-naphthylamide are indicated in parentheses. Paraffin-embedded tissue sections were first treated with 0.3% Triton X-100 for 5 min and then with 0.2 n HCl for 20 min at room temperature. Next, they were treated with proteinase K in phosphate-buffered saline for 10 min at 37 °C, dehydrated in ethanol, and dried. Sections were hybridized at 42 °C for 19 h and then incubated with alkaline phosphatase-conjugated digoxigenin antibody at 37 °C for 1 h. Unbound conjugate was removed by washing in two changes of buffer 1 (0.1 m Tris-HC1, 0.15 m sodium chloride, pH 7.5) followed by one wash in buffer 3 (0.1 m Tris-HC1, 0.1 m sodium chloride, 0.05 m magnesium chloride hexahydrate, pH 9.5). Sections were counterstained with neutral red and mounted using glycerol aqueous mounting mediums. Total RNA was extracted with RNAVzol reagent (Vigorous Biotechnology), and reverse transcription was applied using a Vigoscript first strand cDNA synthesis kit (Vigorous Biotechnology) according to the manufacturer's protocol. The cDNA was analyzed using real time PCR, according to the instructions from the kit. In brief, real time PCR was done using the Bio-Rad IQ™5 multicolor real time PCR detection system and SYBR Premix Ex Taq™ (2×) kit (TaKaRa Japan). The cycling conditions were as follows: 95 °C for 1 min, 40 cycles for 10 s at 95 °C, for 30 s at 55 °C, and for 30 s at 72 °C. SYBR Green fluorescence was measured after each elongation step. At the end of PCR, a melting curve analysis was performed by gradually increasing the temperature from 55 to 95 °C to determine purity. PCR was set up in triplicates. The relative quantification of HPS and HNF1α mRNA was analyzed using the comparative CT method and normalized to that of GAPDH. The following primers were used: HNF1 QP1, 5′-AGTGAGTCCGGGCTTCACAC-3′; HNF1QP2, 5′-TGAAGGTCTCGATGACGCTG-3′; HPSQP1, 5′-CTGGAGATTCCCTTGCGG-3′; HPSQP2, 5′-GTTTTAGCCGTGTAGGGG-3′; GAPDHQP1, 5′-TCAGTGGTGGACCTGACCTG-3′; GAPDHQP2, 5′-TGCTGTAGCCAAATTCGTTG-3′. HNF1α siRNAs were as follows: 5′-UGACAGCACUGCACAGCUUTT-3′ (sense strand) and 5′-AAGCUGUGCAGUGCUGUCATT-3′ (antisense strand). HepG2 cells were grown in 6-well plates to 50% confluence, and HNF1α siRNAs were transfected into HepG2 cells at 100 pmol/well with Vigofect reagent (Vigorous Biotechnology), according to the manufacturer's protocol. The nonspecific RNA duplexes (GenePharam, Shanghai, China) were used in control experiments. Cells were harvested after 48–72 h of incubation, and then real time PCR and Western blot were performed to detect interference effects. For Western blots, 106 cells were lysed with 30 μl of TNT buffer (20 mm Tris-HCl (pH 7.5), 200 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors leupeptin, aprotinin, pepstatin A, chymostatin, and antipain, each at a final concentration of 10 μg/ml). Then 20 μg of protein from each sample was loaded onto the gel. After separation by SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) and probed with various antibodies at the following concentrations: HNF1α (catalog number sc-8986; Santa Cruz Biotechnology), 1:1000; HPS (catalog number MAB1614; R&D Systems), 1:500; and β-actin (catalog number sc-47778; Santa Cruz Biotechnology), 1:1000. Chemiluminescent detection was conducted using Super-signal substrate (Pierce), according to the manufacturer's specifications. Male BALB/c mice (18–22 g body weight) were offered by the Beijing Institute of Radiation Medicine Animal Center (Beijing, China). All surgical procedures were approved by the Animal Care Committee of the Beijing Institute of Radiation Medicine. Animals received humane care according to the criteria outlined in Ref. 34Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research CouncilGuide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC1996: 21-55Google Scholar. All of the mice were pentobarbital-anesthetized (50 mg/kg) before surgery. For partial hepatectomy (PHx), mice were subjected to midventral laparotomy with 70% liver resection, according to Higgins and Anderson (8Higgins G.M. Anderson R.M. Arch. Pathol. 1931; 12: 186-202Google Scholar). The bellies of control animals were cut open without PHx. For nephrectomy, the left kidney was surgically exposed, and then a silk suture was securely tied around the hilus renalis containing the artery, vein, and ureter before the kidney was removed (9Zheng F. Plati A.R. Potier M. Striker G.E. Am. J. Pathol. 2003; 162: 1339-1348Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Sham animals underwent laparotomy without nephrectomy. For splenectomy, a small incision was made in the skin of the left flank. The peritoneum was opened, the splenic artery and vein were ligated with catgut, and the spleen was removed (10Huston J.M. Ochani M. Ballina M.R. Ulloa L. J. Exp. Med. 2006; 203: 1623-1628Crossref PubMed Scopus (487) Google Scholar, 11Wardemann H. Boehm T. Dear N. Carsetti R. J. Exp. Med. 2002; 195: 771-780Crossref PubMed Scopus (200) Google Scholar). For sham-operated mice, the peritoneum was opened and surgically closed. Animals were sacrificed at the indicated time points after surgery. L02 cells were treated with the following cytokines (R&D Systems, Inc. (Minneapolis, MN)): HGF (10 ng/ml), EGF (10 ng/ml), TNFα (10 ng/ml), IL-6 (10 ng/ml), vascular endothelial growth factor (10 ng/ml), or BSA (10 ng/ml) for 24 h. At the end of incubation, luciferase activity was determined as described under “Transfection and Luciferase Assays.” For IL-6 stimulation, L02 cells were grown to 80% confluence, and then the medium was added with various concentrations of IL-6 with or without 1 μm dexamethasone. 24 h later, luciferase activity was measured to examine HPS promoter activity, real time PCR was carried out to explore the endogenous HPS mRNA levels with GAPDH as the internal control, and Western blot was performed to determine the endogenous HPS protein level with β-actin as the internal control. Statistical analyses were performed using SPSS version 12.0 (SPSS, Chicago, IL). Statistical values and results are expressed as mean ± S.D. A p value of <0.05 was considered statistically significant. Student's unpaired t test was used to analyze comparisons between groups. The Spearman test was used to analyze the correlation between parameters. Two-sided Fisher's exact tests were used to analyze the statistical association between clinical-pathological and RNA in situ hybridization variables. HNF1α Is Involved in Tissue-specific Regulation of HPS Gene Expression—The genomic structure for human HPS consists of eight exons separated by seven introns. The translational start site (AUG) is located within exon 2, which is 9891 bp downstream from exon 1. To identify the transcription initiation site(s), 5′-RACE was performed using an HPS-specific primer anchored in exon 6 to amplify total RNA from human liver tissue. Cloning and sequencing of these PCR products revealed two totally distinct DNA sequences. Both sequences extended exon 2 upstream and were located in different regions of exon 1 of the HPS gene. The longer sequence (718 bp; Fig. 1A), extended 54 base pairs into exon 1. We have designated this start site as +1 (Fig. 1B). This sequence was 38 bp shorter than the previously published cDNA HPS sequence in the GenBank™ (accession number NM_004467). To functionally characterize the putative promoter, the 2.4-kb fragment, including 2304 bp of regions upstream of the transcriptional start site, exon 1, and a portion of intron 1, were cloned into a firefly luciferase-expressing vector (pGL3/–2304) (Fig. 1B). Transfection assays were carried out with the human hepatocyte cell line L02 cells and human hepatoma cell line HepG2 cells, and this promoter construct showed substantial transcriptional activity in both cells (Fig. 2A). Furthermore, promoter activities were measured following delivery of reporter vectors in mice by injection into tail veins. The use of this technology helped us study promoter activity in vivo (12Liu F. Song Y.K. Liu D. Gene Ther. 1999; 6: 1258-1266Crossref PubMed Scopus (1456) Google Scholar, 13Andre F.M. Cournil-Henrionnet C. Vernerey D. Opolon P. Min L.M. Gene Ther. 2006; 13: 1619-1627Crossref PubMed Scopus (42) Google Scholar, 14Simone Wattiaux De Coninck M.L. Wattiaux R. Jadot M. J. Gene Med. 2004; 6: 877-883Crossref PubMed Scopus (56) Google Scholar). 24 h after the injection, promoter activity of pGL3/–2304 in the liver was about 18-fold higher than for those transfected with pGL3/Basic plasmid; however, no significant difference was detected in other organs, including the heart, kidney, lungs, and spleen (Fig. 2B). Then we investigated the minimal promoter region by constructing a series of 5′-flanking region deletion fragments, including exon 1 and a portion of intron 1, inserted into plasmids upstream of the luciferase reporter. As shown in Fig. 2C, pGL3/–2304, pGL3/–1654, and pGL3/–1058 constructs showed significant promoter activities to about 18-fold more than pGL3/Basic vector alone. Sequential deletions of the 5′ region up to –454 bp resulted in an obvious decrease in promoter activity, and deletion of an additional 390 bp or more, as found in pGL3/–454, led to an about 15-fold reduction in promoter activity. This finding suggested that there was a strong positive regulator of HPS expression within the –1058 to –454 bp region of the HPS promoter. We searched for potential cis-regulatory element(s) within this 604-bp promoter fragment (between nucleotides –1058 and –454) in the TRANSFAC data base, and this revealed multiple putative binding sites for liver-enriched transcription factors, including HNF3β, CEBPα, and HNF1α (Fig. 2D). According to the search results, four fine deletion mutants (pGL3/–784, pGL3/–614, pGL3/–554, and pGL3/–454), which lacked the HNF3β, C/EBP, and HNF1α binding sites, respectively, were constructed, and reporter activity was analyzed in vivo as well. As shown in Fig. 2E, in contrast to pGL3/–1058, there was no significant difference in pGL3/–784 reporter activity. Deletion of 444 bp (resulting in –614 to +135 bp) substantially enhanced to 42.54-fold over the basal level and to 2-fold over pGL3/–1058, indic