Isolation and functional identification of a novel human hepatic growth factorhepatopoietin Cn

LIVER INJURY/REGENERATION Isolation and Functional Identi?cation of a Novel Human Hepatic Growth Factor:Hepatopoietin Cn Chun-Ping Cui,1*Da-Jin Zhang,1*Bing-Xing Shi,1Shao-Jun Du,1Dan-Li Wu,1Ping Wei,1Gen-Shen Zhong,1

Zi-Kuan Guo,1Yang Liu,2Li-Sheng Wang,1and Chu-Tse Wu1

Hepatic stimulating substance(HSS)was?rst isolated from weanling rat liver in1975and found

to stimulate hepatic DNA synthesis both in vitro and in vivo.Since then,mammalian and human

HSS have been investigated for their potential to treat hepatic diseases.However,the essential

nature in composition and structure of HSS remain puzzling because HSS has not been completely

puri?ed.Heating,ethanolprecipitation,andion-exchangechromatographieshadbeencarried out to

isolate the protein with speci?c stimulating activity from newborn calf liver,and[3H]thymidine

deoxyribose(TdR)/bromodeoxyuridine(BrdU)incorporation and carboxy?uorescein diacetate

succinimidyl ester(CFSE)-based proliferation assay to determine the bioactivity in vitro and in

vivo.We report the puri?cation of a novel30-kDa protein from a crude extract of calf liver HSS.

This protein is a member of the leucine-rich acidic nuclear protein family(LANP)and has been

namedhepatopoietinCn(HPPCn).Studiesofpartiallyhepatectomized(PH)miceshowthatlevels of

HPPCn messenger RNA(mRNA)increase after liver injury.Furthermore,the recombinant

human protein(rhHPPCn)was shown to stimulate hepatic DNA synthesis and activate signaling

pathways involved in hepatocyte proliferation in vitro and in vivo.Conclusion:HPPCn is a novel

hepatic growth factor that plays a role in liver regeneration.(H EPATOLOGY2008;47:986-995.)

T he mammalian liver is characterized by a unique property of responding to physical or chemical in-

juries by regenerating.Liver regeneration is a very complex process,requiring hundreds of substances.Studies using primary hepatocytes led to the identi?cation of several potential hepatocyte growth factors such as hepatocyte growth factor(HGF),transforming growth factor alpha,and transforming growth factor beta.Molecular studies of gene expression cascades showed that more than100immediate-early genes are up-regulated in the regenerating liver.1Spe-ci?c transcription factors and intracellular signaling pathways,such as activator of transcription3(Stat-3),extra-cellular signal regulated kinase(ERK),and sphingosine 1-phosphate/sphingosine kinase(S1P/SPK)2are rapidly ac-tivated in remnant hepatocytes a few minutes after partial hepatectomy(PH).However,it was dif?cult to demonstrate that any of these factors itself had an essential or preferential role in liver regeneration.3

Early studies indicated the existence of hepatic sub-stance capable of stimulating hepatocyte proliferation and liver regeneration.4Later,hepatic-stimulating substance (HSS),a crude extract isolated from rat liver5,6and from human fetal liver7,8was shown by[3H]thymidine deox-tribose(TdR)incorporation to increase synthesis of he-patic DNA in vivo and in vitro.However,the exact composition of HSS remained unknown.Another sub-stance puri?ed from rat liver HSS,augmenter of liver regeneration,was shown to protect the hepatocyte of Eck’s?stula from atrophy but had no effect on cultured hepatocytes and hepatic cell lines.9,10Therefore,we pro-posed that there must be other proteins with stimulatory activity in addition to augmentor of liver regeneration.

Abbreviations:BrdU,bromodeoxyuridine;cDNA,complementary DNA;CFSE, carboxy?uorescein diacetate succinimidyl ester;ERK,extracellular signal regulated kinase;HDGF,hepatoma-derived growth factor;HGF,hepatocyte growth factor; HMGB1,high mobility group box1protein;HPPCn,hepatopoietin Cn;HSS, hepatic stimulation substance;LANP,leucine-rich acidic nuclear protein;LRR, leucine-rich repeat;mRNA,messenger RNA;MS,mass spectrometry;PAGE,poly-acrylamide gel electrophoresis;PBS,phosphate-buffered saline;PCR,polymerase chain reaction;PH,partial hepatectomy;rhHPPCn,recombinant human hepato-poietin Cn;S1P/SPK,sphingosine1-phosphate/sphingosine kinase;SDS,sodium dodecyl sulfate;Stat-3,activator of transcription3;TdR,thymidine deoxyribose. TGF-?:transforming growth factor-?,TGF-?:transforming growth factor-?, From the1Department of Experimental Hematology,Beijing Institute of Radi-ation Medicine,People’s Republic of China;and the2Department of Pharmaceu-tical Engineering,School of Chemical Engineering and Technology,Tianjin University,Tianjin,People’s Republic of China.

Received May12,2007;accepted October29,2007. SupportedbygrantsfromtheNationalHigh-TechR&DProgram(No.2006AA02z182) and the National Basic Research Program of China(No.2004CB518801).

*These authors contributed equally to this study.

Address reprint requests to:Chu-Tse Wu,Professor,Department of Experimental Hematology,Beijing Institute of Radiation Medicine,27Taiping Road,Beijing 100850,People’s Republic of China.E-mail:wuct@85c3a6e0102de2bd97058806;fax:8610-68158311;or Li-Sheng Wang,Professor,Department of Experimental Hematol-ogy,Beijing Institute of Radiation Medicine,27Taiping Road,Beijing100850, People’s Republic of China.E-mail:wangls@nic.bmi.ac.ca;fax:8610-68214653. Copyright?2008by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience(85c3a6e0102de2bd97058806).

DOI10.1002/hep.22126

Potential con?ict of interest:Nothing to report.

986

A30-kDa protein,hepatopoietin Cn(HPPCn),was puri?ed from a crude extract of calf liver HSS.HPPCn was studied for its potential to stimulate DNA synthesis in vivo and in vitro and to determine its role in regeneration of hepatectomized liver.

Materials and Methods

Puri?cation and Sequencing of Calf HPPCn Protein

HSS was puri?ed from calf liver as described.5,6The product was puri?ed by heating,ethanol precipitation, diethylaminoethyl cellulose,and Source15Q(Amersham Biosciences,UK)ion-exchange chromatographies.

The partially puri?ed HSS was applied to nondenatur-ing polyacrylamide gel electrophoresis(PAGE)and stained with imidazole and zinc salts.11The resulting3 bands(approximate sizes44kDa,30kDa,and24kDa) were excised from the gel as previously described11and tested for growth-stimulating activity on hepatoma cell line SMMC7721with[3H]TdR DNA incorporation as-say(described later).The30-kDa band was found to have such activity and was further analyzed after manual exci-sion and digestion in gel with trypsin(Roche,Switzer-land)using the Investigator Progest automated digestor (Genomic Solutions,Ann Arbor,MI).The digested sam-ple was analyzed by nanoLC-MS/MS using a CapLC liq-uid chromatography(Waters Corp.,Milford,MA) coupled with a Q-TOF Ultima hybrid quadruple time-of-?ight mass spectrometry(MS)(Waters Corp.).The MS/MS fragment ion spectra were searched against the PIR and Swiss-Prot databases using the MASCOT data-base search engine(Matrix Science,UK).

Molecular Cloning and Sequence Analysis of Human HPPCn

Human HPPCn fragment was ampli?ed by polymer-ase chain reaction(PCR)using human fetal liver comple-mentary DNA(cDNA)library(gift from professor F.C. He,Beijing Institute of Radiation Medicine)as a template with the following primers:5?-CGGGATCCATG-GAGATGGGCAGACGGATT-3?,incorporating the BamH1site,and5?-AACTGCAGTTTCAAAATAG-GTTATTCCAC-3?,incorporating the Pst1site.Primers were synthesized on the basis of human leucine-rich acidic nuclear protein(LANP)gene sequence(GenBank acces-sion number NM_006305).The PCR products were subcloned into pUC18and sequenced.

Production of Recombinant Human Hepatopoietin Cn

The HPPCn gene was inserted between the BamH1 and Pst1sites of plasmid pET-24a(?),and the recombi-nant gene was expressed in Escherichia coli BL21as inclu-

sion bodies.The puri?ed inclusion bodies were lysed in8

M urea and refolded and puri?ed by HiTrap Ni?-Che-

lating Sepharose fast?ow(GE Healthcare,Sweden)ac-

cording to the manufacturer’s instructions. Expression of HPPCn Regenerating Liver

Animal Treatment.Male C57mice aged6to8weeks

were operated on after an overnight fast.A34%partial

hepatectomy was performed by ligating and excising the

left lobe at its root.12,13Sham-operated control animals

were treated in an identical manner with the omission of

hepatectomy.Mice livers were harvested,and total RNA

samples,total protein samples,and tissue samples were

prepared at0,2,12,24,48,72,96,and120hours after

surgery(n?3mice per group,randomly chosen).The

total RNA and protein samples were prepared using

Trizol reagent(Invitrogen)according to the manufactur-

er’s instructions.

Quantitation of HPPCn Messenger RNA in Regen-

erating Liver Using Real-Time 85c3a6e0102de2bd97058806plementary

DNA was synthesized from liver total RNA isolated from

experimental and control animals using the SYBR Prim-

erScript reverse transcription PCR Kit(Takara Bio,

Shiga,Japan)according to the manufacturer’s instruc-

tions.The ratio of HPPCn and HGF versus?-actin mes-senger RNA(mRNA)expression in experimental and

control animals was determined by real-time PCR.Reac-

tions were performed using Thermal Cycler Dice Real-

Time System(Takara Bio).The HPPCn(106base pairs

[bp])speci?c primers were:5?-cag gac gcc ctc tga tgt-3?

(forward)and5?-gag gtg agg cct acg ttg-3?(reverse).The

HGF(126bp)speci?c primers were:(forward)and5?-cca

ccc tac tgt tgt ttg-3?(reverse).?-actin(109bp)primers were:5?-cta agt cat agt ccg cct aga agc a-3?(forward),and

5?-tgg cac cca gca caa tga a-3?(reverse).Ampli?cations

were carried out in the reaction mixture in25?L con-taining5?L cDNA samples and12.5?L2?SYBR Premix Ex Taq,and the?nal concentration of0.2?mmol/L of each primer pair were added in a program comprising2minutes at50°C,15minutes at95°C,fol-

lowed by40cycles consisting of15seconds at95°C and1

minute at60°C.PCRs on control and experimental

cDNA were performed in triplicate in the same96-well

plate as the standard curves for both the target and refer-

ence genes.Expression of HPPCn and?-actin were cal-culated based on the equations generated from their respective standard curves.The HPPCn and HGF mRNA levels were expressed as a ratio of the fold change in HPPCn expression to the fold change in expression of the reference gene,?-actin.

HEPATOLOGY,Vol.47,No.3,2008CUI,ZHANG,ET AL.987

Western Blot of Total Liver Protein from PH and Sham-Operated Mice.Thirty micrograms total liver protein was separated by10%sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene?uoride membrane(Bio-Rad Laboratories).The blots were probed with anti-HPPCn serum raised in New Zealand White rabbits by immunization with recombinant hu-man HPPCn(rhHPPCn)and af?nity-puri?ed as the im-munoglobulin G fraction on a HiPAC protein A chromatographic column(0.46?10cm;Nihon Gaishi, Tokyo).Each blot was subsequently stripped(62.5mM Tris-HCl,pH6.7,100mM?-mercaptoethanol,and2% SDS for30minutes at50°C)and re-probed with anti-body against mouse?-actin(Santa Cruz Biotechnology). Hybridization of anti-HPPCn or anti–?-actin to the membrane was detected using a chemiluminescence im-munoblotting detection kit(Millipore).The hybridiza-tion signal was measured using a Fujix Bio-Imaging Analyzer,BAS2000(Fuji Photo Film,Japan). Immune Staining.Postoperative liver tissue samples were?xed with4%(w/v)freshly prepared paraformaldehyde in phosphate-buffered saline(PBS),and4-?m-thick sec-tions were cut using a Leica vibratome(Leica,VT1000S, Germany).Nonspeci?c protein binding sites were blocked using2%bovine serum albumin in PBS for1hour followed by incubation of the anti-HPPCn serum in blocking solu-tion overnight at4°C in a humidi?ed chamber.The sections were incubated with secondary antibody labeled with perox-idase,treated with3,3?-diaminobenzidine and hydrogen peroxide,and then visualized by microscopy. Bioactivity Assays of rhHPPCn In Vitro Establishment and Maintenance of Primary Cul-tured Rat Hepatocyte.Hepatocytes were isolated from 200g male Wistar rats by collagenase perfusion as de-scribed.6The isolated hepatocytes,showing more than 90%viability by trypan blue exclusion,were suspended in complete culture medium[Williams’medium E supplemented with10%fetal calf serum,5mM4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid),100 U/mL penicillin,0.1mg/mL streptomycin,10nM dexamethasone,and10nM insulin),diluted to6?105cells/mL,and seeded onto96-well plates(Costar). The monolayers were cultured at37.5°C under a hu-midi?ed atmosphere of5%CO2.

Cultured Cell Lines.Nine different commercially available cell lines were used in the experiment: SMMC7721(hepatoma,human),L02(liver,human), CHO(Ovary,Chinese Hamster),PC-3(prostate adeno-carcinoma,human),ECV304(umbilical cord,human), 293(kidney,human),A549(lung carcinoma,human),B16(skin melanoma,mouse),k562(bone marrow chronic myelogenous leukemia,human).Cells(5?104 cells/mL)were seeded onto96-well plates in complete culture medium without dexamethasone and insulin.The monolayers were cultured at37.5°C under a humidi?ed atmosphere of5%CO2.

Incorporation of[3H]TdR by Cultured Cells.Ac-tivity was assayed as described previously.14Brie?y,cells were plated in96-well plates and incubated for3hours before the addition of various concentrations of rhHP-PCn to the treated cultures.Differences in levels of DNA synthesis in treated versus untreated cells were measured by[3H]TdR incorporation.Approximately36hours after plating,5?Ci/mL[3H]TdR(Amersham Bioscience, UK)was added to each cell culture and incubated for6 hours.After incubation,cells were washed twice with sa-line buffer,trypsinized(0.25%trypsin,0.02%ethyl-enediamine tetraacetic acid)and collected on glass-?ber ?lters with a Brandel cell harvester.The?lters were then assayed for radioactivity in a liquid scintillation counter (Wallac1409,Finland).

Carboxy?uorescein Diacetate,Succinimidyl Ester–Based Proliferation Assay.One molar carboxy?uores-cein diacetate succinimidyl ester(CFSE;Molecular Probes)in warm PBS was added to primary culture hepa-tocytes and incubated for15minutes at37°C under gen-tle shaking and then seeded onto6-well plates(Costar). Cells were then incubated for24hours with0,10,or100 ng/mL rhHPPCn,washed twice with warm PBS,de-tached,counted,and immediately analyzed by?ow cy-tometry.For each sample,we acquired10,000events with a FACS Caliber cytometer(Becton Dickinson). CFSE was detected in FL1with a logarithmic ampli?er. DNA Synthesis Measured by[3H]TdR and Bromodeoxyuridine Incorporation In Vivo

We injected2.5?g rhHPPCn per20g body weight,or saline buffer as a control,into PH animals.After treat-ment,20?Ci[3H]TdR or2mg bromodeoxyuridine (BrdU;Sigma)per20g body weight were injected intra-venously or intraperitoneally,respectively,at20hours or 2hours,respectively,before sacri?ce.Liver tissue was har-vested at different time points(24hours,36hours,48 hours,60hours,72hours,and84hours)post-hepatec-tomy(n?5mice per group).Livers incorporated with [3H]TdR were harvested and DNA was extracted as de-scribed previously.12Incorporation of radioactive nucle-otide was measured in a liquid scintillation counter.Liver sections from mice injected with BrdU were evaluated after immunostaining with a BrdU antibody(Santa Cruz Biotechnology)and a horseradish peroxidase coupled de-tection method.Ten randomly selected62.5-?m2re-

988CUI,ZHANG,ET AL.HEPATOLOGY,March2008

gions were evaluated for BrdU-positive hepatocytes

(?1000hepatocytes per animal).For nonhepatectomized

control,50?g rhHPPCn per20g body weight was in-jected,and rate of DNA synthesis in liver,kidney,cere-

bellum,and spleen were tested.

Protein Kinase Activity Assays

Sphingosine kinase(SPK)activity was measured as de-

scribed previously.15SMMC7721cells were treated with

0,30,60,90,120,or150ng/mL rhHPPCn for30min-

utes or with50ng/mL rhHPPCn for10minutes,30

minutes,1hour,or2hours.Fifty micrograms protein was

extracted as described16from cells and incubated with10?Ci[32P]adenosine triphosphate(PerkinElmer Life Sci-ences,Boston,MA),0.5mM adenosine triphosphate,

and50mM D-e-sphingosine(Sigma)at37°C for30min-

utes in assay buffer(20mM Tris-HCl pH7.5,12mM ?-glycerophosphate,0.25mM ethylenediamine tetraace-tic acid,5%glycerol,1mM sodium pyrophosphate,5 mM sodium?uoride,1mM sodium orthovanadate,1 mM dithiothreitol,1?protease inhibitor cocktail,and 0.5mM4-deoxypyridoxine).Lipids were extracted by adding800?L chloroform:methanol:HCl(100:200:1), vortexing for1minute,and centrifuging.Two-hundred

?fty microliters chloroform and250?L1M KCl were added,and the mixture was vortexed vigorously for5 minutes.Samples were centrifuged for2minutes at 4000g,and the upper phase was removed.The organic phase was dried and suspended in chloroform:methanol (2:1).Lipids were separated by thin-layer chromatogra-phy on Silica Gel60high-performance thin-layer chro-matography plates(Merck,Germany)with1-butanol: acetic acid:water(3:1:1).Radioactive bands were visualized and quanti?ed using a Fujix Bio-Imaging An-alyzer,BAS2000.

ERK1/2and Stat3phosphorylation in cultured cells was analyzed by western blot.SMMC7721cells were treated with0,10,20,50,100,or200ng/mL rhHPPCn for30 minutes or with50ng/mL rhHPPCn for10minutes,30 minutes,1hour,or2hours.Thirty micrograms total cellular protein were extracted after treatment,separated by10% SDS-PAGE,and transferred to polyvinylidine di?uoride membrane(Bio-Rad).The blots were probed with antibod-ies against phos-ERK1/2(tyr204)or phos-Stat3(Santa Cruz).Each blot was subsequently stripped(62.5mM Tris-HCl,pH6.7,100mM?-mercaptoethanol,and2%SDS for30minutes at50°C)and re-probed with antibody to nonactivated ERK1/2or Stat3(Santa Cruz).Hybridization was detected using a chemiluminescence immunoblotting detection kit(Millipore).The hybridization signal was mea-sured using a Fujix Bio-Imaging Analyzer,BAS2000(Fuji Photo Film,Japan).

ERK1/2phosphorylation in regenerating livers was also analyzed by western blot.PH and nonhepatecto-mized mice were injected with2.5?g rhHPPCn per20g body weight,or injected with saline buffer as control. Injections were made1hour,12hours,or24hours after hepatectomy.Animals were sacri?ced1hour after injec-tion,and30?g total liver tissue protein was extracted. Hybridization was performed as described previously. Results

Puri?cation of Calf HSS

The further puri?cation of HSS resulted in a57,714-fold increase in speci?c activity.This puri?ed fraction of HSS was applied to nondenaturing PAGE,and each of3 protein bands of approximate molecular weight44kDa, 30kDa,and28kDa(Fig.1A)were visualized and ex-tracted from the gel after staining with imidazole and zinc 85c3a6e0102de2bd97058806pared with other common stains such as Coo-massie brilliant blue or silver,this method allows ef?cient recovery of intact proteins to further analyze their biolog-ical properties.The3protein species were tested for their ability to in?uence rates of DNA synthesis in vitro.The 30-kDa band caused a10-fold increase in DNA synthesis in the hepatoma cell line SMMC7721over that of saline buffer(P?0.05)(Fig.1B).

The30-kDa protein was digested in-gel,and the amino acid sequence of1tryptic peptide

(LLPQL-Fig.1.Puri?cation of HPPCn.(A)SDS-PAGE of HSS fractions at different stages of puri?cation.M:wide-range protein molecular weight standards.1:partially puri?ed HSS.2:44kDa band recovered from PAGE.3:30kDa band recovered from PAGE.4:28kDa band recovered from PAGE.(B)Determination of bioactivity of different bands in HSS on SMMC7721cells by[3H]TdR incorporation(#P?0.05).(C)The amino acid sequence of HPPCn and linear diagram of the protein.The diagram highlights the leucine-rich region(LRR),acidic tail,and nuclear localiza-tion sequences.

HEPATOLOGY,Vol.47,No.3,2008CUI,ZHANG,ET AL.989

TYLDGYDRDDK)was determined with LC-MS/MS.When compared with amino acid sequences available in the PIR and Swiss-Prot databases,this peptide was 100%identical to residues 135through 150predicted for calf leucine–rich acidic nuclear protein (Bovine LANP,Swiss-Prot accession number P51112).We named this 30-kDa protein Hepatopoietin Cn (HPPCn).

Sequence Analysis of the HPPCn Gene

Using primers derived from the human LANP gene (GenBank accession number NM_006305),we ampli-?ed HPPCn from a human fetal liver cDNA library.Se-quence analysis of the fragment showed 100%identity to Homo sapiens acidic leucine-rich nuclear phosphoprotein 32family member A,which is a member of the LANP family.The sequence of human HPPCn is predicted to have several features by Prosite database (Fig.1C)and is divided into 2domains.The N-terminal two-thirds is a leucine-rich repeat domain (LRR)and shows a high prob-ability of helical conformation according to Chou-Fas-man predictions.17The helical domain has a potential

leucine zipper in the center.The C-terminus of HPPCn is a highly acidic tail,composed of approximately 70%as-partic acid and glutamic acid residues.Additionally,it contains 2potential nuclear localization sequences,each of which is composed of a proline followed by a cluster of basic residues (Fig.1C).The following experiments were carried out for identi?cation of the stimulative activity of rhHPPCn in vitro and in vivo .

HPPCn Expression Pro?le in Hepatectomized Liver To clarify the role of HPPCn in liver regeneration,we measured HPPCn and HGF mRNA expression levels during the postoperative period in PH and sham-oper-ated control mice.The results of real-time reverse tran-scription PCR and western blot showed that the basal level of HPPCn mRNA and protein were low in non-hepatectomied mouse livers (Fig.2).In contrast,HPPCn mRNA levels in PH mouse livers began to rise shortly after the operation,peaked at 2hours and declined there-after (Fig.2A,left).The level of HGF mRNA in mouse livers rise and peaks at 12hours post-PH (Fig.2A,

right),

Fig.2.Expression of HPPCn in regenerating liver.Expression levels of HPPCn mRNA and HPPCn protein in livers of PH mice and sham-operated controls were analyzed with real time PCR,western blot,and immune staining (n ?3mice per group).(A)Levels of HPPCn (left)and HGF (right)mRNA expression over time postsurgery in PH and sham-operated animals using real-time PCR.(B)Western blot showing hybridization to anti-HPPCn antibody and anti–?-actin at various times postsurgery in PH (upper panel)and sham-operated (lower panel)animals.The hybridization intensities were plotted (right panel)by densitometry.(C)Immunohistochemistry showing the stain with anti-HPPCn antibody at 0hours (left)and 12hours (right)postsurgery in PH mice.

990CUI,ZHANG,ET AL.HEPATOLOGY,March 2008

which is consistent with previously described results.18 HPPCn protein levels in livers of PH mice increased over 12hours postoperatively with a subsequent decline(Fig. 2B).Furthermore,immunohistochemistry of liver tissue samples from PH mice and sham-operated controls cor-roborated these results(Fig.2C).

Effect of rhHPPCn on Cell Growth and Proliferation In Vitro

Highly puri?ed rhHPPCn protein was expressed in E.coli BL21as inclusion bodies(data not shown).Its effect on stimulation of hepatocytes was determined by means of [3H]TdR incorporation and CFSE-labeling assay.Stimula-tion of hepatocyte cultures by rhHPPCn measured by [3H]TdR incorporation is shown in Figs.3A 85c3a6e0102de2bd97058806-pared with untreated controls,a dose-dependent stimulation of DNA synthesis was shown in SMMC7721,L02cell lines, and primary cultured hepatocytes treated with rhHPPCn. This stimulation was most pronounced at the100ng/mL concentration of rhHPPCn.Interestingly,the stimulating effect of rhHPPCn on L02was lower than that on SMMC7721(P?0.05),a?nding that is similar to prior ?ndings in studies of HSS.19The speci?city of the activity of rhHPPCn in different cell types is shown in Fig.3B.The only signi?cant effect of rhHPPCn on increased DNA syn-thesis was seen in K562and B16cell lines.The results of these experiments show that rhHPPCn is able to stimulate hepatic cell proliferation in vitro,and that rhHPPCn shows partial cell speci?city.

Additionally,we assessed the effect of rhHPPCn on cell proliferation by?uorescence activated cell sorter anal-ysis using CFSE labeling.In principle,CFSE is retained by the primary cultured liver cells and is shared by the daughter cells at each division,resulting in multimodal ?ow cytometric CFSE histograms,with each cell genera-tion clustering around half the?uorescence intensity of the previous one.20Cells were dyed with CFSE and incu-bated with0,10,or100ng/mL rhHPPCn for24hours as described in Patients and Methods.We measured the per-centages of cells in the?rst3histograms:M1(undivided), M2(divided once),and M3(divided twice).Analysis of the histograms generated by?uorescence activated cell sorter analysis shows that50.41%of untreated cells un-derwent cell division,whereas76.55%and84.55%of cells treated with10ng/mL and100ng/mL rhHPPCn, respectively,underwent cell division(Fig.4B).These re-sults further con?rm the growth-stimulative activity of rhHPPCn on hepatocytes.

Accumulated evidence indicates that cell proliferation is concomitant with the activation of certain kinases and transcription factors.Internal signaling pathways such as S1P/SPK,ERK,and Stat3play a pivotal role in the pro-cess of hepatocyte proliferation.1,2,15,21,22We investigated the effect of rhHPPCn on SPK,ERK1/2,and Stat3in SMMC7721cells.rhHPPCn treatment caused a rapid increase in SPK activity(Fig.5A)and phosphorylation of ERK1/2and Stat3in a dose-dependent and time-depen-dent manner in hepatoma cells(Fig.5B).These results show that rhHPPCn can activate3different kinases re-lated to cell proliferation in SMMC7721cells. Bioactivity Test of rhHPPCn In Vivo

A34%hepatectomy is the minimal resection extent that leads to a detectable increase in DNA synthesis.5,6 In the current experiments,[3H]TdR and BrdU incor-poration assay was used to determine DNA synthesis in regenerating livers,and the results showed that34% hepatectomy led to an evident increase of hepatic DNA synthesis,and reached the peak at48hours after the operation.An intravenous injection of rhHPPCn at

2.5?g/20g body weight intravenous injection yielded

a dramatic increase of hepatic DNA synthesis at48 hours postoperatively,in comparison with saline buffer control(P?0.05)(Fig.6A,C).In contrast,the nor-mal adult liver has an extremely low rate of DNA syn-thesis,and a moderate but statistically signi?cant increase in the incorporation of[3H]TdR into DNA was found at24hours after2.5?g rhHPPCn per20g body weight intravenously into normal adult mice (P?0.05)(Fig.6B),but had no effects on the kidney, cerebellum,and spleen.

Prior investigations showed that PH stimulated an early increase in phosphorylated ERK1/21hour after operation.22In these experiments,rhHPPCn treatment produced a dramatic increase in ERK1/2phosphoryla-tion in PH mice,compared with control mice(Fig.7). Together,the above?ndings support that rhHPPCn has the potential to stimulate hepatic cells in vitro or in vivo, just as native HSS.

Discussion

Here we report a pure protein isolated from calf liver with speci?c proliferation stimulating activity.This pro-tein was puri?ed from a crude extract of HSS and named hepatopoietin Cn(HPPCn).Sequence analysis showed 100%identity to Homo sapiens acidic leucine-rich nuclear phosphoprotein32family member A,which is a member of the LANP 85c3a6e0102de2bd97058806NP proteins typically contain leucine-rich repeat motifs followed by an acidic carboxy-terminal tail and are heat-stable,and are involved in a variety of biological pathways,including signaling,pro-tein degradation,cytoskeletal dynamics,and morphogen-esis,presumably based on the ability of the leucine-rich

HEPATOLOGY,Vol.47,No.3,2008CUI,ZHANG,ET AL.991

repeat (LRR)domains to serve as adapter sites for pro-tein–protein interactions.23,24Previous biochemical stud-ies suggested that human LANP (pp32/I 1PP2A/mapmodulin/HPPCn)has been implicated in a number of cellular processes in both nucleus and cytoplasm,in-cluding proliferation,24differentiation,23caspase-depen-dent and caspase-independent apoptosis,25suppression of transformation in vivo ,26inhibition of protein phospha-tase 2A,27regulation of mRNA traf?cking and stability in association with HuR,28and inhibition of acetyltrans-ferases as part of the INHAT complex.29In this report,we found that rhHPPCn may stimulate the DNA synthesis and proliferation of hepatocytes and hepatoma cell lines and activate kinase pathways including SPK,ERK1/2,and Stat3.Based on previous information and our ?nd-ings of HPPCn bioactivity,we suggest that HPPCn may stimulate the growth of hepatocytes as a growth factor,in addition to acting in the nucleus and cytoplasm.A strain of mice lacking LANP was generated to understand the

role of this protein,but none of abnormalities were ob-served.30In our opinion,the most likely explanation for the apparent normality in mice lacking LANP is the pres-ence of less characterized proteins closely related to

LANP

Fig.3.Determination of bioactivity of rhHPPCn on cultured cell lines.(A)Dose-independent stimulative activity of rhHPPCn on SMMC7721,L02cell lines by [3H]TdR incorporation.(#P ?0.05,*P ?0.01).(B)Effect of 100ng/mL rhHPPCn on proliferation of 8different cell types determined by [3H]TdR incorporation.The cpm values were calculated relative to saline buffer–treated controls and are plotted as fold-increase over controls (#P ?

0.05).

Fig.4.rhHPPCn stimulation of primary cultured rat liver cells.(A)Growth stimulation of rhHPPCn on cultured primary hepatocytes deter-mined by [3H]TdR incorporation.(B)Growth stimulation of rhHPPCn on cultured primary hepatocytes determined by ?uorescence activated cell sorter analysis using CFSE-labeling.M1,M2,and M3represent counts of cells that are undivided,divided once,and divided twice,respectively.The concentration of rhHPPCn used to treat the cells is indicated above each set of histograms.The histogram showed the statistical result of 3identical experiments:cell counts divided once and twice is 50.41%in untreated cells,76.55%in cells treated with 10ng/mL rhHPPCn,and 84.55%in cells treated with 100ng/mL rhHPPCn (#P ?0.05).

????????????????????????????????????????????????????3Fig.5.Effects of rhHPPCn on SPK,Stat3and ERK1/2activities in SMMC7721cells.(A)SMMC7721cells were treated for 30minutes with indicated concentration rhHPPCn and 50ng/mL rhHPPCn for the indi-cated periods.Cell lysates were incubated with sphingosine and [c-32P]a-denosine triphosphate for 30minutes at 37°C.Lipids were then extracted and separated by thin-layer chromatography.Radiolabeled bands were visualized and quanti?ed using a Bio-Imaging Analyzer.(B)The phosphorylation of Stat3and ERK1/2were determined by western blot with antibodies against both species of Stat3and ERK1/2.

992CUI,ZHANG,ET AL.HEPATOLOGY,March 2008

HEPATOLOGY,Vol.47,No.3,2008CUI,ZHANG,ET AL.993

Fig.5.

providing rescue.Unfortunately,those studies did not look at regeneration of the null mice liver.It might be interesting to observe liver regeneration in this LANP null mouse strain in future studies.It would also be interesting to explore the action of HPPCn and the relationship between it and other growth factors or cytokines in liver regeneration using transgenic mice overexpressing HPPCn.

With the availability of the HPPCn gene and its recom-binant product,a series of issues can be examined,such as its production,secretion,and action mechanisms.Because of the potential nuclear localization sequences,we assume that HPPCn is a nuclear factor,but our experimental data show that extracellular HPPCn acts as a growth factor for hepatic cells in culture.In this respect,HPPCn resembles hepatoma-derived growth factor(HDGF)and high-mobility group box1protein(HMGB1).HDGF was originally isolated from the conditioned medium of a human hepatoma–de-rived cell line,HuH-7,and was characterized as a co-activa-tor of both transcription and pre-mRNA splicing,and also as an autocrine and paracrine growth and survival factor.Ex-tracellular HDGF can stimulate the proliferation of cultured hepatoma cells,?broblasts,smooth muscle cells,and endo-thelial cells,but nuclear localization has been shown to be a prerequisite for the mitogenic activity of intracellular HDGF.31-34Similarly,HMGB1,as a cytokine,activates en-dothelial cells,promotes angiogenesis,and initiates in?am-mation,but as a nuclear protein,it enhances the interaction of other proteins with DNA and regulates several families of DNA-binding proteins.35,36Another similarity between HPPCn,HMGB1,and HDGF is their secondary structure. Motifs within their N-terminal domains,named LRR, HMG Box,and HATH,respectively,are involved in pro-tein–protein interaction.Their acidic C-termini,consisting of glutamic acid residues,facilitate their association

with

Fig.6.Effect of rhHPPCn determined by[3H]TdR incorporation in vivo.(A) Effect of rhHPPCn on PH mice(n?5mice per group).The rhHPPCn(2.5?g per 20g body weight)or saline buffer was injected intravenously into PH mice20 hours before sacri?ce.Liver DNA was extracted at the times indicated.Incorpo-ration of radioactive nucleotide was measured,and its value is expressed as cpm per mg DNA.(#P?0.05).(B)Effect of rhHPPCn on nonhepatectomized mice. Twenty hours after rhHPPCn or saline buffer was injected into nonhepatectomized mice,DNA synthesis in liver,kidney,cerebellum,and spleen were tested.The cpm values were determined and shown relative to saline control arbitrarily normalized to100(#P?0.05).(C)Two hours before sacri?ce,rhHPPCn-treated PH,or saline-treated PH,animals were injected with BrdU.Liver samples were harvested at the time points indicated,?xed,sectioned,and processed for BrdU staining(n?5mice per group).Top:representative slides of liver sections from saline control and rhHPPCn-treated mice showing incorporation of BrdU at48 hours post-PH.Positively stained hepatocytes were quanti?ed in each sample by counting the number of cells in at least3different visual?elds per slide.Results are expressed as the average percentage of nuclei stained per1200nuclei counted?SD(#P?

0.05).

Fig.7.Western blot showing phosphorylation of ERK1/2by rhHPPCn

in regenerating livers.Thirty micrograms total tissue protein was extracted

1hour after intravenous injection of2.5?g rhHPPCn/20g body weight

or saline buffer into nonhepatectomized and PH mice.Samples were

taken1,12,and24hours posthepatectomy and analyzed by western

blot with antibody against phos-ERK1/2and ERK1/2.

994CUI,ZHANG,ET AL.HEPATOLOGY,March2008

DNA or histone.These proteins also contain2or3nuclear localization sequences and no signal peptide–like hydropho-bic region.Therefore,we propose that HPPCn might be a bifunctional nuclear protein such as HDGF and HMGB1, with extracellular HPPCn acting as a growth factor on cells, and intracellular HPPCn acting as demonstrated here. Hopefully,future studies will show the secretion and action mechanisms of HPPCn in normal and pathological condi-tions.

In conclusion,we puri?ed from calf liver a novel factor with hepatic stimulatory activity,named HPPCn.We identi?ed the bioactivity of the recombinant human HP-PCn in vitro and in vivo.Future studies of HPPCn will hopefully further our understanding of mechanisms of liver regeneration and lead to potential treatments for liver diseases.

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