hepatic expression of hepatocyte growth factor gene mrna in acute liver failure

Hepatic Expression of Hepatocyte GrowthFactor Gene mRNA in Acute Liver Failure

PHILLIP HARRISON, PhD, MD, CHRISTOPHER GOVE, PhD, and ADRIAN BOMFORD, MD

Hepatocyte growth factor plays a key role in liver regeneration but the role of liver in itssynthesis in acute liver failure is unclear. We therefore measured hepatic expression ofhepatocyte growth factor mRNA in this condition in comparison to H3 histone mRNA, amarker of cellular proliferation. Hepatocyte growth factor mRNA levels were quantified byspecific RNase protection assay in nine patients with acute liver failure and found to besimilar to those in six normal controls. Hepatocyte proliferation, as assessed by H3 histonemRNA expression, was not detected in normal liver but was present in six of nine patientswith acute liver failure (P , 0.05) and was not correlated with expression of hepatocytegrowth factor mRNA (rs 5 20.28). Liver is unlikely to be the source of the high serumhepatocyte growth factor levels observed in acute liver failure.

KEY WORDS: mitogen; liver regeneration; antagonist; messenger RNA; hepatic necrosis.

Hepatocyte growth factor (HGF), a 728-amino acidprotein, is a potent mitogen for rat and human hepa-tocytes in primary culture (1). Its administration trig-gered hepatocyte DNA synthesis in normal rats (2, 3)and enhanced liver growth following partial hepatec-tomy (2–5), portal vein ligation (6), and toxic liverinjury (4). Continuous administration of a neutraliz-ing antibody to HGF in rats following induction ofhepatic necrosis markedly inhibited hepatocyte pro-liferation, providing further evidence that HGF playsa key role in liver regeneration (7).

In the rat, extrahepatic production of HGF appearsto initiate regeneration following liver injury (8), buthepatic synthesis of HGF is thought to sustain livergrowth since increased hepatic expression of HGFmRNA (9, 10), has been reported after both partial

hepatectomy (11, 12) and induction of hepatic necro-sis (13–16). Increased hepatic HGF mRNA levels(17) and HGF immunoreactivity (18) were observedin liver in patients with acute hepatitis, suggesting inthese cases liver might be a principal site of HGFproduction. However, HGF mRNA was detected inonly 1 of 14 cases of acute liver failure (ALF) byreverse transcription-polymerase chain reaction (RT-PCR) (19), suggesting that hepatic HGF synthesismight not contribute to the high serum HGF levelsfound in ALF (20).

In the present study, we measured levels of HGFmRNA in liver in ALF using the sensitive, highlyspecific, quantitative ribonuclease protection assayand compared them to H3 histone mRNA, a markerof cellular proliferation (21, 22). We also measuredthe expression of an alternatively spliced HGF genetranscript which encodes a 290-amino acid polypep-tide comprising the N-terminal region and first twokringles of HGF called HGF-NK2 (23, 24). The trun-cated protein binds to the HGF receptor (24, 25) buthas been found to be an antagonist of HGF-inducedcell proliferation (24, 26). In addition, using ribonu-clease protection mapping analysis, we determined

Manuscript received October 28, 1999; accepted March 2, 2000.From the Academic Department of Hepatology, Institute of

Liver Studies, GKT School of Medicine, Bessemer Road, LondonSE5 9PJ, UK.

Phillip Harrison was supported by a Training Fellowship fromthe Medical Research Council of Great Britain.

Address for reprint requests: Dr. Phillip Harrison, AcademicDepartment of Hepatology, Institute of Liver Studies, GKT Schoolof Medicine, King’s Denmark Hill Campus, Bessemer Road, Lon-don SE5 9PJ, UK.

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the transcription initiation sites of the HGF gene inliver, since preferential utilization of specific sites hasbeen reported in response to acute liver injury in therat (27).

MATERIALS AND METHODS

Liver specimens were obtained at the time of liver trans-plantation from nine patients with ALF (28). Six patients[five females; median age 26 years (range 17–32)] hadacetaminophen-induced liver damage and three had pre-sumed non-A, non-B viral hepatitis (two females; ages 28,36, and 44 years). At the time of liver transplantation, eightpatients had grade IV and one patient grade II encepha-lopathy. The median International Normalised Prothrom-bin Ratio (INR) was 7.6 (range 3.6–15; normal, 0.9–1.1). Inthe acetaminophen overdose cases the median time be-tween overdose and liver transplant was 88 hr (range 76–114). The standard orthotopic liver transplant procedurewas used and patient selection was on the basis of estab-lished criteria for poor prognosis (29). Following removal ofthe diseased liver, small aliquots of tissue from the rightlobe were immediately frozen in liquid nitrogen and storedat 270°C until required. Specimens were also obtainedfrom six viable liver grafts for comparison. This study wasapproved by the Research Ethics Committee, King’s Col-lege Hospital.

RNA Extraction. Total RNA was isolated from the frozentissue according to the method of Chomczynski and Sacchi(30), and the RNA pellet was washed in 4 M lithiumchloride to reduce glycogen contamination.

Ribonuclease Protection Assay. Normal liver RNAprimed with random hexanucleotides was reversed tran-scribed using Moloney murine leukemia virus reverse tran-scriptase (Gibco BRL, Gaithersburg, Maryland), and thefirst-strand cDNA was used as a template in the amplifica-tion of a 668-bp partial cDNA of HGF by PCR. The nestedprimers used in the reaction were: first round, sense strand[nucleotides (nt) 671–691] 59-ATCATACAGAATCAG-GCAAGA-39 and antisense strand (nt 1385–1405) 59-AACGAGAAATAGGGCAATAAT-39; second round,sense strand (nt 700–720) 59-CGCTGGGATCATCAGA-CACCA-39 and antisense strand (nt 1347–1367) 59-TTTCCCGTGTAGCACCAGGGT-39 (10). The amplifiednested PCR product was cloned into the EcoRI–BamHIsites of pGEM-3Z (Promega Ltd, Southampton, UK) andthe plasmid was linearized using the unique BgII restrictionsite (nt 1219) within the HGF cDNA (10).

A cDNA of HGF-NK2 was amplified by PCR fromhuman placental DNA and specificity for the 1.5-kb tran-script was achieved by amplifying part of the 39 UTR. Thelatter is encoded by exon 7b, which is unique to the 1.5-kbHGF-NK2 mRNA (23). The primers were: sense strand (nt939 –958) 59-ACTCTAGATGCGAGACATAACAT-GGGCT-39 and antisense strand (nt 1161–1142) 59-ATGTCGACGGGTAAGGGCCAGCATGTAG-39 (23).The eight nucleotides at the 59 ends of the primers were notspecific for HGF-NK2 but encoded XbaI (sense strand) andSaI (antisense strand) restriction enzyme sites. The XbaI–SalI fragment of the PCR product was cloned into

pGEM-3Z and the vector was linearized using the EcoRIrestriction site within the plasmid’s cloning cassette.

A 360-bp partial cDNA of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was amplified by PCR from re-verse-transcribed normal liver RNA. The nested primerswere: first round, sense strand (nt 61–80) 59-ATGGG-GAAGGTGAAGGTCGG-39 and antisense strand (nt 921–940) 59-TGGAGGAGTGGGTGTCGCTG-39; secondround, sense strand (nt 111–130) 59-ATCGGATCCGGT-CACCAGGGCTGCTTTTA-39 and antisense strand (nt451– 470) 59-ATCTCTAGATGGTTCACACCCATGAC-GAA-39 (31). The nine nucleotides at the 59 ends of thenested primers were not specific for GAPDH but encodedBamHI (sense strand) and XbaI (antisense strand) restric-tion enzyme sites. The BamHI–XbaI fragment of the PCRproduct was cloned into pGEM-3Z, and the vector waslinearized using either the EcoRI restriction site within theplasmid’s cloning cassette or the unique MboI restrictionsite (nt 300) within the GAPDH cDNA. All cDNA insertswere sequenced to confirm the expected templates.

Riboprobes were synthesised in vitro from 0.5 mg oflinearized vector containing the specific cDNA templateand 100 mCi [a-32P]UTP (400 Ci/mmol) (Amersham, UK),using either SP6 or T7 RNA polymerases as appropriate.Cold UTP was included in the transcription reaction for thesynthesis of antisense GAPDH riboprobe to reduce theprobe’s specific activity. The antisense riboprobe for HGFwas 157 nucleotides long, of which 148 nucleotides werecomplementary to a portion of the 6.0-kb HGF mRNAencoding part of the fourth kringle and derived from exons10 and 11. The sense HGF riboprobe was 567 nucleotideslong, 524 nucleotides were identical to part of HGF mRNAderived from exons 6–9, and 166 nucleotides were identicalto part of HGF-NK2 mRNA derived from exons 6 and 7.The antisense riboprobe for HGF-NK2 was 287 nucleotideslong, of which 223 nucleotides were complementary to aportion of the 1.5-kb HGF-NK2 mRNA derived from exon7b. Two antisense riboprobes for GAPDH mRNA weresynthesized, one was 424 nucleotides long, of which 360nucleotides were complementary to GAPDH mRNA, andthe second was 208 nucleotides long, of which 170 nucleo-tides were complementary to GAPDH mRNA.

Liver RNA samples (30 mg) were hybridized at 42°C in80% formamide, 400 mM sodium chloride, 40 mM PIPES,and 1 mM EDTA for 16 hr with riboprobes for either HGF(5 3 105 cpm) or HGF-NK2 (5 3 105 cpm). AntisenseGAPDH riboprobe (1 3 105 cpm) was included in eachreaction to control for the amount of input RNA. In addi-tion, human placental RNA (10 mg) and tRNA (10 mg)samples were also analyzed; the first was used as a positivecontrol and the second to control for any nonspecific pro-tection of the riboprobes. The hybridization products weredigested with 40 mg/ml RNase A and 2 mg/ml RNase T1 for1 hr at 30°C, treated with 50 mg/ml proteinase K, andextracted with phenol–chloroform. The riboprobe/mRNAhybrids were denatured and fractionated by electrophoresison a denaturing 7 M urea/6% polyacrylamide gel. Driedgels were exposed to x-ray film with an intensifying screen at270°C. The amount of specifically protected riboprobe wasquantified by cutting the protected fragment from the poly-acrylamide gel and counting the radioactive content, whichwas then corrected for the expression of GAPDH mRNA in

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the same sample in order to correct for variations in theamount of input RNA.

Northern Blot Analysis. Total liver RNA (30 mg) wasfractionated by gel electrophoresis (1% agarose and 2.2%formaldehyde) and transferred to Genescreen (Du-Pont-NEN, Boston, Massachusetts) in 10 3 SSC (1.5 M sodiumchloride 1 0.15 M trisodium citrate, pH 7.0). The mem-brane was prehybridized for 6 hr at 42°C in a solutioncontaining 50% formamide, 10% dextran sulfate, 0.2%bovine serum albumin, 0.2% polyvinyl-pyrrolidone (MW40,000), 0.2% Ficoll, 50 mM Tris HCl (pH 7.5), 0.1%sodium pyrophosphate, 1% SDS, and 150 mg/ml shearedsalmon sperm DNA. The blot was hybridized overnight at42°C after addition to the prehybridization solution of a32P-labeled cDNA probe for H3 histone. The filter waswashed twice at room temperature for 5 min in 23 SSC plus1% SDS, then twice at 65°C for 30 min in 0.23 SSC plus0.1% SDS and then once at 65°C for 30 mins in 0.23 SSCbefore being exposed to x-ray film with intensifying screensat 270°C. The membrane was subsequently rehybridized toa 32P-labeled 360-bp partial cDNA probe for GAPDH, asdescribed above. Both probes were labeled by the randomprimer labeling procedure using [a-32P]dATP (32).

The 176-bp partial cDNA of H3 histone was amplified byPCR from reverse-transcribed normal liver RNA using:sense strand (nt 87–106 59-CAGGCCGAAGAGAAGGG-GGTA-39 and antisense strand (nt 243–262) 59-TTTCACG-GAGGCGGCCACAGTA-39 (33). The PCR product wascloned into pGEM-3Z, and the insert was sequenced toconfirm the presence of the expected sequence.

Mapping of Transcription Initiation Sites of HGF Gene.The template for the in vitro transcription of the antisenseriboprobe, used in the ribonuclease protection mappinganalysis of the transcription initiation sites of the HGFgene, was prepared as follows: a 1043-bp DNA fragment ofthe HGF gene was amplified by PCR from human placentalDNA, sense strand (nt 2904 to 2885) 59-TCAGGGA-

CAGGCTATGGACA-39 and antisense strand (nt 1120 to1139) 59-AGGAGGAGATGCAGGAGGAC-39 (34). A408-bp SacI–PstI fragment of this PCR product, spanningthe 59-flanking region and first exon, was cloned intopGEM-3Z vector and the plasmid was linearized using theEcoRI restriction site within the cloning cassette. A 435-ntantisense riboprobe was synthesised in vitro and hybridizedwith 30 mg of total RNA from normal and fulminant liver,as described above. In addition, RNA samples from humanplacenta and MRC-5 cells (human embryonic lung fibro-blasts) were analyzed because these are known to be richsources of HGF mRNA. The hybridization products weredigested and fractionated as described above. In order toquantify the use of each transcription initiation site, theprotected fragments from the ribonuclease protection map-ping analysis were cut from the polyacrylamide gel and theradioactive content determined.

Statistical Analysis. All results are expressed as the me-dian and range. The 6.0-kb HGF and 1.5-kb HGF-NK2mRNA levels, corrected for expression of GAPDH mRNA,in the normal and liver-failure samples were comparedusing the Mann-Whitney U test. The levels of H3 histonemRNA (measured by densitometry after 96 hr of autora-diographic exposure) were normalized to that of GAPDHmRNA (3 hr of exposure) in each sample. Any correlationswas analyzed using the Spearman rank correlation coeffi-cient (rs).

RESULTS

The 6.0-kb HGF mRNA was detected in all tissuesamples as a 148-nt protected fragment derived fromthe 157-nt HGF antisense riboprobe (Figure 1). Asmaller fragment was also evident, but it was theproduct of nonspecific protection of the riboprobe,

Fig 1. Expression of HGF mRNA in liver by ribonuclease protection assay. A [32P]RNA probe complementary to HGF mRNA washybridized with 30 mg total liver RNA from patients with acute liver failure due to non-A, non-B hepatitis (lanes 1–3) or acetaminophenoverdose (lanes 10–15) or with normal liver (lanes 4–9). HGF mRNA was detected as a 148-nucleotide protected fragment. Placental RNAwas used as a positive control and tRNA as a negative control. A [32P]RNA probe, complementary to 360 nucleotides of GAPDH mRNA,was used as a loading control.

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since it was also detected in the tRNA sample. TheHGF/GAPDH ratios in ALF [median 1.3 (range 0.6–4.3)] and in the subgroup of those following acetamin-ophen overdose [median 1.1 (range 0.65–4.3)] weresimilar to normal liver [2.5 (range 1.5–2.7)].

The 1.5-kb HGF-NK2 mRNA was detected as twofragments of equal intensity of 223 and 210 nucleo-tides, derived from the 287-nt HGF-NK2 antisenseriboprobe (Figure 2). There was considerable varia-tion in the expression of the 1.5-kb HGF-NK2 mRNAin the patients with ALF [median HGF-NK2/GAPDH ratio 0.37 (range 0.26–3.82)] and, althoughoverall there was no significant difference in levels ofthis mRNA in comparison with the normal liver sam-ples [median 0.30 (range 0.26–0.37)], levels in thesubgroup of those following acetaminophen overdose[median 0.87 (range 0.32–3.82)] were significantlyhigher than normal (P , 0.05). There was no cor-relation between levels of the 6.0-kb HGF mRNAand 1.5-kb HGF-NK2 mRNA transcripts (rs 520.33). No RNA transcripts were detected in any ofthe samples by the sense riboprobe (data not shown).

The HGF/HGF-NK2 ratio was lower in ALF com-pared to the normal liver samples [median 3.4 (range0.5–8.0) vs 7.3 (range 4.4–10.2), respectively, P ,0.05]. In ALF, there was no correlation betweenlevels of the 6.0-kb HGF mRNA and the severity ofdisease, as assessed by the INR and bilirubin (rs 520.43 and rs 5 20.36, respectively), although ex-pression of the 1.5-kb HGF-NK2 mRNA correlatedwith the INR but not the bilirubin (rs 5 0.71, P ,0.05, and rs 5 0.19, respectively). In the acetamin-ophen overdose cases, there was no correlation be-tween the time from overdose to liver transplantation

and the expression of the 6.0-kb HGF mRNA or1.5-kb HGF-NK2 mRNA (rs 5 20.36 and rs 5 0.38,respectively).

The ribonuclease protection mapping of the tran-scription initiation sites of the HGF gene revealed amajor cluster of protected fragments between 99 and103 nt long in all samples and the most intensefragment was 102 nt long (Figure 3). Since the PstIrestriction site is 39 nt downstream of the translationstart site, the major transcription initiation site of theHGF gene mapped to 63 nt upstream of the transla-tion initiation codon. Three minor transcription initi-ation sites were detected upstream of the major site inall samples, centered around 75, 84, and 96 nucleo-tides upstream of the translation initiation codon.One minor site was detected downstream of the ma-jor site in all samples, centered around 50 nt up-stream of the translation initiation codon. There wasno difference in the relative use of the five transcrip-tion initiation sites comparing normal and liver fail-ure samples. The major transcription initiation site ofthe HGF gene detected in both placenta and MRC-5cells by Miyazawa et al, using S1-nuclease analysis(35), accords with a minor rather than the maintranscription initiation site determined in the presentstudy in liver, placenta, and MRC-5 cells. Interest-ingly, this transcription initiation site correspondswith the 59 ends of the reported cDNAs for the 1.5-kbHGF-NK2 mRNA described by Chan et al (24) andMiyazawa et al (23). The most upstream of the tran-scription initiation sites detected in the present studycoincides with both the second transcription initiationsite of the rat HGF gene reported by Okajima et al(27) and the 59 end of the cDNA for human HGF

Fig 2. Expression of HGF-NK2 mRNA in liver by ribonuclease protection assay. A [32P]RNA probe complementary to HGF-NK2 mRNAwas hybridized with 30 mg total liver RNA from patients with acute liver failure due to non-A, non-B hepatitis (lanes 1–3) or acetaminophenoverdose (lanes 10–15) or with normal liver (lanes 4–9). HGF-NK2 mRNA was detected as two protected species of 223 and 210nucleotides. tRNA was used as a negative control. A [32P]RNA probe, complementary to 170 nucleotides of GAPDH mRNA, was used asa loading control.

HARRISON ET AL


isolated by Miyazawa et al from placenta (9). How-ever, no transcription initiation sites were observed inthe 59 flanking region of the gene corresponding tothe 59 end of the HGF cDNA cloned from a livercDNA library by Nakamura et al (10), indicating thatthis clone must have been derived from a HGFmRNA transcript of very low abundance in liver.

Expression of H3 histone mRNA was used to assessthe level of cellular proliferation in each liver. H3histone mRNA was not detected by northern blotanalysis in any of the normal liver specimens, but itwas found in six of nine patients with ALF (P , 0.05)(Figure 4). In the patients, H3 histone mRNA expres-

sion was significantly correlated with the INR (rs 50.94, P , 0.01), and HGF-NK2 mRNA levels (rs 50.76, P , 0.05) but not HGF mRNA expression(rs 5 20.28).

DISCUSSION

Analysis of hepatic expression of the 6.0-kb HGFmRNA by RNase protection assay demonstrates thatlevels are similar in normal controls and patients withestablished ALF due to either acetaminophen over-dose or non-A, non-B hepatitis, suggesting that asustained increase in hepatic HGF synthesis is un-

Fig 3. The Ribonuclease protection mapping analysis of the transcription initiation sitesof the HGF gene. (A) A 435 nucleotide [32P]RNA probe complementary to the regionof the gene 59 to the translation initiation site was hybridized with 30 mg of total RNAfrom normal liver (lane 1) or liver from a patient with acute liver failure due toacetaminophen overdose (lane 2) or with 10 mg total RNA from human placenta (lane3) or MRC-5 cells (lane 4). tRNA was used as a negative control. The size markers werea [g-32P]ATP 59-end-labeled MSP1 digest of pBR322 (New England Biolabs, Beverly,Massachusetts). (B) The [32P]RNA probe used for the ribonuclease protection mappingis shown. The dotted and white boxes are derived from the sequences of the genomic andvector DNA, respectively. The sizes of the protected fragments are indicated.



likely to account for the high serum HGF observed inALF. This finding contrasts with that in animal mod-els of acute liver injury, where high levels of HGFmRNA have been found after acute liver injury, butsupports the results of a study in patients with ALFthat failed to detect HGF mRNA in liver by RT-PCR.

The main transcription initiation site of the HGFgene found in the present study by RNase protectionmapping analysis coincides with that found in rat (27,36) and mouse liver (37, 38). However, in contrast tothe findings in the rat (27), we did not detect anypreferential utilization of transcription initiation sitesin ALF. This findings supports the hypothesis that anincrease in hepatic HGF synthesis is unlikely to ac-count for the high serum HGF found in patients withsevere liver failure requiring transplantation. Onefactor that might explain the difference between thefindings of the present study and the animal models isthe time course of HGF mRNA expression. In ratstreated with carbon tetrachloride, a rise in HGFmRNA expression was detected in nonparenchymalliver cells 5 hr later (13, 16) but levels returned tonormal at 24 hr (16). Since the availability of clinicalmaterial prevented us from measuring HGF mRNAlevels until many hours after the onset of liver injury,a transient rise in expression before liver transplan-tation cannot be excluded.

Other mechanisms might account for the high se-rum HGF in ALF. Animal studies have shown thatother organs synthesize HGF in response to acuteliver injury and HGF mRNA levels markedly in-creased in rat lung following carbon tetrachlorideadministration (39), demonstrating an endocrine, inaddition to a paracrine, mechanism of HGF-inducedliver regeneration. This points to the existence of ahumoral factor that induces HGF production in or-

gans distal to the site of injury (40), although to dateno such factor has been reported in man. Liver is themain organ for the uptake of HGF from serum (41),and in the rat a reduction in hepatocyte mass in-creased the half-life of HGF (42, 43). In ALF, themassive loss of hepatocytes would result in a fall inhepatic clearance and hence could lead to the ob-served rise in serum HGF. This hypothesis is sup-ported by the findings after liver transplantation,where persistently high serum HGF levels were foundin patients with compromised grafts, whereas HGFconcentrations decreased rapidly in patients withgood graft function (44).

Expression of the 1.5-kb HGF-NK2 mRNA haspreviously been reported in human placenta (23) andhuman fibroblast cell lines (24), but this is the firstreport to our knowledge of its expression in liver. Inpatients with liver disease, infiltrating polymorphonu-clear leukocytes and biliary epithelial cells have beenreported to secrete HGF (45), and in animal modelsof toxic liver injury HGF gene expression has beenfound in endothelial cells (16) and Kupffer cells (15).Although at present the site of HGF-NK2 proteinproduction in liver is unknown, it is synthesized bymany cell lines that produce the full-length HGFprotein, and some fibroblasts, such as those fromforeskin, preferentially produce HGF-NK2 (24).HGF gene expression is orchestrated in stromal cellsby extracellular signals (46), but it is not yet knownwhether differential regulation of the alternativelyspliced HGF transcripts occurs at the transcriptionalor posttranscriptional level.

H3 is a core histone that is essential for the chro-mosomal packaging of eukaryotic DNA (47). Levelsof H3 histone mRNA are thought to accurately re-flect the rate of DNA synthesis in cultured cells (21,

Fig 4. Northern blot analysis of H3 histone expression in liver. Samples of total RNA (30 mg) from patients with acute liver failure due tonon-A, non-B hepatitis (patients 1–3), normal liver (samples 4–9), and patients with acute liver failure following acetaminophen overdose(patients 10–15) were fractionated by gel electrophoresis (1% agarose and 2.2% formaldehyde), transferred to Genescreen, and hybridizedwith 32P-labeled human cDNA probes coding for: H3 histone (H3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

HARRISON ET AL


22) and, in regenerating human liver, correlate withthe expression of proliferating cell nuclear antigen(PCNA), another marker of mitosis (48). In thepresent study, high levels of H3 histone mRNA werefound in ALF, supporting the results of two studiesthat found high liver PCNA levels in ALF (49, 50).However, in contrast to the findings of a previousstudy in acute hepatitis (17), we found no correlationbetween hepatic HGF mRNA expression and cellproliferation. Rather, the expression of H3 histonemRNA was significantly correlated with that of HGF-NK2 mRNA, and this relationship is analogous to theone between hepatocyte proliferation and TGF-b1 inALF, where there is strong correlation between liverH3 histone mRNA and TGF-b1 mRNA (51). It hasbeen postulated that the activation of TGF-b, a po-tent inhibitor of liver regeneration, is required duringliver growth to prevent uncontrolled hepatocyte pro-liferation (52). Therefore, the high expression ofHGF-NK2 mRNA in regenerating liver introduces apotential novel mechanism for the control of HGF-induced liver growth in vivo: since the phenotypicexpression of the HGF gene in liver could be deter-mined by the balance between these alternativelyspliced mRNA transcripts encoding HGF agonist andantagonist proteins. However, this is currently specu-lative since there are no data on the expression ofHGF-NK2 protein in tissue. Immunohistochemicalmethods detect both full-length and truncated HGFproteins unless the antibodies are raised only to thelight chain of HGF, and western blot analysis onprotein extracted from diseased liver tissue has notyet been reported.

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HARRISON ET AL


hepatic expression of hepatocyte growth factor gene mrna in acute liver failure

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