ARTICLE |
Correspondence to:
László G. Kömüves, COR Therapeutics, Inc., 256 E. Grand Ave., So. San Francisco, CA 94080. E-mail:
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polypeptide growth factors, including epidermal growth factor (EGF), play a central role in regulating hepatocyte growth both in vivo and in primary culture. To characterize EGF gene expression in the pathogenesis of regenerative cirrhotic fibrosis, we employed biotinylated antisense oligonucleotide probes to localize hepatic mRNA transcripts in situ. In control tissue and regenerative hepatic nodules, EGF receptor (EGFR) mRNA transcripts were expressed constitutively. In contrast, oligonucleotide probes targeting the human EGF coding region showed that EGF transcription was extremely low in control liver but was highly elevated and localized to regenerative hepatic nodules and bile duct epithelia of cirrhotic liver. To determine whether EGF mRNA accumulation accompanied a comparable increase in the EGF peptide, we performed immunohistochemistry using an antibody specific for the nonprocessed peptide aminoterminus. We observed that positive localized EGF staining paralleled its mRNA transcript. These results indicate that EGF upregulation is a characteristic of cirrhotic liver disease and suggest that persistent de novo ligand synthesis and its signaling contribute to an autocrine-mediated hepatocyte proliferation within the regenerative nodule. (J Histochem Cytochem 48:821830, 2000)
Key Words: EGF, EGF receptor, liver cirrhosis, hepatocytes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Liver growth activation occurs through a highly regulated and sequential process, which restores organ mass and capacity after tissue resection or injury ( (TNF-
) and Factor-d interleukin-6 (IL-6) (
(
Hepatocytes express the tyrosine kinase EGF receptor (EGFR) and quickly respond to exogenous EGF in vivo (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Specimens
Liver specimens from three transplant patients with cirrhotic liver disease and control specimens (livers rejected for transplantation) were obtained at surgery according to UCSF policies of the Committee on Human Research at UCSF, in compliance with NIH guidelines. Tissue pieces were immediately placed in ice-cold 4% formaldehyde (freshly prepared from paraformaldehyde), dissolved in Dulbecco's Mg2+/Ca2+-free PBS, and were fixed for 24 hr at 4C. Tissues were dehydrated in increasing ethanol concentrations, cleared in xylenes, and embedded in paraffin by standard methods. Sections (5-µm) were collected on positively charged microscope slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA).
DNA Oligonucleotide Probes
Oligonucleotides were synthesized with an ABI 390 synthesizer. Biotin-dT was incorporated by programming the addition of 5'-dimethoxytrityloxy-5-N((4tbutylbenzoyl)-biotinyl)-aminohexyl) 3'acryimido) 2'deoxy-U-3'-2-cyanoethyl-N,N-diisopropyl)-phosphoramidite (Glen Research; Sterling, VA). After column elution, oligos were deprotected overnight at 58C, dried in vacuo, resuspended in H2O, adjusted to 50% deionized formamide (pH 7.0), heated to 65C for 5 min, and fractionated by electrophoresis on a 15% acrylamide/Bis (30:1), 7 M urea, 1 x TBE gel. Bands were identified by UV shadowing, excised, and eluted in H2O, precipitated twice with 3 volumes of ethanol, and stored in H2O at -20C. Oligo sequences (bT, biotinylated thymidine) were selected from the GeneBank (NCBI) database: human EGF antisense: bttacaaagcactgtbtgtcccaatttgggtbtt (43254295) and abtgtagccaacaacacagbttgcatgcatacttgbtc (34593425); human EGF sense probe btaatggagcaagctbttcatatgccctccta (39153945). The human EGF receptor antisense, ggagcgctgccccggccgtccgg(bT)3 (197220) and sense, ccg-ggacggccggggcagcgctc(bt)3 (476499), were purchased from Research Genetics (Huntsville, AL).
In Situ Localization of EGFR mRNA Using Biotin-labeled Oligonucleotide Probes Detected by ABCPeroxidase
Sections were dewaxed with Safeclear (CMS; Houston, TX), rehydrated in ethanol, washed with PBS, and treated with 0.2 N HCl for 20 min, followed by 1.5% H2O2 for 30 min, then by 0.3% Triton X-100 for 15 min, and incubated with 1 µg/ml proteinase K (Amresco; Solon, OH) for 30 min at 37C. After washing with 0.1 M triethanolamine buffer, pH 8.0, sections were acetylated for 10 min in 0.25% acetic anhydride in triethanolamine buffer, incubated with 40% formamide in 1 x SSC at 37C for 2 hr, and air-dried. One pmole biotinylated probe was diluted in 100 µl hybridization buffer containing 40% formamide, 1 x SSC, 10 x Denhardt's solution, 0.5% SDS, 0.5% sodium pyrophosphate, yeast tRNA (500 µg/ml) in 10 mM Tris buffer, pH 7.4, denatured at 85C, chilled on ice and applied to the sections, and hybridized in a humidified chamber at 40C. After 12 hr the sections were washed at room temperature (RT) (5 ml/slide) with 2 x SSC, followed by 1 x SSC and 0.25 x SSC for 30 min each at 37C. Sections were briefly washed in TBS (10 mM Tris, pH 7.6, containing 500 mM NaCl), blocked with 4% BSA, 0.5% teleostean gelatin, 0.1% Tween-20 in TBS for 30 min, and incubated with ABC peroxidase (Vector; Burlingame, CA) for 30 min, followed by three-min washes in blocking buffer and three-min washes in TBS. Peroxidase activity was revealed with DAB substrate (5 min), purchased from Qual-Tek Laboratories (Santa Barbara, CA). The sections were washed with several changes of distilled water, counterstained lightly with Carazzi's hematoxylin, dehydrated with alcohol and xylenes, and coverslipped.
Localization of EGF Expression by In Situ Hybridization Using Biotin-labeled Oligonucleotide Probes Detected by Tyramide-mediated Signal Amplification
The conditions of pretreatment, hybridization, and posthybridization washes for EGF mRNA were essentially as described above, with the following modifications. Hybridization washes were followed by a brief wash in TBS and sections were blocked with 4% BSA, 0.5% teleostean gelatin, 0.1% Tween-20 in TBS for 30 min. Sections were then incubated with ABCperoxidase (Vector) for 30 min, followed by three 5-min washes in blocking buffer, and were incubated with biotinylated tyramide (Dako; Carpinteria, CA) for 15 min, followed by ABCperoxidase for 15 min. After three 5-min washes with blocking buffer and three 5-min min washes in TBS, peroxidase activity was revealed with DAB substrate for 5 min. Sections were then washed with several changes of distilled water, counterstained lightly with Carazzi's hematoxylin, dehydrated with alcohol and xylenes, and coverslipped. For all hybridizations, a set of controls was used to establish hybridization specificity: (a) sense control probes did not yield signal above background; (b) omission of the biotin-labeled antisense probes resulted in no signal; and (c) omission of ABCperoxidase resulted in no signal, indicating the specificity of the signal amplification procedure.
Immunohistochemical Detection of Pro-EGF Peptide
Antiserum to the human EGF pre-pro-peptide amino acids 121 was a kind gift of Dr. B Mroczkowski and has been characterized elsewhere (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Localization of EGF Receptor mRNA in Control and Cirrhotic Human Liver
To localize the cellular distribution of EGFR gene expression in human liver, we used an antisense EGFR biotinylated oligonucleotide probe (
|
We next compared the EGFR mRNA expression pattern observed in control tissue to sections obtained from advanced cirrhosis. Here, EGFR mRNA staining was evident throughout the cirrhotic tissue (Fig 1C), displaying a higher intensity bias in the smaller nodules and an increased concentration at the nodular periphery (Fig 1C). Moreover, positive signals were evident throughout the highly proliferative bile duct epithelium but were absent from the internodular fibrotic matrix. In all cases, the EGFR mRNA signal was restricted to the cytoplasm (Fig 1D).
Localization of EGF mRNA in Control and Cirrhotic Liver Using Biotin-labeled Oligoprobes Followed by Tyramide-catalyzed Signal Amplification
In previous studies we have demonstrated that a low constitutive level of EGF expression was detectable in rodent liver and was rapidly upregulated in the immediate-early phase of regenerative liver growth (
|
Detection of the EGF Pre-pro-peptide in Human Cirrhotic Liver
We next carried out immunohistochemical localization to determine whether the regenerative parenchyma produced detectable levels of the EGF peptide. Because the hepatocyte EGF receptor can serve as an uptake module for processed EGF (see Discussion), we used an antiserum raised against the pro-peptide region, amino acids 121 (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this work we show that the polypeptide mitogen EGF and its cell surface receptor are expressed differentially in control and cirrhotic human liver. In normal tissue, receptor transcripts appear dispersed throughout the parenchyma, whereas its cognate ligand, i.e., EGF, remains at nondetectable levels. In contrast, in advanced cirrhosis, a continuous receptor synthesis is accompanied by EGF expression that is localized to hepatocytes and proliferative bile epithelia. Furthermore, with an EGF precursor-specific antiserum, our results show that nascent peptide synthesis parallels its mRNA throughout the regenerative tissue. Collectively, these results reveal that EGF and its receptor are synthesized coordinately with nodule expansion and suggest that this co-expression of a mitogenic cell surface receptor and its associated binding ligand may consititute an autocrine growth mechanism of cirrhotic liver disease.
A number of studies have shown that both quiescent and proliferative hepatocytes maintain significant levels of the transmembrane EGF receptor, although its functional role in liver homeostasis has remained controversial (
Our present studies are in agreement with previous work that has shown relatively high receptor transcript levels occurring throughout the control hepatic parenchyma. However, comparison of these results to the staining pattern in cirrhotic sections indicates that receptor transcript density actually increases in localized regions of the diseased tissue, suggesting receptor gene upregulation. Because the fibrotic/parenchymal tissue ratio is relatively high in advanced cirrhosis, final receptor levels, when normalized to accumulated tissue mass, may be decreased overall.
As early as 1973 it was noted that EGF administration to normal liver transiently increases the hepatic labeling index and, more recently, has been shown to directly activate receptor-dependent STAT signaling pathways in situ (
Attempts to correlate changes in circulating pools of EGF with liver growth induction have thus far proved equivocal, and only a limited effect on liver growth has been reported after sialoadenectomy ( (which also binds EGFR) is increased only after the first wave of regenerative cell division (
increases were not observed. This suggests that early signaling pathways are persistent in chronic cirrhosis and may abrogate the sequential downstream pathways observed in the two-thirds HPX model (
The primary focus of our present study was to investigate whether EGF gene expression could be identified in human liver and whether it may exhibit upregulation in human regenerative liver disease. As we have shown here, in contrast to rodent liver, human liver EGF transcripts are virtually undetectable in control liver, with only occasional weak staining in the bile epithelium. In contrast, advanced cirrhotic tissue showed staining for EGF mRNA, which was most prominent at the nodule periphery and notable within presumptive proliferative bile duct cells. Fibroblasts throughout the accumulated internodular matrix were devoid of EGF expression.
The novel identification of human hepatic EGF mRNA expression in cirrhotic liver reported here is probably a result of technical improvements that have enabled the detection of low transcript levels. First, to minimize RNA degradation, tissue obtained at the time of surgery was immediately quenched in fixative, thus minimizing nucleolytic activity that can hinder retrospective analysis of archival samples (
The observed derepression of the EGF gene is accompanied by appearance of the nascent EGF peptide, as detected by immunohistochemistry. EGF is synthesized as a 160-kD transmembrane precursor and is proteolytically processed at the extracellular surface, releasing a mature 6-kD exocrine peptide (
Although the long-term effects of continual hepatic EGF production remain unknown, recent studies have shown that EGFR upregulation is common in hepatic carcinogenesis (
![]() |
Acknowledgments |
---|
Supported in part by a Department of Veterans Affairs Merit Review Grant and NIH grant PO1-AR39448 (LGK).
We thank Dr B. Mroczkowski (Agouron Pharmaceutical; La Jolla, CA) for kindly supplying the EGF N-terminal antiserum and Dr T. Wright and Ms T. Ryan (UCSF) for their help in obtaining tissue samples. We are grateful to members of the Liver Center for helpful comments and to Dr B. Sommers (Glen Research) for advice on oligo design.
Received for publication August 20, 1999; accepted December 17, 1999.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adami HO, Hsing AW, McLaughlin JK (1992) Alcoholism and liver cirrhosis in the etiology of primary liver cancer. Int J Cancer 51:898-902[Medline]
Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:1457-1463
Barnard JA, GravesDeal R, Pittelkow MR, Dubois R, Cook P, Ramsey GW, Bishop PR, Damstrup L, Coffey RJ (1994) Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family. J Biol Chem 269:22817-22822
Bissell DM (1990) Cell-matrix interaction and hepatic fibrosis. Prog Liver Dis 9:143-155[Medline]
Bloch B (1993) Biotinylated probes for in situ hybridization histochemistry: use for mRNA detection. J Histochem Cytochem 41:1751-1754
Block GD, Locker J, Bowen WC, Petersen BE, Katyal S, Strom SC, Riley T, Howard TA, Michalopoulos GK (1996) Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF in a chemically defined (HGM) medium. J Cell Biol 132:1133-1149[Abstract]
Bucher NLF, Patel U, Cohen S (1977) Hormonal factors and liver growth. Adv Enzyme Regul 6:205-213
Burwen SJ, Barker ME, Goldman IS, Hradek GT, Raper SE, Jones AL (1984) Transport of epidermal growth factor by rat liver: evidence for a non-lysosomal pathway. J Cell Biol 99:1259-1265[Abstract]
Carpenter G, Cohen S (1979) Epidermal growth factor. Annu Rev Biochem 48:193-216[Medline]
Chabot JG, Walker P, Pelletier G (1986) Distribution of epidermal growth factor binding sites in the adult rat liver. Am J Physiol 250:G760-764[Medline]
Clark AJL, Ishii S, Richert N, Merlino GT, Pastan I (1985) Epidermal growth factor regulates the expression of its own receptor. Proc Natl Acad Sci USA 82:8374-8378[Abstract]
Cressman DE, Greenbaaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R (1996) Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274:1379-1383
DeCicco LA, Kong J, Ringer DP (1997) Carcinogen-induced alteration in liver epidermal growth factor receptor distribution during the promotion stage of hepatocarcinogenesis in rat. Cancer Lett 111:149-156[Medline]
DeCicco LA, Panzeter PL, Cashman RE, Ringer DP (1996) Changes in the binding capacity of hepatic membranes for epidermal growth factor during multistage hepatocarcinogenesis in rats. Biochem Biophys Res Commun 228:69-74[Medline]
De Jong JS, Van Diest PJ, Van Der Valk P, Baak JPA (1998) Expression of growth factors, growth inhibiting factors and their receptors in invasive breast cancer I: an inventory in search of autocrine and paracrine loops. J Pathol 184:44-52[Medline]
Earp HS, O'Keefe EJ (1981) Epidermal growth factor receptor number decreases during rat liver regeneration. J Clin Invest 67:1580-1583[Medline]
Fausto N (1994) Liver regeneration. In Arias IM, Boyer JL, Jakoby WB, Fausto N, Schachter D, Shafritz DA, eds. The Liver: Biology and Pathobiology. New York, Academic Press, 1059-1084
Fausto N, Laird AD, Webber EM (1995) Liver regeneration 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J 9:1527-1536
Fausto N, Webber EM (1993) Control of liver growth. Crit Rev Euk Gene Express 3:117-135[Medline]
Friedman LL (1993) The cellular basis of hepatic fibrosismechanisms and treatment strategies. N Engl J Med 25:1828-1835
GlynneJones E, Goddard L, Harper ME (1996) Comparative analysis of mRNA and protein expression for epidermal growth receptor and ligands relative to the proliferative index in human prostate tissue. Hum Pathol 27:688-694[Medline]
Gressner AM (1998) The cell biology of liver fibrogenesisan imbalance of proliferation, growth arrest and apoptosis of myofibroblasts. Cell Tissue Res 292:447-452[Medline]
Harada K, Terada T, Nakanuma Y (1996) Detection of transforming growth factor-a protein and messenger RNA in hepatobiliary diseases by immunohistochemical and in situ hybridization techniques. Hum Pathol 27:787-792[Medline]
Ismail T, Howl J, Wheatly M, McMaster P, Neuberger JM, Strain AJ (1991) Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis. Hepatology 14:1076-1082[Medline]
Ishiki Y, Ohnishi H, Muto Y, Matsumoto K, Nakamura T (1992) Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and has a potent anti-hepatitis effect in vivo. Hepatology 16:1227-1235[Medline]
Komminoth P, Adams V, Long AA, Roth J, Saremaslani P, Flury R, Schmid M, Heitz PU (1994) Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR. Pathol Res Pract 190:1017-1025[Medline]
Kruijer W, Skelly H, Botteri R, van der Putten H, Barber J, Verma I, Leffert HL (1986) Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes. J Biol Chem 261:7929-7933
Lambotte L, Saliez A, Triest S, Maiter D, Baranski A, Barker A, Li B (1997) Effect of sialoadenectomy and epidermal growth factor administration on liver regeneration after partial hepatectomy. Hepatology 25:607-612[Medline]
Ljubimova LY, Petrovic LM, Wilson SE, Geller SA, Demetriou AA (1997) Expression of HGF, its receptor c-met, c-myc, and albumin in cirrhotic and neoplastic human liver tissue. J Histochem Cytochem 45:79-87
Marechal H, Jammes H, Rossignol B, Hauduit P (1999) EGF precursor mRNA and membrane-associated EGF precursor protein in rat exorbital lacrimal gland. Am J Physiol 276:C734-746
McGowan JA, Strain AJ, Bucher HL (1981) DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP. J Cell Physiol 108:353-363[Medline]
Mead JE, Fausto N (1989) Transforming growth factor- may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc Natl Acad Sci USA 85:1558-1562
Michalopoulos GK (1990) Liver regeneration: molecular mechanisms of growth control. FASEB J 4:176-187
Michalopoulos GK, De Frances M (1997) Liver regeneration. Science 276:60-66
Milani S, Herbst H, Schuppan D, Stein H, Surrenti C (1991) Transforming growth factors ß1 and ß2 are differentially expressed in fibrotic liver disease. Am J Pathol 139:1221-1229[Abstract]
Mroczkowski B, Reich M (1993) Identification of biologically active epidermal growth factor precursor in human fluids and secretions. Endocrinology 132:417-425[Abstract]
Mullhaupt B, Feren A, Fodor E, Jones A (1994) Liver expression of epidermal growth factor RNA. Rapid increases in immediate early phase of liver regeneration. J Biol Chem 269:19667-19670
Napoli J, Prentice D, Niinami C, Bishop GA, Desmond P, McCaughan GW (1997) Sequential increases in the intra-hepatic expression of epidermal growth factor, basic fibroblast growth factor, and transforming growth factor ß in a bile duct ligated rat model of cirrhosis. Hepatology 26:624-633[Medline]
Oguey D, Marti U, Reichen J (1992) Epidermal growth factor receptor in chronic bile duct obstructed rats: implications for maintenance of hepatocellular mass. Eur J Cell Biol 59:187-195[Medline]
Polimeno L, Azzarone A, Zeng QH, Panella C, Subbotin V, Carr B, Bouzahzah B, Francavilla A, Starzl TE (1995) Cell proliferation and oncogene expression after bile duct ligation in the rat: evidence of a specific growth effect on bile duct cells. Hepatology 21:1070-1078[Medline]
Radinsky R, Bucana CC, Ellis LM, Sanchez R, Cleary KR, Gragiti J, Fidler IJ (1993) A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas. Cancer Res 53:937-943[Abstract]
RuffJamison S, Chen K, Cohen S (1993) Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science 261:1733-1736[Medline]
Schlessinger J (1989) Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci 13:443-447
Speel EJM, Hopman AHN, Komminoth P (1999) Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S). J Histochem Cytochem 47:281-288
St Hilaire RJ, Hradek GT, Jones AL (1983) Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system. Proc Natl Acad Sci USA 80:3797-3801[Abstract]
Thorne BAM, Plowman GD (1994) The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion. Mol Cell Biol 14:1635-1646[Abstract]
Webber EM, Godowski PJ, Fausto N (1994) In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology 2:489-497
Wollenberg GK, Harris L, Farber E, Hayes MA (1989) Inverse relationship between epidermal growth factor induced proliferation and expression of high affinity surface epidermal growth factor receptors in rat hepatocytes. Lab Invest 60:254-259[Medline]
Yamada Y, Kirillova I, Peschon JJ, Fausto N (1997) Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci USA 94:1441-1446