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Basic–Liver, Pancreas, and Biliary Tract

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Basic–Liver, Pancreas, and Biliary Tract GASTROENTEROLOGY 2005;128:424–432 BASIC–LIVER, PANCREAS, AND BILIARY TRACT Amphiregulin: An Early Trigger of Liver Regeneration in Mice CARMEN BERASAIN,* ELENA R. GARCÍA–TREVIJANO,* JOSEFA CASTILLO,* ELENA ERROBA,* DAVID C. LEE,‡ JESÚS PRIETO,* and MATÍAS A. AVILA* *Division of Hepatology and Gene Therapy, Centro de Investigación Médica Aplicada, Facultad de Medicina, Universidad de Navarra, Pamplona, Spain; and ‡Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina This complex response aimed at the restoration of liver- See editorial on page 504. dependent homeostasis is mediated by a network of cytokines, comitogens, and growth factors in a coordi- Background & Aims: Liver regeneration is a unique nated multistep process.2,3 Many of the mediators be- response directed to restore liver mass after resection lieved to be critical in the regenerative response to injury or injury. The survival and proliferative signals trig- or resection in animals are also expressed in humans gered during this process are conveyed by a complex during liver regeneration, thus suggesting the preserva- network of cytokines and growth factors acting in an 4 orderly manner. Activation of the epidermal growth tion of fundamental mechanisms across species. factor receptor is thought to play an important role in Despite the intensive research performed in the past liver regeneration. Amphiregulin is a member of the few decades, the molecules and mechanisms involved in epidermal growth factor family whose expression is the physiological adaptive response to liver injury are not not detectable in healthy liver. We have investigated completely known. We have recently observed that the the expression of amphiregulin in liver injury and its expression of the Wilms’ tumor suppressor 1 (WT1) gene role during liver regeneration after partial hepatec- is induced in the livers of patients with hepatocellular tomy. Methods: Amphiregulin gene expression was damage and in the livers of CCl -treated rats.5 WT1 examined in healthy and cirrhotic human and rat liver, 4 in rodent liver regeneration after partial hepatectomy, encodes a zinc finger transcription factor that can regu- and in primary hepatocytes. The proliferative effects late the expression of a diversity of growth- and differ- and intracellular signaling of amphiregulin were stud- entiation-related genes.6 One of the major physiological ied in isolated hepatocytes. The in vivo role of amphi- targets directly induced by WT1 is amphiregulin (AR).7 regulin in liver regeneration after partial hepatectomy AR is a polypeptide growth factor of the epidermal was analyzed in amphiregulin-null mice. Results: Am- growth factor (EGF) family and a ligand of the EGF phiregulin gene expression is detected in chronically receptor (EGF-R) that was originally isolated from con- injured human and rat liver and is rapidly induced ditioned medium of MCF-7 human breast carcinoma after partial hepatectomy in rodents. Amphiregulin 8 expression is induced in isolated hepatocytes by in- cells treated with phorbol esters. Activation of the EGF-R in the hepatocyte is thought to play an important terleukin 1␤ and prostaglandin E2, but not by hepato- cyte growth factor, interleukin 6, or tumor necrosis role during the early stages of liver regeneration after factor ␣. We show that amphiregulin behaves as a primary mitogen for isolated hepatocytes, acting Abbreviations used in this paper: AR, amphiregulin; BrdU, 5-bromo- through the epidermal growth factor receptor. Finally, 2=-deoxyuridine; CRE, cyclic adenosine monophosphate–responsive el- amphiregulin-null mice display impaired proliferative ement; EGF, epidermal growth factor; EGF-R, epidermal growth factor responses after partial liver resection. Conclusions: receptor; ERK, extracellular regulated kinase; FCS, fetal calf serum; HB-EGF, heparin-binding EGF-like growth factor; HGF, hepatocyte Our findings indicate that amphiregulin is an early- growth factor; IL, interleukin; JNK, c-Jun protein kinase; MEK, extracel- response growth factor that may contribute to the lular regulated kinase kinase; p38 MAPK, p38 mitogen-activated pro- initial phases of liver regeneration. tein kinase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PGE2, prostaglandin E2PH, partial hepatectomy; PI-3K, phosphatidylinositol 3-kinase; TGF, transforming growth factor; TNF, ytoprotective and regenerating mechanisms are set tumor necrosis factor; WT1, Wilms’ tumor suppressor 1. © 2005 by the American Gastroenterological Association Cin motion in the liver after loss of parenchymal mass 0016-5085/05/$30.00 due to partial hepatectomy (PH) or after tissue injury.1–3 doi:10.1053/j.gastro.2004.11.006 February 2005 ROLE OF AMPHIREGULIN IN HEPATOCYTE PROLIFERATION 425 PH, and the levels of EGF-R ligands such as EGF and Isolation, Culture, and Treatment transforming growth factor (TGF)-␣ are increased after of Rat Hepatocytes 1–4 liver resection. The expression of AR is tissue specific: Hepatocytes were isolated from male Wistar rats (150 g) it is most prevalent in human ovary and placenta but is by collagenase (Gibco-BRL, Paisley, UK) perfusion as described undetectable in the healthy and quiescent liver paren- previously.15 Cells were plated onto collagen-coated 6-well dishes chyma.9 Like EGF and TGF-␣, AR is synthesized as a (type I collagen; Collaborative Biomedical, Bedford, MA; 5 ϫ 105 transmembrane precursor that is proteolytically pro- cells per well). Cultures were maintained in minimal essential cessed to the secreted mature form.10 AR shows bifunc- medium supplemented with 10% fetal calf serum (FCS), nones- tional properties, stimulating the proliferation of a vari- sential amino acids, 2 mmol/L glutamine, and antibiotics (all ety of normal cells and inhibiting that of many tumor from Gibco-BRL). After 2 hours, incubation medium was re- cell lines.7,9,11 Together these observations prompted us moved, and cells were refed the same medium with 0.5% FCS. to examine AR gene expression in liver injury and during Where indicated, hepatocytes were treated with interleukin (IL)-1␤ or tumor necrosis factor (TNF)-␣ from Roche (Mann- experimental liver regeneration. We show here that the heim, Germany), hepatocyte growth factor (HGF) or forskolin expression of AR is detected in the liver of cirrhotic from Calbiochem (San Diego, CA), IL-6 from R&D Systems patients and is rapidly induced in rodent liver after PH. (Wiesbaden-Nordenstadt, Germany), or prostaglandin E2 (PGE2) In addition, we show that AR behaves as a primary from Alexis (Carlsbad, CA). mitogen for the hepatocyte. A role for AR in the early steps of liver regeneration is supported by the impaired Transient Transfection of Rat Hepatocytes proliferative response of AR-null mice to PH. Our find- We seeded rat hepatocytes 24 hours before transfection ings identify a novel function for AR in the liver and with the Tfx-50 reagent (Promega, Madison, WI) according to suggest that this molecule may have therapeutic poten- the manufacturer’s instructions, and cells were transfected in tial to stimulate liver regeneration in cases of acute liver duplicate with an equimolar mixture of pCB6 plasmids en- injury. coding the 4 isoforms of WT1 (characterized by the presence or absence of the splice inserts, exon 5, and lysine, threonine, Materials and Methods serine [KTS]) or with an equivalent amount of the empty pCB6 vector, provided by Dr. Jochemsen (Leiden University Patients Medical Center, Leiden, The Netherlands). Transfection effi- Liver tissue samples were from 2 groups of subjects. ciency was monitored by reverse-transcription polymerase The first group comprised control individuals (n ϭ 26; 19 chain reaction (PCR) analysis by using specific primers that men; mean age, 50.8 years; range, 18–73 years) with normal discriminated these 4 isoforms. or minimal changes in the liver. We collected tissue samples at Assay of DNA Synthesis surgery (16 cases) of digestive tumors or from percutaneous liver biopsy performed because of mild alteration of liver Rat hepatocytes were plated at a density of 3 ϫ 104 function tests (10 cases). The second group comprised subjects cells per well in collagen-coated 96-well plates in minimal with liver cirrhosis (n ϭ 29; 24 men; mean age, 56 years; essential medium supplemented with 10% FCS. Five hours range, 36–77 years) that was due to hepatitis C virus infection after plating, medium was changed, and cells were kept in the in 8 cases, alcohol abuse in 13 cases, hepatitis B virus infection absence of serum for 20 hours more. DNA synthesis was in 3 cases, autoimmune hepatitis in 3 cases, and hemochro- measured in response to AR for 30 hours. A pulse of [3H]thy- matosis in 1 case and that was cryptogenic in another case. midine (1 ␮Ci per well; Amersham Biosciences, Piscataway, Associated hepatocellular carcinoma was present in 10 cir- NJ) started 22 hours after AR addition. Cells were harvested, rhotic patients. This study was approved by the Human Re- and [3H]thymidine incorporation was determined in a scintil- search Review Committee of the University of Navarra. lation counter. The effect of AR on DNA synthesis was also tested in the presence of TGF-␤ (Roche) or the extracellular Animal Models regulated kinase kinase (MEK)-1 inhibitor PD98059, the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY-294002, Liver cirrhosis was induced with CCl4 in male Wistar rats (150 g) as previously described.12 Two-thirds PH or sham the c-Jun protein kinase (JNK) inhibitor SP600125, the p38 operations were performed in male Wistar rats (150 g) and mitogen-activated protein kinase (p38 MAPK) inhibitor male ARϩ/ϩ and ARϪ/Ϫ littermate mice13 (20 g) as previously SB202190, and the EGF-R inhibitor PD153035, all from described.14,15 The ARϪ/Ϫ mice, as described previously, are Calbiochem. 13 viable and display no overt phenotype. One hour before RNA Isolation and Analysis of being killed, mice were injected intraperitoneally with 50 Gene Expression mg/kg of 5-bromo-2=-deoxyuridine (BrdU; Sigma, St. Louis, MO). This study was approved by our institution’s Animal Total RNA was extracted by using the TRI Reagent Welfare Committee.
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