1. Introduction. Journal of Hepatology 39 (2003)

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1 Journal of Hepatology 39 (2003) Role of c-kit receptor tyrosine kinase-mediated signal transduction in proliferation of bile epithelial cells in young rats after ligation of bile duct: a study using Ws/Ws c-kit mutant rats Makoto Satake 1,2, Koichi Shimano 1,3, Takashi Yamamoto 4, Atsuhito Okaya 1,2, Teruo Iwasaki 1, Michiko Kakihana 1, Jiro Fujimoto 2, Nobuyuki Terada 1, Tohru Tsujimura 1, * 1 Department of Pathology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo , Japan 2 Department of Surgery, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo , Japan 3 Department of Otolaryngology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo , Japan 4 Department of Clinical Laboratory, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka , Japan Background/Aims: Recently, it has been shown that the c-kit receptor tyrosine kinase (KIT) is expressed in the liver of young rats, and its expression is up-regulated in bile epithelial cells (BEC) after ligation of the common bile duct (BDL). To clarify a role of KIT in BEC, we examined whether BEC of Ws/Ws rats, whose KIT kinase activity was severely impaired, could proliferate in response to bile stasis after BDL. Methods/Results: When 2-week-old control normal (1/1) and Ws/Ws mutant rats underwent BDL, only a few BEC were found in the portal field of livers of Ws/Ws rats, whereas many BEC were found in that of 1/1 rats. Furthermore, Ki-67 immunostaining showed that the proliferative activity of BEC in 2-week-old Ws/Ws rats was much lower than that in 1/1 rats of the same age. In contrast, when 6-week-old 1/1 and Ws/Ws rats underwent BDL, BEC similarly proliferated in the livers of 1/1 and Ws/Ws rats, and the proliferative activity of BEC was comparable. Conclusions: The mechanism whereby BEC proliferate in response to bile stasis after BDL may be different between 2- and 6-week-old rats, and KIT mediated-signal transduction plays a crucial role in the proliferation of immature BEC in young rats. q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: c-kit receptor tyrosine kinase; Ws/Ws rats; Bile epithelial cells; Bile stasis; Oval cells; Liver regeneration 1. Introduction Bile epithelial cells (BEC) and hepatocytes arise embryologically from a common founder cell, the hepatoblast, which has bipotential differentiation capabilities during liver development [1 3]. In rats, BEC are immature at birth and their development continues until 5 weeks of age, whereas the maturation of hepatocytes is completed within 10 days after birth [3 5]. Recently, it has been shown that the expression of c-kit receptor tyrosine kinase (KIT) and its ligand, stem cell factor (SCF), are seen in the liver of young rats and their expression are up-regulated in the liver of young rats up to 5 weeks of age Received 16 October 2002; received in revised form 28 February 2003; accepted 14 March 2003 * Corresponding author. Tel.: þ ; fax: þ address: tohru@hyo-med.ac.jp (T. Tsujimura). after ligation of the common bile duct (BDL). Moreover, immunohistochemical analysis confirmed the expression of KIT and SCF in BEC of the young rats [5]. On the other hand, in the liver of adult rats, the transcripts for KIT and SCF were barely detectable and no increase was observed after BDL [5]. These observations suggest that KITmediated signal transduction plays a more important role in the bile stasis-induced proliferation of immature BEC than in that of mature BEC. Ws is the first and only mutant allele of the c-kit locus in the rat [6]. The Ws mutant allele carries a 12-base pair deletion in the tyrosine kinase domain [7], and c-kit kinase activity is severely impaired in Ws/Ws mutant rats [8]. Ws/Ws rats show hypoplastic anemia and depletion of mast cells and melanocytes [6]. In addition, we recently showed that the development of hepatic oval cells was remarkably suppressed in Ws/Ws rats in the rat 2-acethylaminofluorene/ /03/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi: /s (03)

2 87 partial hepatectomy (AAF/PH) model, in which the AAF administration inhibits hepatocyte proliferation and the PH induces oval cell proliferation [9]. These results indicate that KIT-mediated signal transduction plays a crucial role in hematopoiesis, melanogenesis, and the development of oval cells. Thus, Ws/Ws rats are considered as useful animals for examining the biological role of the SCF/KIT system in vivo. In the present study, to clarify the role of KITmediated signal transduction in the proliferation of immature and mature BEC, we examined the growth of BEC in 2- and 6-week-old control normal (þ/þ) and Ws/Ws rats after BDL. 2. Materials and methods 2.1. Rats and treatments Male þ/þ and Ws/Ws rats were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). The origin of Ws/Ws rats has been described previously [6,7]. The þ/þ and Ws/Ws rats were each divided into two groups. One group was subjected to BDL. BDL was carried out as follows: laparotomy was performed by a longitudinal section, and the common bile duct was isolated, doubly ligated, and resected between the ligatures [4,5]. The other group received a sham operation (SHAM) which was the same procedure excepting ligation and resection of the common bile duct. On day 4 after the operation, bloods were taken from the right ventricle of hearts, and sera were prepared and stored at 2808C until analysis. Livers were promptly removed from the rats for the histological examination. Total bilirubin and alanine aminotransferase (ALT) in the sera were measured by standard laboratory techniques. All surgical procedures were done under sevoflurane anesthesia. All experimental procedures were approved by the Animal Care Committee of Hyogo College of Medicine, and were performed in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science Histological examination Liver tissues were fixed with methacarn (methanol:chloroform:glacial acetic acid, 6:3:1), embedded in paraffin, and cut into 5-mm thick sections; one section was stained with hematoxylin and eosin and the other sections were used for the immunohistological study. For Ki-67 and c-kit immunostaining, sections were heated in 10 mm sodium citrate buffer (ph 6.0) for 10 and 45 min, respectively, to facilitate antigen retrieval. For immunohistochemistry, sections were incubated in 0.3% H 2 O 2 in methanol for 30 min to inactivate endogenous peroxidase, washed with Tris-buffered saline/tween solution (50 mm Tris HCl, 300 mm NaCl, and 0.1% tween 20, ph 7.6), and incubated with normal goat serum or normal rabbit serum to block non-specific binding of antibodies. The sections were then incubated at 48C overnight with either a mouse monoclonal antibody against human cytokeratin (CK)-19 (Novocastra Laboratories, Ltd., Newcastle, UK), that was a marker for BEC, diluted 1:40 in Tris-buffered saline/tween solution, a rabbit polyclonal antibody against human Ki-67 nuclear antigen (Novocastra Laboratories Ltd, Benton Lane, UK), that was expressed on all proliferating cells during late G1, S, M, and G2 phases of the cell cycle, diluted 1:500, or a goat polyclonal antibody against human c- kit peptide (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:25. After washing with Tris-buffered saline/tween solution, the sections were incubated with Histofine Simple Stain MAX-PO(M), MAX-PO(R), or MAX-PO(G) (Nichirei Corporation, Tokyo, Japan) for 60 min, and immunoreacted cells were then visualized with Simple Stain DAB Solution (Nichirei). The sections were lightly counterstained with hematoxylin. The number of CK-19 positive cells was counted in twenty portal fields selected randomly in each CK-19 immunostained specimen, and the value was expressed as the number per portal field ( mm 2 ). BEC (500, 1000) in each Ki-67 immunostained specimen were examined with a light microscope to determine the percentage of BEC expressing Ki-67 antigen (Ki-67 labeling index) Statistical analysis Statistical analysis for the values of serum total bilirubin and ALT was performed by using the non-parametric tests Kruskal Wallis and the corrected Mann Whitney tests for multiple group comparisons. Values are expressed as mean ^ SE, and a two-tailed P, 0:05 was considered statistically significant. Statistical analysis for the number of CK-19 positive cells and Ki-67 labeling index was performed by unpaired, twotailed Student s t-test. A P value below 0.05 was considered significant. 3. Results 3.1. Proliferation of BEC induced by BDL in 2-week-old þ/þ and Ws/Ws rats The concentrations of total bilirubin and the values of ALT of 2-week-old þ/þ and Ws/Ws rats were examined on day 4 after SHAM or BDL. The concentrations of total bilirubin of SHAM-2-week-old þ/þ and Ws/Ws rats were less than 0.3 mg/dl. When BDL was carried out, the concentrations of bilirubin strikingly increased in both BDL-2-week-old þ/þ and Ws/Ws rats, and the values (means ^ SE) were 7.5 ^ 1.1 mg/dl (n ¼ 6) and 7.9 ^ 0.8 mg/dl (n ¼ 6), respectively. There was no significant difference between these two values (Fig. 1). The values (means ^ SE) of ALT in SHAM-2-week-old þ/þ and Ws/Ws rats were 34.3 ^ 1.9 IU/l (n ¼ 3) and 27.8 ^ 2.3 IU/l (n ¼ 3), respectively, with no significant difference between the two values. When BDL was carried out, the values of ALT increased in both 2-week-old þ/þ and Ws/Ws rats, and the values were ^ 20.0 IU/l (n ¼ 6) and ^ 25.0 IU/l (n ¼ 6), respectively. There was no significant difference between the two values (Fig. 1). These results show that BDL induces bile stasis and liver damage similarly in both 2-week-old þ/þ and Ws/Ws rats. Figs. 2A D shows liver sections of 2-week-old þ/þ and Ws/Ws rats on day 4 after BDL. In the liver of BDL-2-weekold þ/þ rats, many BEC were observed in the portal field (Figs. 2A,B). In contrast, far fewer BEC were observed in the liver of BDL-2-week-old Ws/Ws rats (Figs. 2C,D). As shown in Fig. 3, numbers (means ^ SE) of CK-19 positive cells per portal field ( mm 2 ) in SHAM-2-week-old þ/þ and Ws/Ws rats were 25 ^ 1 (n ¼ 3) and 25 ^ 2 (n ¼ 3), respectively. There was no significant difference between these two values. In contrast, numbers of CK-19 positive cells per portal field in BDL-2-week-old þ/þ and Ws/Ws rats were 281 ^ 25 (n ¼ 6) and 56 ^ 4(n ¼ 6), respectively (Fig. 3). These results indicate that the number of CK-19 positive cells per portal field in BDL-2-week-old Ws/Ws rats is significantly lower than that in BDL-2-weekold þ/þ rats. When numbers of CK-19 positive cells per portal field were compared between SHAM- and BDL-2- week-old rats, there was significant difference between

3 88 Fig. 1. Effects of BDL on serum bilirubin and ALT in 1/1 and Ws/Ws rats. (A) Total bilirubin; and (B) ALT. Rats at the age of 2 or 6 weeks underwent SHAM (S) or BDL (B), and the concentrations of serum bilirubin and the values of serum ALT were measured on day 4 after operations. *P <0.05, significant difference by the corrected Mann Whitney tests. these two values in both þ/þ and Ws/Ws rats (Fig. 3). These results show that BEC proliferate remarkably in BDL-2-week-old þ/þ rats, but slightly in BDL-2-week-old Ws/Ws rats. Next, the proportion of proliferating BEC in 2-week-old þ/þ and Ws/Ws rats on day 4 after SHAM or BDL was examined by immunostaining with Ki-67. Ki-67 labeling indices (means ^ SE) in SHAM-2-week-old þ/þ and Ws/Ws rats were 9.1 ^ 0.5 (n ¼ 3) and 9.7 ^ 0.9 (n ¼ 3), respectively. There was no significant difference between these two values. In contrast, Ki-67 labeling indices in BDL-2-week-old þ/þ and Ws/Ws rats were 29.4 ^ 1.1 (n ¼ 6) and 17.3 ^ 1.6 (n ¼ 6), respectively (Fig. 4). These results indicate that the proliferative activity of BEC in BDL-2-week-old Ws/Ws rats is significantly lower than that in BDL-2-week-old þ/þ rats. When Ki-67 labeling indices were compared between SHAM- and BDL-2-week-old rats, there was significant difference between these two values in both þ/þ and Ws/Ws rats (Fig. 4). These results show that the proliferative activity of BEC increases remarkably in 2-week-old þ/þ rats after BDL, but slightly in 2-week-old Ws/Ws rats after BDL Proliferation of BEC induced by BDL in 6-week old þ/þ and Ws/Ws rats The concentrations of total bilirubin in SHAM-6-weekold þ/þ and Ws/Ws rats were under a detectable level. When BDL was carried out, the concentrations of bilirubin (means ^ SE) increased to 6.1 ^ 0.7 mg/dl (n ¼ 6) and 5.8 ^ 0.2 mg/dl (n ¼ 6) in BDL-6-week-old þ/þ and Ws/Ws rats, respectively. There was no significant difference between these two values (Fig. 1). The values (means ^ SE) of ALT in SHAM-6-week-old þ/þ and Ws/Ws rats were 50.0 ^ 2.3 IU/l (n ¼ 3) and 49.3 ^ 1.8 IU/l (n ¼ 3), respectively. When BDL was carried out, the values of ALT increased to ^ 21.0 IU/l (n ¼ 6) and ^ 21.9 IU/l (n ¼ 6) in 6-week-old þ/þ and Ws/Ws rats, respectively. There was no significant difference between these two values (Fig. 1). These results show that BDL induces bile stasis and liver damage similarly in both 6-week-old þ/þ and Ws/Ws rats. Figs. 2E H shows liver sections of 6-week-old þ/þ and Ws/Ws rats on day 4 after BDL. Many BEC and CK-19 positive cells were observed in the portal field of both BDL- 6-week-old þ/þ and Ws/Ws rats. As shown in Fig. 3, numbers of CK-19 positive cells per portal field (means ^ SE) in SHAM-6-week-old þ/þ and Ws/Ws rats were 23 ^ 3 cells (n ¼ 3) and 30 ^ 6 cells (n ¼ 3), respectively, with no significant difference between the two values. When BDL was carried out, the numbers of CK-19 positive cells per portal field increased to 188 ^ 13 cells (n ¼ 6) and 185 ^ 17 cells (n ¼ 6) in BDL-6-week-old þ/þ and Ws/ Ws rats, respectively (Fig. 3). These results indicate that BDL increases the number of CK-19 positive cells in 6-week-old þ/þ and Ws/Ws rats to similar levels. Ki-67 labeling indices (means ^ SE) of BEC in SHAM- 6-week-old þ/þ and Ws/Ws rats were 5.2 ^ 0.4 (n ¼ 3) and 4.7 ^ 0.5 (n ¼ 3), respectively, with no significant difference between the two values. When BDL was carried out, Ki-67 labeling indices increased to 32.4 ^ 1.7 (n ¼ 6) and 31.7 ^ 1.2 (n ¼ 6) in 6-week-old þ/þ and Ws/Ws rats, respectively (Fig. 4). These results show that the proliferative activity of BEC in BDL-6-week-old Ws/Ws rats is similar to that of BDL-6-week-old þ/þ rats Expression of KIT in BEC of 2-week-old rats after BDL We failed to detect the expression of KIT immunohistochemically in BEC of either 2- or 6-week-old þ/þ and

4 89 Fig. 2. Liver sections of 1/1 and Ws/Ws rats that underwent BDL. (A, B) BDL-2-week-old 1/1 rat; (C, D) BDL-2-week-old Ws/Ws rat; (E, F) BDL-6week-old 1/1 rat; and (G, H) BDL-6-week-old Ws/Ws rat. Sections (A, C, E, and G) were stained with hematoxylin-eosin and their adjacent sections (B, D, F, and H) were immunostained with CK-19 antibody (original magnification 3 160). Ws/Ws rats that underwent SHAM. In addition, KIT expression was hardly detectable in BEC of 6-week-old þ /þ and Ws/Ws rats even after BDL (data not shown). However, KIT expression was clearly observed in BEC of both BDL-2-week-old þ /þ and Ws/Ws rats, although the number of BEC of BDL-2-week-old Ws/Ws rats was much lower than that of BDL-2-week-old þ /þ rats (Fig. 5). These results show that KIT expression is similarly

5 90 Fig. 3. Numbers of CK-19 positive cells in the portal field of the livers of 1/1 and Ws/Ws rats. Rats at the age of 2 or 6 weeks underwent SHAM (S) or BDL (B), and numbers of CK-19 positive cells per portal field ( mm 2 ) were counted on day 4 after operations. Bars indicate means 6 SE. *P <0.05, significant difference by unpaired, two-tailed Student s t-test. up-regulated in immature BEC of 2-week-old þ/þ and Ws/ Ws rats after BDL. 4. Discussion In this study, to clarify a role of KIT-mediated signal transduction in BEC, we examined whether BEC of Ws/Ws rats, having impaired c-kit kinase activity due to deletion of 12 bases in the kinase domain [6 8], could proliferate in response to bile stasis after BDL. In the livers of BDL-2- week-old Ws/Ws rats, only a few BEC were observed in the portal field, whereas many BEC were observed in the livers of BDL-2-week-old þ/þ rats. The Ki-67 labeling index of BEC in BDL-2-week-old Ws/Ws rats was also much lower than that of BDL-2-week-old þ/þ rats. Therefore, the SCF/KIT system appears to play a crucial role in the BDLinduced proliferation of immature BEC in 2-week-old rats. KIT-mediated signal transduction probably promotes the cell cycle of immature BEC of young rats. Both the number and the Ki-67 labeling index of BEC in BDL-2-week-old Ws/Ws rats were significantly lower than those in BDL-2-week-old þ/þ rats. However, they were slightly but significantly higher than those in SHAM-2- week-old Ws/Ws rats. The Ws mutant allele of rats carries a 12-base deletion at the kinase domain of the c-kit gene and four amino acids encoded by the deleted 12 bases are located at two amino acids downstream from the tyrosine autophosphorylation site [7]. A weak but detectable level of c-kit kinase activity, however, has been found in the lysate of cultured mast cells derived from bone marrow cells of Ws/Ws rats [8]. Therefore, the residual kinase activity of KIT may participate in the growth of a few immature BEC in 2-week-old Ws/Ws rats after BDL. In the livers of BDL-6-week-old Ws/Ws rats, marked proliferation of BEC was observed, as was the case in BDL- 6-week-old þ/þ rats. The Ki-67 labeling index of BEC of BDL-6-week-old Ws/Ws rats was also similar to that of BDL-6-week-old þ/þ rats. These observations show that the impairment of KIT-mediated signal transduction does not affect the proliferation of mature BEC in 6-week-old rats. Recently, Omori et al. reported that bile ducts newly proliferated after BDL in 10-week-old W/W v and Sl/Sl d mice that have the mutations of KIT and SCF, respectively, to an extent similar to that in þ/þ mice [10]. Therefore, it is likely that KIT-mediated signal transduction is not essential for the proliferation of mature BEC irrespective of animal species. These investigators also showed that the expression of both flt-3 ligand and flt-3 receptor was significantly elevated in 10-week-old þ/þ, W/W v, and Sl/Sl d mice after BDL and that the transcripts for the flt-3 receptor localized in BEC [10]. These findings suggest that signal transduction other than the SCF/KIT system, such as the flt-3 ligand and flt-3 receptor system, may play a role in the proliferation of mature BEC in 6-week-old Ws/Ws rats and 10-week-old W/W v and Sl/Sl d mice after BDL. The SCF/KIT system is believed to play an important role in stem cell biology during hematopoiesis, gametogenesis, and melanogenesis [11 13]. In the adult rat liver, KIT is expressed in oval cells that are regarded to be progeny of the stem cell compartment [9,14,15]. It has been suggested that oval cells originate from the terminal ductule cells connecting the canals of Hering or a population of periductular cells [1,16 19], but a recent report indicates that the bone marrow is a potential source of oval cells [20]. Previously, we have demonstrated that the development of oval cells is remarkably suppressed in Ws/Ws rats in the rat AAF/PH model, suggesting that KIT mediated-signal transduction plays a crucial role in the development of oval Fig. 4. Ki-67 labeling indices of BEC of 1/1 and Ws/Ws rats. Rats at the age of 2 or 6 weeks underwent SHAM (S) or BDL (B), and Ki-67 labeling indices were examined on day 4 after operations. Bars indicate means 6 SE. *P <0.05, significant difference by unpaired, two-tailed Student s t-test.

6 91 up-regulated after BDL. Then, SCF produced by BEC is bind to KIT expressed on BEC in autocrine and/or paracrine fashion(s), leading to activation of the Ras/Raf/MAPK pathway and proliferation of BEC. In older rats, SCF and KIT expression is severely reduced and consequently the Ras/Raf/MAPK pathway is only weakly activated via KITmediated signal transduction; KIT-mediated signal transduction may not participate in BEC proliferation in older rats. In summary, the present study shows that KIT mediatedsignal transduction plays a crucial role in the proliferation of immature BEC, but not mature BEC, after BDL, and suggests that immature BEC respond to bile stasis in a fashion phenotypically similar to that seen in early stages of oval cell activation. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Pancreas Research Foundation of Japan. We thank Dr Hidekazu Asai of Japan SLC (Hamamatsu, Shizuoka, Japan) for his valuable advices and Mr Hirotsugu Kubo, Mr Masato Yagi, and Mr Susumu Tanaka (Hyogo College of Medicine) for their technical assistance. References Fig. 5. Expression of KIT in BEC of BDL-2-week-old rats. (A) BDL-2- week-old 1/1 rat; and (B) BDL-2-week-old Ws/Ws rat. Each liver section of 2-week-old 1/1 and Ws/Ws rats that underwent BDL was immunostained with KIT antibody. Arrows indicate BEC immunoreacted with KIT antibody (original magnification 3 183). cells [9]. The present study demonstrates that the mechanism whereby BEC proliferate in response to bile stasis after BDL is different between 2- and 6-week-old rats, and that KIT mediated-signal transduction plays a crucial role only in the proliferation of immature BEC in young rats. Therefore, it is likely that immature BEC of 2-week-old rats respond to growth stimulus such as that seen following bile stasis in a fashion similar to that seen in early stages of oval cell activation. The SCF/KIT system appears to commonly participate in development of new bile ducts from immature BEC and oval cells from the stem cell compartment. The mechanism whereby KIT activity controls the proliferation of BEC in young rats after BDL is unknown. A series of studies indicated that mitogen-activated protein kinase (MAPK) was involved in KIT-mediated signal transduction [21,22]. In addition, BDL was recently shown to induce BEC proliferation via an activation of MAPK [23]. These results suggest the following mechanism for the proliferation of BEC controlled by KIT. First, in young rats, the expression of SCF and KIT in BEC is [1] Grisham JW, Thorgeirsson SS. Liver stem cells. In: Potten CS, editor. Stem cells. New York: Academic Press; p [2] Le Douarin NM. An experimental analysis of liver development. Med Biol 1975;53: [3] Shiojiri N, Lemire JM, Fausto N. Cell lineages and oval cell progenitors in rat liver development. Cancer Res 1991;51: [4] Van Eyken P, Sciot R, Desmet V. Intrahepatic bile duct development in the rat. Lab Invest 1988;59:52 9. [5] Omori M, Evarts RP, Omori N, Hu Z, Marsden ER, Thorgeirsson SS. Expression of a-fetoprotein and stem cell factor/c-kit system in bile duct ligated young rats. Hepatology 1997;25: [6] Niwa Y, Kasugai T, Ohno K, Morimoto M, Yamazaki M, Dohmae K, et al. Anemic and mast cell depletion in mutant rats that are homozygous at White Spotting (Ws) locus. Blood 1991;78: [7] Tsujimura T, Hirota S, Nomura S, Niwa Y, Yamazaki M, Tono T, et al. Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene. Blood 1991;78: [8] Tei H, Kasugai T, Tsujimura T, Adachi S, Furitsu T, Tohya K, et al. Characterization of cultured mast cells derived from Ws/Ws mast celldeficient rats with a small deletion at tyrosine kinase domain of c-kit. Blood 1994;83: [9] Matsusaka S, Tsujimura T, Toyosaka A, Nakasho K, Sugihara A, Okamoto E, et al. Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model. Hepatology 1999;29: [10] Omori M, Omori N, Evarts RP, Teramoto T, Thorgeirsson SS. Coexpression of flt-3 ligand/flt-3 and SCF/c-kit transduction system in bile-duct-ligated Sl and W mice. Am J Pathol 1997;150: [11] Russell ES. Hereditary anemias of the mouse: a review of geneticists. Adv Genet 1979;20:

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