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From the Departments of Pathology,* Surgery, and Pharmacology, Hyogo College of Medicine, Hyogo; the Department of Biochemistry, Akita University School of Medicine, Akita; and the Institute of Molecular and Cellular Biosciences,¶ University of Tokyo, Tokyo, Japan
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-fetoprotein (AFP) and cytokeratin (CK)-19, respectively.5
They also share characteristics with hematopoietic stem cells, expressing c-kit receptor tyrosine kinase, CD34, and Thy-1.10-13
Oval cells are presumed to be hepatic progenitors in the adult liver and participate in the liver regeneration when the replicative and functional capacity of hepatocytes is impaired.2,3,5-8
However, the molecular mechanism underlying the differentiation of oval cells into hepatocytes remains to be established. Oncostatin M (OSM) is a member of the interleukin-6 cytokine family that includes interleukin-6, interleukin-11, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1, and novel neutrophin-1/B-cell-stimulating factor-3.14-16 Mouse OSM receptor is composed of the gp130 subunit, common to all of the interleukin-6 family cytokines,15 and the OSM-specific subunit17 (hereafter this subunit is called OSM-specific receptor: OSM-R). Ligand binding to the receptor complex activates the Janus tyrosine kinases (Jak1, Jak2, and Tyk2) and the activated Jaks in turn activate downstream pathways, such as SHP-2 tyrosine phosphatase and signal transducer and activator of transcription protein 3.15 Recently, OSM has been shown to induce maturation of mouse fetal hepatocytes derived from the embryonic day 14.5 liver as characterized by the expression of tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, and the accumulation of glycogen.18 OSM in combination with extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma has also been shown to induce tryptophan oxygenase (TO), a liver-specific enzyme expressed by fully matured hepatocytes, in mouse fetal hepatocytes.19 In addition, human fetal hepatocytes, like mouse fetal hepatocytes, have been reported to be induced to differentiate by OSM.20 These results indicate that OSM is a crucial factor for liver development involving maturation of fetal hepatocytes. Because oval cells, like fetal hepatocytes, are believed to represent hepatic progenitors, there is a possibility that OSM may play an important role in liver regeneration involving differentiation of oval cells. To investigate this possibility, we isolated rat OSM-R and OSM cDNA and examined whether OSM induces the differentiation of rat oval cells, OC15-5, into hepatocytes.
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Materials and Methods |
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Oval cells were induced in male Fischer 344 rats of 6 weeks of age (SLC, Hamamatsu, Japan) by the method of the AAF/PH model.11 Briefly, AAF (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was daily administered to rats by gavage at 10 mg/kg body weight for 4 days. On day 5, a standard two-thirds PH was performed, and then the daily administration of AAF at the same dosage continued for 5 days. All animal studies 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.
Preparation of Parenchymal and Nonparenchymal Cells
Parenchymal and nonparenchymal cells of the liver were isolated by the two-step collagenase digestion method with some modifications, as previously described.21
Isolation of Rat OSM-R and OSM cDNA
To obtain rat OSM-R cDNA, RNA was prepared from the liver on day 7 after PH in the AAF/PH model using TRIzol reagent (Life Technologies, Inc., Grand Island, NY), reverse-transcribed by Superscript II reverse transcriptase (RT) (Life Technologies, Inc.) and an anchor primer (5'-GCAATTAACCCTCACTAAAGAATTCAGTCAGTCA(T)17-3'), and amplified by polymerase chain reaction (PCR) using Taq Ex (Takara Shuzou, Kyoto, Japan) and four sets of primers (rFW1 and rRV1, rFW2 and rRV2, rFW3 and rRV3, and rFW4 and rRV4) (Table 1)
with 25 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute. Nested PCR was performed using rFW5 and slica1 as the first set of primers and rFW6 and slica2 as the second set of primers (Table 1)
to obtain the 3'-end of rat OSM-R cDNA. To obtain rat OSM cDNA, RNA was similarly prepared from the fetal liver on embryonic day 14.5, reverse-transcribed, and amplified by PCR using Taq Ex and two sets of primers (FW1 and RV1 and FW2 and RV2) (Table 1)
. Nested PCR was performed using FW3 and slica1 as the first set of primers and FW4 and slica2 as the second set of primers (Table 1)
to obtain the 3'-end of rat OSM cDNA. The products were subcloned into the EcoRV site of Bluescript I KS(–) (Stratagene, La Jolla, CA), and analyzed for nucleotide sequences.
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Northern blot analysis was performed as previously described.21 32P-labeled rat OSM-R and glyceraldehyde-3-phosphate dehydrogenase cDNA were used as probes. Analysis of OSM-R and OSM mRNA in various tissues was conducted using a membrane of rat poly A+ RNA (2 µg) Northern blot-12 major tissues (OriGene Technologies, Inc., Rockville, MD) and the OSM-R and OSM probes.
Number of Oval Cells
Liver tissues were fixed with methacarn, embedded in paraffin, and cut into 5-µm-thick sections. The sections were immunostained with mouse anti-human CK-19 antibody (Novocastra Laboratories, Ltd., Newcastle, UK) diluted 1:40. The number of CK-19-positive oval cells was counted in 10 periportal fields selected randomly in each CK-19-immunostained specimen and expressed as the number per periportal field.
In Situ Hybridization
Liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm-thick sections. One section was used for in situ hybridization and the adjacent section was stained with hematoxylin and eosin. Complementary RNA probes of OSM-R were prepared by using a DIG RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Signals were visualized with a DAKO Gen Point kit (DAKO, Glostrup, Denmark) using the horseradish peroxidase-conjugated anti-digoxigenin antibody (DAKO).
Confocal Laser-Scanning Microscope
For double staining for OSM-R and CK-19, nonparenchymal cells were prepared from the liver on day 7 after PH in the AAF/PH model, cytocentrifuged, and fixed with methacarn. For double staining for OSM and CK-19 and that for OSM and ED2, liver tissues on day 7 after PH in the AAF/PH model were fixed with methacarn, embedded in paraffin, and cut into 5-µm-thick sections. The cytocentrifuged specimens and liver sections were incubated with goat anti-mouse OSM-R antibody (R&D Systems, Minneapolis, MN) diluted 1:50 or goat anti-mouse OSM antibody (R&D Systems) diluted 1:50 at 4°C overnight. Then, they were sequentially incubated with biotin-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 for 60 minutes, horseradish peroxidase-conjugated streptavidin diluted 1:100 for 30 minutes, biotinyl tyramine diluted 1:50 for 10 minutes, and fluorescein isothiocyanate-conjugated streptavidin (DAKO) diluted 1:40 for 30 minutes. After washing, the cytocentrifuged specimens were incubated with mouse anti-human CK-19 antibody diluted 1:40, and the liver sections were incubated with mouse anti-human CK-19 antibody diluted 1:40 or mouse anti-rat ED2 antibody (Serotec Ltd., Oxford, UK) diluted 1:500 for 60 minutes. They were then incubated with R-phycoerythrin-conjugated goat anti-mouse IgG (DAKO) diluted 1:100 for 60 minutes. Co-localization of fluorescein isothiocyanate and R-phycoerythrin was analyzed with a confocal laser-scanning microscope (LSM510; Carl Zeiss Jena GmbH, Jena, Germany).
Flow Cytometry
Cells were incubated with rabbit anti-c-kit antibody (Santa Cruz), rabbit anti-CD34 antibody (Santa Cruz), rabbit anti-Thy-1 antibody (Santa Cruz), or goat anti-OSM-R antibody at 4°C for 30 minutes. After washing, cells were incubated with biotin-conjugated anti-rabbit IgG antibody (Santa Cruz) or biotin-conjugated anti-goat IgG antibody (Santa Cruz). Cells were then stained with R-phycoerythrin-streptavidin (BD PharMingen, San Diego, CA) and analyzed on a FACSCalibur (Becton Dickinson, Los Angeles, CA). All antibodies used were diluted 1:100.
Preparation of Full-Length Rat OSM cDNA and Conditioned Media
The entire coding region of rat OSM cDNA was generated by RT-PCR with a set of 5'-ATGCGGGCTCAGCCTCCACCGAGAA-3' and 5'-TTACCGGGGCACCAGGGACCTGAGCATG-3' primers and was subcloned into the expression vector pEF-BOS. The pEF-BOS DNA with or without rat OSM cDNA (10 µg) was transfected into 293T cells by the calcium phosphate method.22 Transfectants with the pEF-BOS DNA with or without rat OSM cDNA were cultured in Dulbecco’s modification of Eagle’s medium (ICN Biomedicals Inc., Aurora, OH) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) for 2 days, and the media were used as the conditioned medium with OSM or without OSM, respectively. The medium, in which 293T cells treated with calcium phosphate without pEF-BOS DNA were grown for 2 days, was used as the control conditioned medium.
Treatment of OC15-5 Cells with Conditioned Media
OC15-5 cells were plated at a density of 1 x 105 per 100-mm dish and incubated in Dulbecco’s modification of Eagle’s medium supplemented with 10% fetal bovine serum at 37°C for 24 hours. An equal amount of conditioned medium was added to the culture, and incubation continued for 4 days. Viable OC15-5 cells were determined by the trypan blue exclusion method.
Electron Microscopy
Cells were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a JEM-1220 electron microscope (JEOL, Tokyo, Japan).
RT-PCR
Total RNA was reverse-transcribed by Superscript II RT in the presence of random hexamers, and the resultant cDNA was amplified using Taq Ex and a set of sense and anti-sense primers (Table 2)
by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. PCR products were analyzed by electrophoresis in 1.0% agarose gel containing ethidium bromide.
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Cells were lysed in RIPA buffer (10 mmol/L Tris-HCl, pH 7.2, 150 mmol/L NaCl, 1 mmol/L O,O'-bis(2-aminoethyl) ethylene glycol-N,N,N',N'-tetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mmol/L phenylmethyl sulfonyl fluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) on ice for 30 minutes and centrifuged at 15,000 x g for 30 minutes. The supernatant was subjected to sodium dodecylsulfate-10% polyacrylamide gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred onto a nitrocellulose transfer membrane (Nitrobind; Micron Separations, Inc., Westborough, MA). Immunoblotting was then performed with mouse monoclonal antibody against CK-19 (Progen Biotechnik GmbH, Heidelberg, Germany) or rabbit polyclonal antibody against rat albumin (ICN Pharmaceuticals, Inc., Aurora, OH).
Small Interfering RNA (siRNA) and Transfection
To construct a vector expressing siRNA for rat OSM-R, annealed oligonucleotides (5'-ggatcccgTTATGAGTGATAGGATGCTGCttgatatccgGCAGCATCCTATCACTCAT-AAttttttccaaaagctt-3') (OSM-R target sequence in capital letters) were subcloned into pRNAT-U6.1/Neo, an siRNA expression vector (GenScript Corp., Piscataway, NJ). The resultant plasmid was designated pRNAT-U6.1/Neo-OSM-R. pRNAT-U6.1/Neo or pRNAT-U6.1/Neo-OSM-R (10 µg) was transferred into OC15-5 cells (1 x 107) by electroporation (975 mF, 350 V) using Gene Pulser II (Bio-Rad Laboratories, Hercules, CA). Two days after the electroporation, 400 µg/ml of G418 sulfate (geneticin; Life Technologies, Inc.) was added to the culture medium to obtain neomycin-resistant cells.
Statistical Analysis
Statistical analysis was performed by unpaired, two-tailed Student’s t-test. A P value less than 0.05 was considered significant.
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Results |
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Human and mouse OSM-R cDNA were previously isolated,17,23
but rat OSM-R cDNA was not. To examine the expression of OSM-R mRNA in rat oval cells, we attempted to isolate rat OSM-R cDNA. Four overlapping fragments covering most of the OSM-R cDNA were obtained by RT-PCR using the liver RNA on day 7 after PH in the AAF/PH model. The 3'-portion of OSM-R cDNA was obtained by nested PCR using two sets of primers, rFW5-slica1 and rFW6-slica2 (Figure 1A)
. The coding region of rat OSM-R gene showed 89% and 71% homologies to the mouse and human counterparts, respectively.17,23
Analysis of OSM-R mRNA in various tissues revealed that OSM-R mRNA was expressed in all tissues examined, with the liver, skin, and spleen showing strong signals (Figure 2)
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We examined whether the level of OSM-R mRNA was correlated with the number of oval cells developed in the AAF/PH model. As shown in Figure 3A
, the number of CK-19-positive oval cells increased rapidly for 7 days after PH and then gradually decreased. Northern blot analysis showed that OSM-R mRNA was hardly detectable on day 0, but was expressed from day 4 to day 13 after PH. (Figure 3B)
. These results indicate that the level of OSM-R mRNA changed with the number of oval cells.
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. We performed in situ hybridization analysis to identify the type of cells expressing OSM-R mRNA in the liver on day 7 after PH. Signals of OSM-R mRNA were observed in oval cells that were present at the periportal field and the surrounding parenchyma (Figure 3, C and D)
. Furthermore, immunohistochemical double-staining analysis of CK-19 and OSM-R in nonparenchymal cells isolated from the livers on day 7 after PH showed that CK-19-positive oval cells were also positive for OSM-R protein (Figure 3; E to G)
. Expression of OSM
RT-PCR analysis showed the expression of OSM in the livers on days 7, 9, 11, and 13 after PH, when numerous oval cells were generated (data not shown). Because these results suggested that oval cells express OSM as well as OSM-R, we performed immunohistochemical double-staining analysis of CK-19 and OSM in the liver on day 7 after PH. Oval cells positive for CK-19 were found to be positive for OSM (Figure 4; A to C)
. Recently, Kupffer cells were shown to be a major source of OSM in the mouse liver injured by the administration of carbon tetrachloride.24
So, we performed immunohistochemical double-staining analysis of ED2, a marker of Kupffer cells, and OSM in the liver on day 7. Kupffer cells identified by ED2 expression were positive for OSM protein (Figure 4; D to F)
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Human and mouse OSM cDNA were previously isolated,25,26
but rat OSM cDNA was not. To clarify the effect of OSM on rat oval cells, we isolated rat OSM cDNA. Two overlapping fragments covering approximately two thirds of OSM cDNA from the 5'-end were obtained by RT-PCR using the liver RNA from 14.5-day rat embryos. The 3'-portion of OSM cDNA was obtained by nested PCR using two sets of primers, FW3-slica1 and FW4-slica2 (Figure 1B)
. The coding region of rat OSM gene showed 81% and 53% homologies to the mouse and human counterparts, respectively.25,26
OSM mRNA was found to be expressed in the spleen, thymus, and lung, but not other tissues (Figure 2)
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Establishment of the Rat Oval Cell Line OC 15-5
Long-Evans Cinnamon rats carry a defect in the Wilson disease gene, resulting in the accumulation of copper in the liver. Long-Evans Cinnamon rats suffer from acute hepatitis at 
4 months of age, and survivors develop chronic hepatitis with the generation of numerous oval cells.27
We isolated these oval cells and cultured for a long time to establish a cell line, designated as OC15-5. OC15-5 cells grew with a cobblestone appearance (Figure 5A)
and expressed CK-19, AFP, and albumin mRNA as detected by RT-PCR analysis (Figure 5B)
. These cells were also positive for c-kit receptor tyrosine kinase, CD34, and Thy-1, which were new surface markers of oval cells,10-13
and OSM-R (Figure 5C)
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The effect of OSM on OC15-5 cells was examined using the conditioned medium of 293T cells transfected with the pEF-BOS with or without rat OSM cDNA. Medium, in which 293T cells treated with calcium phosphate without pEF-BOS DNA were grown, was used as the control conditioned medium. We first examined the effect of OSM on the growth of OC15-5 cells. OC15-5 cells cultured with the conditioned medium without OSM markedly proliferated as cells cultured with the control conditioned medium. In contrast, OC15-5 cells cultured with the conditioned medium with OSM grew poorly (Figure 6)
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10 µm in size with a high nuclear to cytoplasmic ratio and ovoid nuclei (Figure 7, A and D)
. OC15-5 cells cultured with the conditioned medium without OSM were similar in morphology to those cultured with the control conditioned medium (Figure 7, B and E)
. In contrast, OC15-5 cells cultured with the conditioned medium with OSM were bigger with a decreased nuclear-to-cytoplasmic ratio (Figure 7, C and F)
. In addition, these OSM-treated OC15-5 cells possessed microvilli on the surface, large cytoplasm with developed organelles including mitochondria, lysosome, and rough endoplasmic reticulum, and clear nucleoli in the nuclei, that were characteristics of hepatocytes (Figure 7; G to I)
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. Western blot analysis showed similar changes in CK-19 and albumin expression (Figure 5E)
. These results indicated that OSM induced the differentiation of OC15-5 cells into hepatocytes. Abrogation of the Effect by Rat OSM on OC15-5 Cells by siRNA-Mediated Silencing of OSM-R
We introduced an siRNA construct specifically targeting rat OSM-R into OC15-5 cells and examined whether the siRNA-mediated silencing of OSM-R abrogates the effect of the conditioned medium with OSM. Flow cytometry showed that OSM-R expression in OC15-5 cells was abrogated by the introduction of pRNAT-U6.1/Neo-OSM-R (siRNA), but not pRNAT-U6.1/Neo (vector) (Figure 8A)
. These results indicated that the introduced construct was active and able to suppress the expression of OSM-R in OC15-5 cells. We then examined the effect of the conditioned medium with OSM on the growth of OC15-5 cells introduced with pRNAT-U6.1/Neo or pRNAT-U6.1/Neo-OSM-R. OC15-5 cells carrying pRNAT-U6.1/Neo-OSM-R (siRNA) markedly proliferated in the presence of the conditioned medium with OSM, whereas OC15-5 cells carrying pRNAT-U6.1/Neo (vector) grew poorly (Figure 8B)
. Morphologically, OC15-5 cells carrying pRNAT-U6.1/Neo (vector) became bigger with a decreased nuclear to cytoplasmic ratio in the presence of the conditioned medium with OSM. In contrast, OC15-5 cells carrying pRNAT-U6.1/Neo-OSM-R (siRNA) grew with a cobblestone appearance in the presence of the conditioned medium with OSM (Figure 8C)
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. These results indicated that the effect of the conditioned medium with OSM on OC15-5 cells was abrogated by siRNA-mediated silencing of OSM-R. |
Discussion |
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Next, we isolated the rat OSM cDNA and established a rat oval cell line, OC15-5, to study the role of OSM in the differentiation of oval cells. Rat OSM, like mouse and human OSM,25,26
contains a predicted signal peptide, four -helices, and an AB loop. However, it does not contain four positively charged repeats that are present in the C-terminal region of mouse OSM. Previously, Yoshimura and colleagues26
reported that the conditioned medium of COS cells transfected with C-terminally truncated mouse OSM cDNA lacking four positively charged repeats exhibited biological activity, but that the conditioned medium of cells transfected with the full-length mouse OSM cDNA did not. In this study, we found that the conditioned medium of 293T cells transfected with the full-length rat OSM cDNA, in which four positively charged repeats are not present, possessed biological activity. These results indicate that four positively charged repeats interfere with the activity of OSM and that the truncation of C-terminal region carrying four positively charged repeats is essential for the activation of OSM.
OC15-5 cells express not only the traditional oval cell markers (CK-19 and AFP), but also the new oval cell markers (c-kit receptor tyrosine kinase, CD34, and Thy-1).10-13 OC15-5 cells may be useful to identify the factors that induce the differentiation of oval cells into hepatocytes and study the biological role of oval cells in the liver regeneration. When OC15-5 cells were cultured with the conditioned medium of 293T cells transfected with rat OSM cDNA, their growth was suppressed. The morphology was also changed to become similar to that of hepatocytes. In addition, OC15-5 cells cultured with the conditioned medium with OSM showed decreases in CK-19 and AFP expression, but increases in albumin expression, and were also positive for hepatocyte markers, TAT and TO. These effects of the conditioned medium with OSM were not observed in OC15-5 cells carrying pRNAT-U6.1/Neo-OSM-R, in which OSM-R expression was down-regulated by the introduction of an siRNA construct specifically targeting the rat OSM-R. These results indicate that OSM is important for induction of the differentiation of OC15-5 cells into hepatocytes, and suggest that the OSM/OSM-R system plays a pivotal role in oval cell-mediated liver regeneration. We performed similar experiments using primary oval cells isolated from the livers on day 8 after PH in the AAF/PH model. On incubation with the conditioned medium with OSM, primary oval cells ceased to grow and underwent morphological changes similar to those observed with OC15-5 cells (data not shown). However, analysis of expression of liver-specific proteins by Western blotting could not be performed because of difficulties of obtaining primary oval cells in sufficient quantities. These results, although limited, supported the conclusion derived from OC15-5 cells.
Previously, Kamiya and colleagues18 demonstrated that OSM in combination with glucocorticoid induces differentiation of fetal hepatocytes to express TAT and glucose-6-phosphatase, which are expressed in the perinatal liver. However, no adult liver-specific enzymes, such as TO, were expressed in these cells. These results suggest that OSM renders the characteristics of the neonatal liver, but not of adult hepatocytes, to fetal hepatocytes. In this study, TO mRNA expression was induced in OC15-5 cells by culturing with the conditioned medium of 293T cells transfected with rat OSM cDNA. Several possibilities may be raised to explain the difference between the results of Kamiya and colleagues18 and ours. First, OSM-R-mediated signal transduction is sufficient for the differentiation of oval cells into adult hepatocytes, but not for the maturation of fetal hepatocytes. Second, OSM may induce the maturation of OC15-5, an oval cell line, but not primary oval cells, into adult hepatocytes. Third, 293T cells produce soluble factors required for full maturation of oval cells. This idea is supported by the findings that fetal hepatocytes on embryonic day 14.5 are induced to express TO mRNA by culturing with OSM and extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma.19
Both OSM and hepatocyte growth factor (HGF) are involved in the maturation of fetal hepatocytes.28,29 The OSM-induced hepatic maturation is inhibited by the expression of a dominant-negative form of signal transducer and activator of transcription protein 3, whereas HGF-induced hepatic maturation is not, indicating that OSM and HGF induce the maturation of fetal hepatocytes through different signaling pathways.28 As for the effect on the oval cells, Okano and colleagues30 reported that HGF induces the proliferation of oval cells through the phosphatidylinositol-3 kinase/Akt signaling pathway, and Shiota and colleagues31 reported that HGF accelerates the proliferation of oval cells in the AAF/PH model. On the other hand, the present study showed that OSM induces the differentiation of oval cells into hepatocytes. These results suggest that a combined use of HGF and OSM, that support the proliferation and differentiation of oval cells, respectively, may be of value for the treatment of patients with liver damages.
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Acknowledgements |
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Footnotes |
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Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and for graduate students, Hyogo College of Medicine.
Accepted for publication November 30, 2004.
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