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Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus

Expression of CK18 and αSMA in RL/DMEM cells cultured in DMEM- and DF-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DMEM cells were routinely passaged in DMEM-based medium [DMEM in (A)]. The cells were cultured for 11 days after the replacement of the medium with a DF-based medium [DMEM→DF in (A)]. These cells were then returned to DMEM-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides in the conditions indicated, fixed and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
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Figure 3: Expression of CK18 and αSMA in RL/DMEM cells cultured in DMEM- and DF-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DMEM cells were routinely passaged in DMEM-based medium [DMEM in (A)]. The cells were cultured for 11 days after the replacement of the medium with a DF-based medium [DMEM→DF in (A)]. These cells were then returned to DMEM-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides in the conditions indicated, fixed and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.

Mentions: RL/DMEM cells retained a cobblestone-like morphology and formed an epithelial-like monolayer cell sheet, and yet strongly expressed αSMA (Figure 3A, upper panels). The expression of αSMA is well-defined as a marker of hepatic myofibroblasts, which are principally produced by the transdifferentiation of activated HSC. However, recent studies have revealed that hepatocytes and BECs are able to transdifferentiate into myofibroblasts through the EMT process (Gorrell, 2007). On the basis of these previous findings, it is considered that RL/DMEM cells may have been transformed from parenchymal hepatocytes or hepatoblasts through EMT to myofibroblast-like cells during the immortalization process in DMEM-based medium. In addition, it was reported that the differentiated status of hepatocytes fluctuates in in vitro culture, depending on the signalling pathways triggered by growth factors or the ECM (Godoy et al., 2009). So we examined whether RL/DMEM cells have the capability to undergo reversible transitions between epithelial and mesenchymal phenotypes.


Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Expression of CK18 and αSMA in RL/DMEM cells cultured in DMEM- and DF-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DMEM cells were routinely passaged in DMEM-based medium [DMEM in (A)]. The cells were cultured for 11 days after the replacement of the medium with a DF-based medium [DMEM→DF in (A)]. These cells were then returned to DMEM-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides in the conditions indicated, fixed and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475435&req=5

Figure 3: Expression of CK18 and αSMA in RL/DMEM cells cultured in DMEM- and DF-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DMEM cells were routinely passaged in DMEM-based medium [DMEM in (A)]. The cells were cultured for 11 days after the replacement of the medium with a DF-based medium [DMEM→DF in (A)]. These cells were then returned to DMEM-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides in the conditions indicated, fixed and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
Mentions: RL/DMEM cells retained a cobblestone-like morphology and formed an epithelial-like monolayer cell sheet, and yet strongly expressed αSMA (Figure 3A, upper panels). The expression of αSMA is well-defined as a marker of hepatic myofibroblasts, which are principally produced by the transdifferentiation of activated HSC. However, recent studies have revealed that hepatocytes and BECs are able to transdifferentiate into myofibroblasts through the EMT process (Gorrell, 2007). On the basis of these previous findings, it is considered that RL/DMEM cells may have been transformed from parenchymal hepatocytes or hepatoblasts through EMT to myofibroblast-like cells during the immortalization process in DMEM-based medium. In addition, it was reported that the differentiated status of hepatocytes fluctuates in in vitro culture, depending on the signalling pathways triggered by growth factors or the ECM (Godoy et al., 2009). So we examined whether RL/DMEM cells have the capability to undergo reversible transitions between epithelial and mesenchymal phenotypes.

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus