ORIGINAL PAPER K. Barth Æ J. Reh Æ A. Sturrock Æ M. Kasper Epithelial vs myofibroblast differentiation in immortal rat lung cell lines—modulating effects of bleomycin Accepted: 14 July 2005 / Published online: 27 September 2005 Ó Springer-Verlag 2005 Abstract Two alveolar epithelial cell lines R3/1 and L2 were screened by immunocytochemical and RT-PCR analysis of epithelial and mesenchymal/contractile mar- ker proteins. R3/1 and L2 cells were tested for their sensitivity to bleomycin (BLM), an anticancer drug, which is proposed to induce changes in lung cell differ- entiation. Both epithelial cell lines exhibited a mixed phenotype consisting of epithelial (E-cadherin, aquapo- rin-5 and cytokeratin 8) and myofibroblast-like (vimen- tin, a-SMA and caveolin-3) properties suggesting that the cell lines are arrested in vitro at a certain develop- mental stage during epithelial–mesenchymal transition (EMT). BLM treatment of R3/1 cells resulted in a par- tial reversal of this process modifying the cells in an epithelial direction, e.g., upregulation of E-cadherin, aquaporin-5 and other lung epithelial antigens at the mRNA and protein level. L2 cells showed similar alterations following BLM exposure.Immunohisto- chemical investigation of lung tissue from two different animal models of BLM-induced fibrosis (mouse and rat), revealed no signs of EMT, e.g., myofibroblastic differentiation of alveolar epithelial cells in situ. Immuno- histological analysis of tissue samples of the rat model showed a heterogeneous population of myofibroblasts (a-SMA + /caveolin-3 + , a-SMA - /caveolin-3 + , and a- SMA + /caveolin-3 À ). These results suggest that BLM, on one hand, induces fibrosis and on the other hand possibly suppresses EMT during fibrogenesis. Keywords Pulmonary fibrosis Æ Bleomycin Æ EMT Æ Myofibroblast Æ Caveolin-3 Introduction Epithelial–mesenchymal transition (EMT) is the process by which an epithelial cell becomes a more motile mes- enchymal cell (Condeelis and Segall 2003; Kalluri and Neilson 2003). EMT operates during early embryonic cell layer movements and later during organogenesis (Tosh and Slack 2002; Shook and Keller 2003). During EMT, cell–cell junctions are altered, cells lose epithelial polarity and express the mesenchymal markers vimentin and a-smooth muscle actin (a-SMA), the resulting reorganization of the actin cytoskeleton supports cell migration. EMT is often found during the development of tubulointerstitial kidney fibrosis, a prominent feature of experimental and human diabetic nephropathy determined by de novo expression of the mesenchymal marker a-SMA and the loss of the epithelial marker E-cadherin (Li et al. 2003), particularly in renal proximal tubular cells (Ng et al. 1998; Iwano et al. 2002; reviewed in Manotham et al. 2004). Tubular EMT can be induced by TGF-b (Bottinger and Bitzer 2002; Gore-Hyer et al. 2002), hypoxia (Manotham et al. 2004), and advanced glycation end products signaling through a RAGE-ERK 1/2 MAP kinase pathway (Oldfield et al. 2001; Li et al. 2004). EMT is also well established in epithelial tissue of the liver, mammary gland, pancreas and others (Piek et al. 1999; Shook and Keller 2003). A recent paper has for the first time shown EMT in freshly isolated type II alveolar epithelial (AT II) cells (Yao et al. 2004). In this report, TGF-b induced EMT within a 72 h period as shown by abundant expression of a-SMA, increased levels of col- lagen type I, loss of the epithelial marker E-cadherin and morphological transformation to a myofibroblast. The data were confirmed by Willis et al. (2005), using pri- mary human alveolar epithelial cells in vitro. Further- more, this report presents novel data demonstrating the K. Barth (&) Æ J. Reh Æ M. Kasper Institute of Anatomy, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fiedlerstr. 42, D-01307 Dresden, Germany E-mail: Kathrin.Barth@mailbox.tu-dresden.de Tel.: 0049 351 458 6076 Fax: 0049 351 458 6303 A. Sturrock Department of Internal Medicine, University of Utah School of Medicine, Medical Service, Salt Lake City, UT, USA Histochem Cell Biol (2005) 124: 453–464 DOI 10.1007/s00418-005-0048-2