Conditionally immortalized cell line of inducible metanephric mesenchyme.
BACKGROUND
The mesenchymal-epithelial conversion of metanephric mesenchyme (MM) in the formation of nephronic tubules has long served as a paradigm for inductive signaling in morphogenesis. However, the mechanisms underlying this differentiation have remained an enigma due to insufficient numbers of primary mesenchymal cells that must be isolated manually from animal embryos. To overcome this problem, we have established a conditionally immortalized cell line, the rat-inducible metanephric mesenchyme (RIMM-18) by transfection of primary mesenchymal cells with a vector, encoding an estradiol-dependent E1A-ER fusion protein.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR), luciferase reporter assay, electrophoretic mobility shift assay, immunocytochemical, and immunohistochemical stainings were used to characterize the established cell line.


RESULTS
We demonstrate that in the presence of estradiol, the RIMM-18 cell line proliferates continuously, maintaining mesenchymal characteristics for over 40 passages. These cells are vimentin-positive and cytokeratin-negative. Under inductive conditions in the absence of estradiol, they are responsive to a number of cytokines, which are established inducers of mesenchymal cells in vivo and in vitro [i.e., fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and transforming growth factor-beta 2 (TGF-beta 2)]. We show the presence in RIMM-18 cells of specific protein markers and functionally active signaling pathways required for induction of tubule formation in MM. Furthermore, induced RIMM-18 cells change morphology, acquiring epithelial-like features, and begin to express epithelial markers (e.g., E-cadherin, cytokeratin, gamma-glutamyl-transpeptidase, and secreted frizzled-related protein 2 (sFRP2).


CONCLUSIONS
This preliminary characterization of the RIMM-18 cell line suggests that it will be useful in the study of biochemical and molecular mechanisms of nephronic development and, possibly, of some types of renal cancer such as Wilms' tumor, which caricatures the normal process of kidney development.
Levashova
Zoia B
ZB
Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA. levashovaz@ncifcfr.gov
Plisov
Sergei Y
SY
Perantoni
Alan O
AO
D015415:Biomarkers; D002454:Cell Differentiation; D002461:Cell Line, Transformed / Q000166:cytology* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000196:embryology; D051821:Lymphoid Enhancer-Binding Factor 1; D008648:Mesoderm / Q000166:cytology*; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D015415:Biomarkers; D004268:DNA-Binding Proteins; D051821:Lymphoid Enhancer-Binding Factor 1; C497796:SMAD4 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D015534:Trans-Activators; D014157:Transcription Factors