Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation.
BACKGROUND
Epithelial-mesenchymal transformation (EMT) plays an important role in embryonic development and tumorigenesis and has been described in organ remodeling during fibrogenesis. In the kidney, EMT can be induced efficiently in cultured proximal tubular epithelium by coincubation of transforming growth factor (TGF)-beta1 and epidermal growth factor (EGF). Recently, we also have observed overexpression of basic fibroblast growth factor-2 (FGF-2) protein and mRNA in human kidneys with marked interstitial fibrosis. The aims of the present study were to compare the effects of FGF-2 as a facilitator of EMT in tubular epithelial cells with EGF and TGF-beta1. We analyzed the morphogenic effects of the three cytokines on four different aspects of EMT: cell motility, expression and regulation of cellular markers, synthesis and secretion of extracellular matrix (ECM) proteins as well as matrix degradation.


METHODS
Cell motility was studied by a migration assay and cell differentiation markers were analyzed by immunofluorescence and immunoblots. In addition, regulation of the epithelial adhesion molecule E-cadherin and fibroblast-specific protein 1 (FSP1) were analyzed by luciferase reporter constructs and stable transfections. ELISAs for collagen types I and IV and fibronectin were used for ECM synthesis, and zymograms were utilized for analysis of matrix degradation.


RESULTS
FGF-2 induced cell motility across a tubular basement membrane in two tubular cell lines. All three cytokines induced the expression of vimentin and FSP1, but only FGF-2 and TGF-beta1 reduced cytokeratin expression by immunofluorescence. These effects were most demonstrable in the distal tubular epithelial cell line and were confirmed by immunoblot analyses. Expression of E-cadherin was reduced by 61.5 +/- 3.3% and expression of cytokeratin by 91 +/- 0.5% by TGF-beta1 plus FGF-2. Conversely, the mesenchymal markers alpha-smooth muscle actin (SMA) and FSP1 were induced with FGF-2 by 2.2 +/- 0.1-fold and 6.8 +/- 0.9-fold, respectively. Interestingly, de novo expression of the mesenchymal marker OB-cadherin was induced only by FGF-2 and EGF but not by TGF-beta1. All three cytokines stimulated FSP1 and decreased E-cadherin promoter activity. FGF-2 also induced intracellular fibronectin synthesis but not secretion, the latter of which was stimulated exclusively by TGF-beta1. Finally, zymographic analyses demonstrated that FGF-2 induced MMP-2 activity by 2.6 +/- 0.5-fold and MMP-9 activity by 2.4 +/- 0.1-fold, providing a mechanism for basement membrane disintegration and migratory access of transforming epithelium to the interstitium.


CONCLUSIONS
FGF-2 makes an important contribution to the mechanisms of EMT by stimulating microenvironmental proteases essential for disaggregation of organ-based epithelial units. Furthermore, the expression of epithelial and mesenchymal marker proteins seems to be affected at the promoter level.
Strutz
Frank
F
Department of Nephrology and Rheumatology, Georg-August University Medical Center, Göttingen, Germany. fstrutz@gwdg.de
Zeisberg
Michael
M
Ziyadeh
Fuad N
FN
Yang
Chang-Qing
CQ
Kalluri
Raghu
R
Müller
Gerhard A
GA
Neilson
Eric G
EG
D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D066246:ErbB Receptors / Q000032:analysis; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D017930:Genes, Reporter; D015151:Immunoblotting; D007684:Kidney Tubules / Q000166:cytology* / Q000196:embryology; D008156:Luciferases / Q000235:genetics; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D015415:Biomarkers; D016207:Cytokines; D017468:Receptors, Fibroblast Growth Factor; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D008156:Luciferases; D066246:ErbB Receptors; C496349:Fgfr1 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1