title: TGF-beta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2).
abstract: BACKGROUND
  The prognosis of primary renal disease is often dependent on the degree of tubulointerstitial scarring. Scarring is caused by proliferation and excessive matrix production of renal fibroblasts and possibly other cellular elements. Transforming growth factor-beta (TGF-beta) is the most important cytokine for the induction of matrix synthesis in the kidney. However, its effects on renal fibroblast proliferation have not been determined. We have recently demonstrated that the expression of basic fibroblast growth factor (FGF-2) is robustly up-regulated in human kidneys with tubulointerstitial fibrosis and that FGF-2 is a potent inducer of fibroblast proliferation. The present study examined the interaction between TGF-beta 1 and FGF-2 in human renal fibroblasts.
  
  
  METHODS
  Experiments were performed on a transformed medullary fibroblast line and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-beta 1 were analyzed by Northern blot analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The expression of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and the cdk inhibitor p27(kip1) were determined by immunoblots.
  
  
  RESULTS
  Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-beta 1 mRNA expression, whereas incubation of the cells with TGF-beta 1 induced FGF-2 mRNA up to 3.51 +/- 0.21-fold after six hours. This increase could be blocked almost completely by the addition of cyclohexamide, indicating that the process is in large part dependent on protein synthesis. The up-regulation in FGF-2 mRNA expression was paralleled by de novo detection of FGF-2 protein in the supernatant, peaking after 12 to 24 hours, as determined by Western blot and ELISA, whereas cellular protein was only increased up to 2.1-fold. Interestingly, both methods detected release of FGF-2 protein to the supernatant already at three hours, indicating a role for TGF-beta1 in directly releasing preformed FGF-2. Since TGF-beta 1 induced FGF-2, which results in fibroblast proliferation, we hypothesized that TGF-beta1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of renal fibroblasts with TGF-beta1 resulted in an up to 3.21 +/- 0.28-fold increase in bromodeoxyuridine incorporation and a 1.95 +/- 0.16-fold increase in cell number after 72 hours. This mitogenic effect of TGF-beta1 could be blocked completely by the addition of a neutralizing antibody to FGF-2 or the tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (FGFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epidermal growth factor (EGF) or the tyrphostin B42, which inhibits EGF receptor signal transduction, had no effect. Interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced TGF-beta 1-induced proliferation considerably in primary skin fibroblasts. Finally, TGF-beta1-induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p27(kip1), whose activity is maintained by TGF-beta in epithelial cells, was down-regulated up to 48 hours.
  
  
  CONCLUSIONS
  Our studies demonstrate, to our knowledge for the first time, that TGF-beta1 induces proliferation in human renal fibroblasts and that this process is mediated largely by FGF-2. The induction of proliferation by TGF-beta 1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogenesis.
authors.affiliation: Department of Nephrology and Rheumatology, Georg-August-University Göttingen, Göttingen, Germany. fstrutz@gwdg.de
authors.forename: F
authors.initials: F
authors.lastname: Strutz
authors.forename: M
authors.initials: M
authors.lastname: Zeisberg
authors.forename: A
authors.initials: A
authors.lastname: Renziehausen
authors.forename: B
authors.initials: B
authors.lastname: Raschke
authors.forename: V
authors.initials: V
authors.lastname: Becker
authors.forename: C
authors.initials: C
authors.lastname: van Kooten
authors.forename: G
authors.initials: G
authors.lastname: Müller
mesh_terms: D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000187:drug effects*; D007672:Kidney Cortex / Q000166:cytology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000502:physiology; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D012867:Skin / Q000166:cytology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins; D020032:Tyrphostins; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases