Modulation of tissue plasminogen activator and plasminogen activator inhibitor-1 by transforming growth factor-beta in human retinal glial cells.
OBJECTIVE
The serine proteases tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) and their inhibitor, plasminogen activator inhibitor (PAI)-1, regulate a variety of processes involved in tissue morphogenesis and differentiation. There is much evidence that plasminogen activator-mediated extracellular matrix degradation is an important step in the development of ocular neovascular diseases. The authors investigated whether expression of t-PA, u-PA, and PAI-1 in human retinal glial cells (HRGCs) is influenced by exposure to transforming growth factor (TGF)-beta, a cytokine that regulates the proliferation and differentiation of cells.


METHODS
The extracellular release of t-PA, u-PA, and PAI-1 was measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of HRGC cultures, under basal conditions and after stimulation with TGF-beta at various concentrations (2, 5, 10, or 20 ng/mL). Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze mRNA levels. Smad2 phosphorylation was detected by Western blot analysis.


RESULTS
Under basal conditions, HRGCs secreted considerable amounts of t-PA and PAI-1. Stimulation with TGF-beta resulted in increased synthesis of t-PA and PAI-1 protein in a time- and dose-dependent manner. Moreover, an increased expression of t-PA and PAI-1 mRNA after supplementation with TGF-beta was observed, with maximum expression at 12 hours. In contrast, HRGCs did not respond to TGF-beta with any change of u-PA production, although there were detectable amounts of u-PA mRNA and protein. Phosphorylation of Smad2 was increased after addition of TGF-beta. This effect was partially reversible after treatment with interferon-gamma.


CONCLUSIONS
The production of plasminogen activators and PAI-1 by HRGCs reflects the potential role of these cells in the progression of neovascular ocular diseases. Furthermore, the finding that t-PA and PAI-1 synthesis by HRGCs is mediated by TGF-beta and its downstream effector Smad2 confirms the importance of the TGF-beta signaling pathway in the regulation of interactions between retinal cells and the extracellular matrix.
Schacke
Wolfgang
W
Department of Ophthalmology, Johann Wolfgang Goethe University Hospital, Frankfurt am Main, Germany.
Beck
Karl-Friedrich
KF
Pfeilschifter
Josef
J
Koch
Frank
F
Hattenbach
Lars-Olof
LO
D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D051899:Smad2 Protein; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C497782:SMAD2 protein, human; D015842:Serine Proteinase Inhibitors; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator