May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Increases in furin and MT1–MMP expression are independent of MMP–2 activity in PAF–stimulated corneal myofibroblasts.
Author Affiliations & Notes
  • P. Ottino
    Ophthalmology/Neuroscience,
    LSU Health Sciences Center, New Orleans, LA
  • W.T. Axelrad
    Biochemistry and Molecular Biology,
    LSU Health Sciences Center, New Orleans, LA
  • J.C. He
    Ophthalmology/Neuroscience,
    LSU Health Sciences Center, New Orleans, LA
  • H.E. P. Bazan
    Ophthalmology/Neuroscience,
    LSU Health Sciences Center, New Orleans, LA
  • Footnotes
    Commercial Relationships  P. Ottino, None; W.T. Axelrad, None; J.C. He, None; H.E.P. Bazan, None.
  • Footnotes
    Support  NIH_R01EY04928
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3841. doi:
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      P. Ottino, W.T. Axelrad, J.C. He, H.E. P. Bazan; Increases in furin and MT1–MMP expression are independent of MMP–2 activity in PAF–stimulated corneal myofibroblasts. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3841.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose:Our studies showed that myofibroblasts express the platelet–activating factor (PAF) receptor (He J and Bazan H, ARVO 2003), and that PAF stimulates membrane type–1 MMP (MT1–MMP) gene expression (Axelrad B et al, ARVO 2003). The active form of MT1–MMP functions as an activator of pro–MMP–2. Pro–MT1–MMP activation is thought to be mediated by the intracellular processing protease furin. The objective of this study was to examine whether PAF increases MMP–2 activity through MT1–MMP and what effect PAF has on furin expression in myofibroblasts. Methods:Rabbit corneal myofibroblasts were prepared from fibroblasts seeded at low density (5 cells per mm2) in 100 mm dishes and cultured in DMEM–F12 medium supplemented with 5 % FBS for 5 days. Myofibroblast phenotype was identified by immunodetection of smooth muscle–α–actin (SM–α–actin). Cells were starved overnight in 0.1% FBS–supplemented medium followed by stimulation with 100 nM PAF. For inhibitor studies, cells were pretreated with the PAF antagonists BN50730 (10 µM), CV 3988 (10 µM), or CV 6209 (1 µM) for 45 minutes prior to PAF treatment. Changes in gene expression of MT1–MMP and furin were quantified by Real–Time PCR. Values were normalized to the 18s rRNA and SM–α–actin. Protein expression was assessed by Western blot. MMP–2 activity was determined by gelatin zymography. Results:PAF induced furin gene expression as early as 2 hr (2 fold) with a peak occurring at 8 hours (5.5 fold). This gene induction was followed by an increase in protein expression at 12 hr for furin, while increased levels of MT1–MMP protein were detected at 12 hr with a peak at 24 hr. Pretreatment with PAF antagonists blocked the PAF–induced increases in MT1–MMP and furin expression. Western–blot analysis and gelatin zymography activity assay of PAF–treated myofibroblasts showed no changes in MMP–2 expression when compared to untreated controls. Conclusions: We demonstrate that PAF increased the expression of furin in corneal myofibroblasts through a PAF receptor–mediated mechanism. The time course of gene and protein expression for furin and MT1–MMP suggests a coordinate balance exists between the expression of these enzymes. MMP–2 activity, on the other hand, was unchanged by PAF treatment. Given the link between furin and MT1–MMP activation and MT1–MMPs role in mediating matrix turnover either directly or through MMP–2 activation, these results suggest that in response to the inflammatory mediator PAF, MT1–MMP induction is independent of MMP–2 activity in corneal myofibroblasts. (NIH R01EY04928)

Keywords: cornea: stroma and keratocytes • gene/expression • inflammation 
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