March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Antagonistic Interactions of Interferon- and TGF-β1 within Human Corneal Stromal Fibroblasts
Author Affiliations & Notes
  • William J. O'Brien
    Ophthalmology and Microbiology,
    Medical College of Wisconsin, Milwaukee, Wisconsin
  • Tom Heimann
    Ophthalmology,
    Medical College of Wisconsin, Milwaukee, Wisconsin
  • Farhan Rizvi
    Ophthalmology, Eye Inst/Med Coll of Wisconsin, Milwaukee, Wisconsin
  • Deborah Conklyn
    Ophthalmology and Microbiology,
    Medical College of Wisconsin, Milwaukee, Wisconsin
  • Footnotes
    Commercial Relationships  William J. O'Brien, None; Tom Heimann, None; Farhan Rizvi, None; Deborah Conklyn, None
  • Footnotes
    Support  Supported in part by NIH grants EY R01-017079, EY P30-01931, RR C06-016511 and an unrestricted grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3576. doi:
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      William J. O'Brien, Tom Heimann, Farhan Rizvi, Deborah Conklyn; Antagonistic Interactions of Interferon- and TGF-β1 within Human Corneal Stromal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3576.

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Abstract

Purpose: : We proposed to determine the cellular signaling pathways and the mechanism(s) by which TGF-β1 and IFN-γ regulate transcription of selected genes in human corneal stromal fibroblasts(HCSF).

Methods: : Cultures of HCSF were established from donor corneas following collagenase digestion of the stroma after removal of the epithelium and endothelium. Cells were recovered and grown in DMEM and 5% FBS. To study the regulation of transcription and signaling, cells were plated in DMEM containing 5% defined FBS, serum starved and treated with TGF-β1 and/or pro-inflammatory cytokines; IL1-β, TNF-α and rHuIFN-γ. At various intervals post-treatment, cells were harvested and gene expression was assessed by real-time RT-PCR and western blotting. The genes of interest included NOX NADPH oxidases(NOXs 1,4 and 5), p22phox, cyclooxygenase 2 (COX2), α-smooth muscle actin(α-SMA), nitric oxide synthase 2 (NOS2) and collagen type1, alpha 2(COL1α2).

Results: : TGF-β1 significantly up-regulated transcription of NOX4 (20 to 380 fold)(p= 0.03) and marginally up-regulated transcription of α-SMA(2 to 4 fold)(p=0.01) and COL1α2(2 to 3 fold)(p=0.01) while having little effect on NOX1,NOX5, p22phox, NOS2 and COX2 (≤ 2 fold) as determined by real-time RT PCR when the data was analyzed by the Pfaffl equation and associated statistics. The mixture of pro-inflammatory cytokines as well as low molecular weight inhibitors of TGF-β1 receptor function significantly (p=0.01)reduced steady state levels NOX4 transcripts induced by TGF-β1. Antagonism of TGF-β1 signaling by IFN-γ or the mixture of cytokines was not Smad7 dependent. Mixtures of IL1-β, TNF-α and rHuIFN-γ up-regulated the steady state mRNA pools of NOS2 (20 to 66 fold)(p=0.01),COX 2(5 to 13 fold) and NOX1(p=0.001) while significantly down regulatating NOX 4(p=0.001). This mixture was significantly more effective at reducing the effects of TGF-β1 than any of the cytokines alone. INF-γ significantly increased the phosphorylation of p38 MAP kinase 1hr post-treatment and increased the steady state amounts of the regulatory protein YB-1.

Conclusions: : Steady state pools of NOX4 mRNA were significantly up-regulated in a time and dose dependent fashion by TGF β1. IFN-γ and other proinflammatory cytokines antagonized the regulatory activity of TGF β1 but not through Smad signaling.

Keywords: cornea: stroma and keratocytes • cytokines/chemokines • growth factors/growth factor receptors 
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