May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
FGF–2– and IL–1ß–mediated transcriptional regulation of pro1(I) collagen RNA during endothelial mesenchymal transformation
Author Affiliations & Notes
  • M.K. Ko
    Pathobiology, University of Southern California, Los Angeles, CA
  • E.P. Kay
    Ophthamology, DohenyEye Institute/University of Southern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships  M.K. Ko, None; E.P. Kay, None.
  • Footnotes
    Support  EY06431
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3775. doi:
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      M.K. Ko, E.P. Kay; FGF–2– and IL–1ß–mediated transcriptional regulation of pro1(I) collagen RNA during endothelial mesenchymal transformation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3775.

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

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Abstract

Abstract: : Purpose: To investigate whether FGF–2 or interleukin–1ß (IL–1ß) induced transcription of proα1(I) collagen RNA during endothelial mesenchymal transformation (EMT) in corneal endothelial cells (CECs). Methods: The in vitro EMT model was established by treating cells with FGF–2, IL–1ß, or a combination of the two. The steady–state levels of proα1(I) and proα2(I) collagen RNAs were measured using real–time PCR. Ct values are defined as the cycle number in which fluorescence exceeded a threshold value. Levels were normalized to hypoxanthine–phosphoribosyl–transferase(HPRT) mRNA and converted to a linearized value using the known formula. The relative fold difference is expressed compared with normal CECs. Results: The EMT process was monitored as cells acquired multi–layers of fibroblastic phenotypes: the first passage CECs were modulated to an elongated cell shape in response to treatment with FGF–2 or a combination with IL–1ß. FGF–2 and IL–1ß demonstrated synergistic activities: cells treated with both factors showed much greater modulation. The third passage CECs grown in media containing FGF–2 and IL–1ß acquired multi–layers of fully modulated phenotypes. When transcription of proα1(I) and proα2(I) collagen RNA was determined, in the first passage, the proα1(I) RNA level was increased 5–fold in cells treated with IL–1ß alone and 6–fold in cells treated with FGF–2 and IL–1ß, while the proα1(I) RNA level was slightly decreased in cells treated with FGF–2 alone. On the other hand, the third passage cells treated with FGF–2 or with FGF–2 and IL–1ß demonstrated a more than 24–fold increase in proα1(I) collagen RNA levels. The transcription profiles of proα2(I) RNA were completely different: in first passage cells treated FGF–2, IL–1ß, or FGF–2 and IL–1ß, the levels of proα2(I) RNA were increased by 2.5–fold. Interestingly, as cells were fully modulated by FGF–2 or by both FGF–2 and IL–1ß, the proα2(I) RNA levels were much less than that of the untreated CECs. Conclusions: These data suggest that transcription of proα1(I) collagen RNA is the rate–limiting event during endothelial to mesenchymal transformation.

Keywords: cornea: endothelium • cytokines/chemokines • gene/expression 
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