June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
TGF-β1 modulates the functional expression of the NK-1 receptor in human corneal fibroblasts
Author Affiliations & Notes
  • Sandrine LE ROUX
    Anatomy, Dept. of Integrative Medical Biology, Anatomy, Umeå University, Umeå, Sweden
  • Peter Boman
    Anatomy, Dept. of Integrative Medical Biology, Anatomy, Umeå University, Umeå, Sweden
  • Patrik Danielson
    Anatomy, Dept. of Integrative Medical Biology, Anatomy, Umeå University, Umeå, Sweden
  • Footnotes
    Commercial Relationships Sandrine LE ROUX, None; Peter Boman, None; Patrik Danielson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5228. doi:
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      Sandrine LE ROUX, Peter Boman, Patrik Danielson, ; TGF-β1 modulates the functional expression of the NK-1 receptor in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5228.

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

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Abstract
 
Purpose
 

Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the Neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to be involved in important events in the cornea essential for the proper organization of collagen fibers and the transparency of the stroma. An impaired signaling through NK-1 R could thus disturb the extracellular matrix and impact the vision quality. We hypothesize that TGF-β1 modulates the expression pattern and function of the NK-1 R in human corneal fibroblasts. The purpose of this study was to test that hypothesis.

 
Methods
 

Cultures of primary corneal fibroblasts were set-up with cells derived from healthy corneas, obtained from donated transplantation graft leftovers. Immunocytochemistry for the TGF-β receptor type I (TGF-βRI) and NK-1 R was performed. Gene expression was assessed with qPCR. Western blot was used to confirm the gene expression results at the protein level.

 
Results
 

Expression of TGF-βRI was confirmed on corneal stromal derived fibroblasts. Treating the fibroblasts with TGF-β1 significantly reduced the expression of the NK-1 R gene. Furthermore, immunocytochemistry for NK-1 R showed that the full-length version of the receptor is reduced after treatment by TGF-β1 (figure).

 
Conclusions
 

TGF-β1 downregulates the gene expression of NK-1 R in human corneal fibroblasts, and the results also suggest that it disturbs the signaling pathways triggered by the receptor by reducing the full-length version of it, which might explain the delay in internalization after activation by SP seen with TGF-β1 treatment.

 
 
Immunostaining of human corneal fibroblasts for the inner loop (green; c, f) and the C-terminal (red; b, e) of the NK-1 receptor. Merged pictures in a (of b and c) and d (of e and f), with DAPI added for demonstrating nuclei (bluish). The inner loop is found in both the full-length and the truncated isoform, whereas the C-terminal is only present in the full-length receptor. Untreated cells (a-c) show overlapping immunostainings, whereas cells treated with TGF-β1 (d-f) show reduced staining for the full-length isoform.
 
Immunostaining of human corneal fibroblasts for the inner loop (green; c, f) and the C-terminal (red; b, e) of the NK-1 receptor. Merged pictures in a (of b and c) and d (of e and f), with DAPI added for demonstrating nuclei (bluish). The inner loop is found in both the full-length and the truncated isoform, whereas the C-terminal is only present in the full-length receptor. Untreated cells (a-c) show overlapping immunostainings, whereas cells treated with TGF-β1 (d-f) show reduced staining for the full-length isoform.
 
Keywords: 484 cornea: stroma and keratocytes • 765 wound healing • 480 cornea: basic science  
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