Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes

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 affect important events in the cornea – such as stimulating epithelial cell proliferation – processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis.

Methods: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR).

Results: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor.

Conclusions: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.

KEYWORDS:

• Cornea
• cytokines
• neuropeptides
• stroma
• substance P

Acknowlegments

The authors thank Mr. Peter Boman for technical assistance and scientific advice. We also thank Dr. Maria Brohlin and Ms. Randi Elstad for help in providing the donated corneas from the biobank, as well as to all the OR-staff at the Ophthalmic Surgery Clinic, especially Dr. Berit Byström, of the University Hospital of Umeå for deliverance of graft leftovers.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Financial support was obtained by P.D. from the national Swedish Research Council (grant no. 521-2013-2612), the J.C. Kempe and Seth M. Kempe Memorial Foundations (for S.L.), the Swedish Society of Medicine, the Cronqvist foundation, the foundation Kronprinsessan Margaretas Arbetsnämnd för synskadade (KMA), the foundation Ögonfonden, and Västerbotten County Council (Spjutspetsmedel). Financial support was furthermore provided to P.D. through a regional agreement between Umeå University and Västerbotten County Council (ALF).