Abstract
Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.
Highlights
Lens degeneration in Fpr1Ϫ/Ϫ mice prompted us to search for functional Formyl peptide receptor 1 (FPR1) expression directly on lens epithelial cells
FPR1 Is Expressed by Human Lens Epithelial Cells—FPR1 mRNA was detected by PCR in both FHL 124 (Fig. 1A) and SRA 01/04 human lens epithelial cell lines
Binding of the fluorescent ligand fNLFNYK-Fl (10 nM) to FHL 124 cells was affected by FPR1 shRNA to a similar magnitude (ϳ40% reduction) as FPR1 mRNA (p ϭ 0.0554, Fig. 1C)
Summary
Lens degeneration in Fpr1Ϫ/Ϫ mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. Results: FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr knock-out mice. FPR1 on Lens Epithelial Cells gastric epithelial cells [17] and most recently human retinal pigment epithelial cells [18] In several of these reports, it was proposed that FPR1 may play a role in wound healing and tissue repair, and for some of the epithelial cells this was supported with data from in vitro models of wound healing. For these reasons and to begin to test whether human lens function may be regulated directly by FPR1, we have investigated FPR1 expression and function using human lens epithelial cell lines
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