Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts
Tristan Bourcier1, Tsouria Berbar, Sophie Paquet
1Cornea Bank, EFS AP-HP, EA 3123 Paris 6 University, Paris, France.
Purpose:
The aim of this study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1 (CXCL12), in human corneal fibroblasts.
Methods:
Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1. The functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected in human corneal fibroblasts using reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by western blot analysis.
Results:
[125I]SDF-1 specifically bound to cultured corneal fibroblasts with a KD value of 8.3+/-1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1 induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa protein was detected by western blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4 and SDF-1 mRNAs in human corneal fibroblasts.
Conclusions:
These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1. This latter might exert physiological effects on the cornea and could be involved in pathological conditions such as corneal angiogenesis.
Insights
Cultured human corneal fibroblasts express functional chemokine receptors CXCR4 and its ligand SDF-1. This finding may impact corneal physiology and pathology, including angiogenesis.
Area of Science:
- Ophthalmology
- Cell Biology
- Immunology
Background:
- Chemokines and their receptors play crucial roles in cellular communication and immune responses.
- Chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) are implicated in various physiological and pathological processes.
Purpose of the Study:
- To investigate the expression and functionality of chemokine receptor CXCR4 on human corneal fibroblasts.
- To determine the presence of its specific ligand, SDF-1 (CXCL12), within human corneal fibroblasts.
Main Methods:
- Human corneal fibroblast cultures were established from donor corneas.
- CXCR4 receptor expression and binding affinity were assessed using [125I]SDF-1 binding studies and autoradiography.
- Functional analysis of CXCR4 involved intracellular calcium measurements.
- mRNA and protein expression of CXCR4 and SDF-1 were confirmed via RT-PCR and Western blot analysis.
Main Results:
- Specific binding of [125I]SDF-1 to corneal fibroblasts was observed (KD value of 8.3+/-1.2 nM).
- SDF-1 triggered a rapid intracellular calcium increase, indicating functional CXCR4 signaling.
- Both CXCR4 and SDF-1 mRNA and a 48 kDa CXCR4 protein were detected in corneal fibroblasts.
Conclusions:
- Human corneal fibroblasts express functional CXCR4 chemokine receptors and their ligand SDF-1.
- This expression suggests potential roles for SDF-1 in corneal physiology.
- The findings may be relevant to pathological conditions such as corneal angiogenesis.
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