Characterization and functionality of CXCR4 chemokine receptorand SDF-1 in human corneal fibroblasts

[摘要] Purpose: The aim of this study was to investigate whether culturedhuman corneal fibroblasts express functional chemokine CXCR4 receptorson their cell surface and to determine the presence of its specificligand, SDF-1 (CXCL12), in human corneal fibroblasts.Methods: Human corneal fibroblast cultures were obtained using humandonor corneas. CXCR4 receptors were characterized using binding studiesand autoradiography with [125I]SDF-1. The functionality of CXCR4receptors was assessed by intracellular calcium measurement using adynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected inhuman corneal fibroblasts using reverse transcriptase polymerase chainreaction (RT-PCR). The CXCR4 protein was detected by western blotanalysis.Results: [125I]SDF-1 specifically bound to cultured cornealfibroblasts with a KD value of 8.3±1.2 nM. The presence ofCXCR4 was confirmed by autoradiography of the radioligand on slices ofcorneal stroma. SDF-1 induced a rapid and transient intracellularcalcium increase in cultured corneal fibroblasts that was blocked by thespecific antagonist bicyclam. Moreover, a 48 kDa protein was detected bywestern blot analysis of corneal fibroblast extracts, using a specificCXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4and SDF-1 mRNAs in human corneal fibroblasts.Conclusions: These results indicate for the first time that culturedhuman corneal fibroblasts express the chemokine receptors CXCR4 and itsligand SDF-1. This latter might exert physiological effects on thecornea and could be involved in pathological conditions such as cornealangiogenesis.

[学科分类] 生物化学/生物物理