Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts.
OBJECTIVE
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.
Bourcier
Tristan
T
Cornea Bank, EFS AP-HP, EA 3123 Paris 6 University, Paris, France.
Berbar
Tsouria
T
Paquet
Sophie
S
Rondeau
Nicolas
N
Thomas
Frédéric
F
Borderie
Vincent
V
Laroche
Laurent
L
Rostène
William
W
Haour
France
F
Lombet
Alain
A
D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism*; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D012333:RNA, Messenger / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D012333:RNA, Messenger; D019718:Receptors, CXCR4; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2