Lumican regulates corneal inflammatory responses by modulating Fas-Fas ligand signaling.
OBJECTIVE
The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling. The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries.


METHODS
Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts. Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively. Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA.


RESULTS
Lum-/- -injured corneas showed significantly lower caspase-3/7 activity (apoptosis). Lum-/- -wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta. The Fas protein level, before and after wounding, was dramatically lower in Lum-/- - compared with Lum+/+-injured cornea. However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level. Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican.


CONCLUSIONS
The data suggest that lumican binds FasL and facilitates induction of Fas. Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing.
Vij
Neeraj
N
Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Roberts
Luke
L
Joyce
Sarah
S
Chakravarti
Shukti
S
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D011508:Chondroitin Sulfate Proteoglycans / Q000502:physiology*; D003319:Corneal Stroma / Q000293:injuries / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein; D019084:Fluorescent Antibody Technique, Indirect; D007632:Keratan Sulfate / Q000502:physiology*; D007634:Keratitis / Q000378:metabolism*; D000071234:Lumican; D008264:Macrophages / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009504:Neutrophils / Q000276:immunology; D009195:Peroxidase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D014945:Wound Healing / Q000502:physiology; D019014:fas Receptor
D011508:Chondroitin Sulfate Proteoglycans; D053222:Fas Ligand Protein; C491899:Fas protein, mouse; C505658:Fasl protein, mouse; C000604307:Lum protein, mouse; D000071234:Lumican; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor; D007632:Keratan Sulfate; D009195:Peroxidase; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases