Lumican regulates Fas–Fas ligand mediated apoptosis: implications in the cornea

Abstract

Abstract: : Purpose:Fas–Fas ligand mediated apoptosis plays a pivotal role in maintaining corneal immune privilege. We investigated the role of lumican, a major corneal proteoglycan in regulating apoptosis in cell culture and corneal stroma. Methods: Wildtype (WT) and Lumican–null (Lum^–/–) genotypes were used to obtain primary cultures of mouse embryonic fibroblasts (MEF) and corneal fibroblast (CF) from E14 embryos and corneas respectively. Apoptosis was determined by measuring caspase–3/7 activity and apoptotic cells were identified in corneal sections of postnatal 10–day and 6–week (adult) old mice by TUNEL. Incision stromal wounds were generated in adult mice with a 26 gauge needle. Protein and RNA levels of Fas and Fas ligand were estimated by immunoblotting and RT–PCR respectively. To induce Fas, MEFs were incubated overnight with recombinant Fas ligand followed by assessment of Fas protein levels. To assess potential interactions between lumican and Fas ligand, we explored co–immunoprecipitation of Fas–ligand with anti–lumican. Results:Caspase–3/7 activity was significantly lower in the Lum^–/– MEF and CF than the WT cells. The TUNEL assay showed fewer apoptotic cells in adult corneas before (p=0.025) and after wounding (p=0.056) in the Lum^–/– genotype compared to WT. By immunoblotting we found a dramatic reduction in Fas in MEF, CF and the cornea of Lum^–/– mice, with no difference in the levels of Fas–ligand. To test the possibility that reduced Fas may be due to a deficiency in the presentation of Fas ligand in Lum^–/–, Fas levels were estimated after Fas ligand treatment. While the WT MEF show marked up regulation of Fas after exposure to Fas ligand, Lum^–/– MEF remarkably show no induction of Fas. The failure to induce Fas may be the major cause of the observed decrease in apoptosis associated with lumican–deficiency. Levels of Fas and Fas ligand transcripts were comparable in the Lum^+/+ and Lum^–/– CF indicating the regulation of Fas to be at the protein level. Immunoprecipitation of lumican from MEF and CF protein extracts co–precipitated Fas ligand. Conclusion: Our data suggests that lumican binds the Fas ligand and facilitates the Fas apoptosis cascade. Corneal immune privilege is maintained by Fas–Fas ligand mediated apoptosis of Fas + lymphoid cells invading the stroma, lumican may be an essential regulator of this process.

Keywords: cornea: stroma and keratocytes • apoptosis/cell death • proteoglycans/glycosaminoglycans