Tumor Necrosis Factor–Induced Apoptosis in Corneal Epithelial Cells Is Attenuated by Novel Lacrimal Glycoprotein, Lacritin

Abstract

Abstract: : Purpose: Cell survival is critical during corneal epithelial regeneration after infection or injury and novel lacrimal glycoprotein, lacritin, could be fundamental in cytoprotection and cell proliferation. We set out to investigate the role of lacritin in the stimulation of a putative G–protein coupled receptor (GPCR) dependent pathway regulating corneal epithelial cell turnover– possibly via a increased calcium signaling involving protein kinase C (PKC), and its crosslink with a tumor necrosis factor (TNF–α)–induced apoptosis in a human corneal epithelial cell line (HCE). Methods: Lacritin–induced proliferation and calcium signaling was studied in the human corneal epithelial (HCE) cell line (Araki–Sasaki et al, 1995). HCE cells were grown at 37⁰C in DMEM F/12 supplemented with 10% FBS and 50 µg/ml gentamicin. Each experiment was done in a dose– and time– dependent manner using TNF–α (5–20 ng/ml) and lacritin (1–10 nM). Cell viability was measured spectrophotometrically at an absorbance of 570 nm using the MTT assay. Caspase–8 actvity was detected fluorometrically by measuring cleavage of the fluorogenic substrate IETD–pNA at an excitation wavelength of 405 nm. Lacritin–induced cell proliferation was studied by H³–thymidine incorporation and via fluorescence–based detection of PKC, PLC and cAMP. Calcium signaling was detected by confocal microscopy of Fluo–4 loaded cells. Results: A) TNF–α– induced cell death in HCE cells (32.2 ± 2.6% after 18 hr and 73.7 ±10% after 30 hr) was attenuated by lacritin (10 nM) pretreatment. B) TNF–α induces a three–fold increase in the proapoptotic caspase–8 activity which was completely blocked by lacritin pretreatment. C) Lacritin and its deletion construct (N–24) promote cell proliferation as measured by the thymidine incorporation in HCE cells. Conclusions: The novel prosecretory mitogen, lacritin, promotes cell proliferation and offers cytoprotection to the corneal epithelial cells against TNF–α–induced cell death. Signal transduction events related to this cytoprotection and proliferation of the corneal epithelial cells are under investigation.

Keywords: cornea: epithelium • lacrimal gland • cell death/apoptosis