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S. Kenchegowda, Sr., C. Zhang, N. G. Bazan, H. E. P. Bazan; Stimulation of Neuroprotectin D1(NPD1) Synthesis by Pigment Epithelium Derived Factor (PEDF) in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1970.
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PEDF in presence of Docosahexaenoic acid (DHA) increases corneal nerve regeneration and induces neuroprotectin D1 (NPD1) synthesis after lamellar keratectomy was performed in rabbits (Cortina M, et al, IOVS, in press). In human retinal pigment epithelial (RPE) cells, conversion of DHA to NPD1 is catalyzed by 15-lipoxygenase-1 (15-LOX-1) (Calandria J.M. et al 2009). Here, we investigated if human corneal epithelial cells (HCEC) are able to synthesize NPD1 through 15-LOX-1 when stimulated with PEDF.
HCEC were stimulated with 40 ng/ml of PEDF for 24 and 48 h and the expression of 15-LOX-1 was measured by Western blot. NPD1 synthesis was analyzed by LC-ESI-MS/MS after HCECs were stimulated with PEDF for 24 and 48 h and the cell homogenate was incubated with DHA. In some experiments, the 12/15 LOX inhibitor CDC (10 µM) or the 15-LOX inhibitor PD 146176 (10 µM) were added before stimulation.
PEDF induced a 60-70% increase in 15-LOX expression at 24 h and a 95-100% increase at 48 h. A 14-fold increase in NPD1 synthesis was observed when HCEC were stimulated with PEDF for 24 h. Both the 12/15 LOX and the 15-LOX inhibitor blocked the biosynthesis of NPD1. Although expression of 15-LOX-1 was higher after 48 h of stimulation with PEDF, no significant NPD1 synthesis occurred when HCEC were incubated with PEDF for 48 h.
The results suggest that the mechanism of NPD1 synthesis stimulated by PEDF is not dependent on increase in 15-LOX-1 expression by PEDF, but rather on PEDF’s activation of 15-LOX-1. Future studies will explore possible signaling mechanisms involved in the activation of 15-LOX-1 by PEDF.
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