Abstract
Abstract: :
Purpose: In humans, 9 lipoxygenase genes have been identified. Four of these, 15–lipoxygenase 1 and 2 (15–LOX–1 and 2), 12R–lipoxygase(12R–LO), and epidermal lipoxygenase–3 (ELOX3) are reported to be expressed in epithelial tissues. 15–lipoxygenases have been identified in the corneal epithelium. However, little is known regarding the ocular expression and localization of 12R–LO and ELOX3. Method: Human corneas not suitable for clinical use are obtained from a local eye bank. An SV40 immortalized human corneal epithelial cell line also is used for this study. Oligonucleotide primers for RT–PCR were designed from published cDNA sequences of 15–LOXs, ELOX3, and 12R–LO. The full length cDNA sequences 15–LOXs, ELOX3, and 12R–LO were cloned by PCR and inserted to green fluorescent protein (GFP) expression plasmid. Cultured human corneal epithelial cells were transfected and expression of the lipoxygenase–GFP fusion proteins was detected by laser confocal microscopy. Results: By RT–PCR, appropriate sized bands were detected, cloned, and verified by sequencing for15–LOXs, ELOX3, and 12R–LO messages in human corneas and in the immortalized human corneal epithelial cells. Having confirmed the presence of all four lipoxygenases, transfection studies of lipoxygenase–GFP fusion protein expression were performed. 15–LOX–1–GFP is seen in a diffuse pattern in the cytoplasm and excluded from the nucleus. In contrast 15–LOX–2–GFP expression is seen in a diffuse pattern in the both the cytoplasm and the nucleus. Interestingly, both ELOX3 and 12R–LO are localized to cytoplasm in a speckled pattern. Conclusion: 15–LOXs, ELOX3, and 12R–LO messages are present in human corneal epithelium. The different subcellular expression pattern of the two 15–lipoxygenases indicate that they differing physiologic functions. Similar expression pattern of the ELOX3 and 12R–LO suggest similar physiologic roles.
Keywords: cornea: epithelium • cornea: basic science • eicosanoids