Lipoxygenase mRNA in cultured human epidermal and oral keratinocytes

Abstract

Although 15-lipoxygenase has not been purified from cultured human keratinocytes nor has cDNA coding for the protein been isolated from this source, this enzyme activity is implied by the finding of its stereospecific product in in vitro experiments. Based on two primer pairs derived from human reticulocyte 15-lipoxygenase cDNA, we detected approximately 260 bp and approximately 370 bp cDNA fragments that were indistinguishable by gel electrophoresis and Southern hybridization from those derived from reticulocyte 15-lipoxygenase cDNA. The approximately 260 bp polymerase chain reaction (PCR) fragment from a keratinocyte plasmid cDNA library was cloned and determined, by sequencing, to contain 262 bp that were 100% identical to the corresponding reticulocyte 15-lipoxygenase cDNA. These two reticulocyte-type 15-lipoxygenase PCR fragments were also detected from oral keratinocytes. Using platelet-type 12-lipoxygenase cDNA primers, we derived a 264 bp cDNA fragment from keratinocyte mRNA. By sequence analysis, this fragment was determined to be 99.6% identical to that from platelet-type 12-lipoxygenase cDNA. The same fragment was also observed from two amplified keratinocyte cDNA libraries, and from oral keratinocyte mRNA. This is the first demonstration of reticulocyte-type 15-lipoxygenase cDNA derived from the mRNA of cultured human keratinocytes.