Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

Abstract

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.After the discovery of arachidonate 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) in polymorphonuclear neutrophils the enzyme was identified in other leukocyte lineages (1, 2). Products of the 5-LO pathway, including leukotriene (LT) B4 and the cysteinyl LTs, in addition to their roles as inflammatory agonists, may act as physiological autocrine or paracrine signaling molecules. Until recently 5-LO expression was thought to be restricted to the hematopoietic system. However, attempts to identify the enzyme in other tissues yielded results that support the hypothesis that it is expressed in distinctive extrahematopoietic epithelia. Thus, Natsui et al. (3) Expression of the 5-LO pathway in skin epithelium likewise remains debatable. Several authors using primary skin keratinocytes reported the production of 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4, but subsequent work challenged some of the earlier interpretations (refs. 6-11; for review, see ref. 12).We have used a nontransformed human keratinocyte cell line, HaCaT, and normal human skin keratinocytes (NHKs) as in vitro models to study expression of the 5-LO pathway in extrahematopoietic epithelia. HaCaT cells are derived from a normal skin biopsy and maintain a substantial differentiation potential in culture (13,14). In this report we show that the 5-LO gene is expressed in both HaCaT cells and NHKs under conditions that favor their differentiation. MATERIALS AND METHODS

Functional Coupling between Secretory and Cytosolic Phospholipase A2 Modulates Tumor Necrosis Factor-α- and Interleukin-1β-induced NF-κB Activation

“…TNF-␣ and IL-1␤ increase intracellular calcium levels (83), which could thus contribute to 5-LO activation. However, 5-LO has been found to be active per se in unstimulated post-confluent HaCaT cells (84). Thus, the enzymatic machinery necessary for immediate production of AAgenerated 5-LO metabolites in response to cytokines is present in human HaCaT keratinocytes.…”

Eicosanoids in skin inflammation

“…Cutaneous 5-LOX activity is rather low and has been associated mainly with epidermal keratinocytes [66,67], Langerhans cells [68,69] and infiltrating leukocytes [65]. Interestingly, 5-LOX activity is increased when keratinocytes differentiate [70]. LT and 5-oxo-ETE are potent chemoattractants [64,71] that can contribute to cutaneous inflammation and allergy [72,73], however, the actual formation of peptidoleukotrienes in human skin has been disputed [74].…”

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes

“…PLA # cleaves phospholipids at the sn-2 position to produce arachidonic acid [26]. LPA is known to induce arachidonic acid release from membrane phospholipids [27]. Figures 6a and 6b (Figure 7a).…”

“…If a lipoxygenase product was required for LPA-stimulated H # O # release, addition of this compound would be predicted to alleviate the inhibition by NDGA. HaCaT cells are known to express 5-lipoxygenase [27], which generates 5-HPETE ; 5-HPETE is then converted to 5-HETE, LTB % , or LTC % . Because NDGA has weak anti-oxidant activity, a wellcharacterized specific inhibitor of 5-lipoxygenase, Zileuton [20,21], was used to further assess the role of 5-lipoxygenase activity in LPA-stimulated H # O # release in HaCaT cells.…”

“…A previous study showed that the 5-lipoxygenase products 5-HETE, LTB % and LTC % (all of these are derived from 5-HPETE) could be detected in HaCaT cells [27]. In particular, LTB % and LTC % are produced in HaCaT cells in response to calcium ionophore [30].…”