The wound healing process involves the movement of neighbouring cells to cover the injury area and the subsequent cell proliferation. These steps may be regulated by growth factors, extracellular components and eicosanoids. In this way, arachidonic acid release and prostaglandin E2 (PGE2) production are enhanced in wounded fibroblast cultures and may be involved in the regulation of wound repair process (Lloret & Moreno, 1996; Moreno, 1997). The objective of this study was to determine the role of prostanoids, lipoxygenase and cytochrome P-450 pathway metabolites in the regulation of wound closure of confluent 3T6 fibroblast cultures.
Our results show that the inhibition of the cyclooxygenase and/or cytochrome P-450 pathways significantly decreases the wound closure, whereas that of the lipoxygenase pathway does not modify the wound repair process.
PGE2 is one of the major eicosanoids synthesized by fibroblasts. PGE2 effects are initiated on plasma membrane localized GTP-binding protein-coupled receptors. There are four types of PGE2 receptors (EP1-EP4) (Narumiya et al. 1999). EP1 and EP4 are present in 3T6 fibroblasts. Both EP1 and EP4 receptors mediate PGE2-stimulated 3T6 fibroblast wound closure. Each receptor exerts its effects through changes in a specific cellular pathway. Thus the EP1 receptor increases intracellular calcium concentration, whereas PGE2 interaction with the EP4 receptor raises intracellular cAMP levels. Both second messengers are involved in the control of the cell cycle machinery of 3T6 fibroblasts (Sánchez & Moreno, 2002) and, thus in the wound repair process. On the other hand, we show that ketoconazole, a cytochrome P-450 inhibitor, hinders the wound closure induced by fetal calf serum in wounded 3T6 cell cultures. 12- and 20-Hydroxyeicosatetraenoic acids, which are key arachidonic acid metabolites synthesised by cytochrome P-450, partially, reverse the effects of ketoconazole on the wound repair process. Moreover, our findings suggest that these cytochrome P-450 pathway metabolites participate in the control of calcium. These events may be involved in the control of DNA synthesis, cell growth and the subsequent wound repair process in wounded 3T6 fibroblast cultures.
In summary, the cyclooxygenase and cytochrome P-450 pathway metabolites of arachidonic acid, PGE2 and/or 12- and 20- hydroxyeicosatetraenoic acids respectively, play a pivotal role in 3T6 fibroblast wound closure, and calcium and cAMP are involved in the signalling events induced by these eicosanoids in the wound repair process.
This study was supported by the Spanish Ministry of Education (PM98-0191) and by Generalitat de Catalunya (1999SGR00266). AH 6809 and AH 23848B were kindly provided by Dr S.G. Lister from the Compound Supplies Officer, Glaxo Wellcome, Medicines Research Centre, Stevenage, UK.