Prostaglandin Synthase 2 Expression in Epidermal Growth Factor-Dependent Proliferation of Mouse Keratinocytes

doi:10.1006/abbi.1996.0271

Archives of Biochemistry and Biophysics

Volume 330, Issue 2, 15 June 1996, Pages 419-429

https://doi.org/10.1006/abbi.1996.0271 Get rights and content

Abstract

BALB/c mouse keratinocytes (BALB/MK) are nontumorigenic epithelial cells which are dependent on mouse epidermal growth factor (EGF) for maintaining proliferation in culture. In BALB/MK the oxygenation of both arachidonic acid and linoleic acid was dependent on EGF. EGF stimulated the formation of prostaglandin E₂and prostaglandin F_2αfrom arachidonic acid and 9- and 13-hydroxyoctadecadienoic acid (HODE) from linoleic acid. Analysis of the linoleic acid metabolites determined the ratio of 9-HODE to 13-HODE was approximately 6 to 4, and the 9-HODE was the (R) enantiomer, consistent with metabolism by prostaglandin G/H synthase (PGHS). The formation of these linoleic acid metabolites was sensitive to indomethacin, a PGHS inhibitor. EGF induced the expression of PGHS-2 mRNA after 30 min, which peaked after 1 h, and remained expressed for at least 24 h after the addition of EGF. A less significant increase in the expression of PGHS-1 mRNA occurred 4 h after EGF stimulation. Immunoblot analysis did not detect expression of PGHS-1 protein. However, PGHS-2 protein expression was increased 2 h after EGF exposure and was dependent on EGF. PGHS-2 protein was not transiently expressed as reported with other cell types, but was continually expressed in proliferating cells maintained with EGF at a subconfluent density. Indomethacin significantly attenuated EGF-dependent mitogenesis and cell proliferation. These results suggest that PGHS-2 activity contributes to the proliferative response of BALB/MK to EGF.

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Cited by (21)

G2A plays proinflammatory roles in human keratinocytes under oxidative stress as a receptor for 9-hydroxyoctadecadienoic acid
2008, Journal of Investigative Dermatology
Citation Excerpt :
For instance, free radical oxidation was reported as the main process in producing oxidized derivatives of linoleic acid, including 9-HODE in mouse skin treated with phorbol myristate acetate (Beckman et al., 1994). Furthermore, enzymatic reaction of prostaglandin H-synthase-2 has been found to convert linoleic acid to 9-HODE in mouse keratinocytes and human dermal fibroblasts under stimulation of epidermal growth factor (Loftin and Eling, 1996) and IL-1β (Godessart et al., 1996), respectively. Some of the lipoxygenases and cytochrome P450 enzymes would also be involved in the metabolism of linoleic and arachidonic acids in skin.
G2A is a stress-inducible G protein-coupled receptor for oxidized free fatty acids, such as 9-hydroxyoctadecadienoic acid (HODE). As skin is routinely and pathologically exposed to many oxidative stresses such as UV radiation, chemical agents, and inflammation that might induce both G2A expression and production of G2A ligands, we examined G2A function in human keratinocytes. G2A was expressed in human epidermis, normal human epidermal keratinocytes (NHEK), and an immortalized human keratinocyte cell line (HaCaT). 9(S)-HODE evoked intracellular calcium mobilization and secretion of cytokines, including IL-6, IL-8, and GM-CSF in NHEK cells. These responses became prominent in HaCaT cells by overexpression of G2A. 9(S)-HODE inhibited proliferation of NHEK cells by suppressing DNA synthesis and arresting the cell cycle in the G0/1-phase. On the other hand, 13(S)-HODE, another major oxidative product from linoleate, showed little or no effect on either cytokine secretion or on proliferation in NHEK cells. A small interfering RNA designed to downregulate G2A caused suppression of 9(S)-HODE-induced inhibitory effects on proliferation of NHEK cells. UVB and H₂O₂ induced G2A expression and caused oxidation of linoleate to produce 9-HODE in HaCaT cells. These results suggest that 9-HODE-G2A signaling plays proinflammatory roles in skin under oxidative conditions.
Cyclooxygenase-1-coupled prostaglandin biosynthesis constitutes an essential prerequisite for skin repair
2003, Journal of Investigative Dermatology
Citation Excerpt :
Transgenic animals are characterized by having elevated levels of PGE2 and PGF2α in skin as well as a reduced hair follicle density, sebaceous gland hyperplasia and a hyperplastic scale epidermis resulting from a disturbance in keratinocyte differentiation (Neufang et al, 2001). Accordingly, increased levels of PGE2 have been associated with high proliferation rates in keratinocytes (Pentland and Needleman, 1986), and it is now suggested that keratinocyte proliferation is closely linked to the presence of a functionally active COX-2 (Loftin and Eling, 1996). In line with these in vitro data, COX-2, but not the COX-1 isoenzyme, is reported to be increased in hyperplastic conditions of the skin such as papillomas and carcinomas in the mouse (Müller-Decker et al, 1995;Pentland et al, 1999;Athar et al, 2001), and in keratoses and squamous cell carcinomas in humans (Müller-Decker et al, 1999).
This investigation demonstrated a functional coupling between cyclooxygenase-1 (cox) and prostaglandin E₂/D₂ biosynthesis in murine skin repair. Cyclooxygenase-1 expression decreased transiently after excisional wounding, and this was followed by a marked fall in the rate of prostaglandin E₂/D₂ biosynthesis at the wound site. Expression of cyclooxygenase-1, prostaglandin synthases, and prostaglandin E₂/D₂ production were colocalized in new tissue at the margin of the wound. Although cyclooxygenase-2 expression was strongly induced in granulation tissue on injury, this isoform did not contribute to high prostaglandin E₂/D₂ concentrations in wounds. Accordingly, wound tissue from SC-560-treated mice (selective cyclooxygenase-1 inhibitor) and diclofenac-treated mice (nonselective cyclooxygenase inhibitor), but not celecoxib-treated mice (selective cyclooxygenase-2 inhibitor), and wound tissue from cyclooxygenase-1-deficient animals exhibited a severe loss of prostaglandin E₂/D₂ at the wound site, and this change was associated with an impairment in the normal wound morphology. Topically administered prostaglandin E₂ (dinoprostone) was able to restore normal wound repair to diclofenac-treated mice. In contrast to the presence of an injury-induced cyclooxygenase-2, these data constitute strong evidence that cyclooxygenase-1-coupled prostaglandin E₂/D₂ biosynthesis has a central role in skin repair.
Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2
2002, Prostaglandins and Other Lipid Mediators
The development of mice deficient in either cyclooxygenase-1 (COX-1) or COX-2, as well as mice deficient in both COX isoforms, has provided models to elucidate the physiological and pathophysiological roles of these enzymes. The findings obtained with the COX-deficient mice suggest that COX-2 may be more important than COX-1 for supplying prostaglandins (PGs) to maintain tissue homeostasis. Furthermore, both isoforms may be involved in the development of diseases, such as inflammation and cancer. Therefore, the contribution of each isoform to the prevention or development of disease is more complex than originally described. Studies with the COX-deficient mice suggest that in addition to COX-2-selective inhibition, therapeutic advances may also be achieved with COX-1-selective inhibitors which lack gastrointestinal side effects.
The COX-2 inhibitor NS-398 causes T-cell developmental disruptions independent of COX-2 enzyme inhibition
2001, Cellular Immunology
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the function of cyclooxygenases, COX-1 and COX-2, which catalyze the first step in the synthesis of inflammatory mediators (PGE₂). We sought to understand the roles of cyclooxygenases and NSAIDs in T-cell development. Our data show no significant defects in T-cell development in fetal thymic organ cultures of mice disrupted in both or either COX genes or in mice disrupted in either EP-1 or EP-2 receptor genes. On the other hand, NSAIDs reproducibly caused thymocyte developmental defects. However, the specific effects of the COX-2 inhibitors were not correlated with their potency for inhibition of COX-2 activity. We focused on the NS-398 COX-2 inhibitor and showed that its effects could not be reversed by exogenous PGE₂. Furthermore, NS-398 was inhibitory even when its target, COX-2, was absent. These data show that the T-cell developmental effects of NS-398 are COX-2 and PGE₂ independent.
Is increased arachidonic acid release a cause or a consequence of replicative senescence?
2001, Experimental Gerontology
Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation. During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells. To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis. Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells. Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells. Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis. Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis. So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging.
Cyclooxygenase-2 expression inhibits trophic withdrawal apoptosis in nerve growth factor-differentiated PC12 cells
2000, Journal of Biological Chemistry
Citation Excerpt :
PC12 cells respond to different growth factors in distinct ways; whereas NGF and basic fibroblast growth factor induce the neuronal phenotype, EGF induces mitogenesis in these cells (11). Cox-2 has been implicated in the proliferative response elicited by a number of growth factors including EGF (17-19). It has also been reported, however, that intestinal epithelial cells stably expressing Cox-2 exhibit a cellular phenotype (inhibition of cell proliferation) similar to parental cells following treatment with the growth inhibitory cytokine transforming growth factor-β (20), and, therefore, the influence of Cox-2 on cell growth exhibits a degree of cell type specificity.
Cyclooxygenase-2 (Cox-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being up-regulated by diverse stimuli. Significantly Cox-2 mRNA is absent from rat pheochromocytoma (PC12) cells, both basally and following stimulation with a range of agonists. Using PC12 cells engineered to stably express isopropyl-1-thio-β-d-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis. Cox-2 bioactivity had no effect on nerve growth factor-induced differentiation, epidermal growth factor-induced proliferation, or aromatic l-amino acid decarboxylase expression. However, trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining. The specificity of this effect was confirmed using two pharmacologically distinct nonsteroidal anti-inflammatory drugs, indomethacin and NS398. Investigations showed that the activity of the pro-apoptotic protease caspase-3 was reduced in PCXII cells. This study demonstrates that Cox-2-derived prostaglandins exert cytoprotective effects in trophic factor withdrawal apoptosis and provides evidence that this is, at least in part, due to suppression of caspase-3 activity.

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Regular ArticleProstaglandin Synthase 2 Expression in Epidermal Growth Factor-Dependent Proliferation of Mouse Keratinocytes

Abstract

Regular Article
Prostaglandin Synthase 2 Expression in Epidermal Growth Factor-Dependent Proliferation of Mouse Keratinocytes