In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G 0 /G 1 arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3 (GSK-3), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3. Indeed, DIF-3 dephosphorylated Ser 9 and phosphorylated tyrosine on GSK-3, and it stimulated GSK-3 activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3 by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3 to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G 0 /G 1 arrest in mammalian cells.Differentiation-inducing factors (DIFs) 1 were identified in Dictyostelium discoideum as the morphogens required for stalk cell differentiation of Dictyostelium (1). In the DIF family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first to be identified, and DIF-3, the monochlorinated analogue of DIF-1, is a natural metabolite of DIF-1 in Dictyostelium (2). However, the actions of DIFs are not limited to Dictyostelium. They also have strong effects on mammalian cells. DIF-1 and/or DIF-3 strongly inhibit proliferation and induce differentiation in several leukemia cells, such as the murine erythroleukemia cell line B8, human leukemia cell line K562, and human myeloid leukemia cell line HL-60 (3, 4). DIF-3 has been reported to have the most potent antiproliferative effect on mammalian leukemia cells among DIF analogues examined to date (5). Recently, we found that DIF-1 strongly inhibits proliferation and induces differentiation in human vascular smooth muscle cells, indicating that cells sensitive to DIFs are not limited to transformed cells (6).However, the target molecule (receptor) of DIFs is unknown, and it is not clear even in Dictyostelium how DIFs induce an antiproliferative effect and cell differentiation. DIFs are small hydrophobic molecules and are therefore expected to be able to cross cell membranes without requiring channels or carriers. Also, the rapidity with which DIFs induce prestalk cell-specific gene expression suggests that they directly regulate gene expression. The...
Ligands for peroxisome proliferator-activated receptor gamma, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D(2) (PGD(2)), the metabolic precursor of 15d-PGJ(2), we identified PGD(2) from culture medium of endothelial cells. To study how PGD(2) production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD(2). Endothelial cells expressed the mRNA for the lipocalin-type PGD(2) synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm(2). The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI(2) synthase and thromboxane A(2) synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD(2) and 15d-PGJ(2) into culture medium. These results demonstrate that shear stress promotes PGD(2) production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator-activated receptor gamma ligand is produced in vascular wall in response to blood flow.
Objective-Fluid shear stress induces cyclooxygenase (COX)-2 gene expression in vascular endothelial cells. We investigated the underlying mechanism of this induction. Methods and Results-Exposure of human umbilical vein endothelial cells to laminar shear stress in the physiological range (1 to 30 dyne/cm 2 ) upregulated the expression of COX-2 but not COX-1, a constitutive isozyme of COX. The expression of COX-2 mRNA began to increase within 0.5 hour after the loading of shear stress and reached a maximal level at 4 hours. Roles of the promoter region and the 3Ј-untranslated region in the human COX-2 gene were evaluated by the transient transfection of luciferase reporter vectors into bovine arterial endothelial cells. Shear stress elevated luciferase activity via the region between Ϫ327 and 59 bp. Mutation analysis indicated that cAMP-responsive element (Ϫ59/Ϫ53 bp) was mainly involved in this response. On the other hand, shear stress selectively stabilized COX-2 mRNA. Moreover, shear stress elevated luciferase activity when a 3Ј-untranslated region of COX-2 gene containing 17 copies of the AUUUA mRNA instability motif was inserted into the vector. Key Words: shear stress Ⅲ vascular endothelial cells Ⅲ cyclooxygenase-2 Ⅲ posttranscriptional regulation V ascular endothelial cells are always exposed to a wide variety of biochemical and biomechanical stimuli, including fluid shear stress caused by blood flow. Shear stress modulates several endothelial functions, such as control of vascular tone, maintenance of antithrombotic surfaces, regulation of inflammation, protection against oxidative stresses, and regulation of endothelial cell proliferation and apoptosis. 1 Cyclooxygenase (COX), a rate-limiting enzyme for prostaglandin (PG) biosynthesis, comprises 2 isozymes, COX-1 and COX-2. 2,3 COX-1 is constitutively expressed in most cell species, whereas COX-2 is an inducible enzyme whose expression is regulated differently among cell types. Growing evidence indicates that COX-2 plays a key role in several biological processes, such as inflammation, tumorigenesis, development, and atherogenesis. 4 -9 Laminar shear stress upregulates COX-2 gene expression. 10 COX-2 is involved in lipopolysaccharide-stimulated production of prostacyclin (PGI 2 ) in endothelial cells 11 and is also involved in PGI 2 biosynthesis in healthy humans. 12 Previously, we have reported that shear stress promotes the production of PGD 2 in endothelial cells by stimulating the expression of lipocalintype PGD 2 synthase (L-PGDS), whereas PGI 2 synthase was constitutively expressed but did not respond to shear stress. 13 Therefore, the induction of COX-2 expression by shear stress may be involved in the production of PGI 2 and PGD 2 in endothelial cells. Conclusions-TranscriptionalThree cis-acting elements, namely, the nuclear factor (NF)-B binding site, the NF-interleukin-6 (NF-IL6) binding site, and the cAMP-responsive element (CRE), reside in the region between base pairs Ϫ327 and ϩ59 (Ϫ327/ϩ59) in the human COX-2 gene promoter. Their involveme...
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