Yoji Taba

Transcriptional and Posttranscriptional Regulation of Cyclooxygenase-2 Expression by Fluid Shear Stress in Vascular Endothelial Cells

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/cm2) 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 3′-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 −327 and 59 bp. Mutation analysis indicated that cAMP-responsive element (−59/−53 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 3′-untranslated region of COX-2 gene containing 17 copies of the AUUUA mRNA instability motif was inserted into the vector. Conclusions—Transcriptional activation and posttranscriptional mRNA stabilization contribute to the rapid and sustained expression of COX-2 in response to shear stress.

Fluid Shear Stress Induces Lipocalin-Type Prostaglandin D2 Synthase Expression in Vascular Endothelial Cells

Abstract —Ligands for peroxisome proliferator–activated receptor γ, such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-Δ 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 γ ligand is produced in vascular wall in response to blood flow.

Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library.

To investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells (HUVEC). We used a microcarrier culture system and a combination of subtraction and reverse-subtraction methods to isolate a number of genes upregulated by shear stress. The resultant subtracted library includes several known genes (e.g. MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (e.g. CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel genes. Some responsive to centrifugal force and shear stress (RECS) genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis.

Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site

Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-&kgr;B, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.

Differentiation-Inducing Factor-1, a Morphogen of Dictyostelium, Induces G1 Arrest and Differentiation of Vascular Smooth Muscle Cells

Differentiation-inducing factor-1 (DIF-1) is a morphogen that induces differentiation of DICTYOSTELIUM: Recently, DIF-1 has been shown to inhibit proliferation and induce differentiation in tumor cells, although the underlying mechanisms remain unknown. In this study, we examined the effects of DIF-1 on the proliferation and differentiation of vascular smooth muscle cells, to explore novel therapeutic strategies for atherosclerosis. DIF-1 nearly completely inhibited DNA synthesis and cell division in mitogen-stimulated cells. DIF-1 inhibited the phosphorylation of the retinoblastoma protein and the activities of cyclin-dependent kinase (Cdk) 4, Cdk6, and Cdk2, which phosphorylate the retinoblastoma protein. DIF-1 strongly suppressed the expression of cyclins D1, D2, and D3, as well as those of cyclins E and A, which normally began after that of the D-type cyclins. The mRNAs for the smooth muscle myosin heavy chains SM1 and SM2 were expressed in quiescent cells in primary culture, and these expression levels decreased after mitogenic stimulation. In the presence of DIF-1, the rate of the reduction was significantly decelerated. Moreover, the addition of DIF-1 to dedifferentiated cells induced the expressions of SM1 and SM2, accompanied by a reduction in the level of SMemb, a nonmuscle-type myosin heavy chain. Therefore, DIF-1 seemed to interrupt a very early stage of G(1) probably by suppressing the expressions of the D-type cyclins. Furthermore, this compound may prevent phenotypic modulation and induce differentiation of vascular smooth muscle cells.

Physiology and pharmacology of the prostaglandin J2 family.

The prostaglandin (PG) J(2) family including PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are metabolites of PGD(2). They had been known as powerful inhibitors of cell proliferation and viral replication until 15d-PGJ(2) was found to be a natural ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). Since then, several new pharmacological actions of the PGJ(2) family have been found, such as pro- and anti-apoptotic effects, cell differentiation-inducing effects, and inhibitory effects on inflammatory processes, whether they depend on PPAR gamma or not. We reported that the PGJ(2) family, particularly 15d-PGJ(2), inhibits cell proliferation by reducing the expression of G(1) cyclins and inducing the expression of cyclin-dependent kinase inhibitor p21 and moreover, induces cell differentiation in vascular smooth muscle cells. In vascular endothelial cells, we found that 15d-PGJ(2) inhibits apoptotic cell death at least in part by the induction of the inhibitor of apoptosis protein c-IAP1. More importantly, physiological levels of laminar fluid shear stress loaded on endothelial cells upregulate the expression of lipocalin-type PGD(2) synthase, which converts PGH(2) to PGD(2), the precursor of the PGJ(2) family. Based on these results, we have hypothesized that the PGJ(2) family synthesized in vascular wall plays an important physiological role to protect vascular cells from atherogenic stimuli.