Masao Yoshizumi

Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life.

Nitric oxide (NO), which accounts for the biological properties of endothelium-derived relaxing factor, is generated by NO synthase (NOS). The vascular endothelium contains two types of NOS: one is constitutively expressed (cNOS), and the other is inducible. Endothelium-mediated vasorelaxation is impaired in atherosclerotic vessels. To determine whether tumor necrosis factor (TNF)-alpha, which is commonly found in atherosclerotic lesions, has an effect on NOS message, we measured cNOS mRNA levels in TNF-treated human umbilical vein endothelial cells (HUVECs) by RNA blot analysis with a cNOS cDNA probe. TNF-alpha markedly reduced cNOS mRNA levels in HUVECs in a dose- and time-dependent manner. In response to 3 ng/mL TNF-alpha, cNOS mRNA levels began to decrease at 4 hours and diminished to only 5% of control levels at 24 hours. As little as 0.1 ng/mL TNF-alpha reduced cNOS mRNA levels by 50%. This reduction in cNOS message in response to TNF-alpha depended on protein synthesis as it was blocked by cycloheximide. In nuclear runoff experiments, TNF-alpha did not change the rate of cNOS gene transcription. cNOS mRNA is very stable under basal conditions, with a half-life of 48 hours; however, treatment with TNF-alpha shortened this half-life to 3 hours. TNF-alpha thus appears to decrease cNOS mRNA levels by increasing the rate of mRNA degradation. TNF-induced reductions in cNOS mRNA levels may have an important effect on impaired endothelium-mediated vasorelaxation in atherosclerosis.

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine.

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10–50 μm of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21 ras (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels ofS-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21 ras and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21 ras -MAP kinase pathway may represent one of the mechanisms that mediates homocysteine’s effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.

Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

Human EZF, a Krüppel-like Zinc Finger Protein, Is Expressed in Vascular Endothelial Cells and Contains Transcriptional Activation and Repression Domains

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the β-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the β-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181–388. We also found that amino acids 91–117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Downregulation of vascular endothelial growth factor receptors by tumor necrosis factor-alpha in cultured human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) potently stimulates angiogenesis, whereas TNF-alpha has both pro- and anti-angiogenic activity. By measuring thymidine uptake, we found that TNF-alpha blocked a 2.3-fold increase in DNA synthesis induced by VEGF in human endothelial cells. To explore the possibility that the two interact to regulate endothelial cell proliferation, we examined the effect of TNF-alpha on VEGF receptor expression. In venous and arterial endothelial cells, TNF-alpha potently reduced mRNA transcripts of the two VEGF receptors (KDR/flk-1 and flt-1) in a dose- and time-dependent fashion. TNF-alpha at 1 ng/ml induced maximal inhibition of mRNA expression, which fell by approximately 70% after 24 h. TNF-alpha treatment did not significantly affect the KDR/flk-1 half-life but did decrease its rate of transcription to 40% of control. The decrease in KDR/flk-1 mRNA depended partially on new protein synthesis and was abolished by phorbol ester pretreatment. TNF-alpha decreased the amount of 35S-labeled KDR/flk-1 immunoprecipitated by an antibody specific for KDR/flk-1 to 18% of control. We conclude that TNF-alpha downregulates expression of both VEGF receptors in human endothelial cells and that this effect is transcriptional (at least for KDR/flk-1). These data support the hypothesis that TNF-alpha exerts its antiangiogenic effect in part by modulating the VEGF-specific angiogenic pathway.

Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts.

The regulated expression of cyclins controls the cell cycle. Because cardiomyocytes in adult mammals withdraw permanently from the cell cycle and thus cannot regenerate after injury, we examined cyclin expression during development by comparing cyclin A-E mRNA levels in fetal and adult human hearts. Cyclin B mRNA was detectable in adult hearts, although at a level markedly lower than that in fetal hearts. Levels of cyclin C, D1, D2, D3, and E mRNA were essentially identical in the two groups. In contrast, cyclin A mRNA was undetectable in adult hearts whereas cyclin A mRNA and protein were readily detectable in fetal hearts and cardiomyocytes, respectively. We then measured cyclin A mRNA and protein levels in rat hearts at four stages of development (fetal and 2, 14, and 28 d). Cyclin A mRNA and protein levels decreased quickly after birth (to 37% at day 2) and became undetectable within 14 d, an observation consistent with reports that cardiomyocytes stop replicating in rats by the second to third postnatal week. This disappearance of cyclin A gene expression in human and rat hearts at the time cardiomyocytes become terminally differentiated suggests that cyclin A downregulation is important in the permanent withdrawal of cardiomyocytes from the cell cycle.

The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5 flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.

Suppression of Interleukin-1β-induced Nitric-oxide Synthase Promoter/Enhancer Activity by Transforming Growth Factor-β1 in Vascular Smooth Muscle Cells EVIDENCE FOR MECHANISMS OTHER THAN NF-κB

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-β1 down-regulates inducible NOS after its induction by interleukin (IL)-1β by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5′-flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1β or vehicle. IL-1β increased the activity of the plasmid containing −1485 to +31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1β-responsive elements. Further deletion analysis revealed that a construct containing −234 to +31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1β stimulation. Mutation of the NF-κB site within this region partially reduced IL-1β-inducible activity; however, a large portion of activity remained independent of the NF-κB site. TGF-β1 suppressed promoter/enhancer activity after IL-1β stimulation, and this suppression was complete in the construct with a mutated NF-κB site. In addition, TGF-β1 did not decrease the binding of nuclear proteins to the NF-κB site. These data suggest that the ability of TGF-β1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-κB motif in vascular smooth muscle cells.

Down-regulation of the Cyclin A Promoter by Transforming Growth Factor-β1 Is Associated with a Reduction in Phosphorylated Activating Transcription Factor-1 and Cyclic AMP-responsive Element-binding Protein

Transforming growth factor (TGF)-β1 prevents cell cycle progression by inhibiting several regulators, including cyclin A. To study the mechanisms by which TGF-β1 down-regulates cyclin A gene expression, we transfected reporter plasmids driven by the cyclin A promoter into mink lung epithelial cells in the absence and presence of TGF-β1. The TGF-β1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site, because mutation of this site abolished down-regulation. Surprisingly, although TGF-β1 treatment for 24 h markedly decreased cyclin A promoter activity, it did not decrease the abundance of the ATF-binding proteins ATF-1 and cyclic AMP-responsive binding protein (CREB). However, we detected 90 and 78% reductions (by Western analysis) in phosphorylated CREB and ATF-1, respectively, in mink lung epithelial cells treated with TGF-β1. TGF-β1-induced down-regulation of cyclin A promoter activity was reversed by okadaic acid (a phosphatase inhibitor) and by cotransfection with plasmids expressing the cAMP-dependent protein kinase catalytic subunit or the simian virus small tumor antigen (Sm-t, an inhibitor of PP2A). These data indicate that TGF-β1 may down-regulate cyclin A promoter activity by decreasing phosphorylation of CREB and ATF-1.

Molecular Cloning, Characterization, and Promoter Analysis of the Mouse Crp2/SmLim Gene PREFERENTIAL EXPRESSION OF ITS PROMOTER IN THE VASCULAR SMOOTH MUSCLE CELLS OF TRANSGENIC MICE

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of theCrp2/SmLim gene, we cloned the cDNA and gene of mouseCrp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5′-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5′ of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5′ of the transcription start site, and there are potentialcis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5′-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5′-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases −74 and −39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types.In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.