Novel effects of beraprost sodium on vasculatures.

Beraprost sodium (BPS) is a stable orally active prostacyclin analogue with vasodilatory and anti-platelet effects, and has been widely used as therapeutics for pulmonary artery hypertension and chronic arterial obstruction. In order to elucidate its effects on endothelium, we first examined the short-term effects of BPS on nitric oxide (NO) production and endothelial NO synthase (eNOS) activation using bovine aortic endothelial cells. Short-term treatment of BPS induced NO production as well as eNOS phosphorylation at Ser-1179 mediated via cAMP/protein kinase A (PKA) pathway. The effects of BPS on capillary-like tube formation were next determined using human umbilical vein ECs (HUVECs)/normal human dermal fibroblasts co-culture system. BPS was observed to induce capillary-like tube formation mediated via cAMP/PKA pathway, but not via NO generation. Finally, we performed DNA microarray analyses using RNA extracted from BPS treated HUVECs. Interestingly, BPS up-regulated several genes involved in angiogenesis, anti-atherosclerosis, and endothelial function, while down-regulated several genes involved in atherosclerosis. Our data therefore indicate that BPS may be useful not only for patients with pulmonary artery hypertension and chronic arterial obstruction, but also for general atherosclerotic patients complicated with endothelial dysfunction. Further studies are needed to clarify molecular mechanisms of these BPS effects including the involvement of peroxisome proliferator-activated receptor-delta.

Differential effects among thiazolidinediones on the transcription of thromboxane receptor and angiotensin II type 1 receptor genes.

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands thiazolidinediones (TZDs) have recently been reported to be anti-hypertensive and anti-atherosclerotic. We have previously shown that one of the TZDs troglitazone significantly suppressed the transcription of both thromboxane receptor (TXR) and angiotensin II type 1 receptor (AT1R) genes in vascular smooth muscle cells (VSMCs) by activating PPAR-gamma. In the present study, we compared the effects of troglitazone and other TZDs on the transcription of these genes. TXR and AT1R mRNAs in rat VSMCs were determined by semi-quantitative RT-PCR. Luciferase chimeric constructs containing either the 989-bp rat TXR gene promoter or the 1,969-bp rat AT1R gene promoter were transiently transfected into VSMCs. The cells were incubated with troglitazone, RS-1455 (a derivative of troglitazone which does not contain the hindered phenol resembling alpha-tocopherol), pioglitazone, or rosiglitazone for 12 h before harvesting. mRNA expression levels of TXR and AT1R were significantly decreased by troglitazone in contrast to rosiglitazone. TXR gene and AT1R gene transcription was significantly suppressed by troglitazone in a dose-dependent manner, while RS-1455 was less potent. Pioglitazone and rosiglitazone weakly suppressed the transcription of both genes in a manner almost similar to RS-1455. We have shown that troglitazone suppresses transcription of both the TXR and AT1R genes more potently than other TZDs. The structure of troglitazone and RS-1455 is identical except the hindered phenol, which is recently recognized to function as an antioxidant. Moreover, we have shown that the potency for activating PPAR-gamma is almost identical between troglitazone and RS-1455. We therefore speculate that the strong transcriptional suppression of the TXR and AT1R genes by troglitazone may be mediated in part by its antioxidant effect.

Transcriptional suppression of type 1 angiotensin II receptor gene expression by peroxisome proliferator-activated receptor-gamma in vascular smooth muscle cells.

Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-(Delta)(12,14)-prostaglandin J(2) and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-gamma and activate PPAR-gamma. In the present work, we have studied the effect of PPAR-gamma on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-gamma ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of (3)H-thymidine incorporation into VSMCs was inhibited by PPAR-gamma ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-gamma ligands, and PPAR-gamma overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the -58/-34 region of the AT1R gene promoter was responsible for the suppression; 5) Sp1 overexpression stimulated AT1R gene transcription via the GC-box-related sequence, which was inhibited by additional PPAR-gamma overexpression; 6) electrophoretic mobility shift assay suggested that Sp1 could bind to the GC-box-related sequence whereas PPAR-gamma could not; 7) antibody supershift experiments using VSMC nuclear extracts revealed that protein-DNA complexes formed on the GC-box-related sequence, which were decreased by PPAR-gamma coincubation, were mostly composed of Sp1; and 8) glutathione S-transferase pull-down assay revealed a direct interaction between PPAR-gamma and Sp1. Taken together, it is suggested that activated PPAR-gamma suppresses AT1R gene at a transcriptional level by inhibiting Sp1 via a protein-protein interaction. PPAR-gamma ligands, thus, may inhibit AII-induced cell growth and hypertrophy in VSMCs by AT1R expression suppression and possibly be beneficial for treatment of diabetic patients with hypertension and atherosclerosis.

Renal tubule-specific transcription and chromosomal localization of rat thiazide-sensitive Na-Cl cotransporter gene.

The molecular mechanism underlying the renal expression localization of the thiazide-sensitive Na-Cl cotransporter (TSC) gene was studied. The TSC gene was localized to chromosome 19p12-14. In cultured cells, tissue-specific transcription activity of the 5'-flanking region of the rat rTSC gene (5'FL/rTSC) was demonstrated, and the major promoter region was located between position -580 and -141. To further examine the tissue-specific transcription, transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene were generated. Immunohistochemical analysis clearly showed that LacZ gene expression was co-localized to distal convoluted tubules (DCT) with TSC, indicating that the 5'FL/rTSC regulates the renal tubule-specific TSC expression. Because a transcription factor, HFH-3 (hepatocyte nuclear factor-3/folk head homologue-3), had also been localized to DCT, a possible role of the putative cis-acting element (HFH-3/rTSC, -400/-387 position) for HFH-3 binding in the tissue-specific transcription was examined. Deletion and mutation analyses suggested that transcription of the HFH-3/rTSC was actually responsive to HFH-3, and electrophoretic mobility shift assay showed a direct binding of in vitro synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the rTSC gene is localized to rat chromosome 19p12--24. The transcription regulatory region of the TSC gene confers DCT-specific gene expression. DCT-specific transcription factor HFH-3 may be involved in the renal tubule-specific transcription of TSC gene.

Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction with NRF2.

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor gamma (PPARgamma) in macrophages. The transcription activity of a cloned 5'-flanking region (1.6 kilobases) of the rat TXS gene (5'FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5'FL-TXS were inhibited by PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPARgamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPARgamma effect is located in a region containing the nuclear factor E2 (NF-E2)/AP-1 site (-98/-88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ(2), TRO, or in vitro translated PPARgamma. The transcription suppression by PPARgamma was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPARgamma and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (-98/-88) is the major promoter of 5'FL-TXS which can be suppressed by activated PPARgamma via a protein-protein interaction with NRF2 in macrophages.

Characterization of mouse retinoid X receptor (RXR)-beta gene promoter: negative regulation by tumor necrosis factor (TNF)-alpha.

A genomic clone of mouse retinoid X receptor (RXR)-beta (Rxrb) has recently been isolated and mapped within the H2-K region of the mouse major histocompatibility complex. A putative 250-bp promoter, which is located between Rxrb and H2-Ke4, and may possibly be their common promoter, has also been identified. In order to study the gene regulation of Rxrb, we analyzed the transcriptional function of the Rxrb promoter with chimeric constructs containing the Rxrb promoter fragments fused upstream of a firefly luciferase cDNA, which were transiently transfected into rat GH3 cells. We found that 1) a part of the H2-Ke4 genomic region (1.9-kb), as well as the 250-bp promoter, was transcriptionally active as an Rxrb promoter; 2) tumor necrosis factor (TNF)-alpha significantly repressed the activity of the 250-bp promoter although thyroid hormone, 9-cis retinoic acid, interleukin (IL)-1beta, and IL-6 did not affect the activity; 3) either the change in orientation or point mutations of a consensus NF-kappaB site located in the 250-bp promoter did not affect the repression; 4) SB 203580, a highly specific inhibitor of p38 mitogen-activated protein (MAP) kinase, completely abolished the repression by TNF-alpha. These data suggest that TNF-alpha represses the promoter activity of the 250-bp region, and the repression is mediated by p38 MAP kinase independent of NF-kappaB. We thus have first shown a relation between the retinoic acid receptor and a cytokine TNF-alpha.