Objective-Transforming growth factor-1 (TGF1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGF1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression. Methods and Results-TGF1 induction of SMC gene expression, including smooth muscle protein 22-␣ (SM22␣) and smooth muscle ␣-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGF1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22␣ promoter demonstrated that TGF1 activated the SM22␣ promoter through a CC(A/T-rich) 6 GG (CArG) box, which serves as a serum response factor (SRF)-binding site. TGF1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGF1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22␣ promoter-driven luciferase activity by TGF1, whereas bFGF had no effects on the TGF1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGF1-induced SMC gene expression. Key Words: basic fibroblast growth factor Ⅲ transforming growth factor-1 Ⅲ serum response factor Ⅲ SM22␣ Ⅲ smooth muscle cells P henotypic modulation of smooth muscle cells (SMCs) contributes to development of atherosclerotic and restenotic lesions. There is considerable interest in identifying the various extracellular signals that regulate SMC phenotype and the molecular mechanisms underlying such SMC plasticity. Transforming growth factor-1 (TGF1) is one of the primary differentiation factors for SMCs. 1 TGF1 upregulates several SMC differentiation markers, such as smooth muscle ␣-actin (SM␣-actin), smooth muscle myosin heavy chain, SM22␣, and h1 calponin in vitro. Moreover, TGF1 induces expression of these SMC differentiation maker genes in a variety of nonsmooth muscle precursor cell types in culture, including multipotent embryonic fibroblast (10T1/2 cells) and neural crest cells. 2,3 These results suggest that TGF1 evokes an important signal that induces SMC differentiation. Conclusion-OurIn contrast, basic fibroblast growth factor (bFGF) is one of the most important mitogenic growth factors for SMCs 1 and plays an important role in the onset and development of vascular disease. Several studies indicated that experimental reduction in bFGF expression inhibits SMC proliferation after intimal injury. Lindner et al suggested that in injured arteries, bFGF and FGF receptor-type 1 may be involved in the continued prolifer...
Liver-type fatty acid binding protein (L-FABP) binds with high affinity to hydrophobic molecules including free fatty acid, bile acid and bilirubin, which are potentially nephrotoxic, and is involved in their metabolism mainly in hepatocytes. L-FABP is released into the circulation, and patients with liver damage have an elevated plasma L-FABP level. L-FABP is also present in renal tubules; however, the precise localization of L-FABP and its potential role in the renal tubules are not known. In this study, we examined the cellular and subcellular localization of L-FABP in the rat kidney and tried to determine from where the L-FABP in kidney tissues had originated. Immunohistochemical studies of kidney sections localized L-FABP in the lysosomes of proximal tubule cells (PTC). In rats with carbon tetrachloride (CCl 4 )-induced acute liver injury, we detected high levels of L-FABP in the circulation and in the kidney compared with those in the control rat by immunoblotting, while reverse transcription-polymerase chain reaction showed that the level of L-FABP mRNA expression in the kidney of CCl 4 -treated rats was low and did not differ from that in the control rat. When 35 S-L-FABP was intravenously administered to rats, the kidneys took up 35 S-L-FABP more preferentially than the liver and heart, and histoautoradiography of kidney sections revealed that 35 S-L-FABP was internalized via the apical domains of PTC. Quartz-crystal microbalance analysis revealed that L-FABP bound to megalin, a multiligand endocytotic receptor on PTC, in a Ca 2 þ -dependent manner. Degradation assays using megalin-expressing rat yolk sac tumor-derived L2 cells demonstrated that megalin mediated the cellular uptake and catabolism of 125 I-L-FABP. In conclusion, circulatory L-FABP was found to be filtered by glomeruli and internalized by PTC probably via megalin-mediated endocytosis. These results suggest a novel renal uptake pathway for L-FABP, a carrier of hydrophobic molecules, some of which may exert nephrotoxic effects.
Recent studies have shown that the homeobox gene Hex plays an important role in inducing differentiation of vascular endothelial cells. In this study, we examined the expression of Hex in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Immunohistochemistry showed a marked induction of Hex protein in neointimal VSMCs after balloon injury in rat aorta. Western and reverse transcriptase-polymerase chain reaction analyses demonstrated that Hex was abundantly expressed in cultured VSMCs, whereas it was undetectable in other cell types or in normal aorta. The expression pattern of Hex was similar to that of SMemb/NMHC-B, a nonmuscle isoform of myosin heavy chain that we have previously reported to be a molecular marker of dedifferentiated VSMCs. We next examined the role of Hex in SMemb gene transcription. Promoter analysis demonstrated that the sequence identical to consensus cAMP-responsive element (CRE) located at -481 of the SMemb promoter was critical for Hex responsiveness. Mutant Hex expression vector, which lacks the homeodomain, failed to stimulate SMemb gene transcription, suggesting the requirement of the homeodomain for its transactivation. Elecrophoretic mobility shift assay showed that Hex binds to a consensus binding sequence for homeobox proteins, but not to CRE. Cotransfection of protein kinase A expression vector increased the ability of Hex to stimulate SMemb promoter activity in a CRE-dependent manner. Overexpression of CRE binding protein (CREB), but not Mut-CREB which contains mutation at Ser133, strongly activated Hex-induced SMemb promoter activity. These results suggest that Hex mediates transcriptional induction of the SMemb/NMHC-B gene via its homeodomain, and Hex can function as a transcriptional modulator of CRE-dependent transcription in VSMCs.
Abstract-Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a member of the nuclear receptor superfamily that acts as a key player in adipocyte differentiation, glucose metabolism, and macrophage differentiation. Osteopontin (OPN), a component of extracellular matrix, is elevated during neointimal formation in the vessel wall and is synthesized by macrophages in atherosclerotic plaques. In the present study, we investigated the molecular mechanisms regulating OPN gene expression by PPAR␥ in THP-1 cells, a cell line derived from human monocytic leukemia cells. Northern and Western blot analyses showed that exposure of THP-1 cells to PMA (phorbol 12-myristate 13-acetate) increases OPN mRNA and protein levels in a time-dependent manner. PMA-induced OPN expression was significantly decreased by troglitazone (Tro) and other PPAR␥ ligands. Transient transfection assays of the human OPN promoter/luciferase construct showed that PPAR␥ represses OPN promoter activity, and the PPAR␥-responsive region within the OPN promoter lies between Ϫ1000 and Ϫ970 relative to the transcription start site. Site-specific mutation analysis and electrophoretic mobility shift assays indicated that a homeobox-like A/T-rich sequence between Ϫ990 and Ϫ981, which
Objective-Transforming growth factor-1 (TGF-1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. Methods and Results-TGF-1 induction of the SMC contractile protein SM22␣ gene expression was inhibited by PP1(an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src . PP1 also inhibited the TGF-1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-1-induced expression of SM22␣. Furthermore, we demonstrate that TGF-1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin. Conclusion-Our results suggest that TGF-1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22␣ and PAI-1.
Abstract. Anomaly detection is a promising approach to detecting intruders masquerading as valid users (called masqueraders). It creates a user profile and labels any behavior that deviates from the profile as anomalous. In anomaly detection, a challenging task is modeling a user's dynamic behavior based on sequential data collected from computer systems. In this paper, we propose a novel method, called Eigen co-occurrence matrix (ECM), that models sequences such as UNIX commands and extracts their principal features. We applied the ECM method to a masquerade detection experiment with data from Schonlau et al. We report the results and compare them with results obtained from several conventional methods.
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