Biosynthesis of tumor necrosis factor-␣ (TNF-␣) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-␣ promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-␣ promoter localized enhancer elements in a region (؊182 to ؊37 base pairs (bp)) that were required for optimal transcription of the TNF-␣ gene in response to LPS. Two regions were identified: region I (؊182 to ؊162 bp) contained an overlapping Sp1/Egr-1 site, and region II (؊119 to ؊88) contained CRE and NF-B (designated B3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and B3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and B3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the B3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-␣ promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements.

EGR-1, a transcription factor with important functions in the regulation of growth and differentiation, is highly expressed in brain. Previous studies have shown that EGR-1 suppresses the transformed phenotype. However, the expression and role of EGR-1 in human glioblastoma cells are not yet determined. In this study, we found that the basal expression of the EGR-1 protein is undetectable, but is inducible in four human glioblastoma cell lines. To determine EGR-1 functions, we reexpressed EGR-1 in human glioblastoma U251 cells and found that the secretion of transforming growth factor-␤1 (TGF-␤1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin ( Egr-1 (also known as NGFI-A (1), TIS8, Krox-24, and Zif268) (33) is a member of the immediate-early gene family that encodes a nuclear phosphoprotein. EGR-1 contains three zinc finger motifs that bind and regulate transcription through GCrich elements (GCEs) 1 with a consensus sequence of 5Ј-GCG(T/ G)GGGCG-3Ј (2-4). The promoter regions of many genes, including several growth factors and cytokines, are regulated by EGR-1 (for a review, see Ref. 5). The Egr-1 gene is rapidly and transiently induced by growth factors and other signals and is functionally implicated in cell proliferation and in differentiation processes (6 -8).Egr-1 is broadly expressed during development and in the tissues of adults of many species. It can be found in epithelial tissues, heart, thymus, and central and peripheral nervous systems. The basal expression of the EGR-1 protein in adult rat and mouse brain is high (for a review, see Ref. 9). At a functional level, several in vitro and in vivo studies initially characterized Egr-1 as having a role in the control of cell growth, proliferation, differentiation, and development. However, whether EGR-1 is a participant in disparate activities or is a more central factor regulating coordinate expression of a characteristic phenotype is unclear. Our previous studies have shown that stable re-expression of EGR-1 inhibits transformation in model cells and in several human tumor cell lines (10,11,38,39). The re-expression of Egr-1 in fibrosarcoma HT-1080, glioblastoma U251, and U373 cells leads to decreased DNA synthesis, growth, and tumorigenicity (39). The mechanism of the EGR-1 suppressive function has been studied in detail in fibrosarcoma HT-1080 cells (40 -42). The EGR-1 protein specifically binds the GCE sites of the human TGF-␤1 promoter, transactivates the TGF-␤1 gene, and enhances the expression and secretion of functional TGF-␤1 in the Egr-1-expressing fibrosarcoma cell line, leading to inhibition of cell proliferation and restoration of anchorage-dependent growth (40).TGF-␤1 is the prototype of a large family of cytokines that control cell proliferation, differentiation, adhesion, and extracellular matrix metabolism. TGF-␤1 has been shown to induce fibronectin (FN) expression at both the mRNA and protein levels (12, 24) and promotes net matrix deposition by increasing the expression of specific ECM components such as FN and collagen ...

No abstract

Cellular and molecular mechanisms of methamphetamine (METH)-induced neurotoxicity may involve alterations of cellular redox status and induction of inflammatory genes in endothelial cells. To study these hypotheses, molecular signaling pathways of METH-induced inflammatory responses via activation of redox-sensitive transcription factors were investigated in human brain microvascular endothelial cells (HBMEC). A dose-dependent depletion of total glutathione levels was detected in HBMEC exposed to METH. In addition, electrophoretic mobility shift assay (EMSA) showed significant increases in DNA binding activities of redox-responsive transcription factors, AP-1 and NF-kappaB, in HBMEC treated with METH. METH-mediated AP-1 or NF-kappaB activation was accompanied by induction of transactivation of AP-1 or NF-kappaB, as measured by dual luciferase assay using specific reporter plasmids. Because NF-kappaB and AP-1 are known to regulate expression of inflammatory genes, expression of the gene encoding for tumor necrosis factor-alpha (TNF-alpha) was also studied in METH-treated HBMEC. A dose-dependent overexpression of the TNF-alpha gene was observed in HBMEC treated with METH. The importance of AP-1 and NF-kappaB in METH-induced TNF-alpha gene was confirmed in functional promoter studies using constructs of the TNF-alpha promoter with mutated AP-1 or NF-kappaB sites. These results indicate that METH-induced disturbances in cellular redox status and activation of AP-1 and NF-kappaB can play critical roles in the signaling pathways leading to upregulation of inflammatory genes in human brain endothelial cells.

No abstract