The Transcription Factor EGR-1 Directly Transactivates the Fibronectin Gene and Enhances Attachment of Human Glioblastoma Cell Line U251

Abstract

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 ...

The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFβ1, PTEN, p53, and fibronectin

“…2,12 This possibility was examined in several human glioblastoma cells. Established human cell lines 13 and surgical specimens of glioblastoma 14 or primary cultures of the tumors 15 commonly exhibit low or undetectable levels of Egr1 consistent with a role of Egr1 in the cause or development of glioblastoma. 13,14 Re-expression of Egr1 in several human glioblastoma cell lines such as U251 led to increased expression of TGFb1, fibronectin, and PAI-1.…”

“…Established human cell lines 13 and surgical specimens of glioblastoma 14 or primary cultures of the tumors 15 commonly exhibit low or undetectable levels of Egr1 consistent with a role of Egr1 in the cause or development of glioblastoma. 13,14 Re-expression of Egr1 in several human glioblastoma cell lines such as U251 led to increased expression of TGFb1, fibronectin, and PAI-1. 13 Surprisingly, direct addition of activated TGFb1 to the cells induced expression of PAI-1 but not fibronectin.…”

“…13,14 Re-expression of Egr1 in several human glioblastoma cell lines such as U251 led to increased expression of TGFb1, fibronectin, and PAI-1. 13 Surprisingly, direct addition of activated TGFb1 to the cells induced expression of PAI-1 but not fibronectin. This led to the discovery that fibronectin is a direct target gene of Egr1 via two closely spaced Egr1-binding sequences in the proximal promoter.…”

“…This led to the discovery that fibronectin is a direct target gene of Egr1 via two closely spaced Egr1-binding sequences in the proximal promoter. 13 Both fibronectin and PAI-1 induced by Egr1/TGFb1 are secreted and become part of the extracellular matrix where they greatly increase attachment, an effect that is blocked by addition of RGD-containing peptides or neutralizing antibodies. The 18,19 One mechanism involves the interaction of fibronectin with specific integrins, leading to activation of the protein kinase-B/ Akt (PKB/AkT) pathway 19 (Figure 1).…”

“…24 RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr1 mRNA is always paralleled by the absence of PTEN mRNA, suggesting that Egr1-dependent mechanisms may play a role in the absence of PTEN gene expression that occurs during thyroid cell transformation. 24 Egr1 protein levels are suppressed or absent in a variety of human tumor cell lines 13,28 and tumors, including breast carcinoma, 28 glioblastoma, 14,15 lung cancer, 29,30 and lymphoma. 31 Moreover, in the case of human nonsmall-cell lung cancer, Ferraro et al 32 examined an extensive series of samples from over 125 patients and observed that Egr1 expression predicts both PTEN level and survival to a high degree of significance.…”