Dataset Information

The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences.

ABSTRACT: Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21(WAF1/CIP1) expression in vascular ECs. To elucidate the mechanism through which GAX activates p21(WAF1/CIP1) expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21(WAF1/CIP1). There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21(WAF1/CIP1) promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, approximately 15 kb upstream of the p21(WAF1/CIP1) ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G(0)/G(1) arrest correlates with their ability to transactivate the p21(WAF1/CIP1) promoter. We conclude that GAX activates p21(WAF1/CIP1) through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.

SUBMITTER: Chen Y

PROVIDER: S-EPMC1865102 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences.

Chen Yun Y Leal Alejandro D AD Patel Sejal S Gorski David H DH

The Journal of biological chemistry 20061030 1

Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21(WAF1/CIP1) expression in vascular ECs. To elucidate the mechanism through which GAX activates p21(WAF1/CIP1) expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for th ...[more]

PMID: 17074759

Similar Datasets

Project description:BackgroundLoss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer.MethodsFOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells.ResultsQuantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21(WAF1/CIP1) in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-beta-induced p21(WAF1/CIP1) expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21(WAF1/CIP1) expression. Notably, FOXG1 was able to inhibit the p21(WAF1/CIP1) promoter activity in a p53-independent manner by transient reporter assays.ConclusionOur results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-beta-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21(WAF1/CIP1) transcription.

Dataset Information

The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences.

Publications

The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets