The Homeobox Gene Gax Inhibits Angiogenesis through Inhibition of Nuclear Factor-κB–Dependent Endothelial Cell Gene Expression

Abstract

The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax downregulated numerous nuclear factor-k kB (NF-k kB) target genes and decreased the binding of NF-k kB to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-k kB activity in vascular endothelial cells. Because NF-k kB has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-k kB-dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype. (Cancer Res 2005; 65(4): 1414-24)

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

“…Evidence has accumulated for a role of NF-kB in angiogenic processes during hypoxia and inflammation (Klein et al, 2002;Ko et al, 2002;Kofler et al, 2005;Patel et al, 2005). Our findings suggest that NF-kB signaling can promote its proangiogenic effects by upregulating PlGF transcription.…”

“…Inflammatory cells such as monocytes/macrophages or T lymphocytes are involved in this process by secreting cytokines that exert effects on endothelial cells (Naldini and Carraro, 2005). NF-kB is a key player in the inflammatory response and there are numerous reports on the role of NF-kB in angiogenic processes (Scatena et al, 1998;Malyankar et al, 2000;Klein et al, 2002;Ko et al, 2002;Patel et al, 2005).…”

Overexpression of the growth arrest-specific homeobox gene Gax inhibits proliferation, migration, cell cycle progression, and apoptosis in serum-induced vascular smooth muscle cells

“…Gax expression is decreased during conversion into synthetic VSMCs with abnormal proliferative and migratory activities (Markmann et al, 2003;Han et al, 2006). Recent studies have demonstrated that Gax overexpression decreases the migration of vascular endothelium cells (Patel et al, 2005). However, few studies have investigated the effects of Gax overexpression on the biological behaviors of serum-induced VSMCs (Maillard et al, 1997;Smith et al, 1997;Zeng et al, 2006).…”

“…Overexpression of Gax with adenoviral vectors in vitro can inhibit TGF-b1-induced adventitial fibroblast proliferation, migration, and adherence; Gax overexpression also decreases the number of S phase cells and increases the number of G0-G1 phase cells and promotes cell apoptosis (Liu et al, 2008). Gax is expressed in quiescent vascular endothelial cells and strongly inhibits endothelial cells activation during stimulation with growth factors or tube formation by activating p21 WAF1/CIP1 expression and reducing anti-apoptotic, NF-kB-dependent gene expression, suggesting that Gax is a negative regulator of angiogenesis (Gorski and Leal, 2003;Patel et al, 2005;Chen et al, 2007). In addition, Gax has been shown to be induced at the onset of cellular senescence in keratinocytes (Irelan et al, 2009).…”

The genetic differences between gallbladder and bile duct cancer cell lines

“…It is known that this gene has alternative splicing variants to generate multiple protein isoforms that may play distinct biological roles and that this gene is associated with human cancer (20). The Gax gene is located on 7p21.2 and is one of the homeobox genes that is closely related to angiogenesis (21). The TBX2 gene is located on 17q23.3 and this region is also the site of gains in many cancers (22)(23)(24)(25).…”