Net-targeted mutant mice develop a vascular phenotype and up-regulate egr-1

Abstract

The ternary complex factors (TCFs) Net, Elk-1 and Sap-1 regulate immediate early genes through serum response elements (SREs) in vitro, but, surprisingly, their in vivo roles are unknown. Net is a repressor that is expressed in sites of vasculogenesis during mouse development. We have made gene-targeted mice that express a hypomorphic mutant of Net, Netd, which lacks the Ets DNA-binding domain. Strikingly, homozygous mutant mice develop a vascular defect and up-regulate an immediate early gene implicated in vascular disease, egr-1. They die after birth due to respiratory failure, resulting from the accumulation of chyle in the thoracic cage (chylothorax). The mice have dilated lymphatic vessels (lymphangiectasis) as early as E16.5. Interestingly, they express more egr-1 in heart and pulmonary arteries at E18.5. Net negatively regulates the egr-1 promoter and binds speci®-cally to SRE-5. Egr-1 has been associated with pathologies involving vascular stenosis (e.g. atherosclerosis), and here egr-1 dysfunction could possibly be associated with obstructions that ultimately affect the lymphatics. These results show that Net is involved in vascular biology and egr-1 regulation in vivo. Keywords: egr-1/Elk-3/ERP/Net/Sap-2 IntroductionThe ternary complex factors (TCFs) form a subfamily of Ets-domain transcription factors. The three TCFs, Elk-1, Sap-1 and Net/Sap-2/Erp/Elk-3 (Price et al., 1996;Wasylyk et al., 1998), have four conserved domains, A±D. A is the Ets DNA-binding domain (DBD). The B-box interacts with the serum response factor (SRF). C is a transcriptional activation domain that is stimulated by mitogen-activated protein (MAP) kinase phosphorylation. The D-domain is a MAP kinase-binding site and a nuclear localization signal. The TCFs are nuclear mediators of cellular responses to the activation of MAP kinase pathways. Net differs from the other TCFs in that in basal conditions, in which MAP kinases are not activated, it strongly inhibits transcription. Repression is mediated by two domains, the NID (Maira et al., 1996) and the CID (Criqui-Filipe et al., 1999). The TCFs form ternary complexes with SRF on serum response elements (SREs) of immediate early gene promoters, such as c-fos, egr-1 and jun-B. The SRE is constitutively occupied by factors, and extracellular signals are thought to lead to both phosphorylation of the complex and changes in its composition due to the exchange of TCFs.The in vivo role of the TCFs is poorly understood. They may regulate the expression of immediate early genes in response to various inductive stimuli. The TCFs are expressed in many cell types and tissues (Giovane et al., 1994;Lopez et al., 1994;Magnaghi-Jaulin et al., 1996;Nozaki et al., 1996;Sgambato et al., 1998), but their precise in vivo expression patterns are not well known. Net is expressed during mouse development at E7.5±8.5 in developing vascular primordia, including the yolk sac blood islands, allantoic vessels, heart endocardium and dorsal aortae (Ayadi et al., 2001). Vascular endothelial cell expression persists ...

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

“…However, because ephrinB2 ⌬V/⌬V mutants typically died before 2 weeks of age, a lethality not always seen in other mouse lines with chylothorax (Huang et al, 2000;Ayadi et al, 2001), we reasoned that perhaps these animals had a second problem which contributed to early lethality. During histopathological inspection of postnatal ephrinB2 ⌬V/⌬V mutants, we found that their lungs showed severe airspace enlargement (see below).…”

“…However, the available evidence suggests that the lung abnormalities in ephrinB2 ⌬V/⌬V mice are not a direct result of the abnormal lymphatic function in the same mice (Makinen et al, 2005). Lung airspace enlargement has not been reported in other mouse models of chylothorax (Huang et al, 2000;Ayadi et al, 2001), nor has chylothrorax been reported in mouse models of abnormal alveolar development, suggesting that these two systems can develop independently of each other. Second, examination of mutant lung Tenascin-C immunonreactivity in wild-type lung is distributed as immunoreactive puncta (arrows) with a spidery intervening network.…”

The ternary complex factor Net/Elk-3 participates in the transcriptional response to hypoxia and regulates HIF-1α

“…Net regulates cell migration through repression of PAI-1 , and is required for tumour growth in xenographs (Zheng et al, 2003). Mice that express mutant Net lacking its DNA binding domain exhibit respiratory distress (Ayadi et al, 2001), and delayed wound healing due to impaired angiogenesis (Zheng et al, 2003). We have recently shown that Net is downregulated in hypoxia and is required for the regulation of several genes in response to hypoxia (Gross et al, 2007).…”

Serum Response Factor Contributes Selectively to Lymphocyte Development

“…Recently, members of the TCF family of SRF cofactors have been characterized by knock-out studies. Elk-1 deficient mice display normal immune responses and mildly impaired neuronal gene inactivation (44) and Net mutants show defects in cell migration (45), vasculature development (46), and impaired angiogenesis during wound healing (47). The strongest phenotype with regard to immune functions is observed in SAP-1-null mice where thymocyte development is severely impaired and a decrease in the amount of CD4 ϩ and CD8 ϩ single positive (SP) cells is seen resulting from defective thymocyte positive selection (48).…”