ABSTRACT
Adequate response to low oxygen levels (hypoxia) by hypoxia inducible factor (HIF) is essential for normal development and physiology, but this pathway may also contribute to pathological processes like tumor angiogenesis. Here we show that hypoxia is an inducer of Notch signaling. Hypoxic conditions lead to induction of the Notch ligand Dll4 and the Notch target genes Hey1 and Hey2 in various cell lines. Promoter analysis revealed that Hey1, Hey2 and Dll4 are induced by HIF-1alpha and Notch activation. Hypoxia-induced Notch signaling may also determine endothelial identity. Endothelial progenitor cells (EPCs) contain high amounts of COUP-TFII, a regulator of vein identity, while levels of the arterial regulators Dll4 and Hey2 are low. Hypoxia-mediated upregulation of Dll4 and Hey2 leads to repression of COUP-TFII in eEPCs. Finally, we show that Hey factors are capable of repressing HIF-1alpha-induced gene expression, suggesting a negative feedback loop to prevent excessive hypoxic gene induction. Thus, reduced oxygen levels lead to activation of the Dll4-Notch-Hey2 signaling cascade and subsequent repression of COUP-TFII in endothelial progenitor cells. We propose that this is an important step in the developmental regulation of arterial cell fate decision.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Embryonic Stem Cells/metabolism , Endothelial Cells/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Receptors, Notch/metabolism , Repressor Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Arteries/cytology , Arteries/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/pharmacology , CHO Cells , COUP Transcription Factor II/genetics , COUP Transcription Factor II/metabolism , Calcium-Binding Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Hypoxia , Cell Line , Cricetinae , Cricetulus , Embryonic Stem Cells/cytology , Endothelial Cells/cytology , Feedback , Gene Expression Regulation , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Mice , Models, Biological , Neovascularization, Physiologic , Promoter Regions, Genetic , Receptors, Notch/genetics , Repressor Proteins/genetics , Signal Transduction , Transcriptional ActivationABSTRACT
The Hey basic helix-loop-helix transcription factors are downstream effectors of Notch signaling in the cardiovascular system. Mice lacking Hey2 develop cardiac hypertrophy, often associated with congenital heart defects, whereas combined Hey1/Hey2 deficiency leads to severe vascular defects and embryonic lethality around embryonic day E9.5. The molecular basis of these disorders is poorly understood, however, since target genes of Hey transcription factors in the affected tissues remain elusive. To identify genes regulated by Hey factors we have generated a conditional Hey1 knockout mouse. This strain was used to generate paired Hey2- and Hey1/2-deficient embryonic stem cell lines. Comparison of these cell lines by microarray analysis identified GATA4 and GATA6 as differentially expressed genes. Loss of Hey1/2 leads to elevated GATA4/6 and ANF mRNA levels in embryoid bodies, while forced expression of Hey factors strongly represses expression of the GATA4 and GATA6 promoter in various cell lines. In addition, the promoter activity of the GATA4/6 target gene ANF was inhibited by Hey1, Hey2, and HeyL. Protein interaction and mutation analyses suggest that repression is due to direct binding of Hey proteins to GATA4 and GATA6, blocking their transcriptional activity. In Hey2-deficient fetal hearts we observed elevated mRNA levels of ANF and CARP. Expression of ANF and Hey2 is normally restricted to the trabecular and compact myocardial layer, respectively. Intriguingly, loss of Hey2 leads to ectopic ANF expression in the compact layer, suggesting a direct role for Hey2 in limiting ANF expression in this cardiac compartment.
Subject(s)
Atrial Natriuretic Factor/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Proteins/metabolism , Fetal Heart/metabolism , GATA4 Transcription Factor/genetics , Repressor Proteins/metabolism , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line , DNA, Complementary/genetics , Gene Expression Regulation , Gene Targeting , Helix-Loop-Helix Motifs/genetics , Histone Deacetylases/metabolism , Humans , Mice , Mice, Knockout , Muscle Proteins , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/geneticsABSTRACT
The vertebrate kidney develops through a series of mesenchymal-epithelial interactions between the ureteric bud and the metanephrogenic mesenchyme to form nephrons and the collecting system, which are both embedded in the renal interstitium. The interstitial stromal cells are an essential prerequisite for regular kidney development, but their origin and function is poorly understood. They are found in the kidney periphery and the medulla and are likely derived from the kidney mesenchyme and/or from migrating neural crest cells. During late kidney development, stromal cells are lost through massive apoptosis. We have identified a novel marker of kidney stroma cells, Snep (stromal nidogen extracellular matrix protein), that is additionally expressed in mesenchymal cells of other embryonic tissues and within the nervous system. Of interest, Snep transcripts are also found at sites of embryonic apoptosis. Furthermore, comparative expression analysis of kidney stroma markers suggests that Snep is expressed in a specific subpopulation of stromal cells and may provide environmental cues to support regular development.