Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP‐1 transcription factor is considered as facilitator of hypoxia‐induced VEGF expression through interaction with hypoxia‐inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP‐1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP‐1 subunit junB is a target gene of hypoxia‐induced signaling via NF‐κB. Loss of JunB in various cell types results in severely impaired hypoxia‐induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB‐deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB−/− teratocarcinomas because of severely impaired paracrine‐acting VEGF and the subsequent inability to efficiently recruit host‐derived vessels.

Tumor invasion and metastasis of malignant melanoma have been shown to require proteolytic degradation of the extracellular environment achieved primarily by enzymes of the matrix metalloproteinases (MMP) family. We have earlier shown that increased enzyme activity is localized at the border of tumor cells and the adjacent peritumoral connective tissue, emphasizing the importance of tumor-stroma interactions in the regulation of MMP activity. To confirm the role of stroma-derived MMP-13 in the invasion process, we investigated the invasiveness of melanoma cells upon intradermal injection in mice with complete inactivation of MMP-13. Tumor growth was significantly impaired in mmp-13(-/-) mice and most significant at early time points as compared with wild-type littermates. Moreover, metastasis to various organs was reduced to 17.6 vs 30% in lungs, 2.9 vs 30% in the liver. Strikingly, ablation of MMP-13 completely abrogated formation of metastasis in the heart (0 vs 40%). Notably, decreased tumor growth in mmp-13(-/-) mice was associated with reduced blood vessel density. In addition, decreased blood vessel permeability in the tumors was measured by magnetic resonance imaging of tumor-bearing animals. These data suggest an important role of MMP-13 in tumor growth and an unexpected role in organ-specific metastasis of melanoma cells.

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen levels. By stimulating the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), they trigger the neovascularization of tissues under physiologic and pathologic conditions. Here, we have investigated the endothelial cell-autonomous HIF function in blood vessel growth and development by expressing a dominant-negative HIF mutant (HIFdn) that inhibits the transcriptional responses mediated by both HIF-1 and HIF-2, specifically in endothelial cells of transgenic mice. HIFdn transgenic embryos were growth retarded and died around E11.5. Primitive vascular networks were established, but vascular remodeling in the yolk sac and in the embryo proper was defective, and vascular sprouts failed to invade the neuroepithelium. In addition, heart looping was incomplete, and the ventricles of the heart were thin-walled and lacked trabeculation. Similar cardiovascular defects have been observed in Tie2-deficient mouse embryos. Consistently, HIFdn transgenic embryos expressed reduced levels of the endothelial angiopoietin receptor, Tie-2, whereas other endothelial markers, such as PE-CAM-1, Tie-1, and VE-cadherin were not affected. These results show that HIFs in endothelial cells are essential for embryonic heart and blood vessel development and control angiogenesis and vascular remodeling. IntroductionGrowing tissues have a continuous demand for oxygen and nutrients. Tissue hypoxia efficiently triggers the growth of new blood vessels in both physiologic and pathologic processes. For example, hypoxia stimulates the compensatory formation of blood vessels in coronary heart disease, stroke, or in tumors by upregulating the expression of angiogenic factors such as vascular endothelial growth factor (VEGF). 1,2 It has also been suggested that during embryonic vessel development, regions of relative hypoxia attract growing blood vessels. 3 The cellular responses to low oxygen levels are mediated by transcription factors called hypoxiainducible factors (HIFs). At present, 3 different HIF family members are known: HIF-1, HIF-2, and HIF-3. HIFs are heterodimers composed of 1 of 3 different HIF-␣ subunits, HIF-1␣, 4 HIF-2␣, [5][6][7]8,9 and the common HIF-1␤ (ARNT) subunit. Under normoxic conditions, the HIF-␣ subunits are rapidly degraded. This process involves the posttranslational modification of HIF-␣ by a recently discovered family of prolyl hydroxylases (PHDs). 10 Hydroxylated prolyl residues of the HIF-␣ subunits are recognized by the von Hippel-Lindau protein, a part of the E3 ubiquitin ligase complex that marks the HIF-␣ subunits for degradation by the proteasome. [11][12][13] . Under hypoxic conditions, PHDs are inactive and HIF-␣ subunits accumulate in the nucleus, where they transactivate their target genes. In addition, growth factor signaling modulates HIF activity and stability in a hypoxiaindependent fashion. 10,14 Whereas HIF-1␣ is expressed ubiquitously, HIF-2␣ and HIF-3␣...

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis.