Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene results in highly vascularized tumors, making the VHL tumor syndrome an ideal system to study the mechanisms of angiogenesis. VHL operates along two pathways with the first involving hypoxia-inducible factor-A degradation and downregulation of its proangiogenic target genes vascular endothelial growth factor and platelet-derived growth factor-B, and the second pathway promoting extracellular matrix (ECM) assembly. Secretion of proangiogenic factors was shown to be a primary inducer of angiogenesis. Here, we show that loss of ECM assembly correlates with tumor angiogenesis in VHL disease. Upon inactivation of the VHL-ECM assembly pathway, we observe tumors that are highly vascularized, have a disrupted ECM, and show increased matrix metalloproteinase-2 activity. Loss of the VHL pathway leading to hypoxia-inducible factor-A degradation results in tumors with increased vascular endothelial growth factor levels but with surprisingly low microvessel density, a tightly assembled ECM and low invasive ability. We conclude that loss of ECM integrity could promote and maintain tumor angiogenesis by providing a route for blood vessels to infiltrate tumors.

Neuronal network dysfunction and cognitive decline constitute the most prominent features of Alzheimer’s disease (AD), although mechanisms causing such impairments are yet to be determined. Here we report that virus-mediated delivery of the active spliced transcription factor X-Box binding protein 1s (XBP1s) in the hippocampus rescued spine density, synaptic plasticity and memory function in a mouse model of AD. XBP1s transcriptionally activated Kalirin-7 (Kal7), a protein that controls synaptic plasticity. In addition, we found reduced levels of Kal7 in primary neurons exposed to Aβ oligomers, transgenic mouse models and human AD brains. Short hairpin RNA-mediated knockdown of Kal7 altered synaptic plasticity and memory formation in naive mice. Further, reduction of endogenous Kal7 compromised the beneficial effects of XBP1s in Alzheimer’s model. Hence, our findings reveal that XBP1s is neuroprotective through a mechanism that engages Kal7 pathway with therapeutic implications in AD pathology.

The mechanism by which hypoxia induces gene transcription is now well established. Hypoxia reduces activity of prolyl hydroxylases (PHD) that hydroxylate specific proline residues in the oxygen-dependent degradation domain (ODD) of hypoxia-inducible factor-1␣ (HIF-1␣). As a consequence, HIF-1␣ accumulates and promotes hypoxic tolerance by activating gene transcription. This paper identifies the three forms of PHDs in rats and shows that a period of hypoxia selectively increases expression of PHD-2 mRNAs levels. We developed assays for PHD activity that used (i) the peptidespecific conversion of labeled 2-oxoglutarate into succinate and (ii) the binding of the von Hippel-Lindau protein to a glutathione S-transferase-ODD fusion protein. The two assays indicated a low enzymatic activity in normoxic and hypoxic cells and a rapid increase during reoxygenation. We also developed hydroxyprolinespecific antibodies that recognized hydroxylated forms of a fusion protein (ODD-green fluorescent protein) that combined the ODD domain of HIF-1␣ and the green fluorescent protein. Using this antibody, we demonstrated that reoxygenation induced a rapid hydroxylation of Pro-564, which was followed by a massive degradation of the proteins. The results suggest that a hypoxic upregulation of PHD (presumably PHD-2) acts as a feedback mechanism to stop hypoxic responses in reoxygenated cells. We propose that proline hydroxylation might play a role in hypoxic preconditioning.Cells respond to reduced oxygen tensions by up-regulating the expression of genes involved in angiogenesis (e.g. vascular endothelial growth factor), erythropoiesis (e.g. erythropoietin), and glycolysis. The transcriptional activation of target genes is induced by a common transcription factor, hypoxia inducible factor-1 (HIF-1).1 HIF-1 was first identified as a heterodimeric transactivator that recognizes a specific DNA sequence termed hypoxia-responsive element in the 3Ј-untranslated region of the erythropoietin gene (1). HIF-1 is composed of two subunits, HIF-1␣ and the aryl hydrocarbon nuclear translocator, both of which belong to the large family of basic helix-loop-helix-perarnt-sim transcription factors (2). The mechanism of the hypoxic induction of HIF-1␣ has recently been identified. Under normoxic conditions, specific HIF-1␣ prolyl hydroxylases (PHD) hydroxylate two proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation (ODD) domain of HIF-1␣ (3, 4). The von Hippel-Lindau protein (vHL) E3 ubiquitin ligase complex associates to hydroxylated proline residues and targets HIF-1␣ to proteasomal degradation. Under hypoxic conditions, oxygen becomes rate-limiting for proline hydroxylation. As a consequence, HIF-1␣ accumulates, migrates to the nucleus, and associates with the aryl hydrocarbon nuclear translocator and the complex interacts with hypoxia-responsive element of target genes (5, 6).Less attention has been given to the situation in which hypoxic cells are reoxygenated. Tissue reoxygenation is not an unusual condition. It is frequently...

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.