Elsevier

Neuroscience

Volume 269, 6 June 2014, Pages 112-130
Neuroscience

The high-mobility group I-Y transcription factor is involved in cerebral ischemia and modulates the expression of angiogenic proteins

https://doi.org/10.1016/j.neuroscience.2014.03.036Get rights and content

Highlights

  • We identified HMGIY as a transcription factor (TF) regulated in brain ischemia.

  • Several genes implicated in angiogenesis share HMGIY TF binding sites.

  • Cerebral ischemia promotes changes in HMGIY gene expression.

  • Brain HMGIY interaction profile with other TFs changes under ischemic conditions.

  • HMGIY regulates VEGF and ANGPT-1 expression and promotes endothelial cell viability.

Abstract

The present study aims to identify transcription factors (TFs) contributing to angiogenesis, a mechanism involved in giving plasticity to the brain, as potential therapeutic targets after cerebral ischemia. The promoter sequences from candidate genes involved in angiogenesis were submitted to a comparative analysis by bioinformatics software. High-mobility group I-Y protein (HMGIY) TF characterization in a rat permanent focal cerebral ischemia model was performed by quantitative real time polymerase chain reaction and Western blot for the TF expression profile study. The TF functional study was carried out using a TF–TF interaction array and gene silencing by siRNA in rat brain microvascular endothelial cells. The results showed that the promoters shared a common TF binding site for HMGIY. The expression profile analysis in ischemic rat brain showed an increase in HMGIY mRNA in the acute phase and a progressive overexpression of protein over time post-ischemia. The interaction array analysis revealed that ischemia promotes the interaction of HMGIY with TFs involved in different cerebral plasticity processes. In vitro knockdown studies showed that angiopoietin 1 and vascular endothelial growth factor expression is controlled by HMGIY and that this TF is involved in cell survival in brain endothelial cells. These findings suggest that HMGIY is a potential therapeutic target that could promote brain repair functions after stroke.

Introduction

Despite the discovery of several promising neuroprotective therapies in rodent models of stroke, no therapy other than fibrinolytics has been found to be effective in human clinical trials (Durukan and Tatlisumak, 2007). To address this shortfall in our knowledge, the therapeutic strategy of stroke research has recently been focused on the study of molecular processes involved in brain plasticity in order to reveal the underlying biological bases. Knowledge of these mechanisms could suggest useful therapeutic tools to promote endogenous cerebral repair after brain ischemic damage.

Angiogenesis is a key restorative mechanism in response to ischemia in several non-central nervous system tissues. Autopsy studies have revealed that angiogenesis is partially stimulated in brain ischemia via the hypoxia-inducible factor (HIF) pathways (Krupinski et al., 1994). Although it might seem obvious that a pro-angiogenic therapy in stroke would be beneficial the consequences of such a therapeutic orientation is still unclear. Krupinski et al. demonstrated that the number of new vessels in ischemic penumbral regions correlated with longer survival in ischemic stroke patients, suggesting that active angiogenesis might be beneficial for the ischemic brain (Krupinski et al., 1993). Manoonkittinwongsa et al. found that microvessel density only increased in the ischemic margin adjacent to areas of pan-necrosis and that it was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascular changes, suggesting that ischemia-induced microvessels are formed to facilitate macrophage infiltration and the removal of necrotic brain (Manoonkitiwongsa et al., 2001). Neurovascular remodeling is a key component of recovery after stroke. It is increasingly recognized that neuronal plasticity is dependent on vascular support. Over the past few years, complex mechanisms underlying these endogenous responses in damaged brain parenchyma have increasingly been identified (Navaratna et al., 2009).

Different signaling pathways are initiated in response to ischemia, leading to inflammation, apoptosis, angiogenesis and surveillance (Torregrosa, 2012), which are tightly regulated by a network of different molecules, usually proteins, called transcription factors (TFs). These gene expression modulators do not function in a vacuum but rather interact and cooperate with other transcriptional regulators, components of the general transcriptional machinery, and chromatin-modifying factors (Wu et al., 2010). Detailed study of the transcriptional network will facilitate a more complete understanding of how specific factors regulate the complex angiogenic process in conditions of cerebral ischemia, and better understanding of these pathways should lead to the design of therapies that are specifically aimed at promoting angiogenesis (Navaratna et al., 2009). The present study aims to identify TFs involved in this endogenous therapeutic mechanism in the context of cerebral ischemia. After performing a bioinformatics analysis, we focused the study on the high-mobility group I-Y (HMGIY or HMGA1) TF.

The HMGIY protein belongs to family A (characterized for containing basic DNA-binding domains termed “AT-hooks”), which comprizes four members coded by two genes (HMGA1 and HMGA2) (Fedele et al., 2001, Reeves, 2001, Hock et al., 2007, Catez and Hock, 2010, Maasch et al., 2010). HMGAs are ubiquitous nuclear proteins that bind to DNA and induce structural changes in the chromatin fibers and nucleosome, which regulate the expression of numerous genes in vivo (Reeves, 2001, Hock et al., 2007). They function in the cell as highly connected ‘nodes’ of protein–DNA and protein–protein interactions that influence a diverse array of normal biological processes including growth, proliferation, differentiation, and death (Reeves, 2001, Gerlitz et al., 2009). HMGA are relatively abundant proteins in undifferentiated and proliferating cells of early embryos and undetectable in fully differentiated cells (Hock et al., 2007). Likewise, they participate in pathological processes acting as regulators of viral gene transcription and promoting tumor progression and metastasis when overexpressed in cells. High constitutive HMGA protein levels are among the most consistent features observed in all types of cancers and greater concentrations correlate with increased malignancy (Reeves, 2001, Gerlitz et al., 2009). In the present study we characterize for the first time the HMGIY TF in rat cerebral ischemia and explore its potential angiogenic function in vitro.

Section snippets

Bioinformatics analysis

In order to detect TFs involved in angiogenesis, several genes involved in different processes that promote or inhibit the angiogenic response were selected using the PubMed database as candidate genes. The promoter sequence from the candidate genes was obtained and submitted to bioinformatics analysis in order to find common TF binding sites (TFBS) to identify transcriptional regulatory signals shared among the different co-expressed candidate genes. GenBank, Reference Sequence (RefSeq) and

Candidate co-expressed genes involved in angiogenesis shared TFBS for HMGIY

Eighty-four genes were selected as candidate genes involved in brain angiogenesis processes (Table 3). The bioinformatics analysis of their promoter sequences revealed common TFBS shared by the different co-expressed candidate genes (Table 4, Table 5, Table 6, Table 7, Table 8, Table 9). The candidate genes found to share common TFBSs are shown according to the function and the processes in which they are involved: genes overexpressed in the subventricular zone after ischemia and with

Discussion

In the present study we have identified and characterized HMGIY as a TF regulated in cerebral ischemia. We show that ischemia induces HMGIY interactions with other TFs that modulate brain repair functions and report a new association between HMGIY and the angiogenic proteins VEGF and ANGPT-1 as well as its involvement in the proliferation of endothelial cells.

The knowledge of genes and their transcriptional regulators that participate in giving the brain the potential to compensate for ischemic

Conclusions

The present study has identified HMGIY as a new TF involved in cerebral ischemia. Changes induced by ischemia in the HMGIY TF interaction profile at the acute phase showing an increase of interactions with TFs involved in the modulation and promotion of cerebral repair processes suggest that the genes modulating brain plasticity processes are activated as early as a few hours after stroke. It has also been found that HMGIY regulates both VEGF and ANGPT-1 expression and promotes endothelial cell

Acknowledgments

This work was partially supported by the Centre d’Innovació i Desenvolupament Empresarial (CIDEM) of the Generalitat de Catalunya (grant No. RDITSCON07-1-0006), Grupo Ferrer Internacional SA, Spanish Ministry of Science and Innovation and the European Regional Development Fund (ERDF) (PROFIT grant No. CIT 090000-20008-11), Institut de Salud Carlos III (ISCIII) grant Nos. FIS-PI070322 and ISCIII retics-RENEVAS-RD06/0026/0005 and RD07/0026/2002. We would like to thank Andrew Hughes for performing

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    Present address: Servicio de Neurología, Instituto de Investigación Sanitaria, Hospital Universitario La Princesa, C/ Diego de León, 62, 28006 Madrid, Spain.

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