Matrin 3 as a key regulator of endothelial cell survival

Abstract

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.

Introduction

Matrin 3, an abundant protein of the internal nuclear matrix [1], has emerged as an important regulator of NMDA-induced neuronal death. It seems to be the main substrate of PKA, phosphorylated following extensive NMDA receptor activation and then rapidly degraded [2]. Consistently, blockade of NMDA receptors prevented PKA-induced phosphorylation and degradation of matrin 3 as well as cell apoptosis.

The primary structure deduced for matrin 3 predicts two DNA binding domains with C2H2-type zinc finger domain and two tandem RNA recognition motifs. It has been implicated in the processing of RNAs and retention of hyper-edited RNA within the nucleus [3]. Moreover, matrin 3 has nuclear localization signals and several phosphorylation sites for tyrosine or serine/threonine kinases [4]. Though its activity and mode of action are still unclear, it appears that matrin 3 serves as a scaffolding molecule that interacts with a number of unique nuclear localized proteins [5], [6]. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis.

Recent data provided evidence that there may be a direct link between neuronal death and degeneration of the vasculature. Damage of both types of cells resulting from activation of NMDA receptors by glutamate was described in diabetic retinopathy [7], [8], the retinal ischemia–reperfusion model [9] and the blood–brain barrier [10]. It supported the concept that apart from neurotoxic effects, the increased levels of extracellular glutamate may also be the cause of “endothelial excitotoxicity.” Under certain pathological conditions, endothelial cells can be exposed to excessively high glutamate concentrations released from neurons and astrocytes due to failure or reversal of glutamate transporters or from polymorphonuclear leukocytes in response to inflammatory stimuli. Under ischemic or traumatic conditions, such dramatically increased glutamate concentration in the brain interstitial space has been reported [11]. Endothelial cells appear to contain the NMDA receptors [12] of which expression can be highly upregulated by reactive oxygen species [13]. Hence, both in neurons and endothelial cells, PKA-mediated phosphorylation of matrin 3 induced by the NMDA receptor may serve as a rapid way of transferring information from the membranes to nuclei under physiological conditions and may contribute to cell death under pathological conditions.

Therefore, in the present study we evaluated the significance of matrin 3 for survival of endothelial cells and attempted to explain to what extent it may be implicated in apoptosis or necrosis of these cells.