PKCβII/HuR/VEGF: A new molecular cascade in retinal pericytes for the regulation of VEGF gene expression

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Abstract

Vascular endothelial growth factor (VEGF)-induced new vessels formation is a key event in diabetic retinopathy, a severe progressive multistage pathology. Literature data indicate that protein kinase C (PKC) is involved in the control of VEGF expression, but, so far, no data are available on the molecular pathway underlying this process. Within this context, we suggest the existence of a new molecular cascade, operating in retinal bovine pericytes and involving PKCβII, the mRNA-stabilizing protein HuR, and VEGF. In particular we show that PKCβII activation is responsible, through the RNA-binding protein HuR, for the increase of VEGF protein content and its release in the medium. The specificity of the PKCβII involvement is confirmed by experiments performed with the LY379196 compound, a selective PKCβII inhibitor. Following acute high-glucose insult this pathway seems still functioning, suggesting that a brief exposure to glucose does not compromise this molecular cascade in pericytes. A better understanding on this new pathway could open novel opportunities for the development of innovative pharmacological therapies useful in pathologies where VEGF plays a key role such as in diabetic retinopathy.

Introduction

Retinopathy, one of the major vascular complications occurring in diabetes, generally proceeds through several stages characterized by biochemical and cellular alterations including changes in blood flow and loss of pericytes in the retina, the latter representing one of the earliest events occurring in retinopathy [1]. Typical changes of the first stage of this pathology are the thickening of the basement membrane, hyper-permeability and formation of microaneurysms in the retina. These functional alterations are followed by microvascular occlusions leading to a progressive retinal ischemia that induces the release of the vascular endothelial growth factor (VEGF), also known as vascular permeability factor. Within this context, VEGF is generally thought to sustain the proliferative stage of diabetic retinopathy [2].

Literature data indicate that protein kinase C (PKC), whose activation in the retina is induced by diabetes-associated hyperglycemia (for a review see [3]), is involved in the positive control of VEGF expression. PKC is an ubiquitously expressed family of enzymes implicated in multiple cellular functions [4]. Among the different PKC isoforms, the beta (PKCβ) one seems to be preferentially activated in the retina, thus possibly contributing to the early stages of retinopathy [2]. Up to date, no experimental evidence are available on how PKC exerts its modulation on VEGF expression. A clue comes from the fact that VEGF belongs to the 5–8% of human genes bearing in their 3′ untranslated region of mRNA a specific cis signal able to affect the half-life of the mRNAs themselves. This signal is a consensus sequence called ARE (Adenine and uridine-Rich Element), that governs the decay rates of mRNAs endowed with a rapid response to cell environmental stimuli [5] and corresponds to the docking site for some specific RNA-binding proteins (RBPs). ELAV (as for embryonic lethal abnormal vision) proteins represent the best characterized ARE-binding RBPs. In vertebrates, HuB, HuC and HuD are neuron-specific members of the family (nELAV), while HuR is ubiquitously expressed (see [6]). Moreover, employing a human neuronal cell model, SH-SY5Y neuroblastoma cells, we demonstrated the primary role of PKC in the cascade of nELAV recruitment and mRNA stabilization [7]. These considerations prompted us to investigate whether the ELAV/HuR protein could represent a final target of the signal cascade involving upstream specifically PKCβ and resulting downstream in the stabilization and translation of VEGF mRNA. These events were studied in bovine retinal pericytes. These cells are considered important regulators of vascular development, maturation and remodelling. Indeed, pericyte-endothelial cells coculture systems indicate that pericytes intimately interact with endothelial cells, strongly suggesting that they may play an important role in communication among various cell types and in the maintenance of the microvascular homeostasis [8], [9], [10]. In the developing retina pericytes control vessel sprouting and remodelling, while in the mature retina they principally act as supporting cells and as inhibitors of endothelial proliferation. Therefore the loss of pericytes and the subsequent impairment of functional interactions between pericytes and endothelial cells may be implicated in diabetic retinopathy (see [10], [11]).

A better understanding on the proposed PKCβ/HuR/VEGF cascade could shed light on the biochemical changes which may exert a physiopathological control of VEGF expression before pericyte loss occurs, offering at the same time opportunities for the development of innovative pharmacological therapies for diabetic retinopathy.

Section snippets

Cell cultures

Bovine retinal pericytes were obtained from prof. Alberghina at the University of Catania. Cells were cultured according to the procedures described in Lupo et al. [12]. Briefly, flasks were previously coated with 0.2% gelatin (SIGMA, Italy). Pericytes were grown in Dulbecco's modified minimum essential medium with glutamax-I (DMEM, Invitrogen, Italy) supplemented with 10% fetal calf serum (FCS), penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2 and 95% humidity. Before VEGF medium

PKCβII directly interacts with HuR in retinal pericytes

Firstly, we wanted to investigate, through different approaches, whether PKCβII and HuR do interact following phorbol esters (PMA) treatment. PMA is a PKC activator that is able to induce the translocation of PKC from the cytosol to other cellular compartments. According to our previous experience [7], we confirmed that 15 min of 100 nM PMA exposure promotes PKCβII relocation from the soluble fraction (−45%, p < 0.01, n = 5) to the membrane (+113%, p < 0.01 n = 5) and especially to the cytoskeleton

Discussion

Our data indicate the presence of a new molecular cascade operating in the cytoskeleton of retinal bovine pericytes controlling VEGF expression. Indeed we show that PKCβII activation is able, through the RNA-binding protein ELAV/HuR, to increase VEGF protein levels thus leading to its release in the medium (see Fig. 4). In fact, phorbol esters (PMA) do activate PKCβII that colocalizes with and phosphorylates in serine HuR; this latter in turn binds and stabilizes VEGF mRNA, thus determining an

Acknowledgments

This work was supported by a grant from the Italian Ministero Università Ricerca to A.P. (prot. n° 2004061375_004). The authors wish to thank prof. Mario Alberghina for his helpful suggestions in the design of the experiments.

References (29)

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    We in fact demonstrated the existence, in retinal pericytes, of a novel molecular pathway implicated in the government of VEGF gene expression at post-transcriptional level [26,27]. We also showed that PKCβ is able to activate HuR protein that binds VEGF mRNA, within mRNP complexes, increasing VEGF protein content [26]. These previous studies thus opened the way to a novel pharmacological approach to counteract pathologies involving VEGF modification and to potentially manage the early phase of DR: by silencing HuR, the positive regulator of VEGF expression.

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