PKCβII/HuR/VEGF: A new molecular cascade in retinal pericytes for the regulation of VEGF gene expression
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)
The potential role of PKC beta in diabetic retinopathy and macular edema
Surv Ophthalmol
(2002)- et al.
The role of protein kinase C activation and the vascular complications of diabetes
Pharmacol Res
(2007) - et al.
Happy birthday protein kinase C: past, present and future of a superfamily
Pharmacol Res
(2007) Messenger RNA degradation in eukaryotes
Cell
(1993)- et al.
Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKC{alpha} and the MAPK/ERK cascade
J Lipid Res
(2007) - et al.
t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes
Biochim Biophys Acta
(2001) - et al.
Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays
Methods
(2002) - et al.
Coordinated movement of RACK1 with activated betaII PKC
J Biol Chem
(1999) - et al.
The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting
Int J Biochem Cell Biol
(1996) - et al.
The different facets of protein kinases C: old and new players in neuronal signal transduction pathways
Pharmacol Res
(2006)
Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival
Dev Biol
Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction
Free Radic Biol Med
Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR
J Biol Chem
A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR
J Biol Chem
Cited by (47)
Evidence for novel cell defense mechanisms sustained by dimethyl fumarate in multiple sclerosis patients: the HuR/SOD2 cascade
2022, Multiple Sclerosis and Related DisordersNanosystems based on siRNA silencing HuR expression counteract diabetic retinopathy in rat
2016, Pharmacological ResearchCitation Excerpt :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.
Role of inflammation in diabetic retinopathy: pathogenesis and treatment strategies
2024, Recent Advances in OphthalmologyThe Labyrinthine Landscape of APP Processing: State of the Art and Possible Novel Soluble APP-Related Molecular Players in Traumatic Brain Injury and Neurodegeneration
2023, International Journal of Molecular Sciences