Signaling Mechanisms Involved in PEDF-Mediated Retinoprotection

Abstract

Pigment epithelium-derived factor (PEDF) is involved in signal transduction cascades necessary for protection of the retina. The interaction between PEDF and retinal cells elicits neuroprotective effects in vitro and in vivo. The direct substrates and signaling mechanisms involved in the survival response derived from such interaction are beginning to be revealed. It is of interest to elucidate cell death pathways that are crucial for the retinoprotective response of PEDF for the identification of targets that interfere with retina degeneration with potential therapeutic value. Here we review the molecular pathways triggered by PEDF that are involved in retinal survival activity.

73.1. Introduction

Degeneration of photoreceptor cells and disturbance of the integrity and homeostasis of the retina lead to the development of retinal diseases. The neurosensory retina is in close interaction with the retinal pigment epithelium (RPE) monolayer of cells, which provide structural, functional, and nutritional support to photoreceptors (PRs) (Strauss 2005). RPE and retinal cells are vulnerable to multiple stress conditions, which induce cell death, and have developed protective mechanisms to survive a variety of insults. An approach to counteract injury and favor survival activity involves endogenous molecules that regulate the development and survival of neuronal cells. Pigment epithelium-derived factor (PEDF) is a naturally occurring factor secreted by the RPE to act in protection of PRs and the neural retina (Polato and Becerra 2016). The multifunctional PEDF has cytoprotective effects in retinal and RPE cells in vitro and in vivo. Its neurotrophic activity is mediated through interactions between the neurotrophic domain of PEDF and a central region of the receptor PEDF-R protein (Subramanian et al. 2013; Kenealey et al. 2015). The PEDF/PEDF-R interactions initiate and transport cellular signals from the cell membrane to the nucleus for cytoprotective responses by molecular mechanisms that are beginning to be understood. Small peptides derived from the PEDF neurotrophic region, 17-mer (17-amino acid positions 98–114) and 44-mer (44-amino acid positions 78–121), confer retinoprotection and also trigger intracellular signaling mechanisms for PEDF cell survival (Kenealey et al. 2015; Comitato et al. 2018). Although the multifunctional PEDF regulates signaling pathways involve in several basic cellular processes, here we review the signaling transduction cascades triggered by prosurvival and anti-death activities of PEDF for protection of the retina and RPE.

73.2. PEDF Intracellular Signaling

The first step in the mechanism of action of PEDF neurotrophic activity is interaction of the extracellular PEDF with target cell surfaces, in particular with PEDF-R receptors in plasma membranes. Such interactions trigger changes in gene expression in retinal cells. Several groups have reported that PEDF targets prosurvival pathways involving regulation of apoptosis-inducing factor (AIF)/B-cell lymphoma 2 (Bcl-2), omega-3 fatty acid docosahexaenoic acid (DHA)/docosanoid neuroprotectin D1 (NPD1), mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 serine-threonine protein kinase Akt (PI3k/Akt), and the Janus kinase 2/signal transducers and activators of transcription 3 (JAK/STAT) pathway, for the protection of either the neural retina or the RPE (Table 73.1).

Table 73.1.

Signaling mechanisms involved in PEDF-mediated protection in the retina

Experimental conditions	Insult	Target cell	Time and dose of PEDF treatment	Signaling pathway response	References
In vivo	Pde6 mutation	Inherited RP mouse rd1 model photoreceptors	16 h of intravitreal injection of 6 pmol per eye of PEDF	Decreased intracellular Ca2+, prevented calpains’ activation, suppressed nuclear translocation of AIF, and attenuated Bax activity	Comitato et al. (2018)
	Zaprinast (PDE6 inhibitor)	Mouse 661W	2 h of 10 nM of PEDF	Decreased intracellular Ca2+ and photoreceptor cell death
In vitro	Serum starvation	Rat R28	30 min of 50–250 ng/ml of PEDF	Increased cell survival through STAT3 activation	Eichler et al. (2017)
In vitro	Light damage	Mouse 661W	1–2 h light exposure of 10 nM PEDF	Prevented cell death with induction of PI3k/Akt activity	Rapp et al. (2014)
In vitro	Serum starvation	Rat R28	48 h of 100–2500 ng/mL of PEDF	Prevented nuclear translocation of AIF and photoreceptor apoptosis by induction of Bcl-2 expression in vivo and in vitro	Murakami et al. (2008)
In vivo	Retinal degeneration animal model	Royal College Surgeon (RCS) rat photoreceptors	2 weeks of subretinal injection of SIV-hPEDF in P21 RCS rats
In vitro	Serum starvation	Rat R28	6 h of 100 nM PEDF	Induced Bcl-2 gene expression through the interaction with PEDF-R	Subramanian et al. (2013)
In vitro	Oxidative Stress [H2O2]	Human ARPE-19	2 h of 50 ng/mLof PEDF preconditioning prior to H2O2 or concentrations higher than 25 ng/uL	Suppressed cytotoxicity by induction of ERK1/2 phosphorylation	Tsao et al. (2006)
In vitro	Oxidative Stress [H2O2]	Human ARPE-19	2 h of 50 ng/mL of PEDF preconditioning prior to H2O2 insult	Prevented RPE barrier dysfunction, inhibited p38, and HSP27 phosphorylation	Ho et al. (2006)
In vitro	Oxidative Stress [H2O2]	Primary human RPE	1 h of 250 ng/mL of PEDF prior to H2O2 acute injury	Reduced cytotoxicity and mitochondrial dysfunction with activation of PI3K/Akt pathway	He et al. (2014)
In vitro	TNFα/H2O2	Human ARPE-19	4 h of 50 mg/mL of PEDF	Synergistic effect with DHA in reducing apoptosis by inducing the expression of Bcl-2 family of proteins; synthesis and release of NPD1	Mukherjee et al. (2007a, b)

73.2.1. Retina

The neurotrophic domain of PEDF binds PEDF-R to activate its phospholipase A₂ activity resulting in the release of fatty acids from phospholipids, such as DHA, in the retina and cornea (Subramanian et al. 2013; Pham et al. 2017). The free DHA precedes enhanced docosanoid synthesis and induction of bdnf (brain-derived neutrophic factor), ngf (nerve growth factor), and sema7a (axon growth promoter semaphorin 7a), all of which are known to favor protection and regeneration of corneal neurons from injury (Pham et al. 2017). PEDF/PEDF-R interactions also regulate a calcium-related signaling cascade by DHA-mediated extrusion of intracellular calcium (Comitato et al. 2018). While an increase in Ca²⁺ influx causes PRs degeneration, administration of PEDF or its neurotrophic peptide 17-mer results in extrusion of intracellular Ca²⁺, which in turn suppresses calpain activation to attenuate pro-apoptotic gene Bax activity, followed by upregulation of the anti-apoptotic factor Bcl-2 and restraining nuclear translocation of AIF in a mouse model of retinal degeneration rd1 PRs in vivo, as well as in mouse 661W cells in vitro. In rats, PEDF also upregulates the anti-apoptotic factor Bcl-2 mediated by its interaction with PEDF-R and prevents nuclear translocation of AIF and apoptosis in PRs of Royal College of Surgeons (RCS) rats in vivo and in rat retinal precursor R28 cells in vitro (Murakami et al. 2008; Subramanian et al. 2013; Winokur et al. 2017). Through this novel molecular mechanism PEDF/PEDF-R interactions attenuate the cell death process in retinal cells and particularly in PRs undergoing cell degeneration.

The neuroprotective action of PEDF in mouse-derived cone-like 661W cells also triggers a signaling mechanism that involves phosphorylation of Akt, suggesting an activation of the PI3k/Akt signaling cascade in PRs cells survival (Rapp et al. 2014). PEDF also promotes nuclear accumulation and phosphorylation of STAT3 via PEDF-R in R28 cells under serum-deprived conditions, implying an activation of the JAK/STAT pathway (Eichler et al. 2017). The JAK/STAT pathway promotes neuronal cell survival but can also play a role in cell death, being a cell context-and stimulus-dependent process (Battle and Frank 2002). The positive regulation of PEDF on this pathway likely involves a cross talk with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional signaling pathway, in agreement with PEDF’s induction of prosurvival genes through activation of NF-κB in cerebellar granule cell neurons (Yabe et al. 2005).

73.2.2. RPE

Mammalian MAPK pathways can be divided into three main groups: extracellular-signal-regulated kinases (ERKs), Jun amino-terminal kinases (JNK), and stress-activated protein kinases (p38/SAPKs). Different stimuli activate each cascade to perform a specific individual or shared biological response. PEDF exerts a cytoprotective effect in cultured human RPE (ARPE-19) cells undergoing hydrogen peroxide (H₂O₂) oxidative stress-induced cell death by ERK1/2 activation (Tsao et al. 2006). This positive regulation is mediated by activation of cyclic AMP-responsive element-binding protein (CREB) transcription factor, an effector of the ERK1/2 signaling cascade (Xing et al. 1996). PEDF-protective effects preserve the RPE cell barrier against H₂O₂-mediated oxidative injury by negative regulation of the MAPK/p38 signaling cascade and its effector the 27-kDa heat shock protein signaling (HSP27) (Ho et al. 2006). Hence, MAPK pathway activities could be either positive or negative relative to the stress condition perceived by a cell.

While the main role of mitochondria is to maintain metabolic and energy homeostasis in retinal cells, in several retinal diseases, oxidative injury induces cumulative mitochondrial damage. PEDF can activate PI3k/Akt pathway to reduce cytotoxicity and mitochondrial dysfunction in primary human RPE cells undergoing oxidative insult (He et al. 2014).

Exogenous addition of PEDF also stimulates the release of NPD1 from RPE cells in an apicolateral fashion and prevents nuclear translocation of anti-apoptotic proteins for neuroprotection under oxidative injury (Mukherjee et al. 2007a, b). NPD1 is a cell survival docosanoid that RPE cells synthesize from free DHA released by the activation of the PEDF-R’s phospholipase activity by PEDF and from phagocytosed PR outer segments (Pham et al. 2017; Mukherjee et al. 2007a, b). Among several neurotrophic factors, PEDF is the most efficient factor to promote the synthesis and release of NPD1 from human RPE cells (Mukherjee et al. 2007a, b). Thus NPD1 is considered a bioactive lipid mediator of PEDF action.

73.3. Future Experimental Approaches

PEDF provides extracellular stimuli that trigger molecular signals from the cell membrane to the nucleus to cytoprotect retina cells. The PEDF-mediated regulatory mechanisms and signaling pathways reviewed here provide an insight of downstream targets in stress/survival response in RPE and retinal cells. The identification of potential targets in cellular pathways upon PEDF/PEDF-R interaction will prove useful in the exploration of therapeutic approaches for retinal degenerative disorders. These survivals signaling pathways can be manipulated to prevent pathological retinal degeneration.