Thrombin promotes the expression of Ccnd1 gene in RPE cells through the activation of converging signaling pathways

Highlights

• Ccnd1 gene expression in RPE cells requires the expression of the c-fos gene.

• The joint activation of AP-1 and Ca/CRE leads to Ccnd1 gene expression.

• c-fos and Ccnd1 expression depend on PI3K and PKC, but downstream signaling differ.

• These findings pinpoint pharmacologic targets for preventing PVR.

Abstract

The breakdown of the blood–retina barrier exposes retinal pigment epithelium (RPE) to serum components, thrombin among them. In addition to coagulation, thrombin acting through Protease-Activated Receptors (PARs 1–4) participates in a number of processes including cell proliferation, transformation, and migration. The purpose of this study was to identify interacting signaling pathways by which the activation of PAR1 by thrombin triggers cyclin D1 gene (Ccnd1) expression and the proliferation of RPE cells, characteristic of proliferative vitreoretinopathy (PVR). Our results demonstrate that thrombin induces the expression of the c-fos gene (c-fos), the activation of the (fos/jun) AP-1 site and the expression of Ccnd1, in precise correlation with the activation of CREB. Although the expression of both, c-fos and Ccnd1 requires the activation of conventional PKC isoforms and PI3K, downstream signaling from PI3K differs for both genes. Whereas the expression of c-fos requires PI3K-induced PDK1/Akt activity, that of Ccnd1 is mediated by PDK1-independent PKCζ signaling. Additionally, CREB activation may contribute to the induction of Ccnd1 expression through binding to the Ca/CRE element in the Ccnd1 gene promoter. Since cyclin D1 is a key regulator of cell cycle G1/S phase progression essential for proliferation, these findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

Introduction

The retinal pigment epithelium (RPE), a monolayer of differentiated, quiescent cells located between the neural retina and the choroid, plays a central role in the maintenance of the functional and structural integrity of the neural retina (Strauss, 2005). In addition to its function as the main component of the blood–retina barrier (BRB), the RPE is involved in the trans-epithelial transport of nutriments, the storage and metabolism of vitamin A derivatives, and the renewal of photoreceptor outer segments (Mund, 2006).

A prominent feature of pathological conditions involving BRB breakdown such as ocular trauma, diabetic retinopathy, retinal detachment, retinal hemorrhage or retinal surgery, is the exposure of RPE to serum components, including the serine/threonine protease thrombin generated by the activation of the coagulation cascade, one of the earliest responses to tissue injury. Under these conditions, the release of active factors such as vascular endothelial growth factor (VEGF) and cytokines by activated Müller glia and retinal astrocytes, contributes to further disruption of the BRB (Li et al., 2014) and the promotion of RPE cell proliferation (Hackett et al., 1991, Bastiaans et al., 2014, Palma-Nicolas et al., 2010). Recent findings on this matter show that thrombin activity is increased in the vitreous from PVR patients, and contributes to vitreous-induced production of cytokines/chemokines and growth factors by RPE cells (Bastiaans et al., 2014). These data further support thrombin involvement in the pathogenesis of proliferative vitreoretinopathy (PVR), a human retinal disease that involves the proliferation, de-differentiation and migration of RPE cells into the vitreous and the subsequent assembly of transformed RPE cells into contractile membranes on retinal surface, thus promoting retinal detachment (Nagasaki et al., 1998).

Intracellular thrombin signaling is triggered by the activation of Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular N- terminal domain, which unmasks a new sequence that functions as an intra-molecular ligand. Four members of this family have been identified: PAR1, PAR3 and PAR4, activated by thrombin, and the closely related PAR2, activated by trypsin and other serine-proteases. PAR1 is the prototype of this receptor family, and its cleavage at the Arg41–Ser42 bond by thrombin exposes a new N-terminus (S42FLLRN47) that acts as a tethered ligand (Coughlin, 2000). Synthetic ligands corresponding to the cleaved N-terminus can displace the tethered ligand from the binding site and fully activate PAR1 in an intermolecular mode (Ossovskaya and Bunnett, 2004).

PARs have been linked to the activation of a wide array of physiologic responses by interacting with several GPCR Gα subunits, in particular Gq11α, G12/13α and Gαi, which accounts for the pleiotropic action of its ligands. PAR1 coupling to Gqα activates phospholipase C-β (PLC-β), with the formation of inositol 1,4,5- trisphosphate (IP3) and diacylglycerol (DAG), the endogenous activator of conventional/novel (c/n) Protein Kinase C (PKC) isoforms. Giα inhibits adenylyl cyclase, while the Gβγ subunits can activate phosphoinositide 3-kinase (PI3K) and other lipid modifying enzymes, protein kinases and ion channels. Finally, the α-subunit of G12 and G13 activates Rho GTPases, known to regulate the assembly and organization of the actin cytoskeleton (Ossovskaya and Bunett, 2004).

PAR1 activation promotes intracellular signaling by the Mitogen-Activated Protein Kinase (MAPK) ERK1/2, PKC and Phosphstidylinositol-3 Kinase (PI3K) cascades, all involved in the regulation of the cell cycle regulator cyclin D1. Particularly, thrombin activation of PI3K family of lipid kinases results in the activation of Phosphoinositide-Dependent Kinase 1 (PDK1) and the downstream stimulation of Protein Kinase B (Akt/PKB), which have been shown to participate in the development of proliferative diseases (Kong and Yamori, 2008, Liang and Slingerland, 2003).

We have demonstrated previously that in vitro treatment of RPE cells with α-thrombin induces cell proliferation through the joint activation of PKCζ and MAPK pathways (Palma-Nicolas et al., 2008). More recently, we have also shown the participation of PI3K and Akt in this process (Palma-Nicolas et al., 2008, Parrales et al., 2013, Parrales et al., 2011), in agreement with the involvement of these pathways in mitogenic stimulation (Li and Weinstein, 2006, Liang and Slingerland, 2003).

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases (CDKs) and their target proteins. Immuno-neutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of CDKs, may be rate-limiting for G1 to S phase progression of the cell cycle leading to proliferation. Cyclin D1 forms a complex with CDK4/6 and regulates progression of the early to mid-G1 phase of the cell cycle through the phosphorylation and inactivation of the retinoblastoma protein (Rb) and the activation of the E2F family of transcription factors, which promotes cell cycle progression into the S phase. Since cyclin D1 expression is the rate-limiting step for progression into G1/S, an increase in cyclin D1 expression enhances cell cycle progression and cell proliferation (Blagosklonny and Pardee, 2002). On this line, studies in human RPE cells have demonstrated that the inhibition of cyclin D1 expression significantly prevents serum-induced proliferation (Hecquet et al., 2002).

Although the oncogenic activity of cyclin D1 and its over expression in a number of human cancers has been abundantly documented (Jiang et al., 1993, Musgrove, 2006), more recent studies have revealed that cyclin D1 may modulate the activity of transcription factors and histone deacetylase, and it can also bind to the upstream regulatory region of diverse genes and regulate cancer cell migration and invasion (Pestell, 2013). Together, these functions underline the importance of investigating the cellular receptors and signaling pathways, which control the expression of the cyclin D1 gene (Ccnd1) and highlight the importance of cyclin D1 as a key molecule in thrombin-induced proliferation.

The promoter region of the cyclin D1 gene (Ccnd1) contains a number of potential cis-regulatory elements, such as those bound by AP-1, NFκB, STAT, Sp1, EGR1, and cAMP response element (CRE) binding protein (CREB), which have been implicated in the regulation of cyclin D1 promoter activity (Lee et al., 1999, Klein and Assoian, 2008). Specifically, the consensus AP-1 site, at –934 −928 bp (human promoter) regulated by Fos and Jun (Shen et al., 2008) can be activated by ERK1/2, and PKC, which are activated by thrombin in RPE cells (Lee et al., 1999, Parrales et al., 2010, Soh and Weinstein, 2003). On this matter, recent studies using SV40 antigen have demonstrated that Ccnd1 promoter activity and cell proliferation are driven primarily by an AP-1 binding site at 2954, with additional contribution from a CRE site at 257 (Watanabe et al., 1996). In spite of this knowledge, the signaling pathways involved in the expression of the Ccnd1 gene remain largely undefined.

In order to gain further insight into the molecular mechanisms responsible for thrombin-induced RPE cell proliferation, in the present study we used RPE-J cells as a model to investigate the signaling pathways involved in Ccnd1 expression. Our results show that thrombin promotes Ccnd1 expression through the joint activation of c-fos and CREB, known to interact with Ccnd1 gene promoter. Furthermore, although thrombin-induced transcription of c-fos and the activation of CREB contribute to Ccnd1 transcription, we show that these processes are under the control of distinct upstream regulation. These findings contribute to the elucidation of the complex mechanism leading to RPE cell proliferation and the development of PVR, a major cause of retinal surgery failure (Pastor et al., 2002).