cell biochemistry and function Cell Biochem Funct 2015; 33: 29–36. Published online 16 December 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3082

Up-regulation of semaphorin 4A expression in human retinal pigment epithelial cells by PACAP released from cocultured neural cells Ji-Ae Ko1*, Junko Hirata1, Ken Yamane1, Koh-Hei Sonoda2 and Yoshiaki Kiuchi1 1 2

Department of Ophthalmology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima City, Hiroshima, Japan Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan

Development and homeostasis of multicellular organisms require interactions between neighbouring cells. We recently established an in vitro model of cell–cell interaction based on a collagen vitrigel membrane. We have now examined the role of neural cells in retinal homeostasis by coculture of human retinal pigment epithelial (RPE) cells and neural cells on opposite sides of such a membrane. The neural cells (differentiated PC12 cells) induced up-regulation of semaphorin 4A (Sema4A), a member of the semaphorin family of neural guidance proteins, in RPE (ARPE19) cells. This effect of the neural cells was mimicked by the neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) and was abolished by the PACAP antagonist PACAP(6–38). Coculture with neural cells or stimulation with PACAP also induced the phosphorylation of extracellular-signal-regulated kinase in ARPE19 cells, and this effect of the neural cells was inhibited by PACAP(6–38). Finally, among various cytokines examined, only the amount of interleukin-6 released by cocultures of ARPE19 and neural cells differed from that released by ARPE19 cells cultured alone. Interleukin-6 was not detected in culture supernatants of neural cells, and the reduction in the amount of interleukin-6 released by the cocultures compared with that released by ARPE19 cells alone was prevented by PACAP(6–38). Our findings suggest that PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Development and homeostasis of multicellular organisms require interactions between neighbouring cells. With the use of a coculture system based on a collagen vitrigel membrane, we have now shown that neural cells induce up-regulation of the neural guidance protein Sema4A in RPE cells. This effect of neural cells appears to be mediated by the neuropeptide PACAP. PACAP released from retinal neural cells (photoreceptors or optic nerve cells) may thus regulate Sema4A expression in RPE cells and thereby contribute to the maintenance of retinal structure and function. Copyright © 2014 John Wiley & Sons, Ltd. key words—coculture; retinal pigment epithelial cell; semaphorin 4A; pituitary adenylate cyclase–activating polypeptide (PACAP); interleukin-6; neural cell; retina

INTRODUCTION Retinal degenerative diseases constitute the leading cause of blindness in the industrialized world.1 Retinal pigment epithelium (RPE) cells play an important role in the maintenance of normal retinal morphology and physiology, and the function of these cells is impaired in eye diseases such as age-related macular degeneration2 and retinitis pigmentosa.3 A cascade of events in such diseases eventually results in both RPE and photoreceptor cell death and a consequent permanent loss of vision.4–6 Semaphorins constitute a large, conserved family of secreted or membrane-bound ligands that function as either

*Correspondence to: Ji-Ae Ko, Department of Ophthalmology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Minami-Kasumi, Hiroshima City, Hiroshima 734-8551, Japan. E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

repulsive or attractive cues for neuronal growth cones.7 The semaphorin family of proteins is divided into various classes on the basis of the presence of various functional domains and sequence similarity. Semaphorins of classes 1, 2 and 5 are found in invertebrates, whereas those of classes 3 to 7 are present in vertebrates. Among vertebrate semaphorins, those of classes 4 to 6 are membrane-bound proteins. Among the six members of class 4, semaphorin 4A (Sema4A) and Sema4D mediate interactions between T lymphocytes and antigen-presenting cells.8,9 Class 4 semaphorins are also expressed at high levels in the developing and adult brain, but their precise functions in the nervous system are unclear.8,10,11 Mutations of Sema4A have been found to disrupt the development of retinal photoreceptors and to be a cause of retinitis pigmentosa and cone rod dystrophy.12,13 Sema4A is expressed in retinal ganglion cells, inner retinal neurons and RPE cells during the time at which photoreceptors establish intimate contacts with Received 11 September 2014 Revised 4 November 2014 Accepted 5 November 2014

30

j.-a. ko

the RPE. In RPE cells, Sema4A is localized to the apical membrane.14 These observations suggest that Sema4A may play a role in interactions between RPE cells and photoreceptors. The differentiation and function of many cell types are regulated by cytokines and neurotransmitters via autocrine or paracrine mechanisms. RPE cells have been shown to produce interleukin-1 (IL-1)-like growth factors and to show a proliferative response to several cytokines in vitro.15,16 Various neurotransmitters and neuropeptides including dopamine, neurotensin, γ-aminobutyric acid and glycine have also been detected in the neural retina,17 and these molecules are potential regulators of RPE differentiation or function. Indeed, vasoactive intestinal peptide (VIP) stimulates both the proliferation and differentiation of RPE cells in vitro.18 Pituitary adenylate cyclase–activating polypeptide (PACAP) has been shown to attenuate glutamate-induced neurotoxicity in cultured retinal neurons.19 PACAP is also secreted from differentiated PC12 (rat pheochromocytoma) cells in culture.20,21 Furthermore, PACAP receptor mRNA has been detected in a human RPE cell line (ARPE19), and PACAP inhibited the expression and secretion of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in these cells.22 In addition, PACAP protects human RPE cells from oxidative stress.23 These various observations suggest that PACAP might play an important role in retinal homeostasis. Cell and tissue culture models can provide valuable insight into physiological and pathological processes, with the controlled environment of a culture allowing manipulation of conditions for the analysis of biological mechanisms.24 We have recently applied a coculture system to study the interactions of various cell types—including epithelial cells, stromal fibroblasts and neural cells—in the cornea.25–32 In this system, two cell types of interest are plated on opposite sides of a collagen vitrigel membrane. We have now applied this coculture system to examine the interactions between human RPE (ARPE19) cells and neural cells. Given the difficulties associated with the culture of retinal photoreceptors or optic nerve neurons, we used differentiated PC12 cells as a surrogate to examine the possible effects of such retinal neural cells or factors derived therefrom on RPE cell function. We have found that coculture with neural cells induces the expression of Sema4A and inhibits that of IL-6 in ARPE19 cells and that these effects appear to be mediated by PACAP released from the neural cells. MATERIALS AND METHODS Antibodies and reagents Rabbit polyclonal antibodies to neurofilament-L were obtained from Millipore (Temecula, CA, USA), those to Sema4A were from Abcam (Minneapolis, MN, USA), and those to phosphorylated or total forms of c-Jun NH2terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) were from Cell Signaling Technology Copyright © 2014 John Wiley & Sons, Ltd.

ET AL.

(Danvers, MA, USA). Mouse monoclonal antibodies to αtubulin were obtained from Sigma (St. Louis, MO, USA), and those to extracellular-signal-regulated kinase (ERK) or to phosphorylated ERK were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish-peroxidaseconjugated secondary antibodies were from Promega (Madison, WI, USA), and Alexa-Fluor-488-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes (Carlsbad, CA, USA). PACAP-38 was obtained from Calbiochem (San Diego, CA, USA), and the PACAP antagonist PACAP(6– 38) was from Tocris (Bristol, UK). Neuropeptide Y (NPY), VIP and enkephalin were obtained from Sigma. Cell culture Rat PC12 and human ARPE19 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained under a humidified atmosphere of 5% CO2 at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% foetal bovine serum (FBS) and 5% horse serum or in Dulbecco’s modified Eagle’s medium (DMEM)–F12 (Gibco–Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS respectively. For coculture experiments, PC12 cells were seeded on one side of a collagen vitrigel membrane (diameter of 30 mm; Asahi Technoglass, Tokyo, Japan) in RPMI 1640 and were then cultured for 24 h in a 30-mm culture dish before exposure to the same medium containing recombinant human nerve growth factor (NGF, 10 ng ml 1; R&D Systems, Minneapolis, MN, USA) for 4 to 5 days. The membrane was then inverted in another dish, and ARPE19 cells (1 × 106) were seeded on the empty side in DMEM–F12 supplemented with 10% FBS. The cells were then cultured at 37 °C in a humidified incubator containing 5% CO2. As a control, ARPE19 cells were seeded on a vitrigel membrane without PC12 cells. Culture of ARPE19 cells with neuropeptides ARPE19 cells were seeded on a collagen vitrigel membrane in DMEM–F12 supplemented with 10% FBS and cultured for 24 h. They were then exposed to serum-free medium for 24 h before incubation for 24 h with various neuropeptides—including PACAP, NPY, VIP and enkephalin—in the same medium. Immunofluorescence analysis Cells cultured on a vitrigel membrane were fixed for 15 min at room temperature with 3.7% formalin, washed with Ca2+free and Mg2+-free phosphate-buffered saline [PBS(–)], permeabilized for 5 min with 0.1% Triton X-100 in PBS(–) and incubated for 1 h at room temperature with 1% bovine serum albumin (BSA) in PBS(–). They were then incubated for 1 h with antibodies to neurofilament-L at dilution of 1:200 in PBS(–) containing 1% BSA, washed with PBS(–) and incubated for 1 h with Alexa-Fluor-488-conjugated secondary antibodies at 1:1000 dilution in PBS(–) containing Cell Biochem Funct 2015; 33: 29–36.

UP-REGULATION OF SEMA4A IN RPE CELLS BY PACAP

31

Figure 1. Coculture of RPE (ARPE19) cells and neural (differentiated PC12) cells on a collagen vitrigel membrane. (A) ARPE19 cells were cultured on one side of a collagen vitrigel membrane in the absence (control) or presence of NGF-treated PC12 cells on the other side. (B–D) A vitrigel membrane containing ARPE19 cells on one side and differentiated PC12 cells on the other was examined by phase-contrast microscopy (B) and stained with DAPI (blue fluorescence) for detection of ARPE19 cells as well as subjected to immunofluorescence staining with antibodies to neurofilament-L (green fluorescence) for detection of differentiated PC12 cells (C, D). Scale bar, 50 μm

Figure 2. Expression of semaphorins in ARPE19 cells cultured with or without differentiated PC12 cells. (A) Total RNA isolated from ARPE19 cells cultured for 24 h on a collagen vitrigel membrane in the absence or presence of differentiated PC12 cells was subjected to RT-PCR analysis of mRNAs for Sema3A, Sema3B, Sema4A, Sema4B and G3PDH (internal standard). (B) The abundance of mRNAs for semaphorins in experiments similar to that in panel A was quantified by densitometric scanning of the ethidium-bromide-stained gels and normalized by the corresponding amount of G3PDH mRNA. (C) ARPE19 cells cultured for 24 h on a collagen vitrigel membrane in the absence or presence of differentiated PC12 cells were lysed and subjected to immunoblot analysis with antibodies to Sema4A or to α-tubulin (loading control). (D) The abundance of Sema4A protein in experiments similar to that in panel C was quantified by densitometric scanning of immunoblots and normalized by the corresponding amount of α-tubulin. Data in panels B and D are means ± SEM from three separate experiments. *P < 0.05 for the indicated comparisons (Student’s t-test) Copyright © 2014 John Wiley & Sons, Ltd.

Cell Biochem Funct 2015; 33: 29–36.

32

j.-a. ko

1% BSA. After staining of nuclei with DAPI, the cells were examined with a laser confocal microscope (LSM; Carl Zeiss, Jena, Germany). Reverse transcription polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from ARPE19 cells cultured on a vitrigel membrane with the use of an RNeasy kit (Qiagen, Valencia, CA, USA), and portions (0.5 μg) of the RNA were subjected to RT and PCR analysis with a One-Step RT-PCR kit based on the Platinum Taq system (Invitrogen, Carlsbad, CA, USA). The PCR protocol was designed to maintain amplification in the exponential phase. The sequences of the PCR primers (sense and antisense respectively) were 5′CTGTCTTTTCTGGGGAGTATTACTT-3′ and 5′-AATTAGCACATTCTTTCAGGATGTC-3′for Sema3A, 5′-GACATTGGTACTGAGTGCATGAACT-3′ and 5′-TCCAAA ATACCTTGACAAACTTGGG-3′for Sema3B, 5′-GTCAG ATACTATGCAGGGGATGAAC-3′ and 5′-TCTCCTCGAAGAAGAAGTAGACGAC-3′ for Sema4A, 5′-AAGCT GAACACATCTCCAACTACAC-3′ and 5′-GCAGGAGG ATCTTGATGTAGTTTTG-3′ for Sema4B and 5′-ACCAC AGTCCACGCCATCAC-3′ and 5′-TCCACCACCCTGTT GCTGTA-3′ for glyceraldehyde-3-phosphate dehydrogenase (G3PDH, internal control). The RT and PCR incubation processes were performed with a GeneAmp PCR System

ET AL.

2400-R (PerkinElmer, Foster City, CA, USA). RT was performed at 50 °C for 30 min, and PCR was performed for 25 cycles of incubation at 94 °C for 2 min, 58 °C for 30 s and 72 °C for 1 min. The reaction mixture was then cooled to 4 °C, and the products of amplification were fractionated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. Immunoblot analysis ARPE19 cells cultured on a vitrigel membrane were washed twice with PBS and lysed in 200 μl of a solution containing 150 mM NaCl, 2% sodium dodecyl sulphate (SDS), 5 mM EDTA and 20 mM Tris–HCl (pH 7.5). Cell lysates were fractionated by SDS polyacrylamide gel electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane and exposed consecutively to primary antibodies and horseradish-peroxidase-conjugated secondary antibodies. Immune complexes were detected with enhanced chemiluminescence reagents (GE Healthcare UK, Little Chalfont, UK). Multiplex cytokine assay Culture supernatants were assayed for various cytokines with the use of the Bio-Plex system (Bio-Rad, Hercules, CA, USA).

Figure 3. Effects of neuropeptides on the expression of Sema4A in ARPE19 cells. (A) Total RNA isolated from ARPE19 cells cultured alone on a collagen vitrigel membrane for 24 h in the absence (CONT) or presence of PACAP (1 or 10 μM) or of NPY, VIP or enkephalin (Enk; each at 10 μM) was subjected to RT-PCR analysis of Sema4A and G3PDH mRNAs. (B) The abundance of Sema4A mRNA in experiments similar to that in panel A was quantified by densitometric scanning of the ethidium-bromide-stained gels and normalized by the corresponding amount of G3PDH mRNA. (C) ARPE19 cells cultured as in panel A were lysed and subjected to immunoblot analysis with antibodies to Sema4A or to α-tubulin. (D) The abundance of Sema4A protein in experiments similar to that in panel C was quantified by densitometric scanning of immunoblots and normalized by the corresponding amount of α-tubulin. Data in panels B and D are means ± SEM from three separate experiments. *P < 0.05 versus the corresponding value for ARPE19 cells cultured without neuropeptides (Student’s t-test) Copyright © 2014 John Wiley & Sons, Ltd.

Cell Biochem Funct 2015; 33: 29–36.

UP-REGULATION OF SEMA4A IN RPE CELLS BY PACAP

33

Figure 4. Inhibition of the stimulatory effect of differentiated PC12 cells on Sema4A expression in ARPE19 cells by the PACAP antagonist PACAP(6– 38). (A) ARPE19 cells were cultured on a collagen vitrigel membrane for 24 h in the absence or presence of differentiated PC12 cells and with or without PACAP(6–38) (1 or 10 μM), as indicated. The ARPE19 cells were then lysed and subjected to immunoblot analysis with antibodies to Sema4A or to αtubulin. (B) The abundance of Sema4A in experiments similar to that in panel A was quantified by densitometric scanning of immunoblots and normalized by the corresponding amount of α-tubulin. Data are means ± SEM from three separate experiments. *P < 0.05 versus the value for ARPE19 cells cultured alone (Student’s t-test)

Statistical analysis Quantitative data are presented as means ± SEM from three independent experiments and were analysed with a Student’s t-test. A P value of