Current Eye Research, 2014; 39(5): 487–492 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2013.848900

ORIGINAL ARTICLE

Constitutive Expression of HCA2 in Human Retina and Primary Human Retinal Pigment Epithelial Cells Alice L. Yu1, Kerstin Birke2, Reinhard L. Lorenz3 and Ulrich Welge-Lussen2 1

Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany, 2Department of Ophthalmology, Friedrich-Alexander University, Erlangen, Germany, and 3Institute for Prophylaxis of Cardiovascular Diseases, Ludwig-Maximilians University, Munich, Germany

ABSTRACT Purpose/Aim: HCA2, a receptor of b-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells. Materials and methods: Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis. Results: Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells. Conclusions: Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target. Keywords: AMD, diabetic retinopathy, niacin maculopathy, retina, RPE

INTRODUCTION

activity of lipases, the hydrolysis of triglycerides and the release of free fatty acids. However, physiological plasma levels of niacin are too low to activate HCA2 receptors.3,5 Therefore, further endogenous ligands for the HCA2 receptor were postulated. In 2005, it was found that high levels of the ketone body b-hydroxybutyrate, which is produced by the liver during fasting or starvation, activate HCA2.6 HCA2 activation by b-hydroxybutyrate might function as a negative feedback mechanism to inhibit lipolysis during ketogenic starvation. Interestingly, HCA2 receptors have a very special distribution in the human body. Beside in adipocytes, HCA2 is expressed in keratinocytes and immune cells.1,7–9 In epidermal Langerhans cells, HCA2 activation causes conversion of arachidonic acid to

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Hydroxycarboxylic acid receptor HCA2 (GPR109A; PUMA-G) belongs to the group of G protein-coupled receptors (GPCRs), which are involved in a number of physiological and pathophysiological processes.1 HCA2 is found in both humans and rodents.1,2 With the discovery of HCA2, it was first declared as an orphan transmembrane receptor, since its endogenous ligand was unknown. In 2003, HCA2 was identified as the molecular target of niacin in pharmacologic concentrations.2–4 In pharmacologic terms, niacin is widely known as the first lipid-lowering drug and confers its antilipolytic effects via HCA2 activation.2,5 In adipocytes, niacin binding to HCA2 reduces cAMP levels, the

Received 17 April 2013; revised 9 August 2013; accepted 12 September 2013; published online 11 November 2013 Correspondence: Alice Yu, Ludwig-Maximilians-University, Department of Ophthalmology, Mathildenstr. 8, 80336 Munich, Germany. Tel: +49 89 5160 3811. Fax: +49 89 5160 5160. E-mail: [email protected]

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488 A. L. Yu et al. vasodilator prostaglandin D2 and E2 leading to cutaneous flushing, which is a known side effect of niacin therapy.7,10,11 In contrast, HCA2 activation in keratinocytes leads to a more delayed, prolonged rubor of the skin. Recently, it has been first reported that HCA2 receptors are also expressed in murine retinal pigment epithelial (RPE) cells and human RPE cell lines (ARPE-19), though their function is still unknown.12 In monocytoid cells, niacin stimulates the expression of scavenger receptors and the cellular cholesterol exporter ABCA1.13 These key effectors of reverse cholesterol transport are also involved in the phagocytosis and disposal of shed outer segment membranes by RPE cells.14 We therefore investigated whether HCA2 receptors are expressed in the human retina and in primary human RPE cells, as these receptors might have a pathophysiologic role in various retinopathies.

MATERIALS AND METHODS Immunohistochemistry of Paraffin Sections Donor eyes were cut in quadrants, dehydrated in ascending ethanol series and isopropanol, and embedded in paraffin in an embedding machine (Microm-Heidelberg, Heidelberg, Germany). Twelvemicrometer paraffin sections were cut (Histoslide 200R; Leica, Wetzlar, Germany) and mounted on coverslips (Superfrost; Menzel Glaser, Braunschweig, Germany). Paraffin sections of the posterior pole were deparaffinized and rehydrated in xylol and a descending series of ethanol and rinsed in PBS. For immunochemical staining, rat monoclonal anti-HCA2 primary antibodies (Abcam, Cambridge, UK), diluted 1:250, were applied and incubated overnight at room temperature (RT). The reaction products were visualized by using the Vectastain ABC Elite Kit (Vector Laboratories, Burlingham, CA), treated with DAB as substrate and fixed with enhancer solution for 1 min. Slides were rinsed in PBS, and then stained with Mayers hematoxylin (Sigma, Deisenhofen, Germany). After dehydration, slides were mounted with Permount (Fisher Scientific, New Haven, CT). Slides without primary antibody incubation served as negative controls.

Human RPE Cell Cultures Five human donor eyes were obtained from the eye bank of the Ludwig-Maximilians-University, Munich, Germany, and were processed within 6–24 h after death. The donors’ age ranged between 38 and 62 years. There was no history of eye disease in any of the donors. Methods of securing human tissue were humane, included proper consent and approval, and complied with the tenets of the declaration of

Helsinki. Human retinal pigment epithelial (RPE) cells were harvested following the procedure as described previously.15–17 Epithelial origin was demonstrated by immunohistochemical staining for cytokeratin using a pan-cytokeratin antibody (Sigma).18 The cells were analysed and found free of contaminating macrophages (anti-CD11, Sigma) and endothelial cells (anti-von Willbrand factor, Sigma). Antibodies (Abcam) against RPE-65, a marker for RPE cells, showed the purity of RPE cells. After reaching confluence, primary RPE cells of passage three to five were subcultured and maintained in DMEM with 10% FCS at 37  C and in 5% CO2.

Immunocytochemistry of RPE Cells Cultured RPE cells were grown on microscope slides, fixed with 4% paraformaldehyde (PFA) for 15 min and subsequently washed twice with PBS containing 0.1% Triton X-100. Primary incubation of all samples was performed with rat monoclonal anti-HCA2 primary antibodies (Abcam), diluted 1:250 in PBS, containing 5% bovine serum albumin (BSA) for 4 h at RT. Control samples were incubated with PBS and 5% BSA without primary antibodies. Double-labelling studies were undertaken with rat monoclonal anti-HCA2 antibodies (Abcam) and mouse monoclonal anti-RPE65 antibodies (Abcam), diluted 1:1000 in PBS. Nuclear staining was conducted with 40 ,6-diamidino-2-phenylindole (DAPI) counterstaining. After washing three times, the double-labelled cells were incubated with Alexa 488 conjugated secondary antibodies (diluted 1:2000 in PBS; Mobitec, Go¨ttingen, Germany) and Alexa 555 conjugated secondary antibodies (diluted 1:2000 in PBS; Mobitec), respectively, for 1 h at RT. Cells were then rinsed in PBS and mounted with Kaiser’s glycerin jelly (Merck, Darmstadt, Germany). Slides were examined under a fluorescence microscope (Leica DMR). Representative areas were documented (Leica IM 1000 software).

RNA Isolation and RT-PCR Total RNA was extracted from three different human retina from different donors and grown on 35-mm petri dishes. Structural integrity of the RNA samples was verified by electrophoresis in 1% Tris–acetateEDTA (TAE)-agarose gels. Yield and purity were determined photometrically. After RNA isolation, RNA was transcribed to cDNA via reverse transcriptase. The quality of RNA and cDNA synthesis was demonstrated by amplification of 18S rRNA. The PCR reaction was started with a hot start at 94  C to denature DNA and continued for 36 cycles at an annealing temperature of 58  C. The primers selected were HCA2 forward primer 50 -gggcaaattgtaggcatttc-30 Current Eye Research

HCA2 in Retina and RPE and reverse primer 50 -gaaggccaacaagtcacaagt-30 ; and 18S rRNA forward primer 50 -ctcaacacgggaaacctcac-30 and reverse primer 50 -cgctccaccaactaagaacg-30 .

Protein Extraction and Western-Blot Analysis Retinal pigment epithelial cells from three different RPE cell cultures from different donors were grown on 35-mm tissue culture dishes and were washed twice with ice-cold PBS, collected and lysed in radioimmunoprecipitation assay (RIPA) cell lysis buffer. After centrifugation (19,000 g for 30 min at 4  C) in a microfuge, the supernatants were transferred to fresh tubes and stored at 70  C for future use. The protein content was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Extracted proteins (2 mg) were separated under reducing conditions by electrophoresis using 10% SDS–polyacrylamide gels. Afterwards, semidry blotting transferred the proteins onto a polyvinyl difluoride membrane (Roche), and proteins were probed with a rat monoclonal anti-HCA2 primary antibody (Abcam) as described previously.19 This antibody was used at a dilution of 1:200, respectively. Chemiluminescence

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was detected with the imager (LAS-1000; RayTest, Pforzheim, Germany). Exposure times ranged between 1 and 20 min. Quantification was performed on a computer (AIDA software; RayTest).

RESULTS Localization of HCA2 Protein Expression in Human Retina To determine the expression of HCA2 in human retina, we incubated retinal paraffin sections with HCA2 specific primary antibodies. Cell nuclei were visualized by hematoxylin staining (Figure 1). Negative control sections without primary antibodies showed no reaction (Figure 1A). Staining with primary antibodies demonstrated the expression of HCA2 (Figure 1B) in all strata of the human retina including in the retinal pigment epithelial (RPE) cells.

Immunocytochemistry of RPE Cells Immunocytochemical analysis in primary human RPE cells detected HCA2 expression as seen by the green fluorescent signal (Figure 2B). The DAPI

FIGURE 1 Immunohistochemical staining of retinal paraffin sections. (A) Negative control section without primary antibodies. (B) Retinal section stained with anti-HCA2 antibodies. In all images, cell nuclei were visualized by hematoxylin staining. INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Original magnification: 40.

FIGURE 2 Immunocytochemical staining of primary human RPE cells. (A) Negative control without primary antibodies. (B) RPE cells with anti-HCA2 antibodies. (C) double-labelled RPE cells with anti-HCA2 and anti-RPE65 antibodies. (A–C) DAPI counterstaining to detect cell nuclei. !

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490 A. L. Yu et al. counterstaining depicts cell nuclei in blue. The negative control without primary HCA2 antibody showed no positive signals (Figure 2A). Double-labelling experiments demonstrated coincident staining for HCA2 (green, Figure 2C), and for RPE-65, a RPEspecific marker (red, Figure 2C).

HCA2 mRNA Expression in Human Retina HCA2 mRNA expression was found in human retina by RT-PCR (Figure 3). 18S rRNA served as an internal control. Interestingly, the HCA2 mRNA expression showed variable signal strength in the retina of different human donor eyes (Figure 3).

HCA2 Protein Expression in Primary Human RPE Cells Expression of HCA2 protein was confirmed by western blot in primary human RPE cells, however much lower than b-actin. The signals for HCA2 protein expression were slightly different in different cell lines (Figure 4).

DISCUSSION HCA2 receptors have a very restricted expression pattern in human tissues. They can be found in adipocytes of white and brown adipose tissues, in keratinocytes and monocytes, macrophages and neutrophils.1–3,7,9 Recently, low basal expression of HCA2 was also detected in primary human hepatocytes.20 We could demonstrate that HCA2 are also expressed

FIGURE 3 RT-PCR analysis demonstrated HCA2 mRNA expression in human retina. 18S rRNA served as an internal control.

FIGURE 4 Western-blot analysis showed HCA2 protein expression in primary human RPE cells. b-actin was used as a loading control for Western-blot analysis.

in ocular tissues derived from human donor eyes, such as the human retina and primary human retinal pigment epithelial (RPE) cells. In contrast to murine retina,12 we detected HCA2 in all strata of paraffin section of the human retina. At the mRNA level, there was a clear expression of HCA2 in extracts from human retina. Expression of HCA2 protein could be detected in primary human RPE cells, which confirmed the earlier observation of Gambhir et al.21 However, the reason for the different expression levels of HCA2 in different cell lines is unclear. One possible explanation may result from differences in metabolic status or in inflammatory responses between the donors. In various cellular systems, HCA2 has been characterized as a physiologic b-hydroxybutyrate and pharmacologic niacin receptor.22 Elevated levels of b-hydroxybutyrate can be found during starvation or in poorly controlled diabetes. Therefore, increased HCA2 in the retina might be involved in the pathogenesis of diabetic retinopathy. Previously, it has been shown that activation of the HCA2 receptor also stimulates adiponectin secretion from adipocytes.23 Costagliola et al.24 have found that patients with proliferative diabetic retinopathy have not only a higher level of vascular endothelial growth factor (VEGF) but also a higher level of adiponectin in the aquous humor than control subjects. Intravitreal injection of bevacizumab reduced the levels of both adiponectin and VEGF. Whether or not HCA2 receptors are involved in the elevated adiponectin levels found in the aquous humor of patients with diabetes requires further investigations. Very recently, Gambhir et al.21 reported enhanced retinal HCA2 expression in three mouse models of diabetes and two patients with diabetes compared to controls. As HCA2 agonists reduced TNFa-stimulated IL-6 and Ccl2 expression and NF-B activation, this was interpreted as an anti-inflammatory counterregulation in the retina of diabetics.21 In adipocytes, activation of HCA2 leads to decreased levels of cAMP and thus reduces the release of free fatty acids.2,3 In the RPE, an optimal level of cAMP is required for the nutritive supply of the outer retina.12,25 Therefore, cAMP levels are essential for the maintainance of neuronal integrity in the retina.25 Martin et al.12 have already shown Current Eye Research

HCA2 in Retina and RPE a downregulation of cAMP by niacin or b-hydroxybutyrate in ARPE-19 cells. Further studies are needed to examine whether or not this signalling pathway is also involved in the pathogenesis of diabetic retinopathy or other retinal diseases. In adipocytes, the activation of HCA2 leads to a decreased release of free fatty acids to the liver and thus reduces triglyceride synthesis, VLDL production and LDL-cholesterol levels.2 Decreased levels of plasma triglycerides are associated with increased HDL-cholesterol levels, and niacin has long been known to increase HDL and reduce cardiovascular events and total mortality.26 Lukasova et al.27 have shown that macrophages within atherosclerotic lesions express HCA2 and niacin stimulates the key scavenger receptors and cellular cholesterol exporters to HDL initiating reverse cholesterol transport.13 Thus, progression of atherosclerosis could be reduced by local activation of HCA2 in plaque macrophages contributing to the lipoprotein-altering effects. A number of epidemiological studies have shown that age-related macular degeneration (AMD) shares risk factors with atherosclerosis like hypercholesterolemia.28–32 In a previous article, we demonstrated that LDL and ox-LDL may induce cellular changes in RPE cells in vitro, which resembles the pathological events in AMD.17 However, the role of HCA2 and its ligands on the lipid handling in the RPE is still unknown. As mentioned earlier, HCA2 may stimulate adiponectin secretion in adipocytes.23 In the mouse model, adiponectin may inhibit choroidal neovascularisation.33 Therefore, it may be speculated whether or not activation of HCA2 may have therapeutic effects on the treatment of wet AMD. A very rare ocular condition, in which HCA2 may be involved, is niacin maculopathy, which results from chronic use of niacin at high doses.34–36 Niacin maculopathy is an abnormal accumulation of niacin in the retina. Tachikawa et al.37 have shown that niacin may enter the retina via an Hþ-coupled monocarboxylate transporter at the blood-retina barrier formed by the tight junctions of retinal capillary endothelial cells and RPE. Whether or not HCA2 plays a role in the pathogenesis of niacin maculopathy requires further investigation. In conclusion, we could demonstrate an expression of HCA2 in the human retina and in primary human RPE cells derived from human donor eyes. The functional role of these receptors in the retina is still unknown. However, HCA2 receptors may provide a molecular target in common retinal diseases such as diabetic retinopathy and AMD.

ACKNOWLEDGEMENTS The authors thank Ekaterina Gedova and Agnes Hahn for excellent technical assistance. !

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DECLARATION OF INTEREST The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.

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Constitutive expression of HCA(2) in human retina and primary human retinal pigment epithelial cells.

HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) ...
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