2017, 64 (3), 325-338

PANCREATIC β-cells secrete insulin in response to various metabolic, nervous and hormonal factors and play a pivotal role in systemic glucose homeostasis [1, 2]. Both impaired glucose-stimulated insulin secretion (GSIS) and loss of pancreatic β-cell mass are hallmarks of type-2 diabetes (T2D). However, the impairment of GSIS is a progressive disorder and is generally consid-ered to be a multistep process [3-6]. Pancreatic β-cells are very sensitive to pathophysiological stressors (such as increased levels of reactive oxygen species, cyto-kines and metabolites) and, according to one prevalent view, β-cell dysfunction occurs once the rate of β-cell

Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic β-cells

Stefan Amisten1), 2) *, Israa Mohammad Al-Amily3) *, Arvind Soni3), Ross Hawkes2), Patricio Atanes2), Shanta Jean Persaud2), Patrik Rorsman1), 4) and Albert Salehi3), 4)

1) The Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK2) Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King’s College London, Faculty of Life Sciences &

Medicine, London, UK3) Department of Clinical Science, UMAS, Clinical Research Center, University of Lund, Malmö, Sweden4) Department of Neuroscience and Physiology, Metabolic Research Unit, University of Gothenburg, Gothenburg, Sweden

Abstract. Pancreatic islets express high levels of the orphan G-protein coupled receptor C5C (GPRC5C), the function of which remains to be established. Here we have examined the role of GPRC5C in the regulation of insulin secretion and β-cell survival and proliferation using human and mouse pancreatic islets. The expression of GPRC5C was analysed by RNA-sequencing, qPCR, western blotting and confocal microscopy. Insulin secretion and cell viability were determined by RIA and MTS assays, respectively. GPRC5C mRNA expression and protein level were reduced in the islets from type-2 diabetic donors. RNA sequencing in human islets revealed GPRC5C expression correlated with the expression of genes controlling apoptosis, cell survival and proliferation. A reduction in Gprc5c mRNA and protein expression was observed in islets isolated from old mice (>46 weeks of age) compared to that in islets from newborn (<3 weeks) mice. Down-regulation of Gprc5c led to both moderately reduced glucose-stimulated insulin release and also reduced cAMP content in mouse islets. Potentiation of glucose-stimulated insulin secretion concomitant with enhanced islet cAMP level by all-trans retinoic acid (ATRA) was attenuated upon Gprc5c-KD. ATRA also increased [Ca+2]i in Huh7-cells. Gprc5c over expression in Huh7 cells was associated with increased ERK1/2 activity. Gprc5c-KD in clonal MIN6c4 cells reduced cell proliferation and in murine islets increased apoptosis and the sensitivity of primary islet cells to a cocktail of pro-apoptotic cytokines. Our results demonstrate that agents activating GPRC5C represent a novel modality for the treatment and/or prevention of diabetes by restoring and/or maintaining functional β-cell mass.

Key words: Orphan GPCR, Diabetes, Insulin release, RAIG2, RAIG3

apoptosis exceeds the normal physiological rate [1-7]. These data echo clinical observations indicating that a sustained increase in plasma glucose levels is associ-ated with impaired nutrient-stimulated insulin secre-tion in diabetes-prone individuals [3, 6-9].

Evidence has been presented that daily consumption vitamin A improves pancreatic β-cell function and pre-vents or delays the transition from pre-diabetes to frank T2D [10]. There are also data implicating vitamin A in pancreatic islet development, formation, and function and that postnatal restriction of vitamin A results glu-cose intolerance in adult rodents [11-13].

Many effects of vitamin A are mediated by its oxi-dised metabolite all-trans retinoic acid (ATRA), which has been shown to regulate the transcription of many genes involved in cell growth and differentiation [11, 14], effects that require at least 2-4 hours to occur [11-13]. However, the finding that ATRA influence insulin

Original

©The Japan Endocrine Society

Submitted Jul. 13, 2016; Accepted Nov. 21, 2016 as EJ16-0338Released online in J-STAGE as advance publication Feb. 22, 2017Correspondence to: Albert Salehi, Department of Clinical Science, UMAS, University of Lund, Malmö, Sweden. E-mail: S_Albert.Salehi@med.lu.se* These authors contributed equally to this work.

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the purity of the islets preparation was 70-90%. For the qPCR measurements, clean islets without any detectable attachment of exocrine tissue were selected under a stereomicroscope. Work on human pancreatic islets was approved by the ethical committees at the Universities of Uppsala and Lund, Sweden.

ChemicalsCollagenase (CLS 4) was from Sigma, and fatty acid-

free bovine serum albumin (BSA) was from Boehringer Mannheim. Polyclonal rabbit anti-GPRC5C and HRP-conjugated goat anti-rabbit IgG were from Santa Cruz Biotechnologies. Cy2-conjugated anti-rabbit IgG and Cy5-conjugated anti-guinea pig IgG were from Jackson Immunoresearch Laboratories Inc. The insulin radio-immunoassay was from Millipore. All other chemicals were from Merck AG or Sigma. Gprc5c shRNA and scrambled control lentiviral particles were obtained from Santa Cruz Biotechnology.

Expression of GPRC5A, GPRC5B, GPRC5C and GPCR5D in pancreatic islets

The expression of GPRC5A-D in human and mouse islets was quantified by quantitative real-time PCR (qPCR) using Qiagen’s QuantiFast qPCR kit and QuantiTect primers as described elsewhere [17]. In order to mitigate any potential effects caused by changes in house keeping gene expression in islets from non-diabetic and T2D human donors, the mRNA expression data was normalised against three separate housekeeping genes (GAPDH, HPRT and PPAI).

Expression of GPRC5C in human pancreatic islets analysed by RNA-sequencing

Total RNA was isolated from pancreatic islets from 89 human cadaveric donors, RNA-sequencing sample preparation was performed using Illumina’s TruSeq RNA Sample Preparation Kit. The resulting librar-ies were quality controlled on a 2200 Tapestation (Agilent Technologies) before combining 6 samples into one pool for sequencing on one lane on a Flow cell sequenced on a HiSeq 2000 (Illumina). The out-put reads were aligned to the human reference genome (hg19) with STAR [18, 19] and feature counting was done using feature Counts [20]. Raw data was nor-malised using trimmed mean of M-values and pre-sented as Fragments Per Kilobase of Exon Per Million Fragments Mapped (FPKM) or transformed using into log2 counts per million using the voom-function

secretion during acute exposure (<1 h) suggests that it also signals via a non-genomic mechanism that is independent of transcriptional regulation induced by nuclear retinoid acid receptors [11-13].

We recently reported that the membrane-bound G-protein coupled receptor GPRC5C is highly expressed in pancreatic islets [15]. We have now investigated the expression of GPRC5C in human and mouse pancreatic islets and studied the consequences of GPRC5C down-regulation on glucose-stimulated insulin secretion, insulin content, cell proliferation and islet cell viability. Our data indicate that GPRC5C plays a critical role in the function, survival and pro-liferation of pancreatic β-cells and mediates the acute (non-genomic) effects of ATRA.

Materials and Methods

AnimalsFemale mice of the NMRI strain (B&K, Sollentuna,

Sweden), weighing 25-30 g, were used for the experi-ments on primary islets. The mice were fed a standard pellet diet (B&K) and tap water ad libitum. All animals were housed in metabolic cages with constant temper-ature (22ºC) and 12-h light/dark cycles. All experi-mental protocols and all procedures involving animals were approved by the local animal welfare committee (Lund, Sweden).

Isolation of mouse pancreatic isletsPreparation of mouse pancreatic islets was per-

formed by retrograde injection of a collagenase solu-tion via the biliopancreatic duct. The islets were iso-lated and handpicked under a stereomicroscope at room temperature [16].

Human pancreatic isletsIsolated human pancreatic islets from nondiabetic

and diabetic male and female donors were provided by the Nordic network for clinical islet transplan-tation (Professor O. Korsgren, Uppsala University, Sweden). Donor characteristics are summarised in Supplementary Table 1. Prior to the experiments, the human islets were cultured for 1-5 days in CMRL 1066 (ICN Biomedicals) supplemented with 10 mmol/L HEPES, 2 mmol/L L-glutamine, 50 μg/mL gentami-cin, 0.25 μg/mL fungizone (Gibco), 20 μg/mL cipro-floxacin (Bayer Healthcare) and 10 mmol/L nicotin-amide at 37°C and gassed with 5% CO2. Upon arrival,

327GPRC5C, retinoic acid and β-cell

including 100 μmol/L IBMX. After the incubation, the islets were stored in HCl (100 mmol/L) for 5 min at room temperature followed by sonication. cAMP was measured using a direct cAMP ELISA kit (Enzo Life Sciences) according to the manufacturer’s instructions. The protein concentrations of the islet lysates were measured by a BCA kit (Thermo Fisher Scientific Inc.).

Gprc5c transfection and detection of associated signalSignalling pathways regulated by the orphan recep-

tor GPRC5C were identified using a Cignal 45-Pathway Reporter Array (Qiagen) in Huh7 cells, as the Huh7 cell line is both easy to transfect and expresses rela-tively high levels of native GPRC5C receptor, which suggests that all components required for GPRC5C signaling are also present in the Huh7 cell line. MIN6 cells are not suitable for the Cignal Array assay, as they cannot be transfected at sufficient efficiency for the Cignal array assay. Briefly, a Huh7 cell line sta-bily over-expressing the GPRC5C receptor (+~400% GPRC5C expression as measured by qPCR) was gen-erated by transfecting Huh7 cells with a pcDNA6.2/V5-DEST plasmid (Life Technologies) encoding the full-length ORF of the human GPRC5C receptor (Source Boscience). Signalling pathways activated or inhibited by GRC5C over-expression were then iden-tified following the manufacturer’s instructions. For validation of stimulation of MAPK signaling (i.e. ERK1/2 phosphorylation) in GPRC5C over express-ing Huh7 cells, we used a rabbit anti-phospho-p44/42 primary antibody (Cell Signalling Technologies), and a rabbit anti-GAPDH antibody (New England Biolabs) was used to label the loading control protein GAPDH. Both antibodies were visualized with help of an RDye 800CW conjugated goat anti-rabbit secondary anti-body (LI-COR Biosciences Ltd, UK). Densitometry was performed on 4 independent experiments. The rel-ative expression of each band was quantified and nor-malized to the GAPDH as loading control.

Effects of Ca2+ mobilisation by ATRA in GPRC5C over-expressing Huh7 cells

The effects on Ca2+ mobilisation by Huh7 cells over-expressing the GPRC5C receptor were probed using FLIPR. Briefly, 100,000 Huh7 cells/well were plated out in black walled, clear bottomed plates and left for 48 hours to recover in a humidified incubator at 37°C with 5% CO2. On the day of experimentation media was removed and cells were loaded with 50 µL

(edgeR/limma R-packages). Human islet genes strongly associated (Spearman’s rho>0.8) with the expression of GPRC5C transcript were identified using the “Cor.text” function in the statistical soft-ware R and uploaded into IPA and subjected to an ‘IPA Core Analysis’. Genes correlated with the expression of GPRC5C in human islets and a range of annotated cellular functions known to impact β-cell function and diabetes (cell death and survival, cell cycle, carbohy-drate metabolism, lipid metabolism, glucose tolerance, hormone secretion, hyperglycemia, quantity of islet cells, T2D risk, transport of vesicles) were then identi-fied from the ‘Biological Functions’ analysis of the IPA Core Analysis.

Western blottingLysates of islets (150 islets/vial; 20 µg of total pro-

tein/lane) were analysed by SDS–PAGE using 7.5% SDS-polyacrylamide gels (Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad) and blocked for 1 h at room temperature in 5% (weight/vol) fat-free milk protein. The expression of mouse and human GPRC5C receptor protein relative β-actin was deter-mined using a rabbit-raised polyclonal anti-human and mouse Gprc5c antibody (1:500) and a rabbit anti-β-actin antibody (Sigma) (1:200) that were incu-bated with the membrane for 1 h at room temperature in Tris-buffered saline-Tween 20 (TBST) buffer with 0.01 % (v/v) Tween 20. After several washes in TBST buffer, blots were probed with an HRP-conjugated sec-ondary antibody (Life Technologies) (1:2,000). For the comparison of Gprc5c protein expression between newborn (<3 weeks) and aged mice (>46 weeks), islets (150 islets/vial) were pooled from 5-10 mice in each experiment, due to the low yield of islets from the newborn mice.

Confocal microscopyThe co-expression of Gprc5c with insulin was deter-

mined by immunohistochemistry using Zeiss confocal microscopy software (Zen lite 2012) as described else-where [8, 9] with the polyclonal antibodies targeting insulin (1:400) and GPRC5C (1:200) (see above).

cAMP measurementMouse islets with either normal or Gprc5c-KD were

incubated for 60 min at 1 or 16.7 mmol/L glucose in the absence or presence of RA (5 μmol/L). Each incuba-tion vial contained 50 islets in 1.0 mL of KRB-buffer

328 Amisten et al.

6 days after the recovery period. Briefly, the MIN6c4 cells were washed three times with PBS, pulsed with 1 µCi/well of [methyl-3H] thymidine (Amersham) and incubated for an additional 20 h in complete RPMI medium. The cells were harvested onto glass fibre fil-ters using a FilterMate harvester (Perkin Elmer). The filters were air-dried, and bound radioactivity measured using a liquid scintillation counter (Perkin Elmer). In some experiments proliferation was measured by BrdU colorimetric assay according to the manufacturer’s instructions (Roche, Mannheim, Germany).

Cell viability and apoptosisAfter treatment with Gprc5c shRNA lentiviral par-

ticles (see above), mouse islets were dispersed into single cells using Ca2+ free medium. The islet cells were then cultured with or without a cocktail of pro-apoptotic cytokines (IL-1β, 100 ng/mL; TNFα, 125 ng/mL; and INFγ, 125 ng/mL) for 24 h in RPMI 1640 with 5 mmol/L glucose and 10% FSB supple-mented with 5 μmol/L ATRA. Measurements of cell viability and detection of apoptosis were performed using the MTS reagent kit (Promega) and Cell Death Detection ELISAplus kit (Roche) according to the manufacturer’s instructions.

StatisticsResults are expressed as means ± SEM for the indi-

cated number of observations or illustrated by an observation representative of a result obtained from different experiments (western blots and confocal microscopy). Probability levels of random differences were determined by Student’s t-test or, where appli-cable, the analysis of variance followed by Tukey-Kramers’ multiple comparisons test. A p value <0.05 was considered significant.

Results

Expression of the orphan receptors GPRC5C and its correlation with other gene transcripts in human pancreatic islets

qPCR was used to verify that GPRC5C is one of the most abundantly expressed GPCR mRNAs in human islets (Fig. 1A). Similar results were obtained when the mRNA expression of GPRC5C was determined using RNA-sequencing (n=89 human pancreatic islet preparations, data not shown). A marked and signif-icant correlation between GPRC5C and 1,194 gene

of dye buffer (assay buffer with 2.5 µM Fura-2-AM and 1 mM probenecid) for 1 hour at 37°C before being replaced with 100 µL of assay buffer (10 mM HEPES, 170 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, pH7.4). ATRA (10 µM) or the positive control ATP (10 µM), was added to the cell plate and calcium flux was measured during a 5 minute period using a Flexstation 3 instrument. Ratiometric fluores-cence values were captured and the effects of calcium flux assessed.

Down-regulation of Gprc5cFreshly isolated mouse islets were cultured in 1 mL

RPMI 1670 medium (Statens Veterinärmedicinska Anstalt, Sweden) for 36 h in the presence of a pool of five different Gprc5c shRNA lentiviral particles or scrambled control lentiviral particles according to the manufacturer’s recommendations (Santa Cruz Biotechnology). After transfection, the islets were washed, supplied with fresh RPMI 1670 medium and allowed to recover for 12 h under cell culture condi-tions. The islets were then washed again and assayed for insulin secretion in the absence or presence of test agents as described elsewhere [9]. The degree of target-gene-specific down-regulation of Gprc5c gene and protein expression was determined by qPCR and western blotting as described above. In addi-tion, glucose-stimulated insulin secretion in relation to total insulin and protein content was investigated in MIN6c4 cells following down-regulation of Gprc5c.

Cell proliferationClonal mouse insulin-secreting Min6c4 cells were

seeded at 10 000 cells/well into 48-well plates in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) with GlutaMaxTM (Gibco, USA) con-taining 4.5 g/L glucose and supplemented with 15% fetal calf serum, 50 μg/L streptomycin (Gibco), 75 mg/L penicillin sulphate (Gibco) and 5 μL/mL β-mercaptoethanol (Sigma). The cells were transfected with lentiviral particles targeting Gprc5c as described above. After transfection and a 12 h recovery period, the plates were incubated for 1-6 days at 37°C with 5% ambient CO2. The cells (in individual wells) were harvested by trypsination and counted after 2-6 days in tissue culture using a Bürcker chamber.

The effects of Gprc5c down-regulation on cell pro-liferation were also studied by measuring thymidine incorporation in the DNA of replicating cells 12 h or

329GPRC5C, retinoic acid and β-cell

noreactivity was also detected in insulin- negative islet cells (Fig. 2B).

Expression of the orphan receptors GPRC5A, GPRC5B, GPRC5C and GPRC5D in mouse and human pancreatic islets

We also compared the expression levels of all mem-bers of the GPRC5 family with that of GLP1R, a GPCR with a well-documented function within the pancre-atic islet [21]. The qPCR data showed that GPRC5C is expressed at levels similar to GLP1R in both mouse and human islets (Fig. 2C and 2D). We also found that the mRNA expression of GPRC5C relative to the house-keeping gene GAPDH is roughly ~100% higher in human islets than in mouse islets. The qPCR data also revealed a very low expression of GPRC5A and GPRC5D in mouse and human islets (Fig. 2C and 2D).

We also compared GPRC5C expression by qPCR and western blots in islet obtained from non-diabetic (ND; n=15) and type-2 diabetic (T2D; n=18) donors. Both GPRC5C mRNA expression and protein level were lower in T2D compared to ND islets (Fig. 2E and 2F).

Several studies have implicated Gprc5c in develop-ment/organogenesis [14, 22, 23]. We therefore com-pared the mRNA expression of Gprc5c in islets of newborn (<3 weeks) and older mice (>46 weeks), and found that the Gprc5c expression was 3-fold higher in islets from newborn compared to older mice (Fig. 2G), a finding that was confirmed by western blot analysis (Fig. 2H).

transcripts from human pancreatic islets was found (Supplementary Table 2, Pearson Correlation, ~p<1.1 × 10-23, r>0.8). Ingenuity Pathways Analysis (IPA) revealed that 806 (67.5%) of the genes correlated with GPRC5C mRNA expression have no known functions relevant for islet physiology or diabetes, whereas the remaining 388 genes (32.5%) are involved in regulat-ing (one or several) cellular functions associated with islet physiology and diabetes: cell death and survival (298 genes), cell cycle (71 genes), carbohydrate/lipid metabolism (159 genes) (Supplementary Table 2 and Fig. 1B). No genes with known associations to glu-cose tolerance, hormone secretion, hyperglycemia, quantity of islet cells, T2D risk or transport of vesi-cles were associated with the expression of GPRC5C in human islets.

Gprc5c protein expression in different mouse tissues and pancreatic islets

Protein expression of Gprc5c relative β-actin was determined in mouse brain, lung, heart, liver, kidney and pancreatic islets. Although Gprc5c receptor pro-tein was detected in all the examined tissue homoge-nates, it was expressed at particularly high levels rela-tive to β-actin in the heart and pancreatic islets (Fig. 2A). Next, we determined the cellular distribution of the Gprc5c protein in isolated mouse islets by confo-cal microscopy. As shown in Fig. 2F, 80±3% of the Gprc5-positive cells also contained insulin (Zen 2012 software, Zeiss Microscope). However, Gprc5c immu-

Fig. 1 Expression of 293 GPCRs in human pancreatic islet (A) Mean expression level of 293 GPCRs performed by qPCR in isolated human islets (n=3-4 donors) illustrating that

GPRC5C is one of the most abundantly expressed GPCRs in human islets. (B) Pie chart of functional annotations of the 388 human islet genes strongly associated with the mRNA expression of GPRC5C: 56.4% of the genes with known function (298 genes) regulate cell death and survival, 13.4% (71 genes) regulate cell cycle and 30.1% (159 genes) regulate carbohydrate and lipid metabolism. Please note that several genes were annotated to more than one cellular function category. The remaining 806 strongly associated genes have as yet unknown functions relating to β-cell function and diabetes. Data are based on 89 individual human islet preparations.

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Fig. 2 CPRC5C expression in mouse and human pancreatic islets (A) Gprc5c expression in different mouse tissues analysed by western blotting. A representative image of Gprc5c protein

expression in mouse brain, lung, heart, liver, kidney and pancreatic islets. For comparison, the protein expression of β-actin in each tissue is also shown (B) Confocal microscopy of mouse islets labelled for Gprc5c (green fluorescence) and insulin (red fluorescence) and the overlay of the two (yellow). Scale bar: 20 μm. (C) Expression of Gprc5a, Gprc5b, Gprc5c, Gprc5d and Glp1r mRNA relative to Gapdh mRNA analysed by qPCR in isolated mouse pancreatic islets (n=6 in each group). (D) Comparison of mRNA expression of GPRC5A, GPRC5B, GPRC5C, GPRC5D and GLP1R analysed by qPCR in human islets relative to GAPDH (n=3 donors). (E) mRNA expression of GPRC5C (n=15 in each group) normalised to three separate housekeeping genes (GAPDH, HPRT and PPAI) in human islets from non-diabetic (Non-Diab) and type-2 diabetic (Diab) islet donors. (F) Representative western blot of GPRC5C protein expression in human islets from non-diabetic and diabetic donors. Mean band intensities (means ± SEM) are presented. The blot for the endogenous control protein β-actin is also shown (n=4). (G) Gprc5c mRNA expression relative to Gapdh mRNA in newborn mouse islets plotted relative the mRNA expression of Gprc5c in adult mouse islets (n=5-6 in each group). (H) Fold change differences in intensity of western blot bands showing Gprc5c protein expression relative to β-actin in isolated islets of newborn (<3 weeks) and adult (>46 weeks) mice analysed by qPCR (n=10-16 in each group). * p<0.05, ** p<0.01.

331GPRC5C, retinoic acid and β-cell

overexpressing GPRC5C and, as shown in Fig. 3B, an oscillatory amplification of [Ca2+]i as expressed as relative fluorescent units (RFU) was recorded when ATRA (5 μmol/L) was present. In the following experiment, we also studied the impact of GPRC5C over-expression on signaling pathways by measur-ing the activation or inhibition of pathway specific transcription factors in in Huh7-cells overexpress-ing GPRC5C. Fig. 3C shows that over-expression of GPRC5C is associated with increased activity of transcription factors E2F (Cell cycle pathway), SRE (MAPK/ERK pathway), NFAT (PKC/Ca2+ path-way), PAX6, HNF4 and no significant effect on the PPAR or STAT3, while the pro-inflammatory NFκB transcription factor is markedly suppressed signal-ing from. GPRC5C over-expression in Huh7-cells

Gprc5c activation and down-stream signalling path-ways

Since Gprc5c was first identified as a retinoic acid-induced gene we next examine the impact of ATRA on cAMP content after down-regulation of Gprc5c in mouse islets and also on [Ca2+]i using Huh7-cells overexpressing Gprc5c. A marked reduc-tion in cAMP production was observed when islets were incubated for 60 min at high glucose (16.7 mmol/L) compared with low glucose (1 mmol/L) following Gprc5c-KD (p<0.0005) (Fig. 3A). While ATRA (5 μmol/L) greatly potentiated glucose-induced augmentation of islet cAMP content, no effect was seen in the Gprc5c-KD islets (Fig. 3A).

To examine the possible impact of ATRA on [Ca2+]i in relation to Gprc5c expression, we used Huh7-cells

Fig. 3 CPRC5C activation and down stream signalling molecules (A) The effect of Gprc5c knock-down on isolated mouse islet cAMP content incubated at low (1 mmol/L) or high (16.7 mmol/L)

glucose in the presence or absence of ATRA (5 μmol/L) for 60 min. (B) Addition of ATRA (10 µM) causes increased Ca2+ oscillations in Huh7 cells over-expressing the GPRC5C receptor as measured by FLIPR. (C) Signalling pathways associated with cell cycle progression (E2F), MAPK/ERK (SRE), Ca2+ mobilization (NFAT) and β-cell function (PAX6, HNF4) are more active in cells over-expressing the GPRC5C receptor, whereas the pro-inflammatory transcription factor NFκB is inhibited. GPRC5C over-expression did not affect STAT3 and PPAR transcription factor activity. Data presented as log2 fold change of the mean of transcription factor activity in GPRC5C over-expressing Huh7 cells vs. wt Huh7 cells. (D) Western blots showing phosphorylated ERK1/2 (pERK1/2) relative to GAPDH in wt or HUH7-cells transfected with GPRC5C and densitometry analysis of pERK1/2 bands relative to GAPDH. Mean ± SEM are presented for 4 observations in each group. (E) The proliferative effect on GPRC5C over-expressing Huh7 cells vs. wt were evaluated by BrdU incorporation. Dashed line denotes wt cell proliferation in the absence of FBS. Data are means ± SEM of 4 independent experiments in duplicate. * p<0.05, ** p<0.01, *** p<0.001.

332 Amisten et al.

was associated with an increased phosphorylation of ERK1/2 (Fig. 3D) and also with an increased cell proliferation as measured by BrdU incorporation (Fig. 3E).

The impact of Gprc5c down-regulation on insulin secretion in mouse pancreatic islets

Insulin secretion from intact mouse islets was mea-sured at either low (1 mmol/L) or high (20 mmol/L) glucose from islets treated with lentiviral particles con-taining either scrambled (control) or Gprc5c-specific shRNAs. Down-regulation of Gprc5c did not affect basal insulin secretion but moderately (-30%) reduced insulin secretion evoked by 20 mmol/L (Fig. 4A). The efficiency of silencing was determined by qPCR and western blot. Lentivirally delivered shRNAs target-ing Gprc5c resulted in reduced expression of Gprc5c

mRNA (-87±4% relative to control islets (scrambled siRNA; p<0.001; Fig. 4B)), and a similar reduction of Gprc5c protein expression (Fig. 4C).

Since Gprc5c was first identified as a retinoic acid-induced gene and shares some sequence simi-larity with the metabotropic glutamate receptor fam-ily [22], we compared the effects on insulin secre-tion by exposing islets for 1h to ATRA (0-1,000 μmol/L) and L-glutamate (50 and 1,000 μmol/L) at 8.3 mmol/L glucose (a glucose concentration chosen to facilitate the detection of potentiating effects). In control islets, ATRA stimulated insulin secretion at low concentrations (≤5 μM) whereas no effect was observed at higher ATRA concentrations (Fig. 4D). In Gprc5c-deficient islets, no stimulatory effect of ATRA was observed at low concentrations and at higher con-centrations (>500 μmol/L) ATRA inhibited insulin

Fig. 4 CPRC5C down-regulation and pancreatic islet function (A) The effect of down-regulation of Gprc5c on insulin secretion from isolated islets at low (1 mmol/L) and high (20 mmol/L)

glucose. (B) Efficiency of Gprc5c mRNA down-regulation analysed by qPCR in scrambled control and lentivirus-delivered Gprc5c-down-regulated islets. (C) Representative western blots and densitometric analysis of the western blot bands of Gprc5c protein expression relative the endogenous control protein β-actin in scrambled control islets and in islets treated with Gprc5c specific shRNAs. (D) Concentration-dependent effects of retinoic acid and glutamate (E) on insulin secretion from isolated mouse islets at 8.3 mmol/L glucose in control islets and after down-regulation of Gprc5c (n=6-8 in each group). * p<0.05, ** p<0.01, a p<0.01, *** p<0.001 (ATRA 5 μmol/L vs. 8.3 mmol/L glucose alone in control islets).

333GPRC5C, retinoic acid and β-cell

secretion by ~25% (Fig. 4D). By contrast, no impact of Gpcr5c-depletion on insulin secretion induced by glutamate was observed (Fig. 4E).

The impact of Gprc5c on cell proliferation and cyto-kine-induced apoptosis

The human islet RNA-sequencing data highlighted a correlation between GPRC5C and genes influenc-ing cell proliferation and apoptosis. To investigate the effects of GPRC5C down-regulation on β-cell proliferation, we used mouse Min6c4 insulinoma cells rather than primary mouse β-cells as the latter exhibit a low rate of cell division [5]. As shown in Fig. 5A, there was a significant reduction (~30-50 %) in total Min6c4 cell numbers at days 4 and 6 when Gprc5c had been down-regulated (p<0.05). This effect was further assessed by [3H]-thymidine incorporation. While there was no immediate (at day 0) significant effect on MIN6c4 cell proliferation following Gprc5c down-regulation, there was a marked reduction in thy-midine-incorporation at day 6, indicating a reduction in MIN6c4 cell replication (p<0.05) (Fig. 5B).

We also studied the effect of Gprc5c on glucose-stimulated insulin secretion (GSIS) in Min6c4 cells after on day 0, when cell number was not affected. While basal (at 1 mM glucose) insulin release was not affected, that evoked by high glucose (16.7

mmol/L) was decreased (-26%) following Gprc5c down-regulation (Fig. 5C-D).

To study the effect of ATRA and Gprc5c on cell via-bility (i.e. measuring the reductive capacity of islet cells) and apoptosis, we used mouse primary islet cells, since these cells do not proliferate in vitro, facilitat-ing the analysis of cell viability and apoptosis. After down-regulation of Gprc5c, the islets were dispersed into single cells and cultured with or without a cock-tail of the cytokines IL-1β, TNFα and INFγ (IL-1β, 100 ng/mL; TNFα, 125 ng/mL; and INFγ, 125 ng/mL) in the presence of ATRA (5 μmol/L). Compared to the scrambled controls, where the cytokine-mediated reduction in cell viability was approximately 30%, the sensitivity to the cytokine cocktail was exacerbated fol-lowing Gprc5c down-regulation and cell viability was reduced by >70% (p<0.001 vs. control cells; Fig. 6A). We next investigated the impact of ATRA on islet cells apoptosis after down-regulation of Gprc5c using iso-lated murine islets cultured for 72h at 5 mmol/L glucose ± ATRA (5 μmol/L). The preventive effect of ATRA on islet cell apoptosis was diminished when Gprc5c was down-regulated (Fig. 6B). We ascertained also by qPCR that Gprc5c down-regulation did not have any off-target effects on the expression of the nuclear recep-tors for retinoid acid (i.e. RARα, RARβ and RARγ; Fig. 6C).

Fig. 5 The effect of Gprc5c down-regulation on cell proliferation (A) The effect of Gprc5c down-regulation on Min6c4 cell relative cell numbers counted at time 0 (after 36 h shRNAs treatment

and 12 h recovery period) followed by culture and counting of the cells after 2, 4 and 6 days compared with Min6 cells exposed to scrambled control shRNAs. (B) The impact of Gprc5c down-regulation on [3H]thymidine-incorporation in Min6c4 cells direct after down-regulation period or at day 6 (n=10-12 in each group). (C) The effect of Gprc5c down-regulation (36h + 12h recovery period) on glucose-stimulated insulin release in Min6c4 cells (n=6 in each group). (D) Fold change reduction in glucose-stimulated insulin secretion from (C) (n=6 in each group). * p<0.05, ** p<0.01.

334 Amisten et al.

Discussion

We have recently shown that two members of the GPRC5 family (GPRC5B and GPRC5C) are highly expressed in both mouse and human islets [9, 15]. While activation of GPRC5B leads to reduced β-cell function in terms of a reduced viability and a reduced secretory capacity [9], the functional role of GPRC5C in β-cells has not been investigated to date. Using a range of techniques, we report here that GPRC5C plays an important role in β-cell function and survival. Our RNA sequencing and qPCR data confirm that GPRC5C is highly expressed in human islet cells and at levels comparable to that of the GLP-1 receptor (GLP1R). Interestingly, the GPRC5C mRNA expression is approximately 100% higher in human islets compared to mouse islets, suggesting that GPRC5C might have an even stronger influence on β-cell function in human islets than in mouse islets. The lack of detectable levels of other members of the GPRC5 family (GPRC5A and GPRC5D) argue that these receptors are not involved in pancreatic islet function [24].

The mechanism by which ATRA modulates pancre-atic β-cell function is not known in details. The pres-ent study reveals novel aspects of the interacting role of cAMP and [Ca2+]i as effectors of the rapid action of

ATRA which are markedly dependent on the expres-sion levels of Gprc5c. Our results also show that both metabolic and receptor-mediated cAMP production is disturbed after Gprc5c-KD, indicating both a posi-tive tonic activity of the GPRC5C on β-cell function and also a role for GPRC5C in rapid, non-genomic effects of ATRA in this regards, although consider-able research still needs to be conducted to ascertain whether GPRC5C is the sole receptor accounting for the observed results. The importance of the tonic activ-ity of the GPRC5C receptor, which might explain the anti-apoptotic properties GPRC5C, is further con-firmed by the discovery of a significant increase in the activity of several transcriptional factors with benefi-cial impact on β-cell function, as well as a strong inhi-bition of NFκB activity. As we show here for the first time, overexpression of GPRC5C was associated with an increased activity of ERK1/2. It is well known that ERK1/2 signalling pathway is crucial in cell survival, proliferation and differentiation [8].

Our insulin secretion experiments suggest that Gprc5c has a moderate stimulatory effect on insu-lin secretion in mouse islets and that it might inter-act with ATRA rather than with glutamate, previously proposed to be the natural ligand of GPRC5B [9]. A comparison of the responses to ATRA before and after

Fig. 6 The impact of Gprc5c down-regulation on cell viability (A) The effect of retinoic acid on cell viability in the absence or presence of a mixture of pro-apoptotic cytokines (IL-1β +

INFγ + TNFα) in scrambled control (white columns) and Gprc5c down-regulated (black columns) dispersed mouse islet cells. The islets had been previously cultured for 24h with indicated agents (n=4-6). (B) The impact of ATRA (5 μmol/L) on isolated mouse pancreatic islet cell apoptosis after 24h culture at 5 mmol/L glucose following knock-down of Gprc5c in the islets (n=6-8 in each group). (C) The influence of Gprc5c down-regulation on the expression of nuclear receptors of retinoic acid (Rarα, Rarβ, Rarγ) relative to GAPDH in mouse pancreatic islets analyzed by qPCR after down-regulation of Gprc5c by shRNA treatment (n=6-8). ** p<0.01, *** p<0.001.

335GPRC5C, retinoic acid and β-cell

silencing of Gprc5c indicates that whereas low (stimu-latory) ATRA concentrations (<5 μmol/L) may act via the GPRC5C receptor, higher (inhibitory) concentra-tions of ATRA utilise mechanisms not involving this receptor. In addition, both glucose-induced insulin secretion and β-cell viability and proliferation were reduced in islets with reduced GPRC5C expression. Importantly, glutamate remained capable of potenti-ating glucose-stimulated insulin secretion, indicating that GPRC5C is not required for modulatory effects of glutamate on the mouse pancreatic β-cell.

The strong correlation between GPRC5C and genes implicated in cell death/apoptosis and cell cycle con-trol in human islets supports our experimental data demonstrating an anti-apoptotic and pro-prolifera-tive effect of Gprc5c activation in β-cells, as silenc-ing of Gprc5c in mouse islets and MIN6 cells was markedly associated with pancreatic β-cells apopto-sis and reduced MIN6 cell proliferation. This is in good agreement with our results from Huh7 cells where overexpression of GPRC5C is associated with increased expression of E2F, a transcription factor linked to the cell cycle, and with a potent inhibition of NFκB, a transcription factor associated with inflam-mation and apoptosis. Collectively, these data indi-cate that GPRC5C represents an important regulator of β-cell survival and proliferation, and also moder-ately stimulates insulin secretion. Additional evidence for an important role of GPRC5C in β-cell expansion is provided by the observation that Gprc5c is more abundantly expressed in pancreatic islets of newborn mice (<3 weeks old) compared to older (>46 weeks) mice. It is known that rodent β-cells in fetal and neo-natal islets are not fully developed and continue to dif-ferentiate into functional β-cells almost up to 4 weeks after birth [5, 25]. Since Gprc5c is highly expressed

during the fetal/neonatal stage and it is known to play an important role in embryogenesis [14, 23, 26], it seems likely that Gprc5c influences cell prolifera-tion and thereby contributes to the appropriate expan-sion and maintenance of functional β-cell mass. It is therefore of interest that the levels of GPCR5C pro-tein were reduced in islets from organ donors with type-2 diabetes, which may contribute to the reduc-tion in β-cell mass and insulin secretion associated with type-2 diabetes [4-6]. These observations there-fore raise the exciting possibility that agents targeting GPRC5C may represent a novel therapeutic means of increasing the functional β-cell mass and also mod-estly boost insulin secretion.

Acknowledgements

We thank Britt-Marie Nilsson and Anna Maria Ramsay for skilled technical assistance and Disa Dahlman for the help with cell counting. We also thank the Lund University Diabetes Centre (LUDC) for the provided tissues and information. Supported by grants from Novo Nordisk Foundation, Diabetes & Wellness Foundation, Öresund Diabetes Academy and the EFSD/Lilly Research Fellowship program. SA is a Diabetes UK RD Lawrence Fellow (11/0004172). PR is a Wallenberg Scholar (funded by the Knut and Alice Wallenbergs Stiftelse), a recipient of a Swedish Research Council International Recruitment Grant and holds a Wellcome Trust Senior Investigator Award.

Duality of Interest

The authors declare that there is no duality of inter-est associated with this research paper.

Supplementary Table 1 Characteristics of non-diabetic (ND) and type-2 diabetic (T2D) organ donors

Gender Age BMI Hb1Ac (%)

Male (ND) (82) 55.7 ± 1.5 23.7 ± 0.2 5.5 ± 0.04

Female (ND) (44) 58 ± 1.2 23.2 ± 0.34 5.6 ± 0.06

Male (T2D) (22) 62.6 ± 1.9 26.5 ± 0.9 *** 6.95 ± 0.26 *** Male ND vs. male T2D

Female (T2D) (13) 59.8 ± 3.1 30.2 ± 1.0 *** 6.82 ± 0.16 *** Female ND vs. female T2D

The age, sex, BMI and Hb1Ac are shown. Pancreatic islets from donors with HbA1c <6.2% and BMI of 18.5 to 25.5 were consider non-diabetic (ND) and the islets from donors with the Hb1Ac >6.2% or history of diabetes considered as diabetic islets in our study. *** p<0.001 for male ND vs. male T2D and female ND vs. female T2D.

336 Amisten et al.

PAFAH1B3 CDS 0.88DEDD2 CDS 0.88RBCK1 CC, CDS 0.88FASN CLM, CC, CDS 0.88HYAL2 CLM, CDS 0.88EIF4EBP1 CLM, CC, CDS 0.88JUP CDS 0.88HMG20B CC 0.88EHD1 CLM, CDS 0.88TMEM132A CDS 0.88VPS18 CC 0.88ERF CDS 0.88BAX CLM, CC, CDS 0.88GRINA CLM, CDS 0.88PINK1 CDS 0.87CTBP1 CDS 0.87STK11 CLM, CC, CDS 0.87MTG2 CDS 0.87CC2D1A CDS 0.87WDR81 CDS 0.87OSGIN1 CC, CDS 0.87ADRBK1 CLM, CDS 0.87LRPAP1 CLM, CDS 0.87PLEC CDS 0.87BOK CDS 0.87HDAC11 CC, CDS 0.87AGAP3 CDS 0.87SHISA5 CDS 0.87RXRA CLM, CC, CDS 0.87AKT1S1 CDS 0.87COL18A1 CLM, CDS 0.87SDF2L1 CDS 0.87JUNB CC, CDS 0.87ZYX CDS 0.87TMEM259 CDS 0.87EMD CDS 0.87ABCD1 CLM, CDS 0.87TFPT CDS 0.87UNC119 CDS 0.87RRAS CDS 0.86LZTS2 CDS 0.86AATK CDS 0.86MAFK CDS 0.86H2AFX CDS 0.86LIPE CLM, CDS 0.86MAP3K10 CDS 0.86ZBTB17 CDS 0.86TCF3 CC, CDS 0.86ORAI1 CDS 0.86BIN1 CDS 0.86TRAF7 CDS 0.86NME4 CLM, CDS 0.86PML CLM, CC, CDS 0.86

MBOAT7 CLM, CDS 0.90VAC14 CLM, CDS 0.90PRDX5 CDS 0.90LTBR CDS 0.90PACS2 CDS 0.90BCAR1 CDS 0.90CLCN7 CDS 0.90ARHGDIA CLM, CDS 0.90CLDN3 CDS 0.90ARHGEF1 CDS 0.90IGFBP2 CLM, CDS 0.90MGMT CLM, CC, CDS 0.90MYH14 CC 0.90TRIP6 CDS 0.90SIGIRR CLM, CDS 0.90PIM3 CDS 0.90MAP2K7 CC, CDS 0.90DVL1 CDS 0.90HSPB1 CDS 0.90GNA11 CLM, CDS 0.90LRP5 CLM, CDS 0.90HSF1 CC, CDS 0.90MRPL41 CDS 0.90ISG15 CDS 0.89FASTK CDS 0.89DPM3 CLM, CDS 0.89CST3 CDS 0.89ADRM1 CDS 0.89LGALS3BP CDS 0.89IRAK1 CLM, CDS 0.89ST14 CDS 0.89HSPBP1 CDS 0.89SLC9A3R2 CLM, CDS 0.89LRWD1 CC 0.89TICAM1 CDS 0.89ELL CLM, CDS 0.89MXD4 CC 0.89BRAT1 CDS 0.89SLC25A10 CDS 0.89RUVBL2 CC, CDS 0.89GATAD2A CDS 0.89ARAF CC, CDS 0.89EEF1D CDS 0.88INTS1 CDS 0.88MAP1S CDS 0.88SCRIB CDS 0.88UBE2S CDS 0.88PVRL2 CDS 0.88TRAF2 CC, CDS 0.88E4F1 CC, CDS 0.88NME3 CDS 0.88PRKD2 CDS 0.88BOP1 CC 0.88

Supplementary Table 2 Association (rho>0.80) of human islet genes known to regulate carbohydrate and lipid metabolism (CLM), cell cycle (CC) and cell death and survival (CDS) with the expression in human islets of GPRC5C

Symbol Annotated function rhoCOMT CLM, CDS 0.95GNB2 CDS 0.95SHARPIN CDS 0.95PKP3 CDS 0.94NELFB CC, CDS 0.94GADD45GIP1 CDS 0.94MAP3K11 CC, CDS 0.94FZR1 CC, CDS 0.94CACFD1 CDS 0.94STUB1 CDS 0.94NINJ1 CDS 0.94NACC1 CDS 0.93NAGLU CLM, CDS 0.93FURIN CDS 0.93PAK4 CDS 0.93TSPO CLM, CDS 0.93KATNB1 CDS 0.93NR1H2 CLM, CDS 0.92SIRT6 CLM, CDS 0.92GRK6 CDS 0.92LMNA CLM, CC, CDS 0.92XAB2 CDS 0.92SYNGR2 CDS 0.92GNPTG CDS 0.92APRT CDS 0.92SCYL1 CDS 0.92ATP13A2 CDS 0.92CDC34 CDS 0.92MPG CLM, CC, CDS 0.92MDK CDS 0.92KIFC3 CDS 0.92ATAD3A CDS 0.92NAA38 CDS 0.92BSG CLM, CDS 0.92GPX1 CLM, CDS 0.91FKBP8 CDS 0.91MAP2K2 CDS 0.91TNIP2 CDS 0.91MVP CLM, CDS 0.91GPC1 CLM, CC, CDS 0.91PHLDA3 CDS 0.91DPP7 CDS 0.91SLC4A2 CDS 0.91PSMB10 CDS 0.91TADA3 CDS 0.91PNPLA2 CLM, CDS 0.91ARSA CLM, CDS 0.91DDAH2 CLM, CDS 0.91NLRX1 CDS 0.91PKN1 CDS 0.91GRN CDS 0.91AGPAT2 CLM, CDS 0.91PIN1 CC, CDS 0.91

337GPRC5C, retinoic acid and β-cell

NCOR2 CLM, CDS 0.82CD14 CLM, CDS 0.82NFIC CDS 0.82EHMT1 CC, CDS 0.82INS CLM, CC, CDS 0.82CTRB2 CDS 0.82SOD3 CLM, CDS 0.82BBC3 CLM, CDS 0.82INPP5E CLM, CDS 0.82DYRK1B CC 0.82CDK9 CDS 0.82ACHE CLM, CDS 0.82IER3 CC, CDS 0.81MAP1LC3A CDS 0.81ATN1 CDS 0.81C1orf159 CDS 0.81PPP1R9B CDS 0.81PIEZO1 CDS 0.81RPS19 CDS 0.81SLC4A3 CDS 0.81PTOV1 CC 0.81NAPA CDS 0.81COX8A CDS 0.81MCOLN1 CDS 0.81SREBF1 CLM, CC, CDS 0.81ADAM8 CDS 0.81RAC3 CDS 0.81IDUA CLM, CDS 0.81NSMF CDS 0.81FBXL15 CC 0.81HSPG2 CDS 0.81HDAC5 CC, CDS 0.81MAPK7 CC, CDS 0.81GDF15 CLM, CC, CDS 0.80UBE2M CDS 0.80RPS6KB2 CLM, CDS 0.80FDXR CLM, CDS 0.80THOC6 CDS 0.80MT2A CDS 0.80DAB2IP CLM, CDS 0.80RAPGEF1 CDS 0.80CDR2L CDS 0.80NRTN CDS 0.80PLIN3 CLM, CDS 0.80MAD1L1 CDS 0.80MNT CDS 0.80CD151 CDS 0.80CBX4 CDS 0.80Bonferoni-corrected p values for all listed genes < 1e-23.

MEN1 CLM, CC, CDS 0.84GLTSCR2 CDS 0.84RHOG CDS 0.84ENDOG CDS 0.84HIGD2A CDS 0.84GLIS2 CDS 0.84PDXK CDS 0.84CEBPB CLM, CC, CDS 0.84POLD4 CC 0.84USE1 CDS 0.84PLEKHF1 CDS 0.84RAD9A CC, CDS 0.84ZBTB7A CLM, CDS 0.84AXIN1 CDS 0.84GSTP1 CLM, CDS 0.84TRADD CDS 0.84SIRT7 CDS 0.84TNFRSF14 CC, CDS 0.83NACC2 CDS 0.83KEAP1 CDS 0.83SIVA1 CDS 0.83FAU CDS 0.83COMMD4 CDS 0.83A4GALT CLM, CDS 0.83NCK2 CDS 0.83PDLIM4 CDS 0.83POMC CLM, CC, CDS 0.83NOC2L CDS 0.83ABCB8 CDS 0.83RARA CLM, CC, CDS 0.83TNFSF12 CLM, CDS 0.83PKD1 CLM, CC, CDS 0.83DOT1L CC, CDS 0.83TSC2 CLM, CC, CDS 0.83TFAP4 CC, CDS 0.83BAP1 CC, CDS 0.83DVL2 CDS 0.83MVK CLM, CDS 0.83EIF3G CDS 0.83BCL3 CC, CDS 0.83JAG2 CDS 0.83FBXO2 CDS 0.83DPEP1 CDS 0.83IMPDH1 CDS 0.83BRD4 CC, CDS 0.83CAMK2N2 CDS 0.83KDM6B CDS 0.82TFEB CLM, CDS 0.82KLHDC8B CC 0.82FAAH CLM, CDS 0.82C9orf114 CDS 0.82SPHK2 CLM, CC, CDS 0.82KMT2B CLM, CDS 0.82

Symbol Annotated function rhoOAZ1 CDS 0.86ID3 CC, CDS 0.86SUN2 CC, CDS 0.86CDC37 CDS 0.86CYBA CDS 0.86REPIN1 CDS 0.86ID1 CC, CDS 0.86RABL6 CDS 0.86MTA1 CDS 0.86C1orf86 CC 0.86FGFR4 CLM, CDS 0.86BRMS1 CLM, CDS 0.86CSK CDS 0.86LAMA5 CDS 0.86PIAS4 CDS 0.86BCL2L12 CDS 0.86HPN CLM, CDS 0.86ADAM15 CDS 0.86CTSD CLM, CDS 0.86DPP9 CDS 0.86AKT2 CLM, CDS 0.85LONP1 CDS 0.85VPS28 CDS 0.85CAPN10 CLM, CDS 0.85NR0B2 CLM, CDS 0.85ZNF512B CDS 0.85HNF1A CLM, CC, CDS 0.85BRF1 CDS 0.85PELP1 CDS 0.85BRD1 CDS 0.85PLCD3 CDS 0.85CIZ1 CC 0.85HIRA CC 0.85HS1BP3 CDS 0.85TNFRSF12A CLM, CDS 0.85FOXK2 CDS 0.85TRPM4 CDS 0.85TRIM28 CDS 0.85RPTOR CLM, CC, CDS 0.85GPX4 CLM, CDS 0.85IRF7 CDS 0.85DNAJC5 CDS 0.85HLA-F CDS 0.85HIP1R CDS 0.85GADD45G CC, CDS 0.85AGRN CDS 0.84TXNRD2 CDS 0.84SPHK1 CLM, CC, CDS 0.84ADCK3 CDS 0.84POR CLM, CDS 0.84SLC12A7 CDS 0.84PPM1F CDS 0.84KDM4B CC 0.84

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