The constitutive activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent pathway

Molecular and Cellular Endocrinology 299 (2009) 232–239

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The constitutive activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent pathway Pui Ngan Lau a , Kevin B.S. Chow a , Chi-Bun Chan b,1 , Christopher H.K. Cheng b , Helen Wise a,∗ a b

Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

a r t i c l e

i n f o

Article history: Received 27 August 2008 Received in revised form 10 December 2008 Accepted 11 December 2008 Keywords: Ghrelin receptors Constitutive activity Apoptosis

a b s t r a c t The ghrelin receptor (GHS-R1a) displays a high level of constitutive signaling through a phospholipase C/protein kinase C-dependent pathway. Therefore, we have investigated the role of agonist-dependent and agonist-independent signaling of GHS-R1a in apoptosis using the seabream GHS-R1a stably expressed in human embryonic kidney 293 cells (HEK-sbGHS-R1a cells). Cadmium-induced activation of caspase3 was significantly attenuated in HEK-sbGHS-R1a cells compared to wild-type HEK293 cells, while the apoptotic responses to the protein kinase C inhibitor staurosporine were similar. GHS-R1a ligands had no effect on caspase-3 activation or on cell proliferation. Concentrations of the inverse agonist [d-Arg1 ,dPhe5 ,d-Trp7,9 ,Leu11 ]-substance P sufficient to inhibit constitutive inositol phosphate generation did not enhance caspase-3 activity, suggesting a possible role of phosphatidylcholine-specific phospholipase C in the anti-apoptotic activity of GHS-R1a. In conclusion, our data suggests that the constitutive activity of sbGHS-R1a may be sufficient alone to attenuate apoptosis via a protein kinase C-dependent pathway. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Ghrelin is a 28-amino acid peptide with an acylated octanoic acid at Ser3 which is essential for activation of the ghrelin receptor (Kojima et al., 1999). Ghrelin, and other growth hormone secretagogues such as GHRP-6, stimulate a Gq/11-protein coupled receptor (Howard et al., 1996) now known as the ghrelin receptor or GHSR1a (Davenport et al., 2005). In addition to GHS-R1a, there is wide spread distribution of a splice variant comprising the first five predicted transmembrane regions of GHS-R1a, known as GHS-R1b (Howard et al., 1996). The truncated ghrelin receptor polypeptide (GHS-R1b) does not bind ghrelin but acts as a dominant-negative for the expression of GHS-R1a at the cell surface and therefore has a marked effect on the constitutive (agonist-independent) signaling properties of GHS-R1a when co-transfected into human embryonic kidney (HEK) 293 cells (Chu et al., 2007; Leung et al., 2007). The ghrelin receptor is unusual in displaying extremely high levels of constitutive activity (Holst et al., 2003), and it has been proposed that a lack of constitutive activity is responsible for the altered growth status in persons expressing novel variants of the GHSR gene (Holst and Schwartz, 2006).

∗ Corresponding author. Tel.: +852 2609 6849; fax: +852 2603 5139. E-mail address: [email protected] (H. Wise). 1 Current address: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA. 0303-7207/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2008.12.006

There is much conflicting information in the literature concerning the role of ghrelin as a proliferative agent, in part because not all ghrelin-stimulated responses are mediated through GHSR1a (van der Lely et al., 2004). For example, in myocardium there are at least three functional receptors for ghrelin: GHSR1a, GHS-Ru (unknown) and CD36 which is a glycoprotein type B scavenger receptor (Cao et al., 2006). But if we focus on cellular responses which are attributable to ghrelin stimulation of GHS-R1a (i.e. assays where nonacylated ghrelin was inactive and/or GHS-R1a was identified by protein or mRNA expression), we see that ghrelin stimulates proliferation which can be inhibited by [dArg1 ,d-Phe5 ,d-Trp7,9 ,Leu11 ]-substance P (SPa) or d-Lys(3)-GHRP-6 in rat and human primary cell cultures (Andreis et al., 2003; Li et al., 2007; Sato et al., 2006; Zhang et al., 2005) and in cell lines (Jeffery et al., 2002; Kim et al., 2005; Nanzer et al., 2004). And, while ghrelin either has no effect on apoptosis (Andreis et al., 2003) or enhances apoptotic deletion (Belloni et al., 2004), the predominant effect appears to be the ability to inhibit apoptosis, as shown in adipocytes (Kim et al., 2004), pancreatic cells (Andreis et al., 2003) and in rat hypothalamic cell cultures (Chung et al., 2007). In addition, GHRP-6 also inhibits apoptosis in rat central nervous system (Delgado-Rubín de Célix et al., 2006; Frago et al., 2002). Since the discovery of GHS-R1a and GHS-R1b, researchers have attempted to correlate the relative expression of these proteins (at the mRNA level) with the state of malignancy of human tumors. For example, (i) somatotroph adenomas had significantly higher GHSR1b mRNA copy number compared to normal pituitary (Korbonits et al., 2001), (ii) prostate cancer cell lines express GHS-R1a and

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GHS-R1b, whereas normal prostate only has GHS-R1a (Jeffery et al., 2002), (iii) 50% of human endocrine pancreatic tumors express GHS-R1b and 50% of these also contain GHS-R1a, with the majority of these tumors expressing GHS-R1a also expressed ghrelin (such co-expression was not seen in normal endocrine pancreas) (Volante et al., 2002), (iv) human pancreatic adenocarcinoma cell lines express both GHS-R1a and GHS-R1b (Duxbury et al., 2003), (v) in contrast to normal lung, a human bronchial neuroendocrine tumor expresses ghrelin, GHS-R1a and GHS-R1b (Arnaldi et al., 2003), and (vi) the ratio of ghrelin to GHS-R1a increases in highly differentiated testicular tumors (Gaytan et al., 2004). Whether or not the GHS-R1b polypeptide functions as a dominant-negative mutant of GHS-R1a under physiological conditions remains to be determined, but it has been proposed to form a heterodimer complex with neurotensin receptor 1 and thus function as a receptor for neuromedin U in non-small cell lung cancer cells (Takahashi et al., 2006). Thus, the presence of GHS-R1a and/or GHS-R1b may contribute to the tumor potential of cells. Seabream GHS-R1a shares 60% amino acid identity with mammalian GHSRs and has higher affinity for GHRP-6 than for human ghrelin (Chan and Cheng, 2004). Therefore, having established a cell line stably expressing seabream GHS-R1a (HEK-sbGHS-R1a) (Chan and Cheng, 2004), we chose this system to investigate the role of GHS-R1a and GHS-R1b in regulating apoptosis and proliferation. As we have shown previously that GHS-R1b behaves as a dominantnegative mutant of GHS-R1a and can attenuate the constitutive activity of GHS-R1a (Leung et al., 2007), and that d-Lys(3)-GHRP6 can inhibit constitutive activation of ERK1/2 in HEK-sbGHS-R1a cells (Chan et al., 2004), we have used these HEK-sbGHS-R1a cells to study the role of agonist-dependent and agonist-independent signaling through sbGHS-R1a in the regulation of apoptosis and proliferation. In order to determine the role of sbGHS-R1a signaling through the Gq/phospholipase C/protein kinase C pathway, we have compared the effect of apoptosis inducers predicted to be protein kinase C-dependent (staurosporine, Ozawa et al. (1999)) and protein kinase C-independent (cadmium, Hamada et al. (1996)). Here we show that the constitutive activity of sbGHS-R1a, when stably expressed in HEK293 cells, appears to attenuate apoptosis via a protein kinase C-dependent pathway but does not affect proliferation of these cells. 2. Materials and methods 2.1. Reagents GHRP-6 and [d-Arg1 ,d-Phe5 ,d-Trp7,9 ,Leu11 ]-substance P were obtained from Phoenix Pharmaceutical Inc. (Beijing, China). d-Lys(3)-GHRP-6 was obtained from Bachem AG (Bubendorf, Switzerland). Unless specified, all other compounds were supplied by Biomol (Plymouth Meeting, PA), Invitrogen (Carlsbad, CA) or Sigma Chemical Co. (St. Louis, MO). 2.2. Cell culture and transfection HEK293 cells were purchased from American Type Culture Collection (Manassas, VA) and were maintained in DMEM containing 100 i.u./ml penicillin, 100 ␮g/ml streptomycin and 10% (v/v) foetal bovine serum, and were incubated in a humidified atmosphere of 5% CO2 /95% air at 37 ◦ C. A clonally selected HEK293 cell line stably expressing sbGHS-R1a was developed as previously described (Chan and Cheng, 2004) and was maintained in DMEM containing 500 ␮g/ml G418. Cells were transfected at 80% confluency in culture plates using LipofectAMINE 2000 liposome reagent and Opti-mem I reduced serum medium (Invitrogen, Carlsbad, CA) for 5 h, according to the manufacturer’s instructions, and all cells were assayed 48 h post-transfection. 2.3. Preparation of epitope-tagged sbGHS-R1a and sbGHS-R1b The HA-sbGHS-R1a and HA-sbGHS-R1b were constructed by PCR amplification from seabream pituitary cDNA. Gene specific primers were designed flanking the entire coding regions of sbGHS-R1a and sbGHS-R1b respectively, with the appropriate restriction enzyme cutting sites (Kpn I and Xba I) engineered at the 5 ends of the primers. A HA tag was introduced to the N-terminal of the receptors by incorporat-

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ing the HA sequence to the forward primers. The primers for HA-sbGHS-R1a were sbGHS-R1a-start-HA (a forward primer having the sequence: GAG GTA CCG AAA TGT ATC CAT ATG ATG TTC CAG ATT ATG CTC CCT CTT GGC CAA ATC TC) and sbGHS-R1astop (a reverse primer having the sequence: CAC ATC TAG ATT AGA AGC TGA TTG TGG A). The primers for HA-sbGHS-R1b were sbGHS-R1a-start-HA (the same as for sbGHS-R1a) and sbGHS-R1b-stop (a reverse primer having the sequence: CAC ATC TAG ACT ACA TGG ATA AAG TTA TG). PCR was carried out in a 50 ␮l final volume containing 50 mM KCl, 10 mM Tris–HCl at pH 9, 1.5 mM MgCl2 , 0.1% Triton X-100, 0.02% bovine serum albumin (BSA), 200 ␮M dNTP, 0.2 pmol primers, 2.5U Taq polymerase, and 1 ␮l cDNA. The reaction was performed with annealing temperature of 50 ◦ C for 25 cycles. The PCR product was size separated on an agarose gel, gene-cleaned, unidirectionally subcloned into the eukaryotic expression vector pcDNA3.1(+)/Zeo, and all modifications were confirmed by DNA sequencing. 2.4. Measurement of sbGHS-R1a and sbGHS-R1b mRNA Quantitative real time PCR was performed to quantify the expression of sbGHS-R1a and sbGHS-R1b, essentially as described previously for human ghrelin receptors (Leung et al., 2007). Oligonucleotide primers were: 5 GTGGGAATGAATGGGACTGG-3 as the forward primer for both sbGHS-R1a and sbGHS-R1b, and 5 -GCCAACACCACCACCACCAAC-3 for the sbGHS-R1a reverse primer, and 5 -GGATAAAGTTATGCGTGTGCG-3 for the sbGHS-R1b reverse primer. PCR reactions were performed in duplicate in three independent experiments using the ABI Prism 7700 Sequence Detector System (Applied Biosystems, Foster City, CA) in a total volume of 25 ␮l containing Sybr green PCR buffer (Applied Biosystems, UK), MgCl2 , dNTP blend, AmpliTaq Gold, AmpErase UNG primers, water and diluted cDNA template. Negative controls contained template without performing reverse transcription. The PCR conditions were: 50 ◦ C for 2 min and denaturing at 95 ◦ C for 10 min, followed by 40 cycles at 94 ◦ C for 45 s and 64 ◦ C for 45 s. Absolute quantification of transcripts was performed against standard curves obtained by amplification of serially diluted solutions of plasmid clones containing sbGHS-R1a and sbGHS-R1b sequences as templates. Levels of mRNA expression were normalized against total RNA (Whelan et al., 2003). 2.5. Caspase-3 assay For the comparison of caspase-3 activity in HEK-sbGHS-R1a and HEK-sbGHS-R1b cells, cells were seeded in 6-well culture plates at 8 × 105 cells per well, and maintained in DMEM plus 10% fetal bovine serum. After 24 h, the cells were transfected and apoptosis inducers added 48 h post-transfection. After 8 h incubation of triplicate wells with apoptosis inducer or control solution, the medium was removed and cells were washed and harvested in PBS. Cell pellets were lysed (lysis buffer: 50 mM Hepes, 1 mM DTT, 0.1 mM EDTA, 0.1% CHAPS, pH 7.4), centrifuged at 10,000 × g for 10 min at 4 ◦ C, and lysates assayed for caspase-3 activity. Activated caspase-3 cleaves a specific caspase-3 I substrate (Calbiochem, San Diego, CA) to release p-nitroaniline which was detected by absorbance at 405 nm in a 96-well plate ELISA reader (Universal Microplate Reader Elx800, Bio-Tek Instruments Inc., Winooski, VT). The protein content of the cell lysates was determined using a Micro BCATM Protein Assay Kit (Pierce, Rockford, IL) and caspase-3 specific activity was then determined. To compare the effect of GHS-R1a ligands, cells were seeded as above. Cells were incubated with DMEM or SPa (1 ␮M) or d-Lys(3)-GHRP-6 (100 ␮M) for 30 min before DMEM or GHRP-6 (100 ␮M), then caspase-3 activity was measured in duplicate wells after 24 h incubation with DMEM (control), 10 ␮M staurosporine (stau), or 25 ␮M cadmium. 2.6. DNA fragmentation Cells were seeded in 6-well culture plates at 8 × 105 cells per well. The next day, cells were incubated for 8, 16 or 24 h with apoptosis inducer or control (0.1% DMSO) solution, then harvested with the addition of lysis buffer (Promega, Madison, IN). Genomic DNA was isolated using Wizard SV Genomic DNA Purification System (Promega, Madison, IN). DNA (2 ␮g) was electrophoresed on a 1.5% agarose gel and the pattern of DNA fragmentation was photographed with a ChemiDoc XRS (Bio-Rad, Hercules, CA) after staining with GelRed (Biotium Inc., Hayward, CA). 2.7. Cell viability assay Cells were seeded in 96-well culture plates, pretreated with poly-d-lysine (10 ␮g/ml), at 104 cells per well, then maintained and transfected as described for the caspase-3 assay. The conversion of 2-(4 ,5 -dimethyl-2 -thiazolyl)-3-(4 sulfophenyl) (MTS) solution to a colored formazan product in metabolically active cells was determined, in duplicate, by incubation for 4 h in a CO2 incubator, and the absorbance at 570 nm was recorded with a 96-well plate ELISA reader (Universal Microplate Reader Elx800, Bio-Tek Instruments Inc., Winooski, VT). 2.8. Cell proliferation assay Cells were seeded in 12-well culture plates, pretreated with poly-d-lysine (10 ␮g/ml), at 105 cells per well, and cultured in DMEM plus 10% fetal bovine

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Table 1 Expression of sbGHS-R1a and sbGHS-R1b in transfected cell lines. mRNA copy/␮g total RNA (×109 )

Cell type

Transfection material

sbGHS-R1a

sbGHS-R1b

HEK293

pcDNA3.1/Zeo HA-sbGHS-R1a HA-sbGHS-R1b

ND 81 ± 13* ND

ND ND 10 ± 2

HEK-sbGHS-R1a

pcDNA3.1/Zeo HA-sbGHS-R1a HA-sbGHS-R1b

38 ± 6** 243 ± 51 37 ± 4**

ND ND 32 ± 9

Cells were transfected with control vector pcDNA3.1/Zeo, HA-sbGHS-R1a or HAsbGHS-R1b at 0.25 ␮g/ml plus control vector pcDNA3.1/Zeo (0.75 ␮g/ml). Messenger RNA expression was determined by real time PCR and data shown are means ± SEM of 3 independent experiments, each performed in duplicate. ND = not detected. * P < 0.05 compared with HEK-sbGHS-R1a cells transfected with HA-sbGHS-R1a. ** P < 0.01 compared with HEK-sbGHS-R1a cells transfected with HA-sbGHS-R1a.

serum with the addition of 0–100 ␮M GHRP-6, 1 ␮M SPa or 100 ␮M d-Lys(3)GHRP-6 to duplicate wells as appropriate. Medium and drugs were replaced every 24 h, as described by Jeffery et al. (2002). Cells were harvested by trypsinization and counted using a hemocytometer slide. The proportion of viable cells was determined using the trypan blue exclusion test. Trypan blue (0.4%) was added to cells immediately before counting, with non-viable cells showing blue cytoplasm. 2.9. [3 H]inositol phosphates accumulation assay [3 H]inositol phosphates ([3 H]IP) accumulation was assayed by incubating [3 H]myo-inositol-labelled cells, in duplicate, with test compounds for 60 min at 37 ◦ C in 1 ml HEPES-buffered saline containing 20 mM LiCl, as described previously (Chow et al., 2001). [3 H]IP was separated from the [3 H]inositol fraction by column chromatography and the production of [3 H]IP was expressed as the ratio of [3 H]IP to the total [3 H]inositol-containing fraction (i.e. [[3 H]IP/([3 H]IP + [3 H]inositol)] × 100). 2.10. Statistical analysis All analyses were performed using the GraphPad Prism software version 5.0 (GraphPad Software Inc., San Diego, CA). Values reported are mean ± SEM. Comparisons between groups were made using analysis of variance (ANOVA) with Bonferroni’s posttests. Statistical significance was taken as P < 0.05.

3. Results 3.1. Apoptosis in HEK293 cells expressing sbGHS-R1a and sbGHS-R1b In order to study the relative effects of sbGHS-R1a and sbGHSR1b, we performed preliminary experiments which indicated that transfection of HEK293 cells with 0.25 ␮g/ml HA-sbGHS-R1a would result in a similar mRNA expression level of sbGHS-R1a as found in HEK-sbGHS-R1a cells. The same concentration of HA-sbGHSR1b cDNA also produced a similar mRNA expression as sbGHS-R1a (Table 1), although it is unknown how these mRNA levels relate to actual protein expression. As might be expected, significantly higher sbGHS-R1a mRNA expression was obtained when HEKsbGHS-R1a cells were transiently transfected with HA-sbGHS-R1a when compared with wild-type HEK293 cells transiently expressing HA-sbGHS-R1a (Table 1). Caspases play an essential role during apoptotic cell death (Nicholson, 1999), therefore the effect of apoptosis inducers to increase caspase-3 activity was determined after 8 h incubation. Both staurosporine and cadmium significantly increased caspase-3 activity in control HEK293 cells, and in HEK293 cells transiently expressing HA-sbGHS-R1a or HA-sbGHS-R1b (Fig. 1a). In contrast, only staurosporine, but not cadmium, significantly activated caspase-3 activity in HEK-sbGHS-R1a cells (Fig. 1b). In addition, HEK-sbGHS-R1a cells appeared more resistant to the effects of cadmium when compared to wild-type HEK293 cells (P < 0.001). When analyzed for DNA fragmentation as further evidence of apoptosis (Wyllie, 1980), we found that staurosporine produced a

Fig. 1. A comparison of caspase-3 activity in HEK293 and HEK-sbGHS-R1a cells, and the effect of transient expression of sbGHS-R1a or sbGHS-R1b in these cells. Cells were transfected with control vector pcDNA3.1/Zeo (pcDNA), HA-sbGHS-R1a (GHSR1a) or HA-sbGHS-R1b (GHS-R1b) at 0.25 ␮g/ml plus control vector (0.75 ␮g/ml). Caspase-3 specific activity was assayed 8 h after incubation with control medium (open bar), 10 ␮M staurosporine (black bar), or 25 ␮M cadmium (grey bar). * P < 0.05 compared with own control group. Data shown are means + SEM of 3 independent experiments, each performed in triplicate.

similar time course of DNA fragmentation in both HEK293 cells and in HEK-sbGHS-R1a cells (Fig. 2). Furthermore, cell viability was not compromised by either staurosporine (10 ␮M) or cadmium (25 ␮M) after 8 h in serum-containing medium, whereas Triton X-100 (0.1%, v/v) significantly decreased cell viability in both HEK293 and HEKsbGHS-R1a cells (Fig. 3).

3.2. GHS-R1a ligands did not affect caspase-3 activity in HEK-sbGHS-R1a cells When tested after 24 h, staurosporine again significantly increased caspase-3 activity in HEK-sbGHS-R1a cells, while cadmium did not (Fig. 4). However neither the GHS-R1a agonist (GHRP-6) nor the GHS-R1a inverse agonist (SPa, 1 ␮M) (Holst et al., 2003) had any effect on staurosporine or cadmium-induced caspase-3 activity (Fig. 4a). Increasing the concentration of SPa to 10 ␮M to produce a greater inhibition of basal [3 H]IP production (Table 2) failed to increase caspase-3 activation in HEK-sbGHS-R1a cells (Fig. 4b). The GHS-R1a antagonist d-Lys(3)-GHRP-6 (Smith et al., 1993) has an estimated pA2 value of 5.69 (Kojima et al., 1999) and is commonly tested at 100 ␮M to achieve complete inhibition of ghrelin-stimulated responses (Carreira et al., 2006; Glavaski-Joksimovic et al., 2003). d-Lys(3)-GHRP-6 failed to have any effect on staurosporine or cadmium-induced caspase-3 activity (Fig. 4c).

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Fig. 2. Effect of staurosporine on DNA fragmentation. HEK293 and HEK-sbGHS-R1a cells were incubated with staurosporine (10 ␮M) from 0 to 24 h, then DNA fragmentation was analyzed on a 1.5% agarose gel. Data shown is representative of one of three independent experiments.

Fig. 4. Pretreatment of HEK-sbGHS-R1a cells with GHS-R1a agonist or antagonists does not affect caspase-3 activity. (a) Cells were incubated with DMEM or SPa (1 ␮M) for 30 min before DMEM or GHRP-6 (100 ␮M), then caspase-3 activity was measured after 24 h incubation with DMEM (control), 10 ␮M staurosporine (stau), or 25 ␮M cadmium. Ligands tested: DMEM (open bars), SPa (horizontal bars), GHRP-6 (vertical bars) and SPa + GHRP-6 (crossed bars). Caspase-3 specific activity is expressed relative to the control cell group within each experiment (0.028 ± 0.008 pmol/min/␮g). (b) Cells were incubated with DMEM or SPa (10 ␮M) for 30 min, then caspase-3 activity was measured after 24 h incubation with DMEM (control), 10 ␮M staurosporine (stau), or 25 ␮M cadmium. Ligands tested: DMEM (open bars), SPa (filled bars). Caspase-3 specific activity is expressed relative to the control cell group within each experiment (0.046 ± 0.002 pmol/min/␮g). (c) Cells were incubated with DMEM or dLys(3)-GHRP-6 (100 ␮M) for 30 min, then caspase-3 activity was measured after 24 h incubation with DMEM (control), 10 ␮M staurosporine (stau), or 25 ␮M cadmium. Ligands tested: DMEM (open bars), d-Lys(3)-GHRP-6 (filled bars). Caspase-3 specific activity is expressed relative to the control cell group within each experiment (0.059 ± 0.011 pmol/min/␮g). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with own control (DMEM) group. Data shown are means + SEM of 3 independent experiments, each performed in triplicate.

Fig. 3. Short-term activation of caspase-3 is not associated with loss of cell viability. Cells were transfected with control vector pcDNA3.1/Zeo (pcDNA), HA-sbGHSR1a (GHS-R1a) or HA-sbGHS-R1b (GHS-R1b) at 0.25 ␮g/ml plus control vector pcDNA3.1/Zeo (0.75 ␮g/ml). To match conditions in Fig. 1, the MTT assay was performed 8 h after incubation with control medium (open bar), 10 ␮M staurosporine (black bar), 25 ␮M cadmium (grey bar), or 0.1% Triton X-100 as the positive control (stripped bar). * P < 0.001 compared with control group. Absorbance (570 nm) values for control cells: HEK293 1.11 ± 0.15 and HEK-sbGHS-R1a 1.18 ± 0.10. Data shown are means + SEM of 3 independent experiments, each performed in duplicate. Data is expressed relative to the control group of pcDNA3.1/Zeo-transfected cells.

3.3. GHS-R1a ligands did not affect proliferation of HEK-sbGHS-R1a cells GHRP-6 maximally stimulated phospholipase C activity in HEKsbGHS-R1a at concentrations approximately 10 ␮M (see Fig. 6). Therefore, we tested the ability of GHRP-6 at 1–100 ␮M to increase proliferation of HEK-sbGHS-R1a cells in the presence of 10% fetal bovine serum to match the caspase-3 studies. Because any proliferative effect of GHRP-6 might by attenuated by the co-presence

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of growth factors in the serum, and because the effect of GHSR ligands might be attenuated by serum, we additionally conducted these experiments in the absence of serum and in comparison to wild-type HEK293 cells. Data in Fig. 5a indicates a failure of GHRP6 to affect proliferation of HEK-sbGHS-R1a cells in the presence or absence of serum. As predicted, proliferation of wild-type HEK293 cells was not affected by GHRP-6. When the effect of the inverse agonist SPa was investigated, a new batch of HEK-sbGHS-R1a cells was tested and it is noticeable that cells at greater passage number have a higher degree of proliferation (compare Fig. 5a and b). SPa (1 ␮M) had no effect on cell proliferation when assayed after 3 days in any of the cell groups studied. The GHS-R1a antagonist d-Lys(3)-GHRP-6 also had no significant effect on cell proliferation (Fig. 5c). None of the drug treatments had any significant effect on the viability of the three cell groups. The percentage of viable cells in the control groups were 99 ± 1% in HEK-sbGHS-R1a cells (plus serum), 98 ± 1% in HEK-sbGHS-R1a cells (serum-free), and 99 ± 1% in HEK293 cells (serum-free) for experiments in Fig. 5a. Similar viability values were obtained in experiments shown in Fig. 5b and c. 3.4. Constitutive activation of phospholipase C by sbGHS-R1a As expected, GHRP-6 did not increase [3 H]IP production in HEK293 cells transfected with control vector or HA-sbGHS-R1b (Fig. 6a and c). In contrast, both constitutive (basal) and agonist (GHRP-6)-stimulated phospholipase C activation was readily detected in cells expressing sbGHS-R1a (Fig. 6b, d, e and f), with an EC50 of 0.86 ± 0.53 ␮M, and could be inhibited to a similar extent by SPa (Table 2). The ghrelin receptor inverse agonist, SPa (1 ␮M), inhibited constitutive [3 H]IP production by approximately 27% and produced a approximate 3-fold increase in EC50 values for GHRP-6. SPa (10 ␮M) produced a more dramatic 73–75% inhibition of basal [3 H]IP production; P < 0.001 (Table 2). Overall, there was no direct correlation between sbGHS-R1a mRNA expression (Table 1) and basal [3 H]IP production (Table 2). Indeed, phospholipase C activity appeared to be higher in cells transiently transfected with HA-sbGHS-R1a compared with cells stably expressing sbGHS-R1a. When transiently co-expressed, GHS-R1b acts as a dominant-negative for the expression of GHS-R1a and decreases constitutive activation of phospholipase C (Leung et al., 2007), but sbGHS-R1b did not appear to inhibit stably expressed sbGHS-R1a in this current study. Fig. 5. The lack of effect of GHS-R1a ligands on proliferation of HEK-sbGHS-R1a and HEK293 cells. Cells were incubated in the presence or absence of serum for 3 days, then harvested and counted. (a) Effect of 0–100 ␮M GHRP-6. (b) Effect of 1 ␮M SPa (horizontal bars) or DMEM (open bars). (c) Effect of 100 ␮M d-Lys(3)GHRP-6 (horizontal bars) or DMEM (open bars). Data shown are means + SEM of 3 independent experiments, each performed in duplicate.

4. Discussion As wild-type HEK293 cells do not express human GHS-R1a receptors (unpublished observations) or human GHS-R1b (Leung et al., 2007), they provide an ideal tool to study the relative roles of the ghrelin receptor and its mutant receptor polypeptide GHSR1b. The truncated ghrelin receptor polypeptide behaves as a dominant-negative mutant of the ghrelin receptor when transiently

Table 2 Effect of GHS-R1a inverse agonist on constitutive phospholipase C activity in HEK293 cells expressing sbGHS-R1a. Cell type

Transfection material

Basal PLC activity ([IP/(IP + INOS)] × 100)

Effect of SPa on basal PLC activity %Inhibition at 1 ␮M

HEK293 HEK-sbGHS-R1a HEK-sbGHS-R1a HEK-sbGHS-R1a

HA-sbGHS-R1a pcDNA3.1/Zeo HA-sbGHS-R1a HA-sbGHS-R1b

62 24 66 37

± ± ± ±

7 12 11 13

14 25 23 45

± ± ± ±

5 15 7 6**

%Inhibition at 10 ␮M 73 73 74 75

± ± ± ±

1*** 4*** 4*** 2***

Cells were transfected with control vector pcDNA3.1/Zeo, HA-sbGHS-R1a or HA-sbGHS-R1b at 0.25 ␮g/ml plus control vector pcDNA3.1/Zeo (0.75 ␮g/ml). Data shown are means ± SEM of 3 independent experiments, each performed in duplicate. ** P < 0.01 compared with basal PLC activity. *** P < 0.001 compared with basal PLC activity.

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Fig. 6. GHRP-6-stimulated phospholipase C activity in HEK293 and HEK-sbGHS-R1a cells transiently expressing HA-sbGHS-R1a or HA-sbGHS-R1b. Cells were transfected with control vector pcDNA3.1/Zeo (pcDNA), HA-sbGHS-R1a or HA-sbGHS-R1b at 0.25 ␮g/ml plus control vector pcDNA3.1/Zeo (0.75 ␮g/ml). (a and c) Phospholipase C activity was determined in the presence (filled bars) and absence (open bars) of 100 ␮M GHRP-6. (b, d, e and f) Phospholipase C activity was determined in response to GHRP-6 () in the presence of 1 ␮M SPa (䊉). Data shown are means and SEM of 3 independent experiments, each performed in duplicate.

transfected into HEK293 cells (Leung et al., 2007). Both ghrelinstimulated [3 H]IP production and ERK1/2 activity were unaffected by GHS-R1b, whereas the constitutive activation of phospholipase C, but not of ERK1/2, was significantly attenuated (Chu et al., 2007). Taken together, these results suggested that any upregulation of GHS-R1b might preferentially attenuate functional activity dependent on the constitutive activation of ghrelin receptors, while leaving ghrelin-dependent signaling unaffected. Given the growing evidence supporting a role for the ghrelin/GHS-R1a axis in cancer (Arnaldi et al., 2003; Duxbury et al., 2003; Gaytan et al., 2004; Jeffery et al., 2002; Korbonits et al., 2001; Volante et al., 2002), and in particular the possible autocrine nature of this pathway (Jeffery et al., 2003), we have attempted to assess the contribution of constitutive activation and agonist-stimulated activation of ghrelin receptors stably expressed in HEK293 cells, and to assess the effect of GHS-R1b on these responses. The results show that eight hours incubation with staurosporine or cadmium increases caspase-3 activity in HEK293 cells without loss of cell viability. The short incubation time and protective effect of serum (López et al., 2003) may have acted to limit the signs of decreased cell viability at this time point. Importantly, we found that cadmium-stimulated caspase-3 activity in HEK-sbGHSR1a cells was attenuated compared to its effect in wild-type HEK293 cells. These results suggest that stable expression of sbGHS-R1a in HEK293 cells provides anti-apoptotic properties to these cells and makes them more able to resist apoptotic injury. However, this advantage is lost when the apoptotic stimulus is dependent on the inhibition of protein kinase C. Despite similar levels of sbGHS-R1a mRNA expression in transiently transfected HEK293 cells and HEK-sbGHS-R1a cells, only cells stably expressing sbGHS-R1a possessed this anti-apoptotic property against cadmium. The addition of the HA-tag did not affect the ability of sbGHS-R1a to increase [3 H]IP production (data not shown) and would therefore not be expected to account for the differences observed here. The demonstration that stably expressed sbGHS-R1a attenuates cadmium-induced apoptosis but not staurosporine-induced apoptosis complies with our knowledge of GHS-R1a as primarily coupling to Gq/11-proteins (Chu et al., 2007; Howard et al., 1996) with subsequent activation of protein kinase C (Camina et al., 2007). We and others have shown that GHS-R1a activates ERK1/2 in a protein kinase C-dependent manner (Camina

et al., 2007; Chu et al., 2007; Mousseaux et al., 2006), and ghrelinmediated inhibition of apoptosis in 3T3-L1 preadipocytes and rat hypothalamic neurons is attenuated by inhibition of protein kinase C (Chung et al., 2007; Kim et al., 2004). Therefore the anti-apoptotic properties of stably expressed sbGHS-R1a cannot be demonstrated against staurosporine because staurosporine blocks protein kinase C and therefore blocks sbGHS-R1a-dependent cell signaling. Akt (also known as protein kinase B) is well recognized as an anti-apoptotic factor (New et al., 2007) and would be predicted to be activated in GHS-R1a-expressing cells. However, our previous studies of HEK293 cells transiently expressing GHS-R1a failed to show evidence of elevated levels of phosphorylated Akt (Chu et al., 2007). This lack of signaling of GHS-R1a through Akt may reflect the distinct differences we have observed herein between cells stably and transiently expressing sbGHS-R1a. In transiently transfected cells, ghrelin stimulated ERK1/2 phosphorylation was G-protein-dependent and ␤-arrestin-independent and involved a novel protein kinase C isoform which could utilize diacylglycerol derived from phosphatidylcholine (Chu et al., 2007). In contrast, Camina et al. (2007) have identified three distinct cell signaling options for GHS-R1a when stably expressed in HEK293 cells. The anti-apoptotic actions of pERK1/2 have been equated with a cytoplasmic (␤-arrestin-dependent) rather than nuclear localization of pERK1/2 (Ajenjo et al., 2004), suggesting again that stably transfected sbGHS-R1a may have alternative signaling options when compared to transiently expressed sbGHS-R1a. Thus, our studies in HEK-sbGHS-R1a cells further demonstrate that agonist-independent activity of sbGHS-R1a also provides anti-apoptotic properties to HEK293 cells. Although we have not demonstrated a lack of ghrelin production by HEK293 cells, any such endogenous human ghrelin receptor agonist is unlikely to stimulate sbGHS-R1a (Chan et al., 2004) and we can therefore conclude that HEK293 cells can be protected from apoptosis by the constitutive activity of stably expressed sbGHS-R1a. Although we have shown previously that GHS-R1b can decrease constitutive activation of phospholipase C when co-transfected with GHS-R1a into HEK293 cells (Leung et al., 2007), HA-sbGHS-R1b did not decrease [3 H]IP production when transiently transfected into HEK293 cells stably expressing sbGHS-R1a in the current study and did not affect apoptotic responses in sbGHS-R1a-expressing cells. Therefore, further studies are required to better understand the relationship between

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transiently expressed and stably expressed GHS-R1a and GHS-R1b in HEK293 cells. Despite the ability of SPa (10 ␮M) to decrease constitutive [3 H]IP production by 74%, this effect was insufficient to allow one to observe any change in caspase-3 activity in HEK-sbGHS-R1a cells in the presence of this inverse agonist. Similarly, the higher levels of constitutive [3 H]IP production in HEK-sbGHS-R1a transiently expressing HA-sbGHS-R1a did not confer additional anti-apoptotic activity. These anomalies may be explained by our previous observations suggesting that ghrelin receptor-dependent generation of inositol phosphates may be independent of activation of ERK1/2, with the latter dependent on phosphatidylcholine-dependent phospholipase C and activation of novel isoforms of protein kinase C (Chu et al., 2007). Therefore, the constitutive anti-apoptotic activity seen in HEK-sbGHS-R1a cells may be mediated via a phosphatidylcholine-dependent phospholipase C/protein kinase C pathway rather than a phosphatidylinositol-dependent phospholipase C/protein kinase C pathway. Ghrelin produced a bell-shaped concentration-response curve for proliferation in PC3 (prostate carcinoma) cells, peaking at 5 nM (Jeffery et al., 2002). However, neither GHRP-6, SPa nor d-Lys(3)GHRP-6 influenced proliferation of HEK-sbGHS-R1a cells, even at concentrations of GHRP-6 shown to maximally activate [3 H]IP production. Although increased cell proliferation and decreased apoptosis can both contribute to an increase in cell survival, these two properties do not necessarily go hand in hand. Thus, ghrelin can stimulate proliferation of rat adrenal zona glomerulosa cells without any significant effect on the apoptotic deletion rate (Andreis et al., 2003). In the present study we observed the converse situation with GHS-R1a ligands failing to influence proliferation of HEK-sbGHS-R1a cells while the constitutive activity of sbGHS-R1a seemed sufficient to provide some degree of anti-apoptotic activity. The potency of GHRP-6 and SPa were lower than expected from studies of human GHS-R1a (Holst et al., 2005). Although we have cloned the gene for seabream ghrelin (Yeung et al., 2006), we have yet to identify the chemical nature of the physiological ligand and perform comparative studies with human ghrelin/GHS-R1a. Nevertheless, there is a high degree of conservancy of GHS-R1a among species (Howard et al., 1996) which might suggest functional similarities between seabream and human GHS-R1a. In conclusion, sbGHS-R1a appears to display constitutive antiapoptotic activity when stably expressed in HEK293 cells, and the lack of relationship between caspase-3 activation, cell proliferation, and [3 H]IP generation suggests a possible role for phosphatidylcholine-specific phospholipase C/protein C pathway in regulating cell survival. In addition, we found no evidence for an involvement of agonist-dependent or agonist-independent signaling of sbGHS-R1a in regulating proliferation of HEK-sbGHS-R1a cells. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement Funding: This work was fully supported by a grant from The Chinese University of Hong Kong (CUHK 2005.1.092). References Ajenjo, N., Canon, E., Sanchez-Perez, I., Matallanas, D., Leon, J., Perona, R., Crespo, P., 2004. Subcellular localization determines the protective effects of activated ERK2 against distinct apoptogenic stimuli in myeloid leukemia cells. J. Biol. Chem. 279, 32813–32823. Andreis, P.G., Malendowicz, L.K., Trejter, M., Neri, G., Spinazzi, R., Rossi, G.P., Nussdorfer, G.G., 2003. Ghrelin and growth hormone secretagogue receptor are

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