The Endogenous Growth Hormone Secretagogue (Ghrelin) Is Synthesized and Secreted by Chondrocytes
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内分泌学杂志 2005年第3期
Departments of Physiology (J.E.C., C.D.), Morphological Sciences (T.G.-C., M.B., R.G.), and Medicine, Molecular Endocrinology Section (F.F.C.), University of Santiago de Compostela; and Complexo Hospitalario Universitario de Santiago (O.G., M.O., J.J.G.-R.), Research Area, Laboratory 4, NEIRID Lab (NeuroEndocrine Interactions in Rheumatology and Inflammatory Diseases), and Complexo Hospitalario Universitario de Santiago (F.L.), Research Area, Laboratory 1, Molecular and Cellular Cardiology Unit, Santiago de Compostela 15706, Spain; and Beth Israel Deaconess Medical Center (M.B.G.), Division of Rheumatology, New England Baptist Bone and Joint Institute, Harvard Institutes of Medicine, Boston, Massachusetts 02115-5713
Address all correspondence and requests for reprints to: Oreste Gualillo, Ph.D., Santiago University Clinical Hospital, Research Area, Research Laboratory 4, Trav. Choupana, sn, 15706 Santiago de Compostela, Spain. E-mail: gualillo@usc.es.
Abstract
Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), is a recently isolated hormone, prevalently expressed in stomach but also in other tissues such as hypothalamus and placenta. This novel acylated peptide acts at a central level to stimulate GH secretion and, notably, to regulate food intake. However, the existence of further, as yet unknown, effects or presence of ghrelin in peripheral tissues cannot be ruled out. In this report, we provide clear evidence for the expression of ghrelin peptide and mRNA in human, mouse, and rat chondrocytes. Immunoreactive ghrelin was identified by immunohistochemistry in rat cartilage, being localized prevalently in proliferative and maturative zone of the epiphyseal growth plate, and in mouse and human chondrocytic cell lines. Moreover, ghrelin mRNA was detected by RT-PCR and confirmed by Southern analysis in rat cartilage as well as in mouse and human chondrocytes cell lines. Ghrelin mRNA expression has been studied in rat along early life development showing a stable profile of expression throughout. Although ghrelin expression in chondrocytes suggests the presence of an unexpected autocrine/paracrine pathway, we failed to identify the functional GH secretagogue receptor type 1A by RT-PCR. On the other hand, binding analysis with 125I ghrelin suggests the presence of specific receptors different from the 1A isotype. Scatchard analysis revealed the presence of two receptors with respectively high and low affinity. Finally, ghrelin, in vitro, was able to significantly stimulate cAMP production and inhibits chondrocytes metabolic activity both in human and murine chondrocytes. In addition, ghrelin is able to actively decrease both spontaneous or insulin-induced long chain fatty acid uptake in human and mouse chondrocytes. This study is the first to provide evidence for the presence of this novel peptide in chondrocytes and suggests novel potential roles for this newly recognized component of the GH axis in cartilage metabolism.
Introduction
THE GROWTH OF the long bones is driven by the cellular activity of chondrocytes within the growth plate. Small germinal chondrocytes in the resting zone enter the cell cycle and undergo successive mitotic divisions to form columns of chondrocytes in the proliferative zone. The proliferative and hypertrophic layers of the growth plate distinguish anatomically these phases of growth. Until recently, the major regulation of the growth plate was thought to be due to systemic hormones, mainly those related to the GH-IGF-I axis (1, 2). However, it is likely that locally produced peptide factors play an important autocrine and/or paracrine role in ensuring that skeletal development and growth progress correctly. Investigations in vitro and in vivo have demonstrated the importance of the peptide growth factors IGF-I, TGF-?, and basic fibroblast growth factor in modulating cell proliferation, extracellular matrix biosynthesis, and changes in morphology associated with the progressive stages of chondrocyte differentiation (3). Recently, ghrelin, a new member of the GH axis, has been identified. This new hormone is a gastric orexigenic peptide and a ligand for the GHS-R [GH secretagogue (GHS) receptor] that potently stimulates GH release both in vivo and in vitro (4, 5, 6). Several previous studies, performed with synthetic analogs of ghrelin, suggest specific actions on bone tissues. Namely, hexarelin treatment inhibits expression of the markers of bone resorption in rats (7), and ipamorelin induced longitudinal bone growth in rats (8). In humans, several studies show that nonpeptidyl GHSs are capable of actions on bone tissue. For example, MK-677 treatment induces potent increases in biochemical markers of bone formation and bone resorption (9, 10).
The aim of this work was to verify whether ghrelin and its cognate receptor, GHS-R, were expressed in chondrocytes. In the current study, ghrelin and its mRNA expression was assessed in human, rat, and mouse chondrocytes by means of immunohistochemistry, RT-PCR, and Southern analysis. In addition, binding studies were performed to assess the expression of specific ghrelin receptors at the plasma membrane. Finally, we evaluated whether ghrelin was able to exert some biological effect by testing cAMP modulation, cell metabolic activity, and long chain fatty acid uptake on a human immortalized chondrocyte cell line upon ghrelin stimulation.
Materials and Methods
Cell culture
The ATDC-5 murine chondrogenic cell line was a kind gift from Dr. Agamennon E. Grigoriadis (Department of Orthodontics and Pediatric Dentistry & Craniofacial Development, King’s College London, Guy’s Hospital, London, UK). Cells were cultured in monolayer as described previously (11) in a standard medium of DMEM Ham’s F12 supplemented with 5% fetal calf serum (FCS), 10 μg/ml human insulin, 10 μg/ml human transferrin, and 3 x 10–8 M sodium selenite and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin. For longer term culture, up to 21 d, the media were changed to -MEM with the same additives to induce hypertrophic differentiation. The immortalized human juvenile costal chondrocytes cell lines T/C-28a2, C-20/A4, and C-28/I2 were cultured in DMEM/ F12 as described previously (12) supplemented with 10% FCS, L-glutamine, and antibiotics. Cells were grown in monolayer and passaged every 5–6 d unless otherwise specified.
Immunohistochemistry
Specimens of rat epiphyseal growth plate, mouse and human tissues, and cell cultures were immersion-fixed in 4% buffered formaldehyde for 24 h, dehydrated, and embedded in paraffin by standard procedure. Cartilage tissues were decalcified in EDTA acid buffer for 2 h (MicroStain, Bologna, Italy). Sections 5-μm thick were mounted on Histobond adhesion microslides (Mariefeld, Lauda-Konigshofen, Germany) and deparaffined before analysis by immunohistochemistry. Antigen retrieval was carried out for antighrelin using a microwave oven for three cycles of 5 min each at 750 W in 0.001 M sodium citrate buffer (pH 6.0). The polyclonal IGF-I antibody was used without unmasking procedure. Samples were immunostained with a rabbit polyclonal ghrelin antibody (a generous gift of Dr. M. Kojima), anti-IGF-I. Sections were consecutively incubated in: 1) antighrelin at a dilution of 1:500, anti-IGF-I at a dilution of 1:250 (1 h at room temperature); 2) 3% hydrogen peroxide (Merck, Darmstadt, Germany) to block endogenous peroxidase (10 min); 3) goat antirabbit Igs conjugated to peroxidase-labeled dextran polymer (Dako EnVision peroxidase rabbit; Dako, Carpinteria, CA); 4) 3,3'-diamino-benzidine-tetrahydrochloride (DAB) solution prepared by dissolving one DAB-buffer tablet (Merck) in 10 ml of distilled water (10 min). Between steps, sections were washed twice for 5 min with TBS [0.05 M Tris buffer (pH 7.6), containing 0.3 M NaCl] and after step 4 with distilled water. All dilutions were made in TBS except the dilution of the primary antisera (step 1) where the TBS was substituted by an antibody diluent (Dakoppats). TC-28/A2 (human cell line) and ATDC5 (mouse cell line) chondrocyte cells were fixed by the following procedure: 15 min in 10% buffered formalin, 5 min in PBS [0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCl], 4 min in –20 C methanol, 2 min in –20 C acetone and 5 min twice in PBS. Epitope retrieval was done by microwaving at 750 W for 10 min in 0.01 M sodium citrate buffer. In this case, we used antighrelin at a dilution of 1:50. Negative controls were performed by either : 1) preadsorbing the antighrelin antibody with the homologous antigen (ghrelin 10 nM) for 24 h at 4 C; or 2) omitting any essential step of the reaction. Sections were consecutively incubated as described previously (13).
Tissue explant and RNA preparation.
Rat tissues were obtained from Sprague Dawley rats at different ages. Cartilage was carefully dissected from fetal tibia epiphyseal growth plate cartilage and from 1-, 15-, and 21-d-old Sprague Dawley rats. Animals were killed under anesthesia, according to protocols approved by the Animal Care Committee of the University of Santiago de Compostela. The tibia growth plate and adjacent articular cartilage from rats were dissected shortly after death as described previously (14), minimizing all possible contaminations from other tissues. RNA was isolated from frozen or freshly explanted tissues by Trizol method as described previously (15). Briefly, tissues were homogenized using a Polytron homogenizer in 1000 μl of Trizol LS reagent (Invitrogen Life Technologies, Carlsbad, CA) and, after recovery by isopropanol precipitation, the total RNA was measured with a spectrophotometer (Beckman Inc., Fullerton, CA) at 260 nm.
RT-PCR and Southern analysis.
Two micrograms of total RNA were used to perform RT-PCR. cDNAs were synthesized using 200 U of Moloney murine leukemia reverse transcriptase (Invitrogen Life Technologies) and 6 ml of deoxynucleotide triphosphates (dNTPs) mix (10 mM of each dNTP), 6 μl of first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 (Invitrogen Life Technologies)] 1.5 μl of 50 mM MgCl2, 0.17 μl of random hexamers solution [3 μg/ml (Invitrogen Life Technologies)], 0.25 μl of RNase Out [recombinant ribonuclease inhibitor 40 U/ml (Invitrogen Life Technologies)], in a total volume of 30 μl. Reaction mixtures were incubated at 37 C for 50 min and at 42 C for 15 min. The reverse transcription (RT) reaction was terminated by heating at 95 C for 5 min and subsequently quick-chilled on ice. Three microliters of RT reaction were used for PCR amplification. The amplification conditions were as follows: 5 μl of PCR buffer [200 mM Tris-HCl pH 8.4 and 500 mM KCl (Invitrogen Life Technologies)], 1.5 μl of 50 mM MgCl2, 4 μl of dNTPs mix, 150 ng of specific upstream and downstream primers (13, 15) [sequences of specific primers has been reported in Table 1], and 1.25 U of Taq DNA Polymerase (Invitrogen Life Technologies)]. The amplification profile for rat, human and mouse ghrelin was: denaturation at 98 C for 15 sec, annealing at 60 C for 60 sec, and extension at 72 C for 1 min. Thirty-five-cycle amplification was completed with an additional step at 72 C for 10 min. The amplification was performed in an automatic thermal cycler (Mastercycler gradient; Eppendorf AG, Hamburg, Germany). To check the quality of mRNA in each sample, human, mouse, and rat ghrelin RNA samples were amplified together with respectively housekeeping genes human and mouse hypoxanthine guanine phosphoribosyl transferase (hHPRT, mHPRT) and rat ?-actin. Multiple positive and negative controls were performed for RT-PCR. Negative controls consisted of omitting the RT reaction for each sample or amplifying samples of RT reaction without Moloney murine leukemia virus reverse transcriptase. All negative controls resulted in no bands after amplification. The identity of the amplimer for ghrelin was confirmed by performing the RT-PCR together with positive controls (stomach RNA). PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, examined with UV light and visualized with a Typhoon 9410 Documentation System (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). To confirm authenticity of the amplimers, Southern blot analysis was carried out. Hybridization of nylon transferred resolved amplicons was performed using 32P cDNA-specific probes for ghrelin. The probes were prepared by random prime labeling of the ghrelin cDNAs subcloned in EcoRI double restriction site of a pBS-SK (2) expression vector. Ghrelin cDNAs were purified from plasmid expression vector using the Sephaglas BandPrep Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). The resolved amplicons were transferred by capillary blotting onto a charged nylon membrane and fixed with UV light. The membranes were hybridized with 32P-labeled rat or human ghrelin (1 x 106 cpm/ml) at 44 C for 18 h. After removing the excess of labeled probe by washing, membranes were exposed to autoradiography, and the sizes of the bands determined by comparison with molecular weight marker on the ethidium bromide-stained gels.
TABLE 1. Primers pairs
Real-time PCR
Real-time PCR assay was carried out with SYBR Green system using a LightCycler rapid thermal cycler system (LightCycler, Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. PCR parameters were as follows: an initial denaturation at 96 C for 20 sec followed by 40 cycles at 96 C for 2 sec; 60 C for 15 sec, and 72 C for 15 sec. Fold differences for gene expression were calculated by the comparative Ct (threshold cycle) method. This method compares test samples to a calibrator sample and uses results obtained with a uniformly expressed control gene to correct for differences in the amount of RNA present in the two samples being compared with generate a Ct value. Specificity of real time RT-PCR amplification of each primer pair was confirmed by analyzing PCR products by agarose gel electrophoresis and by melting curve analysis.
Ghrelin RIA
Ghrelin levels in ATDC5 murine chondrocytes, T/C-28a2, C20/A4 and C-28/I2 human immortalized chondrocytes culture media were determined using specific RIA kits from Phoenix Pharmaceuticals (Belmont, CA), according materials and protocol provided by the supplier. Ghrelin determination was performed in serum-free conditions after a starvation of 24 h.
Binding analysis
Hormone binding to the T/C-28a2 cells was performed in six-well plates as previously described (16). Cells were seeded at a density of 500,000 cell/well and with a viability, evaluated by trypan blue exclusion method, of greater than 90%. Cells were grown until a confluence of 70–80% and before binding were carefully washed with DMEM/NutF12 and incubated in this serum-free medium overnight. Subsequently, plates were put on ice and washed with ice cold PBS. In a final volume of one ml of binding buffer (PBS with 1% wt/vol of BSA, fraction V, Sigma Chemical Co., St. Louis, MO) whole cells were incubated with 90000 cpm of 125I ghrelin (Amersham Pharmacia Biotech Ltd.) with a specific activity of 2000 Ci/mmol. Incubation was carried out at 4 C to avoid internalization of the receptor and/or ligand and overestimation of the binding capacity. At the end of the incubation, unbound label was removed by two PBS washes; 1 ml of 1 N NaOH was added, and radioactivity in lysates was measured using a counter (1261; LKB-Wallac, Gaithersburg, MD). Specific binding was determined by subtracting the amount of 125I ghrelin in the presence of excess of unlabeled ghrelin (3 μM). For Scatchard analysis, increasing amounts of unlabeled recombinant human ghrelin (0.03–30 nM) were added to compete with 125I human ghrelin for binding in saturating conditions. Ligand software (Elsevier-Biosoft, Cambridge, UK) was used to calculate the dissociation constant (Kd) and binding capacity (17).
cAMP determination
Intracellular cAMP was determined by direct scintillation proximity assay of acid extracts from cells grown in 24-well cluster plates and treated with different doses of ghrelin (1, 10, 50, and 100 nM), according materials and protocols provided by the supplier (RPA 538; Amersham Biosciences).
Cell metabolic activity using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay.
Cell metabolic activity was examined using a colorimetric assay based on the MTT labeling reagent (Roche Molecular Biochemicals, Barcelona, Spain). Assays were performed according instructions and protocol provided by the manufacturer. Briefly, the TC/28 a2, C20/A4 and C28/I2 were seeded in 96-well plates (8000 cell/well, in 0.2 ml of complete medium). After complete adhesion, cells were incubated with serum-free medium and stimulated with human recombinant ghrelin (0.1, 1.0, 10, and 100 nM). After 48 h, the MTT labeling reagent solution (final concentration 0.5 mg/ml) was added to each well, and incubations continued for 4 h at 37 C. After this incubation, 100 μl of solubilization solution (SDS 10% in HCl 0.01 M) were added to the wells until complete solubilization of the purple formazan crystals. Spectrophotometrical absorbance was measured using a microtiter ELISA reader at 550 nm (Multiskan EX, Labsystem, Helsinki, Finland). Data are reported as mean ± SEM values of at least three independent determinations. All experiments were done at least four times, each time with three or more independent observations. Statistical analysis was performed by ANOVA, and multiple comparisons were made by Student-Newman-Keul’s test with Instat computerized package.
Fatty acid uptake (BODIPY 3823) assay
4,4-Difluoro-5-methyl-4-bora-3a-diaza-3-indacene-3-dodecanoic acid (BODIPY 3823, Molecular Probes, Inc., Eugene, OR)-labeled fatty acid uptake was used for measuring long-chain fatty acid uptake in human immortalized chondrocytes T/C-28a2 and in murine ATDC5 chondrocytes. Cells were treated with ghrelin 100 nM for 2 h, washed with DMEM, and incubated with PBS containing 20 μM BSA (fatty acid free) and 10 μM BODIPY 3823 (Molecular Probes) for 2 min at 37 C. Cells were then washed extensively at 4 C with PBS containing 0.1% BSA to remove surface-associated BODIPY. Cells were suspended in DMEM buffered with HEPES to pH 7.5 for cytometric analysis. Cell emission of BODIPY was measured by flow cytometry with a FACS Calibur Becton Dickinson (BD; San Jose, CA) flow cytometer and results were analyzed using the software CellQuest (BD).
Results
Immunohistochemistry
As shown in Fig. 1A, immunoreactive ghrelin was localized in rat epiphyseal growth plate, where it was expressed in the majority of the chondrocytes in the proliferation and maturation zones and only occasionally in the hypertrophic and provisional calcification area. A similar pattern was observed for IGF-I immunostaining (Fig. 1B). Ghrelin was found in the cytoplasm, whereas the nuclei were negative. IGF-I-positive staining was more intense around the nuclei. No immunoreactivity was shown when the primary antibody was preadsorbed with the homologous antigen or when the primary antibody or other incubation steps were substituted by TBS (Fig. 1C).
FIG. 1. A, Ghrelin immunoreactivity in rat epiphyseal growth plate. Ghrelin was expressed in the majority of the chondrocytes in the proliferation and maturation zones and only occasionally in the hypertrophic and provisional calcification area. A similar pattern was observed for IGF-I. B, Immunostaining for IGF-I. Ghrelin immunopositivity was found in ATDC5 mouse cell line (D) and in human immortalized chondrocytes (E). C and F, Negative controls obtained by preadsorbing primary antibody with the homologous antigen.
ATDC-5 chondrogenic mouse cell line (Fig. 1D) and the human chondrocyte cell line TC-28A2 (Fig. 1E) showed intense ghrelin-positive staining. Immunoreactivity was localized in the cytoplasm of the cells. No immunopositivity was observed when the primary antibody was preadsorbed with the homologous antigen or when any essential incubation step was substituted by TBS (Fig. 1F).
RT-PCR and Southern analysis
Ghrelin mRNA and GHS-R expression.
As shown in Fig. 2A, ghrelin mRNA was expressed in the human chondrocyte cell lines (C20/A4 and C28/I2). Identity of amplimers was confirmed by Southern hybridization (see lower panels of Fig. 2, A–C). Moreover, ghrelin mRNA expression was also identified in the mouse chondrogenic cell line ATDC-5 (Fig. 2B) as well as in tibial growth plate cartilage of male rats of different ages (Fig. 2C).
FIG. 2. Ghrelin mRNA expression in human immortalized chondrocyte cell lines C20/a4 and C28/I2 (A), mouse chondrogenic ATDC5 cell lines (B), and rat growth plate cartilage of different age rats (C). Identity of amplimers was confirmed by Southern analysis.
We studied also expression of GHS-R type 1A in human immortalized chondrocytes and in rat growth plate of rats at different ages. We failed in identifying mRNA for GHS-R 1A in either tissue, as shown in Fig. 3, despite its presence in the positive controls, namely human and rat pituitary mRNA.
FIG. 3. RT-PCR for GHS-R 1-a in human immortalized chondrocytes (A) TC28A2, C20/a4 and C28I2. The housekeeping gene used for checking RT-PCR was human HPRT. Expression of GHS-R 1A in rat growth plate of rats of different ages (B). Qualitative and quantitative controls for rat samples were performed using ?-actin amplification (C).
Secretion of ghrelin by cultured chondrocytes
The RIA-measured concentration of ghrelin following 24-h starvation in cell culture medium was 4.19 ± 0.24 pg/ml in ATDC5 murine chondrocyte (n = 4) cell line. In human immortalized chondrocytes T/C-28a2, C20/A4, and C-28/I2 were, respectively, 10.85 ± 0.39 pg/ml (n = 4), 11.717 ± 0.26 pg/ml (n = 4), and 11.203 ± 0.75 pg/ml (n = 4).
Ghrelin binding analysis
Because GHS-1A receptors were not expressed in chondrocytes, we have further evaluated whether ghrelin could bind specifically to the cell surface of human immortalized chondrocytes. The analysis of binding of [125I] ghrelin to whole cells is presented in Fig. 4. Scatchard analysis showed that the Kd for high affinity receptor was: 3.83 x 10–9 M with a maximum binding capacity (Bmax) of 7.82 x 10–10 M. Moreover, these data suggest the presence of a low-affinity isotype receptor for ghrelin with high levels of expression, different from the isotype GHS-R 1A, with a Kd = 1.32 x 10–8 and a Bmax = 2.85 x 10–9.
FIG. 4. Scatchard analysis of binding data from the competition studies between [125I]ghrelin and unlabeled ghrelin using T/C-28a2 human immortalized chondrocytes.
cAMP determination
Because ghrelin has been demonstrated to exert its biologic effects in various cells through the stimulation of cAMP-mediated biochemical pathways, the ability of ghrelin to induce a biological response was investigated as a function of cAMP concentration in articular immortalized chondrocytes. As shown in Fig. 5, a 15-min exposure of chondrocytes to ghrelin (1–100 nM) elevated intracellular cAMP concentration in a dose-dependent fashion. As positive control, human and murine chondrocytes were treated with forskolin, a direct activator of adenylate cyclase.
FIG. 5. Dose-dependent accumulation of cAMP in response to different doses of ghrelin and forskolin (F), treatment for 15 min in T/C-28a2 human immortalized chondrocytes (gray bars), and ATDC5 murine chondrocytes (black bars). F was used as a direct activator of adenylate cyclase.
MTT metabolic assay
To evaluate whether the immortalized human chondrocytes could demonstrate a physiologically relevant response to ghrelin, cell metabolic activity was analyzed using the MTT assay. As shown in Fig. 6, human recombinant ghrelin was able to significantly inhibit cell metabolic activity in a dose-dependent manner in all three human immortalized juvenile costal chondrocyte cell lines. These cell lines showed a significant response to FCS, whereas ghrelin stimulation caused a significant decrease (40%) in cell metabolic activity when the cells were incubated with human recombinant ghrelin in serum-free conditions. To note, cell cycle analysis performed by flow cytometry with propidium iodide staining upon ghrelin stimulation did not show any alteration in the percentage of hypodiploid cells (apoptotic), G0/G1 and S/M cell cycle phases in comparison to untreated control cells, excluding in such a way a potential cytotoxic effect of ghrelin (data not shown). In keeping, preliminary data obtained by cDNA microarray analysis (Genechip Human Genome U133A; Affymetrix, Santa Clara, CA) of human immortalized chondrocytes indicated that ghrelin was able to significantly decrease gene expression of relevant genes involved in the cellular RedOx system such as reduced nicotinamide adenine dinucleotide phosphate oxidase 1 and cytochrome p450p9, without affecting genes related to cell-cycle progression. In addition, real-time PCR confirmed array analysis and showed that upon ghrelin treatment (100 nM) cytochrome P450p9 decreased its expression by 21% vs. control, whereas reduced nicotinamide adenine dinucleotide phosphate oxidase 1 is almost undetectable after ghrelin treatment.
FIG. 6. Human recombinant ghrelin inhibits basal metabolic activity, evaluated by the MTT colorimetric method, in human immortalized chondrocytes in a dose-dependent manner. *, P < 0.05; **, P < 0.01. As positive control, metabolic activity was evaluated in presence of 10% (vol/vol) fetal bovine serum.
Fatty acid uptake (BODIPY 3823) assay
To determine whether ghrelin was able to modulate fatty acid uptake in chondrocytes, we used flow cytometry to examine the effect of ghrelin on fatty acid transport in chondrocytes. We measured the uptake of BODIPY 3823, a fluorescent-saturated LCFA analog at the immortalized human chondrocytes and murine chondrocytes. Cells were incubated with 100 nM ghrelin for 2 h; then BODIPY 3823 was added for another 2 min and washed with PBS buffer twice. The flow cytometry was used to examine fatty acid uptake. The results (Fig. 7) showed that ghrelin significantly decrease BODIPY uptake (23.6% ± 1.76 in human immortalized chondrocyte and 16.34% ± 0.87 in murine chondrogenic cell line). In addition, ghrelin was able to completely revert the insulin-induced BODIPY 3823 uptake in human chondrocytes, whereas the effect, although significant, was partial in murine chondrocytes.
FIG. 7. Flow cytometry analysis of long chain fatty acid uptake in human and murine chondrocytes. Bars represent percentage of BODIPY 3823 basal uptake upon 2 h treatment with ghrelin (100 nM), insulin (10 nM), or a combination of the two hormones.
Discussion
Cartilage, a connective tissue of diverse embryonic origins, serves multiple prenatal and postnatal functions. This tissue provides structural support for the early embryo, forms templates for developing endochondral bones, provides for postnatal growth of the skeleton, and participates in the repair of fractured bones during endochondral ossification.
Chondrocytes represent a target tissue for a number of molecules that are integrated into the developmental program of the skeleton. Evidence is growing that several endocrine factors, locally produced in cartilage, may also act by a paracrine and/or autocrine mechanism. Thus, in support of this hypothesis are reports confirming expression of IGF-I (18), TGF-? (19), basic fibroblast growth factors (20), and other locally produced factors involved in chondrocyte metabolism. It is clear that the local microenvironment influences key cartilage processes such as the cell cycle, extracellular matrix biosynthesis, and changes in morphology associated with the progressive stages of chondrocyte differentiation.
In the present work, and to the best of our knowledge, for the first time, the expression of ghrelin, the endogenous GHS, has been identified unambiguously in human, mouse, and rat chondrocytes. Ghrelin is a novel GH-releasing peptide, isolated from the stomach, that is the endogenous ligand for GHS-R. In addition to its action as GHS, recent studies have shown that ghrelin causes a positive energy balance by stimulating food intake and decreasing fat use through a GH-independent mechanism (21, 22). We identified ghrelin in growth plate cartilage of embryonic and postnatal rat and in cultured chondrocytes derived from mouse, rat, and human cartilage by several immunological and molecular approaches. In keeping with these data, we have further explored the possibility that ghrelin was secreted by chondrocytes. As expected, mouse ATDC5 cells and human chondrocytes cell lines cultured in vitro produced immunoreactive ghrelin, determined by specific RIA, in serum-free supernatants. Our results suggested that ghrelin has a specific role in cartilage function because its expression is confined to the proliferative and maturation zones, a cartilage anatomic compartment in which regulation and coordination of the chondrocyte cell metabolic rate is essential to normal skeletal morphogenesis. Because the presence of ghrelin in cartilage proliferation zone suggested a potential role as growth factor for chondrocytes, we tested the effect of ghrelin treatment on chondrocytes metabolic activity and cell cycle in cultured immortalized human chondrocytes. Surprisingly, ghrelin acts as an inhibitor of cell metabolic activity in cultures of all human immortalized chondrocyte cell lines tested, as evaluated by MTT assay. In addition, flow cytometry studies revealed that ghrelin did not affect cell cycle in chondrocytes, in any of the cell cycle phases, suggesting that the effect of ghrelin is focused on metabolic rate modulation and it is not due to a cytotoxic action. This hypothesis is further confirmed by data assessing gene expression. This analysis clearly indicated that several genes encoding for key enzymes of the RedOx cellular system are down-regulated upon ghrelin treatment. To date, some of these enzymes are known to be involved into the production of reactive oxygen species that normally act as detrimental factors of cartilage integrity. So, it seems plausible that ghrelin could function as a counterregulatory signal controlling the expression of some inflammatory mediators in cartilage. On the other hand, we have demonstrated that ghrelin is able to increase cAMP production in both human and murine chondrocytes in a dose-responsive manner. The role of cAMP in the metabolism of mammalian cartilage has been extensively studied but remains in part undefined. At any rate, several published data indicate that increase of cAMP in chondrocytes is related with an augment of sulfated proteoglycan and hyaluronate synthesis (23, 24, 25, 26). Intriguingly, in our experimental set ghrelin was able to stimulate cAMP production but also induced a significant increase in the gene expression of chondroitin sulfate type IV (data obtained by DNA array experiments). To date, cAMP increase is linked with chondrocytes apoptosis (27), the final stage of endochondral bone growth. Therefore, it is reasonable to hypothesize that ghrelin in cartilage could participate in chondrocyte metabolism by promoting hypertrophy processes by increasing proteoglycan synthesis and programmed cell death, thus clearing the way for osteoblastic bone formation. However, this hypothesis should be further explored.
Another aspect arising from our work is that ghrelin was able to significantly inhibit fatty acid uptake. It is well known that fatty acids are macronutrients that serve as precursor to a wide family of eicosanoids. These factors plays a significant role not only in joint physiology but also in the pathogenesis of joint disorders. Although any available data demonstrated that ghrelin is able to modulate prostaglandins synthesis, the observation that the hormone down-regulates fatty acid uptake in chondrocytes, limiting precursor availability and thereby inhibiting prostaglandin and/or leukotrienes synthesis, might therefore be considered as a very attractive hypothesis for a potential therapeutic intervention of articular degenerative inflammatory diseases such as osteoarthritis and rheumatoid arthritis.
Another issue arising from our results is the presence of specific receptors for ghrelin different from the classic 1A receptor subtype, the unique GHS-R form that is able to transduce signals (28). Our results obtained by RT-PCR indicate that GHS-R type 1A is absent in chondrocytes, but binding studies suggest the presence of at least two receptors, respectively, of high and low affinity. This result is in agreement with other previously published results that hypothesize that multiple ghrelin receptors may be expressed (29, 30). For instance, CD36, a fatty acid receptor, has been identified recently as a myocardial receptor for hexarelin, a synthetic peptidyl GHS. However, neither ghrelin nor the synthetic nonpeptidyl GHS, MK-0677, recognize this receptor (31, 32). Finally, although ghrelin have been shown to exert important biological effects in different tissues, its physiological relevance is still unclear. Deletion of the ghrelin gene in mice has been reported not to impair either growth or appetite (33), although in other studies it has been shown that constitutive absence of ghrelin prevents high-fat diet-induced obesity (34). Furthermore, transgenic rats expressing an antisense GHS-R mRNA under the control of the promoter for tyrosine hydroxylase, thus selectively attenuating GHS-R protein expression in the Arc, had lower body weight and less adipose tissue than did control rats. GH secretion and plasma IGF-I levels were reduced in female transgenic rats, indicating that the ghrelin system plays a major physiological role in the regulation of GH secretion, food intake, and adiposity (35). Although these discrepancies in genetically modified animals are due to interspecies differences, genetic background or experimental conditions remains to be established.
In conclusion, our study suggests that ghrelin may have a significant role in regulating chondrocyte metabolism in the growth plate because it is localized in the proliferative and maturation zones, inhibits cell metabolic rate, promotes cAMP accumulation and inhibits basal and induced fatty acid uptake in chondrocyte cultures. Thus, ghrelin produced by chondrocytes could influence, by an autocrine/paracrine pathway, the synthesis of factors that selectively promote osteogenesis, or alternatively, modulate the biosynthesis of eicosanoids in the cartilage. In addition, we cannot exclude that ghrelin could induce or repress genes whose roles in cartilage physiology or pathology are yet to be defined. Thus, identification of changes exerted by ghrelin may provide the basis for future studies on the molecular effects of this novel member of the GH axis on chondrocyte biology.
Acknowledgments
The authors thank Dr. Juan Vi?uela (Santiago University Clinical Hospital, Laboratory of Clinical Analysis, Immunology Section) and gratefully acknowledge his help in flow cytometric analysis experiments.
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Address all correspondence and requests for reprints to: Oreste Gualillo, Ph.D., Santiago University Clinical Hospital, Research Area, Research Laboratory 4, Trav. Choupana, sn, 15706 Santiago de Compostela, Spain. E-mail: gualillo@usc.es.
Abstract
Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), is a recently isolated hormone, prevalently expressed in stomach but also in other tissues such as hypothalamus and placenta. This novel acylated peptide acts at a central level to stimulate GH secretion and, notably, to regulate food intake. However, the existence of further, as yet unknown, effects or presence of ghrelin in peripheral tissues cannot be ruled out. In this report, we provide clear evidence for the expression of ghrelin peptide and mRNA in human, mouse, and rat chondrocytes. Immunoreactive ghrelin was identified by immunohistochemistry in rat cartilage, being localized prevalently in proliferative and maturative zone of the epiphyseal growth plate, and in mouse and human chondrocytic cell lines. Moreover, ghrelin mRNA was detected by RT-PCR and confirmed by Southern analysis in rat cartilage as well as in mouse and human chondrocytes cell lines. Ghrelin mRNA expression has been studied in rat along early life development showing a stable profile of expression throughout. Although ghrelin expression in chondrocytes suggests the presence of an unexpected autocrine/paracrine pathway, we failed to identify the functional GH secretagogue receptor type 1A by RT-PCR. On the other hand, binding analysis with 125I ghrelin suggests the presence of specific receptors different from the 1A isotype. Scatchard analysis revealed the presence of two receptors with respectively high and low affinity. Finally, ghrelin, in vitro, was able to significantly stimulate cAMP production and inhibits chondrocytes metabolic activity both in human and murine chondrocytes. In addition, ghrelin is able to actively decrease both spontaneous or insulin-induced long chain fatty acid uptake in human and mouse chondrocytes. This study is the first to provide evidence for the presence of this novel peptide in chondrocytes and suggests novel potential roles for this newly recognized component of the GH axis in cartilage metabolism.
Introduction
THE GROWTH OF the long bones is driven by the cellular activity of chondrocytes within the growth plate. Small germinal chondrocytes in the resting zone enter the cell cycle and undergo successive mitotic divisions to form columns of chondrocytes in the proliferative zone. The proliferative and hypertrophic layers of the growth plate distinguish anatomically these phases of growth. Until recently, the major regulation of the growth plate was thought to be due to systemic hormones, mainly those related to the GH-IGF-I axis (1, 2). However, it is likely that locally produced peptide factors play an important autocrine and/or paracrine role in ensuring that skeletal development and growth progress correctly. Investigations in vitro and in vivo have demonstrated the importance of the peptide growth factors IGF-I, TGF-?, and basic fibroblast growth factor in modulating cell proliferation, extracellular matrix biosynthesis, and changes in morphology associated with the progressive stages of chondrocyte differentiation (3). Recently, ghrelin, a new member of the GH axis, has been identified. This new hormone is a gastric orexigenic peptide and a ligand for the GHS-R [GH secretagogue (GHS) receptor] that potently stimulates GH release both in vivo and in vitro (4, 5, 6). Several previous studies, performed with synthetic analogs of ghrelin, suggest specific actions on bone tissues. Namely, hexarelin treatment inhibits expression of the markers of bone resorption in rats (7), and ipamorelin induced longitudinal bone growth in rats (8). In humans, several studies show that nonpeptidyl GHSs are capable of actions on bone tissue. For example, MK-677 treatment induces potent increases in biochemical markers of bone formation and bone resorption (9, 10).
The aim of this work was to verify whether ghrelin and its cognate receptor, GHS-R, were expressed in chondrocytes. In the current study, ghrelin and its mRNA expression was assessed in human, rat, and mouse chondrocytes by means of immunohistochemistry, RT-PCR, and Southern analysis. In addition, binding studies were performed to assess the expression of specific ghrelin receptors at the plasma membrane. Finally, we evaluated whether ghrelin was able to exert some biological effect by testing cAMP modulation, cell metabolic activity, and long chain fatty acid uptake on a human immortalized chondrocyte cell line upon ghrelin stimulation.
Materials and Methods
Cell culture
The ATDC-5 murine chondrogenic cell line was a kind gift from Dr. Agamennon E. Grigoriadis (Department of Orthodontics and Pediatric Dentistry & Craniofacial Development, King’s College London, Guy’s Hospital, London, UK). Cells were cultured in monolayer as described previously (11) in a standard medium of DMEM Ham’s F12 supplemented with 5% fetal calf serum (FCS), 10 μg/ml human insulin, 10 μg/ml human transferrin, and 3 x 10–8 M sodium selenite and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin. For longer term culture, up to 21 d, the media were changed to -MEM with the same additives to induce hypertrophic differentiation. The immortalized human juvenile costal chondrocytes cell lines T/C-28a2, C-20/A4, and C-28/I2 were cultured in DMEM/ F12 as described previously (12) supplemented with 10% FCS, L-glutamine, and antibiotics. Cells were grown in monolayer and passaged every 5–6 d unless otherwise specified.
Immunohistochemistry
Specimens of rat epiphyseal growth plate, mouse and human tissues, and cell cultures were immersion-fixed in 4% buffered formaldehyde for 24 h, dehydrated, and embedded in paraffin by standard procedure. Cartilage tissues were decalcified in EDTA acid buffer for 2 h (MicroStain, Bologna, Italy). Sections 5-μm thick were mounted on Histobond adhesion microslides (Mariefeld, Lauda-Konigshofen, Germany) and deparaffined before analysis by immunohistochemistry. Antigen retrieval was carried out for antighrelin using a microwave oven for three cycles of 5 min each at 750 W in 0.001 M sodium citrate buffer (pH 6.0). The polyclonal IGF-I antibody was used without unmasking procedure. Samples were immunostained with a rabbit polyclonal ghrelin antibody (a generous gift of Dr. M. Kojima), anti-IGF-I. Sections were consecutively incubated in: 1) antighrelin at a dilution of 1:500, anti-IGF-I at a dilution of 1:250 (1 h at room temperature); 2) 3% hydrogen peroxide (Merck, Darmstadt, Germany) to block endogenous peroxidase (10 min); 3) goat antirabbit Igs conjugated to peroxidase-labeled dextran polymer (Dako EnVision peroxidase rabbit; Dako, Carpinteria, CA); 4) 3,3'-diamino-benzidine-tetrahydrochloride (DAB) solution prepared by dissolving one DAB-buffer tablet (Merck) in 10 ml of distilled water (10 min). Between steps, sections were washed twice for 5 min with TBS [0.05 M Tris buffer (pH 7.6), containing 0.3 M NaCl] and after step 4 with distilled water. All dilutions were made in TBS except the dilution of the primary antisera (step 1) where the TBS was substituted by an antibody diluent (Dakoppats). TC-28/A2 (human cell line) and ATDC5 (mouse cell line) chondrocyte cells were fixed by the following procedure: 15 min in 10% buffered formalin, 5 min in PBS [0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCl], 4 min in –20 C methanol, 2 min in –20 C acetone and 5 min twice in PBS. Epitope retrieval was done by microwaving at 750 W for 10 min in 0.01 M sodium citrate buffer. In this case, we used antighrelin at a dilution of 1:50. Negative controls were performed by either : 1) preadsorbing the antighrelin antibody with the homologous antigen (ghrelin 10 nM) for 24 h at 4 C; or 2) omitting any essential step of the reaction. Sections were consecutively incubated as described previously (13).
Tissue explant and RNA preparation.
Rat tissues were obtained from Sprague Dawley rats at different ages. Cartilage was carefully dissected from fetal tibia epiphyseal growth plate cartilage and from 1-, 15-, and 21-d-old Sprague Dawley rats. Animals were killed under anesthesia, according to protocols approved by the Animal Care Committee of the University of Santiago de Compostela. The tibia growth plate and adjacent articular cartilage from rats were dissected shortly after death as described previously (14), minimizing all possible contaminations from other tissues. RNA was isolated from frozen or freshly explanted tissues by Trizol method as described previously (15). Briefly, tissues were homogenized using a Polytron homogenizer in 1000 μl of Trizol LS reagent (Invitrogen Life Technologies, Carlsbad, CA) and, after recovery by isopropanol precipitation, the total RNA was measured with a spectrophotometer (Beckman Inc., Fullerton, CA) at 260 nm.
RT-PCR and Southern analysis.
Two micrograms of total RNA were used to perform RT-PCR. cDNAs were synthesized using 200 U of Moloney murine leukemia reverse transcriptase (Invitrogen Life Technologies) and 6 ml of deoxynucleotide triphosphates (dNTPs) mix (10 mM of each dNTP), 6 μl of first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 (Invitrogen Life Technologies)] 1.5 μl of 50 mM MgCl2, 0.17 μl of random hexamers solution [3 μg/ml (Invitrogen Life Technologies)], 0.25 μl of RNase Out [recombinant ribonuclease inhibitor 40 U/ml (Invitrogen Life Technologies)], in a total volume of 30 μl. Reaction mixtures were incubated at 37 C for 50 min and at 42 C for 15 min. The reverse transcription (RT) reaction was terminated by heating at 95 C for 5 min and subsequently quick-chilled on ice. Three microliters of RT reaction were used for PCR amplification. The amplification conditions were as follows: 5 μl of PCR buffer [200 mM Tris-HCl pH 8.4 and 500 mM KCl (Invitrogen Life Technologies)], 1.5 μl of 50 mM MgCl2, 4 μl of dNTPs mix, 150 ng of specific upstream and downstream primers (13, 15) [sequences of specific primers has been reported in Table 1], and 1.25 U of Taq DNA Polymerase (Invitrogen Life Technologies)]. The amplification profile for rat, human and mouse ghrelin was: denaturation at 98 C for 15 sec, annealing at 60 C for 60 sec, and extension at 72 C for 1 min. Thirty-five-cycle amplification was completed with an additional step at 72 C for 10 min. The amplification was performed in an automatic thermal cycler (Mastercycler gradient; Eppendorf AG, Hamburg, Germany). To check the quality of mRNA in each sample, human, mouse, and rat ghrelin RNA samples were amplified together with respectively housekeeping genes human and mouse hypoxanthine guanine phosphoribosyl transferase (hHPRT, mHPRT) and rat ?-actin. Multiple positive and negative controls were performed for RT-PCR. Negative controls consisted of omitting the RT reaction for each sample or amplifying samples of RT reaction without Moloney murine leukemia virus reverse transcriptase. All negative controls resulted in no bands after amplification. The identity of the amplimer for ghrelin was confirmed by performing the RT-PCR together with positive controls (stomach RNA). PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, examined with UV light and visualized with a Typhoon 9410 Documentation System (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). To confirm authenticity of the amplimers, Southern blot analysis was carried out. Hybridization of nylon transferred resolved amplicons was performed using 32P cDNA-specific probes for ghrelin. The probes were prepared by random prime labeling of the ghrelin cDNAs subcloned in EcoRI double restriction site of a pBS-SK (2) expression vector. Ghrelin cDNAs were purified from plasmid expression vector using the Sephaglas BandPrep Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). The resolved amplicons were transferred by capillary blotting onto a charged nylon membrane and fixed with UV light. The membranes were hybridized with 32P-labeled rat or human ghrelin (1 x 106 cpm/ml) at 44 C for 18 h. After removing the excess of labeled probe by washing, membranes were exposed to autoradiography, and the sizes of the bands determined by comparison with molecular weight marker on the ethidium bromide-stained gels.
TABLE 1. Primers pairs
Real-time PCR
Real-time PCR assay was carried out with SYBR Green system using a LightCycler rapid thermal cycler system (LightCycler, Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. PCR parameters were as follows: an initial denaturation at 96 C for 20 sec followed by 40 cycles at 96 C for 2 sec; 60 C for 15 sec, and 72 C for 15 sec. Fold differences for gene expression were calculated by the comparative Ct (threshold cycle) method. This method compares test samples to a calibrator sample and uses results obtained with a uniformly expressed control gene to correct for differences in the amount of RNA present in the two samples being compared with generate a Ct value. Specificity of real time RT-PCR amplification of each primer pair was confirmed by analyzing PCR products by agarose gel electrophoresis and by melting curve analysis.
Ghrelin RIA
Ghrelin levels in ATDC5 murine chondrocytes, T/C-28a2, C20/A4 and C-28/I2 human immortalized chondrocytes culture media were determined using specific RIA kits from Phoenix Pharmaceuticals (Belmont, CA), according materials and protocol provided by the supplier. Ghrelin determination was performed in serum-free conditions after a starvation of 24 h.
Binding analysis
Hormone binding to the T/C-28a2 cells was performed in six-well plates as previously described (16). Cells were seeded at a density of 500,000 cell/well and with a viability, evaluated by trypan blue exclusion method, of greater than 90%. Cells were grown until a confluence of 70–80% and before binding were carefully washed with DMEM/NutF12 and incubated in this serum-free medium overnight. Subsequently, plates were put on ice and washed with ice cold PBS. In a final volume of one ml of binding buffer (PBS with 1% wt/vol of BSA, fraction V, Sigma Chemical Co., St. Louis, MO) whole cells were incubated with 90000 cpm of 125I ghrelin (Amersham Pharmacia Biotech Ltd.) with a specific activity of 2000 Ci/mmol. Incubation was carried out at 4 C to avoid internalization of the receptor and/or ligand and overestimation of the binding capacity. At the end of the incubation, unbound label was removed by two PBS washes; 1 ml of 1 N NaOH was added, and radioactivity in lysates was measured using a counter (1261; LKB-Wallac, Gaithersburg, MD). Specific binding was determined by subtracting the amount of 125I ghrelin in the presence of excess of unlabeled ghrelin (3 μM). For Scatchard analysis, increasing amounts of unlabeled recombinant human ghrelin (0.03–30 nM) were added to compete with 125I human ghrelin for binding in saturating conditions. Ligand software (Elsevier-Biosoft, Cambridge, UK) was used to calculate the dissociation constant (Kd) and binding capacity (17).
cAMP determination
Intracellular cAMP was determined by direct scintillation proximity assay of acid extracts from cells grown in 24-well cluster plates and treated with different doses of ghrelin (1, 10, 50, and 100 nM), according materials and protocols provided by the supplier (RPA 538; Amersham Biosciences).
Cell metabolic activity using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay.
Cell metabolic activity was examined using a colorimetric assay based on the MTT labeling reagent (Roche Molecular Biochemicals, Barcelona, Spain). Assays were performed according instructions and protocol provided by the manufacturer. Briefly, the TC/28 a2, C20/A4 and C28/I2 were seeded in 96-well plates (8000 cell/well, in 0.2 ml of complete medium). After complete adhesion, cells were incubated with serum-free medium and stimulated with human recombinant ghrelin (0.1, 1.0, 10, and 100 nM). After 48 h, the MTT labeling reagent solution (final concentration 0.5 mg/ml) was added to each well, and incubations continued for 4 h at 37 C. After this incubation, 100 μl of solubilization solution (SDS 10% in HCl 0.01 M) were added to the wells until complete solubilization of the purple formazan crystals. Spectrophotometrical absorbance was measured using a microtiter ELISA reader at 550 nm (Multiskan EX, Labsystem, Helsinki, Finland). Data are reported as mean ± SEM values of at least three independent determinations. All experiments were done at least four times, each time with three or more independent observations. Statistical analysis was performed by ANOVA, and multiple comparisons were made by Student-Newman-Keul’s test with Instat computerized package.
Fatty acid uptake (BODIPY 3823) assay
4,4-Difluoro-5-methyl-4-bora-3a-diaza-3-indacene-3-dodecanoic acid (BODIPY 3823, Molecular Probes, Inc., Eugene, OR)-labeled fatty acid uptake was used for measuring long-chain fatty acid uptake in human immortalized chondrocytes T/C-28a2 and in murine ATDC5 chondrocytes. Cells were treated with ghrelin 100 nM for 2 h, washed with DMEM, and incubated with PBS containing 20 μM BSA (fatty acid free) and 10 μM BODIPY 3823 (Molecular Probes) for 2 min at 37 C. Cells were then washed extensively at 4 C with PBS containing 0.1% BSA to remove surface-associated BODIPY. Cells were suspended in DMEM buffered with HEPES to pH 7.5 for cytometric analysis. Cell emission of BODIPY was measured by flow cytometry with a FACS Calibur Becton Dickinson (BD; San Jose, CA) flow cytometer and results were analyzed using the software CellQuest (BD).
Results
Immunohistochemistry
As shown in Fig. 1A, immunoreactive ghrelin was localized in rat epiphyseal growth plate, where it was expressed in the majority of the chondrocytes in the proliferation and maturation zones and only occasionally in the hypertrophic and provisional calcification area. A similar pattern was observed for IGF-I immunostaining (Fig. 1B). Ghrelin was found in the cytoplasm, whereas the nuclei were negative. IGF-I-positive staining was more intense around the nuclei. No immunoreactivity was shown when the primary antibody was preadsorbed with the homologous antigen or when the primary antibody or other incubation steps were substituted by TBS (Fig. 1C).
FIG. 1. A, Ghrelin immunoreactivity in rat epiphyseal growth plate. Ghrelin was expressed in the majority of the chondrocytes in the proliferation and maturation zones and only occasionally in the hypertrophic and provisional calcification area. A similar pattern was observed for IGF-I. B, Immunostaining for IGF-I. Ghrelin immunopositivity was found in ATDC5 mouse cell line (D) and in human immortalized chondrocytes (E). C and F, Negative controls obtained by preadsorbing primary antibody with the homologous antigen.
ATDC-5 chondrogenic mouse cell line (Fig. 1D) and the human chondrocyte cell line TC-28A2 (Fig. 1E) showed intense ghrelin-positive staining. Immunoreactivity was localized in the cytoplasm of the cells. No immunopositivity was observed when the primary antibody was preadsorbed with the homologous antigen or when any essential incubation step was substituted by TBS (Fig. 1F).
RT-PCR and Southern analysis
Ghrelin mRNA and GHS-R expression.
As shown in Fig. 2A, ghrelin mRNA was expressed in the human chondrocyte cell lines (C20/A4 and C28/I2). Identity of amplimers was confirmed by Southern hybridization (see lower panels of Fig. 2, A–C). Moreover, ghrelin mRNA expression was also identified in the mouse chondrogenic cell line ATDC-5 (Fig. 2B) as well as in tibial growth plate cartilage of male rats of different ages (Fig. 2C).
FIG. 2. Ghrelin mRNA expression in human immortalized chondrocyte cell lines C20/a4 and C28/I2 (A), mouse chondrogenic ATDC5 cell lines (B), and rat growth plate cartilage of different age rats (C). Identity of amplimers was confirmed by Southern analysis.
We studied also expression of GHS-R type 1A in human immortalized chondrocytes and in rat growth plate of rats at different ages. We failed in identifying mRNA for GHS-R 1A in either tissue, as shown in Fig. 3, despite its presence in the positive controls, namely human and rat pituitary mRNA.
FIG. 3. RT-PCR for GHS-R 1-a in human immortalized chondrocytes (A) TC28A2, C20/a4 and C28I2. The housekeeping gene used for checking RT-PCR was human HPRT. Expression of GHS-R 1A in rat growth plate of rats of different ages (B). Qualitative and quantitative controls for rat samples were performed using ?-actin amplification (C).
Secretion of ghrelin by cultured chondrocytes
The RIA-measured concentration of ghrelin following 24-h starvation in cell culture medium was 4.19 ± 0.24 pg/ml in ATDC5 murine chondrocyte (n = 4) cell line. In human immortalized chondrocytes T/C-28a2, C20/A4, and C-28/I2 were, respectively, 10.85 ± 0.39 pg/ml (n = 4), 11.717 ± 0.26 pg/ml (n = 4), and 11.203 ± 0.75 pg/ml (n = 4).
Ghrelin binding analysis
Because GHS-1A receptors were not expressed in chondrocytes, we have further evaluated whether ghrelin could bind specifically to the cell surface of human immortalized chondrocytes. The analysis of binding of [125I] ghrelin to whole cells is presented in Fig. 4. Scatchard analysis showed that the Kd for high affinity receptor was: 3.83 x 10–9 M with a maximum binding capacity (Bmax) of 7.82 x 10–10 M. Moreover, these data suggest the presence of a low-affinity isotype receptor for ghrelin with high levels of expression, different from the isotype GHS-R 1A, with a Kd = 1.32 x 10–8 and a Bmax = 2.85 x 10–9.
FIG. 4. Scatchard analysis of binding data from the competition studies between [125I]ghrelin and unlabeled ghrelin using T/C-28a2 human immortalized chondrocytes.
cAMP determination
Because ghrelin has been demonstrated to exert its biologic effects in various cells through the stimulation of cAMP-mediated biochemical pathways, the ability of ghrelin to induce a biological response was investigated as a function of cAMP concentration in articular immortalized chondrocytes. As shown in Fig. 5, a 15-min exposure of chondrocytes to ghrelin (1–100 nM) elevated intracellular cAMP concentration in a dose-dependent fashion. As positive control, human and murine chondrocytes were treated with forskolin, a direct activator of adenylate cyclase.
FIG. 5. Dose-dependent accumulation of cAMP in response to different doses of ghrelin and forskolin (F), treatment for 15 min in T/C-28a2 human immortalized chondrocytes (gray bars), and ATDC5 murine chondrocytes (black bars). F was used as a direct activator of adenylate cyclase.
MTT metabolic assay
To evaluate whether the immortalized human chondrocytes could demonstrate a physiologically relevant response to ghrelin, cell metabolic activity was analyzed using the MTT assay. As shown in Fig. 6, human recombinant ghrelin was able to significantly inhibit cell metabolic activity in a dose-dependent manner in all three human immortalized juvenile costal chondrocyte cell lines. These cell lines showed a significant response to FCS, whereas ghrelin stimulation caused a significant decrease (40%) in cell metabolic activity when the cells were incubated with human recombinant ghrelin in serum-free conditions. To note, cell cycle analysis performed by flow cytometry with propidium iodide staining upon ghrelin stimulation did not show any alteration in the percentage of hypodiploid cells (apoptotic), G0/G1 and S/M cell cycle phases in comparison to untreated control cells, excluding in such a way a potential cytotoxic effect of ghrelin (data not shown). In keeping, preliminary data obtained by cDNA microarray analysis (Genechip Human Genome U133A; Affymetrix, Santa Clara, CA) of human immortalized chondrocytes indicated that ghrelin was able to significantly decrease gene expression of relevant genes involved in the cellular RedOx system such as reduced nicotinamide adenine dinucleotide phosphate oxidase 1 and cytochrome p450p9, without affecting genes related to cell-cycle progression. In addition, real-time PCR confirmed array analysis and showed that upon ghrelin treatment (100 nM) cytochrome P450p9 decreased its expression by 21% vs. control, whereas reduced nicotinamide adenine dinucleotide phosphate oxidase 1 is almost undetectable after ghrelin treatment.
FIG. 6. Human recombinant ghrelin inhibits basal metabolic activity, evaluated by the MTT colorimetric method, in human immortalized chondrocytes in a dose-dependent manner. *, P < 0.05; **, P < 0.01. As positive control, metabolic activity was evaluated in presence of 10% (vol/vol) fetal bovine serum.
Fatty acid uptake (BODIPY 3823) assay
To determine whether ghrelin was able to modulate fatty acid uptake in chondrocytes, we used flow cytometry to examine the effect of ghrelin on fatty acid transport in chondrocytes. We measured the uptake of BODIPY 3823, a fluorescent-saturated LCFA analog at the immortalized human chondrocytes and murine chondrocytes. Cells were incubated with 100 nM ghrelin for 2 h; then BODIPY 3823 was added for another 2 min and washed with PBS buffer twice. The flow cytometry was used to examine fatty acid uptake. The results (Fig. 7) showed that ghrelin significantly decrease BODIPY uptake (23.6% ± 1.76 in human immortalized chondrocyte and 16.34% ± 0.87 in murine chondrogenic cell line). In addition, ghrelin was able to completely revert the insulin-induced BODIPY 3823 uptake in human chondrocytes, whereas the effect, although significant, was partial in murine chondrocytes.
FIG. 7. Flow cytometry analysis of long chain fatty acid uptake in human and murine chondrocytes. Bars represent percentage of BODIPY 3823 basal uptake upon 2 h treatment with ghrelin (100 nM), insulin (10 nM), or a combination of the two hormones.
Discussion
Cartilage, a connective tissue of diverse embryonic origins, serves multiple prenatal and postnatal functions. This tissue provides structural support for the early embryo, forms templates for developing endochondral bones, provides for postnatal growth of the skeleton, and participates in the repair of fractured bones during endochondral ossification.
Chondrocytes represent a target tissue for a number of molecules that are integrated into the developmental program of the skeleton. Evidence is growing that several endocrine factors, locally produced in cartilage, may also act by a paracrine and/or autocrine mechanism. Thus, in support of this hypothesis are reports confirming expression of IGF-I (18), TGF-? (19), basic fibroblast growth factors (20), and other locally produced factors involved in chondrocyte metabolism. It is clear that the local microenvironment influences key cartilage processes such as the cell cycle, extracellular matrix biosynthesis, and changes in morphology associated with the progressive stages of chondrocyte differentiation.
In the present work, and to the best of our knowledge, for the first time, the expression of ghrelin, the endogenous GHS, has been identified unambiguously in human, mouse, and rat chondrocytes. Ghrelin is a novel GH-releasing peptide, isolated from the stomach, that is the endogenous ligand for GHS-R. In addition to its action as GHS, recent studies have shown that ghrelin causes a positive energy balance by stimulating food intake and decreasing fat use through a GH-independent mechanism (21, 22). We identified ghrelin in growth plate cartilage of embryonic and postnatal rat and in cultured chondrocytes derived from mouse, rat, and human cartilage by several immunological and molecular approaches. In keeping with these data, we have further explored the possibility that ghrelin was secreted by chondrocytes. As expected, mouse ATDC5 cells and human chondrocytes cell lines cultured in vitro produced immunoreactive ghrelin, determined by specific RIA, in serum-free supernatants. Our results suggested that ghrelin has a specific role in cartilage function because its expression is confined to the proliferative and maturation zones, a cartilage anatomic compartment in which regulation and coordination of the chondrocyte cell metabolic rate is essential to normal skeletal morphogenesis. Because the presence of ghrelin in cartilage proliferation zone suggested a potential role as growth factor for chondrocytes, we tested the effect of ghrelin treatment on chondrocytes metabolic activity and cell cycle in cultured immortalized human chondrocytes. Surprisingly, ghrelin acts as an inhibitor of cell metabolic activity in cultures of all human immortalized chondrocyte cell lines tested, as evaluated by MTT assay. In addition, flow cytometry studies revealed that ghrelin did not affect cell cycle in chondrocytes, in any of the cell cycle phases, suggesting that the effect of ghrelin is focused on metabolic rate modulation and it is not due to a cytotoxic action. This hypothesis is further confirmed by data assessing gene expression. This analysis clearly indicated that several genes encoding for key enzymes of the RedOx cellular system are down-regulated upon ghrelin treatment. To date, some of these enzymes are known to be involved into the production of reactive oxygen species that normally act as detrimental factors of cartilage integrity. So, it seems plausible that ghrelin could function as a counterregulatory signal controlling the expression of some inflammatory mediators in cartilage. On the other hand, we have demonstrated that ghrelin is able to increase cAMP production in both human and murine chondrocytes in a dose-responsive manner. The role of cAMP in the metabolism of mammalian cartilage has been extensively studied but remains in part undefined. At any rate, several published data indicate that increase of cAMP in chondrocytes is related with an augment of sulfated proteoglycan and hyaluronate synthesis (23, 24, 25, 26). Intriguingly, in our experimental set ghrelin was able to stimulate cAMP production but also induced a significant increase in the gene expression of chondroitin sulfate type IV (data obtained by DNA array experiments). To date, cAMP increase is linked with chondrocytes apoptosis (27), the final stage of endochondral bone growth. Therefore, it is reasonable to hypothesize that ghrelin in cartilage could participate in chondrocyte metabolism by promoting hypertrophy processes by increasing proteoglycan synthesis and programmed cell death, thus clearing the way for osteoblastic bone formation. However, this hypothesis should be further explored.
Another aspect arising from our work is that ghrelin was able to significantly inhibit fatty acid uptake. It is well known that fatty acids are macronutrients that serve as precursor to a wide family of eicosanoids. These factors plays a significant role not only in joint physiology but also in the pathogenesis of joint disorders. Although any available data demonstrated that ghrelin is able to modulate prostaglandins synthesis, the observation that the hormone down-regulates fatty acid uptake in chondrocytes, limiting precursor availability and thereby inhibiting prostaglandin and/or leukotrienes synthesis, might therefore be considered as a very attractive hypothesis for a potential therapeutic intervention of articular degenerative inflammatory diseases such as osteoarthritis and rheumatoid arthritis.
Another issue arising from our results is the presence of specific receptors for ghrelin different from the classic 1A receptor subtype, the unique GHS-R form that is able to transduce signals (28). Our results obtained by RT-PCR indicate that GHS-R type 1A is absent in chondrocytes, but binding studies suggest the presence of at least two receptors, respectively, of high and low affinity. This result is in agreement with other previously published results that hypothesize that multiple ghrelin receptors may be expressed (29, 30). For instance, CD36, a fatty acid receptor, has been identified recently as a myocardial receptor for hexarelin, a synthetic peptidyl GHS. However, neither ghrelin nor the synthetic nonpeptidyl GHS, MK-0677, recognize this receptor (31, 32). Finally, although ghrelin have been shown to exert important biological effects in different tissues, its physiological relevance is still unclear. Deletion of the ghrelin gene in mice has been reported not to impair either growth or appetite (33), although in other studies it has been shown that constitutive absence of ghrelin prevents high-fat diet-induced obesity (34). Furthermore, transgenic rats expressing an antisense GHS-R mRNA under the control of the promoter for tyrosine hydroxylase, thus selectively attenuating GHS-R protein expression in the Arc, had lower body weight and less adipose tissue than did control rats. GH secretion and plasma IGF-I levels were reduced in female transgenic rats, indicating that the ghrelin system plays a major physiological role in the regulation of GH secretion, food intake, and adiposity (35). Although these discrepancies in genetically modified animals are due to interspecies differences, genetic background or experimental conditions remains to be established.
In conclusion, our study suggests that ghrelin may have a significant role in regulating chondrocyte metabolism in the growth plate because it is localized in the proliferative and maturation zones, inhibits cell metabolic rate, promotes cAMP accumulation and inhibits basal and induced fatty acid uptake in chondrocyte cultures. Thus, ghrelin produced by chondrocytes could influence, by an autocrine/paracrine pathway, the synthesis of factors that selectively promote osteogenesis, or alternatively, modulate the biosynthesis of eicosanoids in the cartilage. In addition, we cannot exclude that ghrelin could induce or repress genes whose roles in cartilage physiology or pathology are yet to be defined. Thus, identification of changes exerted by ghrelin may provide the basis for future studies on the molecular effects of this novel member of the GH axis on chondrocyte biology.
Acknowledgments
The authors thank Dr. Juan Vi?uela (Santiago University Clinical Hospital, Laboratory of Clinical Analysis, Immunology Section) and gratefully acknowledge his help in flow cytometric analysis experiments.
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