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Insulin-like Growth Factor I Regulates Apoptosis in Condylar Cartilage
T. Yokota1,
H. Shimokawa2,
S. Shibata3,
K. Itoh1,
Y. Baba1,
K. Ohya2,
K. Ohyama1 and
S. Suzuki*,1
1 Section of Maxillofacial Orthognathics, Department of Maxillofacial Restoration, Division of Maxillofacial/Neck Reconstruction, and
2 Section of Pharmacology, Department of Hard Tissue Engineering, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan; and
3 Oral Histology, Graduate School of Dentistry, Health Science University of Hokkaido, Japan
Correspondence: * corresponding author, s-suzuki.mort{at}tmd.ac.jp
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ABSTRACT
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Endogenous insulin-like growth factor-I (IGF-I) is known to affect the growth and development of condylar cartilage. However, the critical effect of IGF-I on cell survival is still unknown. We hypothesized that endogenous IGF-I could regulate the survival of cells of the mandibular condylar cartilage. Mandibular condyles dissected from 12-day-old rats were cultured for 1, 3, and 5 days in medium containing antisense oligodeoxynucleotide (AS-ODN) for IGF-I. Real-time RT-PCR analysis showed that the levels of IGF-I and IGF binding protein (IGFBP)3 mRNAs in the AS-ODN group were significantly decreased. After 3 days’ culture, the number of necrotic cells was observed in the undifferentiated mesenchymal cell layer. These cells were TUNEL-positive and confirmed to be apoptotic by electron microscopic observation. Immunoblotting revealed that expression of cleaved caspase3 was increased with AS-ODN. These results may suggest that the cells in the undifferentiated mesenchymal cell layer of the mandibular condyle require IGF-I for survival.
Key Words: IGF-I • mandibular condylar cartilage • rat • apoptosis • antisense-ODN
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INTRODUCTION
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Insulin-like growth factor I (IGF-I) has been identified as a somatomedin, a mediator of growth hormone, and strongly affects general growth and development (Svoboda et al., 1980; Schoenle et al., 1982; Bikle et al., 2001). It has been reported, by conditional inactivation of the IGF-I gene in the liver, that the liver is the main source of circulating IGF-I; however, locally produced IGF-I is more crucial than circulating IGF-I for post-natal growth and development (Sjögren et al., 1999; Yakar et al., 1999). The production of IGF-I in the mandibular condyle has been confirmed by immunohistological and molecular biological techniques (Maor et al., 1993a; Visnapuu et al., 2002). In organ cultures of the mandibular condyle, exogenous IGF-I significantly increased the uptake of 3H-thymidine and 35S-sulfate into condylar cartilage (Maor et al., 1993a). In in vivo experiments, local administration of IGF-I into the articular cavity of the temporomandibular joint accelerated endochondral bone growth of the mandibular condyle (Suzuki et al., 2004). Recently, in the condylar cartilage of young rats fitted with a mandibular propulsive appliance, which produced induction of the mandibular condyle, IGF-I mRNA expression was increased (Hajjar et al., 2003), indicating that IGF-I plays a role as a mediator when mechanical stimulation promotes local growth of the mandibular condyle. These results revealed that both exogenous and endogenous IGF-I affect the growth and development of condylar cartilage.
In addition to cell proliferation and differentiation, various roles for IGF-I—such as the regulation of cell survival and maintenance of the character of the differentiated cells—have been reported (Politi et al., 2001; Lawlor et al., 2000; LeRoith and Roberts, 2003). However, the critical effects of IGF-I on the cell survival of the mandibular condyle are still unknown. We hypothesized that endogenous IGF-I could regulate the survival of cells of the mandibular condylar cartilage. To examine this hypothesis, we assessed the cell survival of mandibular condyle when cultured in serum-free medium, where IGF-I production was blocked by an antisense method.
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MATERIALS & METHODS
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Experimental Animals
Twelve-day-old Sprague-Dawley-strain rats were used. Bilateral mandibular condyles were excised under pentobarbital anesthesia, and then used in an organ culture. All techniques used in this study were approved by the Animal Study Committee of the Tokyo Medical and Dental University (Approval No. 0040296).
Design of Oligodeoxynucleotides
The antisense oligonucleotide (AS-ODN) for IGF-I was designed to contain the translation initiation site of exon 1 of the rat IGF-I gene (Shimatsu and Rotwein, 1987; Castro-Alamancos and Torres-Aleman, 1994). The AS-ODN for IGF-I (5'-ACT GCT GAT TTT CCC CAT-3') and S-ODN for IGF-I (5'-ATG GGG AAA ATC AGC AGT-3') were synthesized with phosphorothioate modifications and purified by HPLC (Fasmac Corp., Kanagawa, Japan).
Organ Culture of Mandibular Condyles
Dissected mandibular condyles were cultured in a humidified atmosphere of 5% CO2 in air at 37° C, in Ham’s F-12 medium (Gibco, Paisley, UK), pH 7.4, supplemented with L-glutamate (300 µg/mL) (Wako, Osaka, Japan), glycine (50 µg/mL) (Wako), ascorbic acid (100 µg/mL) (Wako), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Gibco) by a modified Trowell-type organ culture system. The AS-ODN and S-ODN were mixed with a cationic reagent, Oligofectamine (Invitrogen, Carlsbad, CA, USA), incubated at room temperature for 1 hr, and then added to the culture medium at a final concentration of 30 µM. In the ODN-free culture, only Oligofectamine was added to the culture medium. The medium was changed daily.
Real-time RT-PCR
The total RNA was extracted from combined samples of 6 mandibular condyles cultured for 3 days with ISOGEN (Nippon Gene, Tokyo, Japan). After spectrophotometric measurement of the RNA, total RNA in diethyl pyrocarbonate (DEPC)-treated water was reverse-transcribed to cDNA by means of a First-Strand Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). For the quantitative real-time reverse-transcription polymerase chain-reaction (RT-PCR) analysis of IGF-I, IGFBP3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, a LightCycler System and reagents (Roche) were used with a double-strand DNA binding dye, SYBR Green 1, according to the manufacturer’s instructions. The housekeeping gene GAPDH was used to ensure that various mRNA levels were normalized against the total mRNA content in the samples. Amplified products were represented as the ratio of the respective PCR product/GAPDH PCR product. The following primers were chosen: GAPDH, forward, 5'-ACCACAGTCCATGCCATCAC-3', reverse, 5'-TCCACCACCCTGTTGCTGTA-3'; IGF-1, forward, 5'-TCGGCCTCATAATACCCACTCTG-3', reverse, 5'-CCGAGCTGGTAAAGGTGAGCA-3'; and IGFBP3, forward, 5'-AAACAGTGTCGCCCTTCCAAA-3', reverse, 5'-TAAGTG GCACAGCGGTATCTA-3'.
Histological Observations
Mandibular condyles were cultured for 1, 3, or 5 days. The specimens were fixed in Karnovsky’s fixative solution, decalcified with 8% formic acid, and embedded in water-soluble resin (HistoResin; Kulzer, Hereaus, Germany). Sagittal sections in the central region of the condyle with a thickness of 2 µm were prepared and stained with 0.1% toluidine blue for histological observation. Apoptotic cells were detected immunohistochemically by the TdT-mediated biotinylated dUTP nick-end-labeling (TUNEL) method, with a commercially available kit (Cell death detecting kit, Gibco, Paisley, UK). After culture, explants were fixed with 4% paraformaldehyde, then decalcified with 10% EDTA, and embedded in water-soluble resin. Thin sections with a thickness of 2 µm were prepared. For electron microscopic observation, specimens were immediately immersed in 5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperature. The explants were post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, at 4° C for 3 hrs, and then dehydrated in a graded series of ethanol and embedded in Epon 812 (TAAB, Janning, Vanves, France). For electron microscopic examination, ultrathin sections of condyles were prepared and stained with uranyl acetate and lead citrate, and examined with a Hitachi-800 electron microscope (Hitachi Ltd., Tokyo, Japan).
Immunoblotting
The amount of protein extracted from mandibular condyles cultured for 3 days was determined with the use of a dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A 20-µg quantity of protein from each sample was fractionated on a 12% SDS-PAGE gel (Invitrogen, Carlsbad, CA, USA) and transferred to a 0.22-µm PVDF membrane. We used the BenchMark Protein Ladder (Invitrogen) to estimate the molecular weight of the protein bands. We stained the gel with Coomassie staining to confirm equal amounts of protein loading to each well. For immunostaining, after being blocked with Blocking Agent (Amersham, Piscataway, NJ, USA), the membranes were probed for 1 hr at room temperature with primary polyclonal antibodies against caspase 3 (1:100 dilution) (Chemicon International, Temecula, CA, USA). The membranes were rinsed in PBS containing 0.5% Tween 20, and exposed to HRP-conjugated secondary antibodies (Cell Signaling, Frankfurt-am-Main, Germany) for 1 hr at room temperature. Specific bands were visualized by means of the ECL Western Blotting Detection Kit (Amersham).
Statistical Analysis
Results are presented as the mean ± standard deviation. Differences among groups were tested by one-way ANOVA, with p values calculated by the Bonferroni/Dunn post-test. A p value < 0.05 was considered significant.
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RESULTS
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Real-time RT-PCR Analysis of Gene Expression
We used a specific AS-ODN for IGF-I to determine the role of IGF-I in the cell survival of condylar cartilage cultured for 3 days. Quantitative analysis of mRNA levels of IGF-I showed that mRNA level in the AS-ODN group decreased significantly, to average 57%, compared with those in the ODN-free group, and averaged 65% in the S-ODN group. There was no significant difference between control and S-ODN groups (Fig. 1A ). These findings suggest that the AS-ODN specifically inhibited the expression of the IGF-I gene.
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Figure 1. Real-time RT-PCR analysis of IGF-1 and IGFBP3 mRNA in the mandibular condyles cultured for 3 days. (A) The mRNA expression of IGF-1 in the AS-ODN group was significantly lower than that in the S-ODN and control groups. (B) The mRNA expression of IGFBP3 in the AS-ODN group was significantly lower than that in the S-ODN and ODN-free groups. Values are means ± SD, derived from triplicate arrays. *p < 0.05 vs. the ODN-free and S-ODN groups.
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In addition, quantitative analysis of mRNA levels of IGFBP3 showed that the mRNA level in the AS-ODN group was significantly decreased, to average 61%, compared with that in the ODN-free group, and averaged 56% in the S-ODN group. There was no significant difference between the ODN-free and S-ODN groups (Fig. 1B ).
Histological Observation
On day 1 of culture, there were no significant differences between the AS-ODN group and the S-ODN group in the cultured mandibular condyles, and each of the groups showed an almost normal histology (Figs. 2A-a, 2A-b). In the mandibular condyles of the AS-ODN group cultured for 3 or 5 days, a great many cells denatured due to nuclear condensation and considerable cell debris were observed in the undifferentiated mesenchymal cell layer directly beneath the articular fibrous layer (Figs. 2A-d, 2A-f, 2B-b), but not in the S-ODN and ODN-free groups (Figs. 2A-c, 2A-e, 2B-a, 2B-c). In the mandibular condyles of the S-ODN and ODN-free groups, very small numbers of TUNEL-positive cells were observed (Figs. 2B-d, 2B-f). In contrast, in the AS-ODN group, many TUNEL-positive cells were observed in the undifferentiated mesenchymal cell layer (Fig. 2B-e).
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Figure 2. Histological observation. (A) Toluidine blue staining of the mandibular condyle for 1 day (a,b), 3 days (c,d), or 5 days (e,f), treated with IGF-1 AS-ODN (b,d,f) or S-ODN (a,c,e). The condylar cartilage was subdivided into the articular fibrous layer (A), the undifferentiated mesenchymal cell layer (U), and chondrocytes (C). A great many dead cells due to nuclear condensation (arrows) were observed in the undifferentiated mesenchymal cell layer directly beneath the articular fibrous layer (d,f). (a-f) Toluidine blue staining. (B) Toluidine blue staining (a,b,c) and TUNEL staining (d,e,f) of three-day-cultured mandibular condyles treated with ODN-free (a,d), AS-ODN (b,e), and S-ODN (c,f). Many TUNEL-positive cells (large arrows) were observed in the undifferentiated mesenchymal cell layer (b), and nuclear condensation (arrows) was also observed (e). (a-c) Toluidine blue staining. (d-f) TUNEL staining. Scale bars: 10 µm.
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In the cells of the undifferentiated mesenchymal cell layer of the S-ODN group, an ultrastructural morphology with minimal heterochromatin was observed (Fig. 3A). In contrast, typical signs of apoptosis, including crescent-shaped condensed chromatin, were observed in the AS-ODN group (Fig. 3B).
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Figure 3. Electron microscopic images of the cells in the undifferentiated mesenchymal cell layer on day 3 of culture. (A) Electron microscopic image of the S-ODN group. Little heterochromatin (asterisks) was observed. (B) Electron microscopic image of the AS-ODN group. Chromatin condensation (asterisks) was detected in the nucleus and shrunken cytoplasm (arrows). Scale bars: 5 µm.
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Immunoblotting
Since activation of caspase 3 appears to be a key step in apoptotic cell death (Wolf et al., 1999), the activity of this enzyme was examined in the mandibular condylar cartilage treated with IGF-I AS-ODN. We found that AS-ODN increased cleavage products of caspase 3 (17-kDa fragment), compared with the ODN-free and S-ODN groups (Fig. 4B). Almost equal amounts of protein were loaded in each sample (Fig. 4A).
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Figure 4. Immunoblotting for cleaved caspase 3. Mandibular condyles were cultured for 3 days in medium containing AS-ODN, S-ODN, or no ODN. Extracted protein was analyzed by immunoblotting analysis with an antibody specific for the cleaved caspase 3. (A) Almost equal amounts of protein were loaded in each sample well. (B) The cleaved products of caspase 3 were increased after treatment with AS-ODN, compared with the ODN-free and S-ODN groups.
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DISCUSSION
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IGF-I mRNA was expressed in cells of the undifferentiated mesenchymal cell layer in the rat mandibular condyle (Visnapuu et al., 2002). This cell layer has been called the chondroprogenitor cell layer, based on its stages of differentiation (Silbermann et al., 1983), and high proliferative activity has been found in this cell layer in an autoradiographic study (Blackwood, 1966; Luder et al., 1988). A previous study demonstrated that the IGF-I receptor protein was distributed in the mandibular condyle of one- to 21-day-old rats, and particularly strong expression was observed both in the undifferentiated mesenchymal cell layer and in the upper layer of hypertrophic chondrocytes, although its distribution was throughout the entire layer of the mandibular condyle cartilage (Visnapuu et al., 2001). Taken together, these studies present a picture of the intrinsic IGF-I in the mandibular condyle as produced mainly in the chondroprogenitor cells, and the production may be controlled in an autocrine/paracrine manner.
The action of the IGFs in both the circulation and tissues is tightly regulated by a family of IGF binding proteins (IGFBPs), of which 6 distinct IGFBPs have been identified (Jones and Clemmons, 1995). Of these IGFBPs, IGFBP3 is the major circulating IGFBP present during postnatal life (Longobardi et al., 2003). It has been reported that IGF-I induced IGFBP3 gene expression through P42/44 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways (Kiepe et al., 2005) in growth plate chondrocyte culture. In the developing condylar cartilage of the rat, IGFBP3 protein is expressed in the undifferentiated, chondroblast, and chondrocyte cell layers (Hajjar et al., 2006). It is suggested that IGFBP3 mRNA may also be expressed in other cell layers such as the undifferentiated cell layer. The inhibition of IGFBP3 mRNA expression by AS-ODN treatment indicates that, following the reduction of the mRNA level, the protein level of IGF-I in condylar cartilage was decreased.
In this study, AS-ODN treatment increased the apoptosis of the chondroprogenitor cells, suggesting that the decrease in autocrine/paracrine stimulation by IGF-I causes apoptosis in the chondroprogenitor cells. Moreover, apoptosis was observed primarily in the chondroprogenitor cell layer, but not in mature chondrocytes or hypertrophic chondrocytes, even though they possess IGF-I receptors. This suggests that cell survival of the chondroprogenitor cell layer may strongly depend on the effects of IGF-I, compared with differentiated cells. According to a study of mandibular condylar culture, which used a neutralizing antibody against IGF-I, denatured cells showing pyknosis were found in the murine mandibular condyle (Maor et al., 1993b). Since the neutralizing antibody against IGF-I inhibits the activity of intrinsic IGF-I, the cells that showed pyknosis in the present study may reflect the apoptosis of chondroprogenitor cells, as shown in previous studies. Further experiments, such as a rescue experiment, should be conducted to confirm the knockdown effect of IGF-I.
The mechanism of the induction of apoptosis through the depression of IGF-I activity in chondroprogenitor cells of the mandibular condyles is still unclear. Apoptosis of chondrocytes induced by NO in the articular cartilage was rescued by the addition of IGF-I, and these effects were mediated through a PI3K and Akt pathway (Oh and Chun, 2003). In this experiment, the cleaved caspase 3 in condylar cartilage was increased by AS-ODN treatment. Activation of caspase 3 appears to be a key step in apoptotic cell death (Wolf et al., 1999), and dexamethasone-induced suppression of the PI3K and Akt pathway causes the activation of caspases in proliferative chondrocytes in vitro (Chrysis et al., 2005). Our finding of increased cleaved caspase 3, when IGF-I mRNA expression was significantly reduced by AS-ODN, supports the hypothesis that IGF-I inhibits the activation of apoptotic pathways in the mandibular condylar cartilage. Further studies are needed to analyze the signaling pathway by which IGF-I inhibits apoptosis in the mandibular condyle.
The apoptosis induced in the undifferentiated mesenchymal cells of the mandibular condyle is correlated with some pathologic states, such as osteoarthritis. Indeed, apoptosis is known to occur in articular cartilage in human osteoarthritis (Hashimoto et al., 1998). Apoptosis in the mandibular condyle might be related to pathological shortening of the mandibular ramus, as seen in condylolysis (Susami et al., 1992) and temporomandibular disorders with deformation of the condyle, which includes osteoarthritis. The anti-apoptotic function of IGF-I may recover various pathological conditions of the condyle caused by apoptosis. Further studies are needed to examine the correlation between the onset of apoptosis in the mandibular condyle and the role of IGF-I with respect to temporomandibular disorders and growth impairment of the mandibular condyle.
In conclusion, the results of the present study suggest that AS-ODN against IGF-I mRNA induced apoptosis in the undifferentiated mesenchymal cells of the mandibular condyle. These observations indicate that IGF-I is an important endogenous factor that regulates apoptosis in the undifferentiated mesenchymal cells of the mandibular condyle.
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ACKNOWLEDGMENTS
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This work was supported by a Grant-in-Aid for Scientific Research (No. 10671926) from the Japanese Ministry of Education, Science, Culture and Sports.
Received for publication January 31, 2007.
Revision received November 1, 2007.
Accepted for publication November 5, 2007.
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Journal of Dental Research, Vol. 87, No. 2,
159-163 (2008)
DOI: 10.1177/154405910808700216
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ABSTRACT
INTRODUCTION