RESEARCH ARTICLE
C-28/I2 and T/C-28a2 chondrocytes as well as human primary articular chondrocytes express sex hormone and insulin receptors—Useful cells in study of cartilage metabolism

https://doi.org/10.1016/j.aanat.2010.09.005Get rights and content

Summary

Sex hormones and insulin have been implicated in articular cartilage metabolism. To supplement previous findings on the regulation of matrix synthesis with 17β-estradiol and insulin and to find a possible model to study cartilage metabolism in vitro, we evaluated the expression of estrogen receptors α and β (ERα, ERβ), androgen receptor (AR) and insulin receptor (IR), in immortalized C-28/I2 and T/C-28a2 chondrocytes and in human primary articular cartilage cells. Chondrocytes were treated with increasing concentrations of 17β-estradiol, dihydrotestosterone or insulin and analyzed by means of RT-PCR and Western blotting. Both cell lines as well as human articular chondrocytes expressed ER α and β, AR and IR at mRNA and protein levels. In immortalized C-28/I2 chondrocytes, we showed that increasing concentrations of 17β-estradiol diminished the 95 kDa band of IR. Since 17β-estradiol suppresses insulin-induced proline incorporation and type II collagen synthesis, as we have previously demonstrated, our findings give the first clue that 17β-estradiol may have negative effects on cartilage anabolism triggered by insulin during hormonal imbalance. Compared to chondrocytes cultured without hormones, immunostaining for ERα/β, AR and IR was decreased in both cell lines after incubation of cells with the receptor-specific hormones. It can be assumed that C-28/I2 and T/C-28a2 chondrocytes interact with the respective hormones. Our findings provide a reproducible model for investigating sex hormone and insulin receptors, which are present in low concentrations in articular chondrocytes, in the tissue-specific context of cartilage metabolism.

Introduction

Receptors for estrogens (Chen et al., 2004) and androgens (Abu et al., 1997) have been described in osteoblasts. The beneficial action of sex hormones on bone maintenance is well established (Manolagas et al., 2002, Jin and Tian, 2002, Liang and Liao, 2004, Zallone, 2006). This is carried by receptors for estrogens and androgens in osteoblasts. The presence of sex hormone receptors has also been well established in chondrocytes of growth plate (Egerbacher et al., 2002, Nilsson et al., 2003, Bonnelye et al., 2007), laryngeal (Holt et al., 1986, Sassoon et al., 1986, Claassen et al., 2006a) and costal cartilage (Itagane et al., 1991). However, the action of sex hormones on cartilage has hardly been elucidated and the clinical impact of sex hormones in articular cartilage is completely unclear to date.

The incidence of osteoarthritis (OA) increases with age in both men and women. However, women seem to be protected against OA until menopause (Spector and Champion, 1989, Spector et al., 1991, Spector et al., 1997, Wluka et al., 2000). Estrogens have been considered possible factors in the predisposition to postmenopausal OA, and estrogen depletion or altered metabolism has been regarded as among the risk factors for OA (For a review see Gokhale et al., 2004). By contrast, men seem to lack protection against OA beginning at the age of 30. Very early reports in mice revealed a detrimental effect of testosterone on articular cartilage (Silberberg and Silberberg, 1961, Silberberg and Silberberg, 1963). On the other hand, recent clinical studies have shown a positive correlation between testosterone and tibial cartilage volume (Cicuttini et al., 2003, Hanna et al., 2005). Due to hormonal changes in mid-life and the so-called metabolic syndrome, insulin may also play a role in modifying articular cartilage metabolism (Athanasiou et al., 1999). Recently, we showed that 17β-estradiol suppresses the anabolic effects of insulin in female bovine articular chondrocytes (Claassen et al., 2006b).

To gain further insights into a possible involvement of sex hormones and insulin in articular cartilage metabolism, we evaluated the expression of receptors of the above-mentioned hormones at the mRNA and protein levels. Because previous studies to investigate hormonal influences on human articular cartilage metabolism have provided variable results due to the uncontrolled sources of cartilage and insufficient numbers of cells obtained from random procedures, we used und studied the immortalized chondrocyte cell lines, C-28/I2 and T/C-28a2, which have already been shown to be a reproducible cell culture model of human origin (Goldring et al., 1994, Loeser et al., 2000, Goldring, 2004). Furthermore, we compared our results with the respective ones in human primary articular chondrocytes. Due to our recent finding that 17β-estradiol influenced the anabolic insulin effect (Claassen et al., 2006b), we incubated immortalized chondrocytes C-28/I2 and T/C-28a2 with physiological and supraphysiological doses of 17β-estradiol and with insulin followed by examination of insulin receptor (estrogen receptor) expresssion.

Section snippets

Cell culture

The immortalized chondrocyte cell lines, C-28/I2 and T/C-28a2, originated from cells isolated from rib cartilage of a 15-year-old female and were transduced with simian virus 40 (SV 40) containing the large T-antigen (Goldring et al., 1994). Chondrocytes of both cell lines were seeded at a density of 100,000 cells/cm2 in 25 cm2 flasks or on glass coverslips and cultured in DMEM/F-12 medium (Gibco 11039) containing 10% fetal calf serum (Seromed S0115), 100 U/ml penicillin/streptomycin (Seromed

Results

Since our previous experiments, in which ethanol was used as a solvent for 17β-estradiol, showed responses in cartilage cells, we used two controls in the present experiments. In the first control, C1, cells were incubated with serum-free medium. In the second control, C2, cells were incubated with serum-free medium containing ethanol or PBS/NaOH, the solvent or vehicle for sex hormones or insulin, respectively.

Discussion

Several lines of evidence suggest that hormones like insulin, and in particular sex hormones like 17β-estradiol and dihydrotestosterone, are involved in the pathogenesis of OA. Previous immunohistochemical studies have shown that ERα is expressed in human articular chondrocytes (Claassen et al., 2001). Because it is difficult to obtain healthy human articular cartilage, particularly in larger amounts, for cell culture experiments, we analyzed the expression of sex hormone and insulin receptors

Acknowledgements

We would like to thank Dr. J. Askaa (DAKO, Denmark) for providing us with antibody to ERβ, clone 8D5, and M. Beall for editing the English.

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