Current Topics in Pharmacological Research on Bone Metabolism: Molecular Basis of Ectopic Bone Formation Induced by Mechanical Stress

Journal of Pharmacological Sciences

J Pharmacol Sci 100, 201 – 204 (2006)

©2006 The Japanese Pharmacological Society

Forum Minireview

Current Topics in Pharmacological Research on Bone Metabolism: Molecular Basis of Ectopic Bone Formation Induced by Mechanical Stress Ken-Ichi Furukawa1,* 1

Department of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

Received November 24, 2005; Accepted January 29, 2006

Abstract. Ectopic bone formation (EBF) is frequently found in various tissues and affects the prognosis of diseases accompanied by EBF. Although the mechanism of EBF remains unclear, several local factors that influence the progression of EBF have been proposed. We have been focusing on the role of mechanical stress as a local factor in EBF in spinal ligament tissues, that is, ossification of the posterior longitudinal ligament (OPLL), which causes serious neurological deficiencies. Transcriptome analyses revealed that the expressions of several marker genes related to bone remodeling were enhanced after exposure of ligament cells derived from OPLL patients (OPLL cells) to cyclic stretching as a type of mechanical stress. However, no significant alterations in gene expressions were detected after cyclic stretching of ligament cells derived from non-OPLL patients. OPLL cells exposed to cyclic stretching released several autocrine / paracrine factors that are known to mediate bone remodeling. These results suggest that OPLL cells have been transformed into cells that are highly sensitive to mechanical stress, which may induce the progression of OPLL. These observations provide information regarding the role of mechanical stress in the process of EBF. Keywords: spinal ligament, ectopic bone formation, mechanical stress, microarray, transcriptome analysis

Introduction

progression of OPLL from the molecular aspect.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligament. It is a common disease in Japan and throughout Asia (1). OPLL compresses the spinal cord and its roots, leading to various degrees of neurological symptoms, from discomfort to severe myelopathy. As an established therapy, surgical treatment is often applied to OPLL patients, although it is still associated with problems such as a higher risk of neurological complications (2). Therefore, the development of a safe and effective drug therapy is required. However, the mechanism involved in promoting the ossification remains to be elucidated. In this review, we will discuss a possible role for mechanical stress in the

Characterization of spinal ligament cells derived from OPLL patients Spinal ligament cells derived from OPLL patients (OPLL cells) have been reported to possess several phenotypic characteristics of osteoblasts, that is, in vitro calcification and high alkaline phosphatase (ALP) activity in cultures (3), compared with normal spinal ligament cells. OPLL cells, but not non-OPLL cells (i.e., cells derived from other cervical diseases with no relation to OPLL), respond to transforming growth factor-β (TGF-β) (4), bone morphogenetic protein-2 (BMP-2) (5), insulin-like growth factor-I (6), connective tissue growth factor (7), prostaglandin I2 (PGI2) (8), and parathyroid hormone (9). An immunohistochemical study of ossified ligament tissues from OPLL patients showed enhanced expressions of osteogenic protein1/ BMP-7 and its receptors (type-IA, -IB, and -II recep-

*Corresponding author. [email protected] Published online in J-STAGE: March 4, 2006 DOI: 10.1254/jphs.FMJ05004X4

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tors) (10). To confirm the characteristic differences between OPLL and non-OPLL cells, we tested whether these cells exhibit mineralization upon culture in osteogenic medium. After exposure to osteogenic medium, the matrix around OPLL cells began to mineralize and crystals appeared on the collagen fibers within 4 weeks accompanied by high ALP activity. In contrast, nonOPLL cells did not show these morphological changes. These observations are consistent with the hypothesis that OPLL cells have been transformed into osteoprogenitor cells.

to the bottom of a deformable culture chamber (Fig. 1). The expressions of various marker genes related to bone remodeling were up-regulated by cyclic stretching in OPLL cells (Table 1), but not in non-OPLL cells. These results suggest that OPLL cells have a higher sensitivity to mechanical stress than non-OPLL cells and that the genetic backgrounds of OPLL and non-OPLL patients differ.

Responses of OPLL cells to mechanical stress

Members of the fos-jun proto-oncogene family involved in the specification and modification of morphogenetic plans are thought to be modulated by various cytokines (16). These cytokines and their receptors have been suggested to mediate the progression of OPLL (5, 7, 8, 10, 17 – 21). For example, TGF-β stimulates ALP activity through the induction of connective tissue growth factor (7). A transcriptome analysis revealed that cyclic stretching enhanced their expressions in OPLL cells (Table 1). Addition of BMP-2 or conditioned medium from OPLL cells exposed to cyclic stretching enhanced the expression of ALP in OPLL cells, and this effect was significantly blocked by an anti-BMP antibody (20). As suggested by the cyclic stretch-induced PGI2 synthase expression (Table 1), enhanced release of prostacyclin was observed in OPLL cells (8). Furthermore, addition of beraprost, a stable

OPLL is often associated with concurrent ossification of other spinal ligaments. It has been regarded as one of the manifestations of diffuse idiopathic skeletal hyperostosis (DISH) (11) and ankylosing spinal hyperostosis (12). Therefore, systemic factors have been considered to play roles in the pathogenesis of OPLL. On the other hand, several lines of clinical evidence have suggested that mechanical stress acting on the ligaments is important as one of the local factors for the progression of OPLL (13 – 15). To explore the latter possibility, we investigated the effect of mechanical stress on ligament cells by comprehensive analyses, including DNA microarray and differential display RT-PCR methods. As a mechanical stress that may act on ligament tissues, cyclic stretching was loaded on ligament cells attached

Mechanical stress-induced production and release of autocrine / paracrine factors

Fig. 1. Application of mechanical stress by uniaxial cyclic stretching. Ligament cells were cultured on a deformable silicon chamber coated with gelatin. After reaching confluence, the cells were subjected to uniaxial cyclic stretching. After different periods of cyclic stretching, gene expression analyses were performed.

Ectopic Bone Formation in Ligament

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Table 1. Increases in the expressions of genes related to bone metabolism analyzed by a cDNA microarray Gene name Collagen, type I, alpha 1

Mean ratio 3.12

Collagen, type V, alpha 2

2.14

Collagen, type VIII, alpha 1

3.35

Collagen, type IX, alpha 3

3.22

Collagen, type XI, alpha 1

2.30

Collagen, type XI, alpha 2

3.03

Collagen, type XVIII, alpha 1

3.27

Secreted phosphoprotein 1 (osteopontin)

2.19

Osteocalcin

2.19

Cadherin 11, type 2

2.89

Bone morphogenetic protein 1

3.28

Bone morphogenetic protein 2

2.41

Bone morphogenetic protein 4

2.04

Bone morphogenetic protein 5

4.39

Bone morphogenetic protein 6

2.12

Bone morphogenetic protein receptor, type IA

2.20

Bone morphogenetic protein receptor, type IB

2.56

Bone morphogenetic protein receptor, type II

2.37

Connective tissue growth factor

3.20

Insulin-like growth factor 1

2.61

Insulin-like growth factor 1 receptor

3.39

Insulin-like growth factor 2 receptor

3.42

Parathyroid hormone

3.09

Parathyroid hormone receptor 1

2.10

Vitamin D (1,25-dihydroxyvitamin D3) receptor

2.94

Estrogen receptor 1

3.02

Estrogen-related receptor gamma

3.98

Calcitonin /calcitonin-related polypeptide, alpha

2.01

Vascular endothelial growth factor

3.33

Transforming growth factor, beta 1

2.97

Transforming growth factor, beta 2

2.76

Transforming growth factor, beta receptor I

2.73

Alkaline phosphatase, liver /bone/kidney

2.14

Alkaline phosphatase

2.08

Prostaglandin I2 (prostacyclin) synthase

2.38

Prostaglandin I2 (prostacyclin) receptor (IP)

2.38

Mean ratio: ratio of the signal of a stretched sample to that of a nonstretched sample in four OPLL cells.

analog of prostacyclin, stimulated the expressions of osteogenic marker genes. Beraprost induced an oscillation of the intracellular calcium concentration in OPLL cells (Fig. 2), and c-fos activation and drugs that inhibited this oscillation concomitantly diminished the expressions of osteogenic marker genes and c-fos activation induced by beraprost (K.-I. Furukawa et al., unpublished observation, 2005). These observations

Fig. 2. Effects of beraprost on the intracellular calcium concentrations in OPLL and non-OPLL cells. Fura-2-loaded ligament cells were stimulated with 10 µM beraprost and the ratio of the fluorescence intensities of the cells after excitation at 340 and 380 nm (F340/F380) was recorded as an index of the intracellular calcium concentration in the cells.

suggest that mechanical stress accelerates the progression of OPLL via the production of various cytokines that initiate the activation of a signaling pathway through c-fos and intracellular calcium mobilization. Conclusion Mechanical stress is thought to play a key role in the progression of OPLL, at least in part through promoting the autocrine / paracrine mechanism of several cytokines involved in this lesion. The effects of mechanical stress on OPLL cells are presumed to be an initial step in the mechanically-induced ossification processes. These data may provide some insights into the role of mechanical stress in the ectopic bone formation found in other tissues. Acknowledgments I thank Drs. Yuji Yamamoto, Masahiko Tanno, Koei Iwasaki, Hirotaka Ohishi, Tomohiro Iwasawa, and Miki Hashimoto as well as Professors Satoshi Toh and Shigeru Motomura of Hirosaki University School of Medicine for their technical assistance and valuable discussions. I also acknowledge Associate Professor Ituro Inoue of the Institute of Medical Science, Tokyo University, for his support and valuable suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports, and Culture of Japan and by a Health and Labour Science Research Grant from the Ministry of

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Health, Labour, and Welfare. I also thank The Karohji Memorial Aid for Medical Study and Hirosaki University Educational Improvemental Promotional Aid for financial support.

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