We established four types of mouse models exhibiting various speeds of OA progression. By applying a mouse genomics approach to the models, molecular backgrounds in various stages of OA development can be clarified.

Objective. By producing instability in mouse knee joints, we attempted to determine the involvement of runt-related transcription factor 2 (RUNX-2), which is required for chondrocyte hypertrophy, in the development of osteoarthritis (OA).Methods. An experimental mouse OA model was created by surgical transection of the medial collateral ligament and resection of the medial meniscus of the knee joints of heterozygous RUNX-2-deficient (Runx2 ؉/؊ ) mice and wild-type littermates. Cartilage destruction and osteophyte formation in the medial tibial cartilage were compared by histologic and radiographic analyses. Localization of type X collagen and matrix metalloproteinase 13 (MMP-13) was examined by immunohistochemistry. Localization of RUNX-2 was determined by X-Gal staining in heterozygous RUNX-2-deficient mice with the lacZ gene insertion at the Runx2-deletion site (Runx2 ؉/lacZ ). Messenger RNA levels of type X collagen, MMP-13, and RUNX-2 were examined by real-time reverse transcriptasepolymerase chain reaction analysis.Results. RUNX-2 was induced in the articular cartilage of wild-type mice at the early stage of OA, almost simultaneously with type X collagen but earlier than MMP-13. Runx2 ؉/؊ and Runx2 ؉/lacZ mice showed normal skeletal development and articular cartilage; however, after induction of knee joint instability, they exhibited decreased cartilage destruction and osteophyte formation, along with reduced type X collagen and MMP-13 expression, as compared with wild-type mice.Conclusion. RUNX-2 contributes to the pathogenesis of OA through chondrocyte hypertrophy and matrix breakdown after the induction of joint instability.Osteoarthritis (OA), a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation, is a major cause of disability in the elderly. Despite significant demand for more information, risk factors for this disease, as identified by epidemiologic studies, have to date been limited to age, obesity, trauma history, occupation, and sex (1,2). Since these factors are closely related to the accumulation of mechanical loading on joints, mechanical instability of the joints may play some role in OA pathogenesis. In an effort to clarify the mechanisms whereby joint instability leads to the development of OA, experimental animal models of OA induced by producing instability in the joints by surgical intervention have been developed in dogs, rabbits, guinea pigs, and rats (3-9). Due to recent progress in mouse genomics and the availability of transgenic and knockout mice, the mouse is currently the ideal animal for molecular study. Using a microsurgical technique to produce instability in the knee joints of mice, we established models of mechanical instabilityinduced OA that were reproducible and resembled OA in humans (10).

The Komeda miniature rat Ishikawa (KMI) is a naturally occurring mutant caused by an autosomal recessive mutation mri, which exhibits longitudinal growth retardation. Here we identified the mri mutation as a deletion in the rat gene encoding cGMP-dependent protein kinase type II (cGKII). KMIs showed an expanded growth plate and impaired bone healing with abnormal accumulation of postmitotic but nonhypertrophic chondrocytes. Ex vivo culture of KMI chondrocytes reproduced the differentiation impairment, which was restored by introducing the adenovirus-mediated cGKII gene. The expression of Sox9, an inhibitory regulator of hypertrophic differentiation, persisted in the nuclei of postmitotic chondrocytes of the KMI growth plate. Transfection experiments in culture systems revealed that cGKII attenuated the Sox9 functions to induce the chondrogenic differentiation and to inhibit the hypertrophic differentiation of chondrocytes. This attenuation of Sox9 was due to the cGKII inhibition of nuclear entry of Sox9. The impaired differentiation of cultured KMI chondrocytes was restored by the silencing of Sox9 through RNA interference. Hence, the present study for the first time shed light on a novel role of cGKII as a molecular switch, coupling the cessation of proliferation and the start of hypertrophic differentiation of chondrocytes through attenuation of Sox9 function.

This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-B ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage.Fibroblast growth factors (FGFs) 1 are potent mitogens for a wide variety of cells of mesenchymal and neuroectodermal origin (1). FGFs also play a role in the differentiation of a variety of cells and are involved in morphogenesis, angiogenesis, and development. The FGF family now consists of 23 members, FGF-1 to FGF-23, and there are 4 structurally related highaffinity receptors (FGFR1 to FGFR4) belonging to receptor tyrosine kinases that have an intrinsic protein tyrosine kinase activity and elicit tyrosine autophosphorylation of the receptor (1, 2). Recent reports showing that mutations of FGFRs cause several genetic diseases with severe impairment of bone and cartilage formation, such as achondroplasia (3, 4) and thanatophoric dysplasia type II (5), indicate the essential role of FGF signalings on bone and cartilage metabolism.Among FGFs, FGF-2 is well known as a potent regulator of functions of bone and cartilage cells. It is produced by cells of osteoblastic lineage, accumulated in bone matrix, and acts as an autocrine/paracrine factor for bone cells (6 -8). We and others have reported that the exogenous application of FGF-2 has stimulatory effects on bone formation in several in vivo models as a pharmacological action (9 -11). In addition, the Fgf-2-deficient mouse exhibits decreased bone mass and bone formation, although these changes were rather moderate (12). Paradoxically, FGF-2 is also known as a potent stimulator of bone resorption (13-17) and is involved in joint destruction of rheumatoid arthritis patients (18). The stimulatory effect of FGF-2 on osteoclast formation is mediated by the induction of cyclooxygenase-2, a main regulatory enzyme for prostagland...

The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects.

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3-E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP-2, -3, -9, -11, -13, and -14 or TIMP-1, -2, and -3 was detected in MC3T3-E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25-dihydroxyvitamin D(3), prostaglandin E(2), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) were added to the cell cultures, MMP-13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP-3 and -9 or TIMP-1 and -3 were found to increase mainly by the cytokines IL-1beta and TNF-alpha. BB94, a nonselective MMP inhibitor, neutralized the (45)Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP-13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP-3 and -9 or TIMP-1 and -3 might modulate matrix degradation by local cytokines only.

Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1 ؊/؊ mice). Among 15 IRS-1 ؊/؊ mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1 ؊/؊ fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1 ؊/؊ chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-Istimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1 ؊/؊ culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF-2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF-2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS-398, a selective cyclo-oxygenase 2 (COX-2) inhibitor.