Kazuhisa Nakashima

The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation

We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.

Regulatory mechanisms in the pathways of cartilage and bone formation.

Three transcription factors of the Sox family have essential roles in different steps of the chondrocyte differentiation pathway. Because the transcription factor Cbfa1, which is needed for osteoblast differentiation, also stimulates hypertrophic chondrocyte maturation, it links the chondrocyte and osteoblast differentiation pathways in endochondral bone formation. Signaling molecules, including Indian Hedgehog, PTHrP and FGFs, also establish essential links either between these pathways, between steps in these pathways or between signaling molecules and transcription factors, so that a more comprehensive view of endochondral bone formation is emerging.

NFAT and Osterix cooperatively regulate bone formation

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Transcriptional mechanisms in osteoblast differentiation and bone formation.

Osteoblasts, the cells responsible for bone formation, differentiate from mesenchymal cells. Here, we discuss transcription factors that are involved in regulating the multistep molecular pathway of osteoblast differentiation. Runx2 and Osx, a newly identified zinc-finger-containing protein, are transcription factors that are expressed selectively and at high levels in osteoblasts. Null mutations of either leads to a complete absence of bone in mice. Runx2 plus its companion subunit Cbf beta are needed for an early step in this pathway, whereas Osx is required for a subsequent step, namely the differentiation of preosteoblasts into fully functioning osteoblasts. The finding that Osx-null cells acquire a chondrocyte phenotype implies that Osx is a negative regulator of Sox9 and of the chondrocyte phenotype. This leads to the hypothesis that Osx might have a role in the segregation of osteoblasts from osteochondroprogenitors. We also discuss recent progress in studies of other transcription factors that affect skeletal patterning and development.

Unloading Induces Osteoblastic Cell Suppression and Osteoclastic Cell Activation to Lead to Bone Loss via Sympathetic Nervous System

Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts.

Potent mitogenic effects of parathyroid hormone (PTH) on embryonic chick and rabbit chondrocytes. Differential effects of age on growth, proteoglycan, and cyclic AMP responses of chondrocytes to PTH.

The effect of PTH on chondrocyte proliferation as a function of cartilage age was examined. PTH[1-34] induced a 12- to 15-fold increase in the efficiency of colony formation in soft agar by chondrocytes from embryonic 13- to 19-d-old chickens and fetal 25-d-old rabbits with a 10-fold increase in their DNA content. It also caused a 2.5-fold increase in [3H]thymidine incorporation into DNA in fetal 25-d-old rabbit chondrocytes. No mitogenic responses to PTH were observed, however, in postnatal 7- to 21-d-old chick chondrocytes or postnatal 21-d-old rabbit chondrocytes. This age dependency was observed only with PTH: fibroblast growth factor, epidermal growth factor, and insulin stimulated chondrocyte proliferation irrespective of cartilage age. The absence of a mitogenic effect in postnatal chondrocytes was not due to a decrease in number or a reduction in affinity of receptors for PTH. PTH also increased [35S]sulfate incorporation into proteoglycans and the cyclic AMP level in fetal and postnatal chondrocytes, but at 100-fold higher concentrations (10(-8)-10(-7) M) than those (10(-10)-10(-9) M) required for the stimulation of cell division. These results suggest that PTH is a potent mitogen for embryonic chondrocytes, and that its mitogenic effect disappears selectively after birth.

Angiotensin II Type 2 Receptor Blockade Increases Bone Mass

Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass.

Transient receptor potential vanilloid 4 deficiency suppresses unloading‐induced bone loss

Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading‐induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading‐induced bone loss. Hind limb unloading induced osteopenia in wild‐type mice. In contrast, TRPV4 deficiency suppressed such unloading‐induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading‐induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading‐induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading‐induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading‐induced bone loss. J. Cell. Physiol. 216: 47–53, 2008.

Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein–induced bone formation

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)–induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2–induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.