Sunao Takeshita

TNF-α induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand

While TNF-alpha is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-alpha alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-kappaB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-alpha and RANKL markedly potentiate NF-kappaB and stress-activated protein kinase/c-Jun NH(2)-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-alpha prompts robust osteoclast formation in chimeric animals in which ss-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-alpha alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-alpha to promote osteoclastogenesis, TNF-alpha appears to be a more convenient target in arresting inflammatory osteolysis.

Identification and Characterization of a Novel Protein, Periostin, with Restricted Expression to Periosteum and Periodontal Ligament and Increased Expression by Transforming Growth Factor β

We had previously identified the cDNA for a novel protein called osteoblast‐specific factor 2 (OSF‐2) from an MC3T3‐E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF‐2, the protein was renamed “periostin.” Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast‐like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C‐terminal domain, possibly caused by alternative splicing events. Reverse transcription‐ polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin‐Fc recombinant protein supported attachment and spreading of MC3T3‐E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to βig‐h3, a molecule induced by transforming growth factor β (TGF‐β) that promotes the adhesion and spreading of fibroblasts. Because TGF‐β has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF‐β increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.

Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts.

Osteoclasts are thought to belong to a macrophage lineage. However, the nature of common precursors of osteoclasts and macrophages remains to be investigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage‐colony‐stimulating factor (M‐CSF) stimulated the proliferation of bone marrow macrophages in a dose‐dependent manner and these M‐CSF‐dependent bone marrow macrophage (MDBM) cells efficiently differentiated into the tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the presence of soluble RANKL (sRANKL) and M‐CSF in the in vitro culture. The macrophage‐like cell line TMC16 was established from tsA58 (temperature‐sensitive SV40 large T‐antigen) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and smaller (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [MNCs]) on day 4. The surface expression of macrophage marker CD14 was down‐regulated and that of CD43 was up‐regulated on pOC, analyzed by flow cytometry. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, and TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cells did not express these genes. However, the osteopontin (OPN) gene was strongly expressed in MDBM cells and this expression became weakened after differentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, and OPN, suggesting that the TMC16 cell line is immortalized at a stage slightly differentiated from MDBM cells. Furthermore, cell sorting analysis revealed that osteoclast early progenitors in bone marrow cells are preferentially present in the Mac‐1− F4/80dull population, which differentiated into MDBM cells (the osteoclast progenitor) expressing Mac‐1+ F4/80int, suggesting that M‐CSF plays roles of a differentiation factor as well as a growth factor for osteoclast early progenitors. These results showed the transition of morphology, surface markers, and gene expression from the early to mature stage in osteoclast differentiation. We propose three differentiation stages in the osteoclast lineage: the pro‐osteoclast (spindle‐shaped macrophage cells), the pre‐osteoclast (small round mononucleated TRAP‐positive cells), and the mature osteoclast (multinucleated TRAP‐positive cells) stage.

NF-κB p50 and p52 Regulate Receptor Activator of NF-κB Ligand (RANKL) and Tumor Necrosis Factor-induced Osteoclast Precursor Differentiation by Activating c-Fos and NFATc1

Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-κB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-κB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-κB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-κB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-κB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-κB should prevent RANKL- and TNF-induced bone resorption.

Coordination of PGC-1β and iron uptake in mitochondrial biogenesis and osteoclast activation

Osteoclasts are acid-secreting polykaryons that have high energy demands and contain abundant mitochondria. How mitochondrial biogenesis is integrated with osteoclast differentiation is unknown. We found that the transcription of Ppargc1b, which encodes peroxisome proliferator–activated receptor-γ coactivator 1β (PGC-1β), was induced during osteoclast differentiation by cAMP response element–binding protein (CREB) as a result of reactive oxygen species. Knockdown of Ppargc1b in vitro inhibited osteoclast differentiation and mitochondria biogenesis, whereas deletion of the Ppargc1b gene in mice resulted in increased bone mass due to impaired osteoclast function. We also observed defects in PGC-1β–deficient osteoblasts. Owing to the heightened iron demand in osteoclast development, transferrin receptor 1 (TfR1) expression was induced post-transcriptionally via iron regulatory protein 2. TfR1-mediated iron uptake promoted osteoclast differentiation and bone-resorbing activity, associated with the induction of mitochondrial respiration, production of reactive oxygen species and accelerated Ppargc1b transcription. Iron chelation inhibited osteoclastic bone resorption and protected against bone loss following estrogen deficiency resulting from ovariectomy. These data establish mitochondrial biogenesis orchestrated by PGC-1β, coupled with iron uptake through TfR1 and iron supply to mitochondrial respiratory proteins, as a fundamental pathway linked to osteoclast activation and bone metabolism.

Marrow Stromal Cells and Osteoclast Precursors Differentially Contribute to TNF-α-Induced Osteoclastogenesis In Vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-κB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-α-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-α. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-α treatment or whether the recipient animals possess TNF-α-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-α requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-α increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-α is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-α depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-α may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.

Activation of Renin-Angiotensin System Induces Osteoporosis Independently of Hypertension

Hypertension and osteoporosis are two major age‐related disorders; however, the underlying molecular mechanism for this comorbidity is not known. The renin‐angiotensin system (RAS) plays a central role in the control of blood pressure and has been an important target of antihypertensive drugs. Using a chimeric RAS model of transgenic THM (Tsukuba hypertensive mouse) expressing both the human renin and human angiotensinogen genes, we showed in this study that activation of RAS induces high turnover osteoporosis with accelerated bone resorption. Transgenic mice that express only the human renin gene were normotensive and yet exhibited a low bone mass, suggesting that osteoporosis occurs independently of the development of hypertension per se. Ex vivo cultures showed that angiotensin II (AngII) acted on osteoblasts and not directly on osteoclast precursor cells and increased osteoclastogenesis‐supporting cytokines, RANKL and vascular endothelial growth factor (VEGF), thereby stimulating the formation of osteoclasts. Knockdown of AT2 receptor inhibited the AngII activity, whereas silencing of the AT1 receptor paradoxically enhanced it, suggesting a functional interaction between the two AngII receptors on the osteoblastic cell surface. Finally, treatment of THM mice with an ACE inhibitor, enalapril, improved osteoporosis and hypertension, whereas treatment with losartan, an angiotensin receptor blockers specific for AT1, resulted in exacerbation of the low bone mass phenotype. Thus, blocking the synthesis of AngII may be an effective treatment of osteoporosis and hypertension, especially for those afflicted with both conditions.

The Transition of Cadherin Expression in Osteoblast Differentiation from Mesenchymal Cells: Consistent Expression of Cadherin‐11 in Osteoblast Lineage

Osteoblasts are derived originally from pluripotent mesenchymal stem cells on migration into the bone matrix. To elucidate the contribution of classical cadherins in this differentiation pathway, we developed a new protocol for their analysis and studied their specific expressions in various cell lines of the mesenchymal lineage, including osteoblasts. N‐cadherin was expressed constitutively in all cell lines examined except an osteocyte‐like cell line whereas cadherin‐11 was expressed selectively in preosteoblast and preadipocyte cell lines. P‐cadherin also was expressed in primary cultures of calvarial cells and mature osteoblasts at a relatively low level compared with N‐cadherin and cadherin‐11. M‐cadherin was expressed only in a premyoblast cell line. We observed the transition of cadherin expression from M‐cadherin to cadherin‐11 in the premyoblast cell line when osteogenic differentiation was induced by treatment with bone morphogenetic protein 2 (BMP‐2), while the expression of N‐cadherin remained unchanged. In contrast, when a preadipocyte cell line, which shows a similar pattern of cadherin expression to osteoblasts, was induced to undergo adipogenic differentiation, the expression of N‐cadherin and cadherin‐11 was decreased. These observations characterize the cadherin expression profile of mesenchymal lineage cells, especially osteoblasts, which regularly express cadherin‐11. Cadherin‐11 may affect cell sorting, alignment, and separation through differentiation.

Osteoclast-secreted CTHRC1 in the coupling of bone resorption to formation

Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule serves as a coupling factor for bone resorption to formation. We found that CTHRC1, a protein secreted by mature bone-resorbing osteoclasts, targets stromal cells to stimulate osteogenesis. Cthrc1 expression was robustly induced when mature osteoclasts were placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increased in a high-turnover state (such as that induced by RANKL injections in vivo), but decreased in conditions associated with suppressed bone turnover (such as with aging and after alendronate treatment). Targeted deletion of Cthrc1 in mice eliminated Cthrc1 expression in bone, whereas its deficiency in osteoblasts did not exert any significant effect. Osteoclast-specific deletion of Cthrc1 resulted in osteopenia due to reduced bone formation and impaired the coupling process after resorption induced by RANKL injections, impairing bone mass recovery. These data demonstrate that CTHRC1 is an osteoclast-secreted coupling factor that regulates bone remodeling.