Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL-RANK interaction

It is well known that thyroid hormone excess causes bone loss. However, the precise mechanism of bone loss by thyroid hormone still remains unclear. When T(3) was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of indomethacin and IL-6 Ab. T(3) increased the expression of osteoprotegerin (OPG) messenger RNA (mRNA), but not of receptor activator of nuclear factor kappaB ligand (RANKL) in unfractionated bone cells, suggesting that the stimulatory effect of T(3) on osteoclast formation was not mediated by the RANKL/OPG system. We next examined the direct effect of T(3) on osteoclast precursors in the absence of osteoblasts, using hemopoietic blast cells derived from spleen cells. T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors. OPG did not inhibit T(3)-induced osteoclast formation from osteoclast precursor cells. The polymerase chain reaction (PCR) product corresponding in size to the mouse T(3) receptor alpha1 cDNA was detected in osteoclast precursors from mouse hemopoietic blast cells as well as mouse heart and mouse osteoblastic cell line MC3T3-E1 cells, suggesting that T(3) directly stimulated osteoclast-like cell formation from osteoclast precursors in the absence of osteoblasts. Further, T(3) increased the expression of c-Fos mRNA at 15 min and 24 h and Fra-1 mRNA at 2 and 6 h in osteoclast precursors. Consistent with the increased expression of c-Fos mRNA observed by RT-PCR, the activation of c-Fos occurred in osteoclast precursor cells stimulated by T(3), while the activation of neither NF-kappaB nor MAPKs was observed by immunoblot analysis. Antisense oligodeoxynucleotides (as-ODN) complementary to c-Fos mRNA at 1 microM significantly inhibited T(3)-induced osteoclast-like cell formation from osteoclast precursors in the absence of stromal cells while sense-ODN did not affect T(3)-induced osteoclast-like cell formation. These results indicate that T(3) directly stimulates osteoclast differentiation at least in part by up-regulation of c-fos protein in osteoclast precursor cells.     

Activated MEK Stimulates Expression of AP-1 Components Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but Requires a PI3-Kinase Signal To Stimulate DNA Synthesis

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.     

An Inducible,Ligand-Independent Receptor Activator of NF-κB Gene to Control Osteoclast Differentiation from Monocytic Precursors

Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.     

An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry

Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G(0)-to-G(1) transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1- and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G(1). Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G(0)-to-G(1) transition of the cell cycle.     

Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins.

We have determined the different Fos/Jun complexes present in Swiss 3T3 cells either following serum stimulation of quiescent cells or during exponential growth by immunoprecipitation analyses. We have shown that while c-Fos is the major Fos protein associated with the Jun proteins (c-Jun, JunB, and JunD) soon after serum stimulation, at later times Fra-1 and Fra-2 are the predominant Fos proteins associated with the different Jun proteins. During exponential growth, the synthesis of Fra-1 and Fra-2 is maintained at a significant level, in contrast to c-Fos and FosB, which are expressed at very low or undetectable levels. Consequently, Fra-1 and Fra-2 are the main Fos proteins complexed with the Jun proteins in asynchronously growing cells. To determine whether the Fos proteins are differentially required during the G0-to-G1 transition and exponential growth for the entrance into S phase, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, and Fra-2. We have found that while the activities of c-Fos and FosB are required mostly during the G0-to-G1 transition, Fra-1 and Fra-2 are involved both in the G0-to-G1 transition and in asynchronous growth.     

Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation

Osteoblasts/stromal cells support the formation of osteoclast-like cells (OCL) from osteoclast progenitor cells via expressing a membrane-associated protein, osteoclast differentiation factor (ODF), in the presence of osteotropic factors, whereas the cells secrete a substantial amount of osteoclastogenesis inhibitory factor (OCIF) in the unstimulated state. There are both OCL formation-supporting and the nonsupporting cell lines in osteoblasts/stromal cell lineages. The mechanism that divides osteoblasts/stromal cell lines into the two types is not known. The present study reports that OCL formation-supporting cell line ST2 showed a greatly increased level of ODF mRNA, whereas their OCIF mRNA was drastically diminished in the presence of 1alpha, 25(OH)2-dihydroxyvitamin D3 or prostaglandin E2. In contrast, MC3T3-E1 cells lacking OCL formation-supporting ability did not show a decrease in OCIF mRNA in response to the factors, despite a similar increase in ODF mRNA as ST2 cells. However, inactivated MC3T3-E1 cells secreting nothing supported OCL formation in coculture with human promyelocytic cells, HL60. On the contrary, ST2 cells did not support OCL formation from HL60 cells when cocultured in medium conditioned by 1alpha, 25(OH)2 vitamin D3-treated MC3T3-E1. These findings indicate that reciprocal gene expression of ODF and OCIF in osteoblasts/stromal cells is essential for supporting OCL formation.     

Expression and possible role of PVR/CD155/Necl-5 in osteoclastogenesis

Osteoclasts, the bone-resorbing cells, are differentiated from hematopoietic precursors via two-step cell–cell interactions. One is the interaction between the osteoclast precursor and the stromal cell to initiate differentiation. The other is the interaction among osteoclast precursors to form multinucleated osteoclasts. Recently, the poliovirus receptor (PVR, CD155, Necl-5) was reported to play important roles in cell adhesion and migration. However, there are no reports of PVR in osteoclastogenesis. In this paper, we examined the expression of PVR and its ligand, DNAX accessory molecule-1 (DNAM-1, CD226), in osteoclast precursors, mature osteoclasts, and stromal cells. We found that the PVR was constitutively expressed in both osteoclast cells and stromal cells. The expression of PVR was not changed at various stages of osteoclast formation. In contrast, the expression of DNAM-1 was observed in mononuclear cells and was down-regulated during osteoclastogenesis. Moreover, multinucleated osteoclast formation was inhibited by treatment with the extracellular domain of DNAM-1 (ED-DNAM-1) as a soluble ligand for PVR, but mononuclear preosteoclast formation was not affected. Especially, during the 7-day cultivation, osteoclast formation was suppressed by the treatment with ED-DNAM-1 on days 6 and 7, when the mononuclear preosteoclasts fused into multinucleated osteoclasts. This suppression was abrogated partially by a small interfering RNA specific for PVR. These results suggest that, at least in part, the binding of PVR with DNAM-1 negatively regulates osteoclast formation. Furthermore, our results indicate that the cellular fusion process may be inhibited by the PVR-mediated signaling.     

MHC class II transactivator negatively regulates RANKL-mediated osteoclast differentiation by downregulating NFATc1 and OSCAR

Nuclear factor of activated T cells (NFAT) c1 plays a key role in receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and function via induction of osteoclast-specific target genes including osteoclast-associated receptor (OSCAR), cathepsin K, and tartrate-resistant acid phosphatase. To elucidate which downstream target genes are regulated by NFATc1 during osteoclastogenesis, we used microarray analyses to examine gene expression profiles in the context of bone marrow-derived macrophages overexpressing a constitutively active form of NFATc1. Herein, we demonstrate that MHC class II transactivator (CIITA) is up-regulated downstream of NFATc1. Overexpression of CIITA in osteoclast precursors attenuates RANKL-induced osteoclast formation through down-regulation of NFATc1 and OSCAR. Epigenetic overexpression of CIITA regulates NFATc1 and OSCAR by competing with c-Fos and NFATc1 for CBP/p300 binding sites. Furthermore, silencing of CIITA by RNA interference in osteoclast precursors enhances osteoclast formation as well as NFATc1 and OSCAR expression. Taken together, our data reveal that CIITA can act as a modulator of RANKL-induced osteoclastogenesis.     

Selective cytotoxicity of ascochlorin in ER-negative human breast cancer cell lines

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.