Nacksung Kim

TRANCE, a TNF Family Member, Activates Akt/PKB through a Signaling Complex Involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.

v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation.

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H+) ATPase (v-ATPase) V0 domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.

Osteoclast differentiation independent of the TRANCE–RANK–TRAF6 axis

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor κB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE–RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-α in the presence of cofactors such as TGF-β. We provide direct evidence against the current paradigm that the TRANCE–RANK–TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Inhibition of Histone Deacetylation Blocks Cardiac Hypertrophy Induced by Angiotensin II Infusion and Aortic Banding

Background— A number of distinct stress signaling pathways in myocardium cause cardiac hypertrophy and heart failure. Class II histone deacetylases (HDACs) antagonize several stress-induced pathways and hypertrophy. However, cardiac hypertrophy induced by transgenic overexpression of the homeodomain only protein, HOP, can be prevented by the nonspecific HDAC inhibitors trichostatin A and valproic acid, suggesting that alternate targets that oppose class II HDAC function might exist in myocardium. We tested the effects of several HDAC inhibitors, including a class I HDAC-selective inhibitor, SK-7041, on cardiac hypertrophy induced by angiotensin II (Ang II) treatment or aortic banding (AB). Methods and Results— Cardiac hypertrophy was induced by chronic infusion of Ang II or by AB in mice or rats and evaluated by determining the ratio of heart weight to body weight or to tibia length, cross-sectional area, or echocardiogram. Cardiac hypertrophy induced by Ang II or AB for 2 weeks was significantly reduced by simultaneous administration of trichostatin A, valproic acid, or SK-7041. Echocardiogram revealed that exaggerated left ventricular systolic dimensions were relieved by HDAC inhibitors. HDAC inhibitors partially reversed preestablished cardiac hypertrophy and improved survival of AB mice. The expressions of atrial natriuretic factor, &agr;-tubulin, &bgr;-myosin heavy chain, and interstitial fibrosis were reduced by HDAC inhibition. Conclusions— These results suggest that the predominant effect of HDAC inhibition, mainly mediated by class I HDACs, is to prevent cardiac hypertrophy in response to a broad range of agonist and stretch stimuli.

A novel member of the leukocyte receptor complex regulates osteoclast differentiation.

Osteoclasts (OCs) are multinucleated cells that resorb bone and are essential for bone homeostasis. They develop from hematopoietic cells of the myelomonocytic lineage. OC formation requires cell-to-cell interactions with osteoblasts and can be achieved by coculturing bone marrow precursor cells with osteoblasts/stromal cells. Two of the key factors mediating the osteoblast-induced osteoclastogenesis are macrophage–colony stimulating factor (M-CSF) and the tumor necrosis factor (TNF) family member TNF–related activation-induced cytokine (TRANCE) that are produced by osteoblasts/stromal cells in response to various bone resorbing hormones. In addition, other factors produced by osteoblasts/stromal cells further influence osteoclastogenesis. Here we report the identification and characterization of OC-associated receptor (OSCAR), a novel member of the leukocyte receptor complex (LRC)-encoded family expressed specifically in OCs. Genes in the LRC produce immunoglobulin (Ig)-like surface receptors and play critical roles in the regulation of both innate and adaptive immune responses. Different from the previously characterized members of the LRC complex, OSCAR expression is detected specifically in preosteoclasts or mature OCs. Its putative–ligand (OSCAR-L) is expressed primarily in osteoblasts/stromal cells. Moreover, addition of a soluble form of OSCAR in coculture with osteoblasts inhibits the formation of OCs from bone marrow precursor cells in the presence of bone-resorbing factors, indicating that OSCAR may be an important bone-specific regulator of OC differentiation. In addition, this study suggests that LRC-encoded genes may have evolved to regulate the physiology of cells beyond those of the immune system.

Stimulation by Toll-Like Receptors Inhibits Osteoclast Differentiation

Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-κB activation and up-regulated TNF-α production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone.

The Mechanism of Osteoclast Differentiation Induced by IL-1

IL-1 is a potent cytokine that can induce bone erosion in inflammatory sites such as rheumatoid joint regions via activation of osteoclasts. Not only is IL-1 capable of activating osteoclasts, but it is also a key cytokine involved in the differentiation, multinucleation, and survival of osteoclasts. Herein, we show that IL-1 has the potential to drive osteoclast differentiation via a receptor activator of NF-κB ligand (RANKL)/RANK-independent mechanism. Although IL-1 has a synergistic effect on RANKL-induced osteoclast formation, IL-1 alone cannot induce osteoclast differentiation from osteoclast precursors (bone marrow-derived macrophages (BMMs)) due to a lack of IL-1 signaling potential in these cells. However, we demonstrate that overexpression of the IL-1RI receptor in BMMs or induction of IL-1RI by c-Fos overexpression enables IL-1 alone to induce the formation of authentic osteoclasts by a RANKL/RANK-independent mechanism. The expression of IL-1RI is up-regulated by RANKL via c-Fos and NFATc1. Furthermore, the addition of IL-1 to IL-1RI overexpressing BMMs (IL-1/IL-1RI) strongly activates NF-κB, JNK, p38, and ERK which is a hallmark gene activation profile of osteoclastogenesis. Interestingly, IL-1/IL-1RI does not induce expression of c-Fos or NFATc1 during osteoclast differentiation, although basal levels of c-Fos and NFATc1 seem to be required. Rather, IL-1/IL-1RI strongly activates MITF, which subsequently induces osteoclast-specific genes such as osteoclast-associated receptor and tartrate-resistant acid phosphatase. Together, these results reveal that IL-1 has the potential to induce osteoclast differentiation via activation of microphthalmia transcription factor under specific microenvironmental conditions.

Regulation of NFATc1 in Osteoclast Differentiation.

Osteoclasts are unique cells that degrade the bone matrix. These large multinucleated cells differentiate from the monocyte/macrophage lineage upon stimulation by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL). Activation of transcription factors such as microphthalmia transcription factor (MITF), c-Fos, NF-κB, and nuclear factor-activated T cells c1 (NFATc1) is required for sufficient osteoclast differentiation. In particular, NFATc1 plays the role of a master transcription regulator of osteoclast differentiation. To date, several mechanisms, including transcription, methylation, ubiquitination, acetylation, and non-coding RNAs, have been shown to regulate expression and activation of NFATc1. In this review, we have summarized the various mechanisms that control NFATc1 regulation during osteoclast differentiation.

Microphthalmia transcription factor and PU.1 synergistically induce the leukocyte receptor osteoclast-associated receptor gene expression.

We have recently reported the identification of a novel member of the leukocyte receptor family, osteoclast-associated receptor (OSCAR), which has two Ig-like domains and functions as a bone-specific regulator of osteoclast differentiation. Here, we have cloned the OSCAR promoter region to examine its regulation by transcription factors. The 1.7-kb promoter region of the mouse OSCAR gene contains two potential E-box elements for microphthalmia transcription factor (MITF) and three putative PU.1 sites. MITF or PU.1 alone activates the OSCAR reporter construct 5–6-fold, and the combination of MITF and PU.1 synergistically activates the OSCAR reporter activity up to 110-fold. The mRNA expression patterns of MITF, PU.1, and OSCAR in TRANCE-treated (RAW 264.7) or TRANCE/M-CSF-treated cells (primary osteoclasts) reveal that MITF mRNA expression is induced at a much earlier time point than OSCAR gene expression. In contrast to MITF, PU.1 mRNA levels remain relatively constant at all time points, suggesting that TRANCE-induced MITF, not PU.1 expression, is one of the critical regulatory mechanisms for optimal OSCAR expression during osteoclastogenesis. In addition, we have shown that the combination of MITF and constitutively active MKK6-expressing plasmids synergistically activates OSCAR reporter activity. Taken together, our results strongly suggest that PU.1 and MITF transcription factors synergistically activate OSCAR gene expression. Moreover, the activation of OSCAR gene expression by PU.1/MITF is further enhanced by the TRANCE-induced MKK6/p38 signaling cascade.

Akt Induces Osteoclast Differentiation through Regulating the GSK3β/NFATc1 Signaling Cascade

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P3 and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3β (phospho-GSK3β) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3β attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3β, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3β attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3β/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.