Receptor activator of NF-B ligand (RANKL) and its receptor activator of NF-B (RANK) play pivotal roles in osteoclast differentiation and function. However, the structural determinants of the RANK that mediate osteoclast formation and function have not been definitively identified. To address this issue, we developed a chimeric receptor approach that permits a structure/function study of the RANK cytoplasmic domain in osteoclasts. Using this approach, we examined the role of six RANK putative tumor necrosis factor receptorassociated factor-binding motifs ( initiates NF-B, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 signaling pathways and PVQEET 559 -564 activates NF-B and p38 pathways in osteoclasts, whereas PVQEQG 604 -609 is only capable of activating NF-B pathway. Significantly, the revelation of these functional RANK cytoplasmic motifs has not only laid a foundation for further delineating RANK signaling pathways in osteoclasts, but, more importantly, these RANK motifs themselves represent potential therapeutic targets for bone disorders such as osteoporosis.Osteoclasts, the principal bone-resorbing cells, play a pivotal role in skeleton development and maintenance (1). Osteoclasts are derived from mononuclear precursors of monocyte/macrophage lineage upon stimulation of two key factors: monocyte/ macrophage colony-stimulating factor (M-CSF) 1 and receptor activator of nuclear factor B (RANKL, also known as OPG ligand/osteoclast differentiation factor/TNF-related activationinduced cytokine) (1-3). RANKL was identified as a member of the TNF superfamily independently by several groups in the late 1990s (4 -7). RANKL regulates both osteoclast formation and function by binding to its receptor RANK expressed on osteoclast precursors and mature osteoclasts (4, 8, 9). The essential role of both RANKL and RANK in the osteoclastogenic process has been well demonstrated by the findings that mice lacking the gene for either protein develop osteopetrosis caused by failure to form osteoclasts (10 -12).RANK was identified as a member of the TNF receptor family (6). Members of the TNF receptor family are characterized by a lack of intrinsic enzymatic activity, and thus they usually transduce intracellular signals by recruiting various adaptor proteins such as TNF receptor-associated factors (TRAFs) through the specific motifs in their cytoplasmic domains (13-15). Since the unraveling of the RANKL/RANK system, enormous efforts have been undertaken to elucidate RANK-initiated intracellular signaling. Particularly, many of the previous works have been focused on characterizing the receptor-proximal signaling events (8, 16 -20), which represent the initial and critical component of intracellular signaling pathways initiated by membrane-bound receptors. Although these studies have mapped RANK cytoplasmic regions capable of interacting with TRAF proteins by various in vitro binding assays (8, 16 -20), the physiological relevance of these data to osteoclast biology remains largely unexplored.In the pre...

Chloroplast biogenesis is indispensable for proper plant development and environmental acclimation. In a screen for mutants affected in photosynthesis, we identified the protein phosphatase7-like (pp7l) mutant, which displayed delayed chloroplast development in cotyledons and young leaves. PP7L, PP7, and PP7-long constitute a subfamily of phosphoprotein phosphatases. PP7 is thought to transduce a blue-light signal perceived by crys and phy a that induces expression of SIGMA FACTOR5 (SIG5). We observed that, like PP7, PP7L was predominantly localized to the nucleus in Arabidopsis (Arabidopsis thaliana), and the pp7l phenotype was similar to that of the sig6 mutant. However, SIG6 expression was unaltered in pp7l mutants. Instead, loss of PP7L compromised translation and ribosomal RNA (rRNA) maturation in chloroplasts, pointing to a distinct mechanism influencing chloroplast development. Promoters of genes deregulated in pp7l-1 were enriched in PHYTOCHROME-INTERACTING FACTOR (PIF)-binding motifs and the transcriptome of pp7l-1 resembled those of pif and CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) signalosome complex (csn) mutants. However, pif and csn mutants, as well as cop1, cryptochromes (cry)1 cry2, and phytochromes (phy)A phyB mutants, do not share the pp7l photosynthesis phenotype. PhyB protein levels were elevated in pp7l mutants, but phyB overexpression plants did not resemble pp7l. These results indicate that PP7L operates through a different pathway and that the control of greening and photosystem biogenesis can be separated. The lack of PP7L increased susceptibility to salt and high-light stress, whereas PP7L overexpression conferred resistance to highlight stress. Strikingly, PP7L was specifically recruited to Brassicales for the regulation of chloroplast development. This study adds another player involved in chloroplast biogenesis.

Receptor activator of NF-B (RANK) ligand (RANKL) and its receptor RANK play an essential role in osteoclastogenesis and osteoclast function by activating several signaling pathways. However, several lines of evidence suggest that RANK also transmits an unidentified signaling pathway(s) essential for osteoclastogenesis. To identify the novel pathway(s), we carried out a detailed structure/function study of the RANK cytoplasmic domain. A series of studies using numerous deletion/point mutants elucidated a specific 4-amino acid motif ( 535 IVVY 538 ) essential for osteoclastogenesis. This novel motif plays a crucial role in committing macrophages to the osteoclast lineage but is not implicated in osteoclast function or survival. Moreover, this motif does not activate the known RANK signaling pathways, indicating that it initiates a novel pathway(s). The identification of the novel motif not only provides critical insight into RANK signaling in osteoclastogenesis, but more importantly, the RANK motif and its downstream signaling pathways may represent specific therapeutic targets for various bone diseases, including postmenopausal osteoporosis.Receptor activator of nuclear factor B ligand (RANKL), 3 also known as OPGL (osteoprotegerin (OPG) ligand)/ODF (osteoclast differentiation factor)/TRANCE (tumor necrosis factor (TNF)-related activationinduced cytokine), was identified as a member of the TNF superfamily independently by two bone groups (1, 2) and two immunology groups (3, 4) in the late 1990s. To date, RANKL has been shown to play pivotal roles in regulating various biological processes such as bone homeostasis (2, 5), immune function (3, 6) and mammary gland development (7).In bone, RANKL is not only an essential regulator of osteoclast formation (osteoclastogenesis) but also a potent modulator of osteoclast function and survival (5,8,9). RANKL exerts its effects on osteoclast formation and function by binding to its receptor RANK (receptor activator of nuclear factor B) expressed on osteoclast precursors and mature osteoclasts (1, 10, 11). RANKL also has a decoy receptor, OPG, which inhibits RANKL action by competing with RANK for binding RANKL (5,12). The essential role of both RANKL and RANK in the osteoclastogenic process has been unambiguously demonstrated by the findings that mice lacking the gene for either protein develop osteopetrosis due to failure to form osteoclasts (13)(14)(15).Because the unraveling of the RANKL/RANK system, enormous efforts have been focused on elucidating RANK-initiated intracellular signaling. RANK was identified as a member of the TNF receptor (TNFR) family (3). Members of the TNFR family lack intrinsic enzymatic activity, and hence, they transduce intracellular signals by recruiting various adaptor proteins including TNF receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic domain (16,17). Six TRAF proteins (TRAF1, -2, -3, -4, -5, and -6) have been identified so far, and they play important but distinct roles in TNFR member-induced intracellular sign...

Tumor necrosis factor-␣ (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-B (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY 535-538 motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.TNF, a proinflammatory cytokine produced by many cells including fibroblasts, endothelial cells, and macrophages, plays a critical role in immune and inflammatory responses (1). Consequently, aberration in TNF expression and function has been associated with many autoimmune disorders and inflammatory complications (2). Moreover, this potent proinflammatory cytokine has also been shown to be a pro-osteoclastogenic factor and thus promotes bone loss in postmenopausal osteoporosis (3) and inflammatory conditions such as rheumatoid arthritis (4) and periodontitis (5) by stimulating osteoclast formation and function.Osteoclasts, the sole bone-resorbing cells in the body, are multinucleated giant cells derived from mononuclear cells of the monocyte-macrophage lineage upon stimulation by the macrophage/monocyte colony-stimulating factor (M-CSF) 2 and RANKL (6). RANKL (also known as OPGL, ODF, and TRANCE), identified as a member of the TNF superfamily (7, 8), stimulates osteoclast formation and function by activating its receptor RANK, a member of the TNF receptor (TNFR) superfamily (8). RANKL also has a soluble decoy receptor, osteoprotegerin (OPG), which inhibits RANKL functions by competing with RANK for binding RANKL (9). Members of the TNFR superfamily primarily employ TNF re...