Hypoxia-induced SUMOylation of E3 ligase HAF determines specific activation of HIF2 in clear-cell renal cell carcinoma.

Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity.

A TRAF2 binding independent region of TNFR2 is responsible for TRAF2 depletion and enhancement of cytotoxicity driven by TNFR1.

Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity.

Ubiquitin-specific protease 25 regulates TLR4-dependent innate immune responses through deubiquitination of the adaptor protein TRAF3.

Protein ubiquitination plays a critical role in Toll-like receptor (TLR) signaling and innate immunity. Although several E3 ubiquitin ligases have been identified downstream of TLRs, the regulation of protein deubiquitination in TLR-triggered innate immune responses is poorly understood. We identified ubiquitin-specific protease 25 (USP25) as a regulator of TLR signaling. USP25 was recruited to the TLR4 signaling complex, and it associated with the adaptor proteins tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF6 after stimulation of TLR4 with its ligand lipopolysaccharide (LPS). USP25 specifically reversed the Lys(48)-linked ubiquitination of TRAF3 that was mediated by the E3 ubiquitin ligase cIAP2 (cellular inhibitor of apoptosis 2). Deficiency in USP25 enhanced the extent of ubiquitination of TRAF3 and accelerated its degradation after TLR4 activation, which potentiated TLR4-induced activation of NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase) signaling, but inhibited activation of the transcription factor IRF3 (interferon regulatory factor 3). USP25-deficient mice exhibited increased susceptibility to LPS-induced septic shock compared to their wild-type counterparts, which was associated with enhanced production of proinflammatory cytokines and decreased production of interferon-α. Thus, by inhibiting the degradation of TRAF3 during TLR4 activation, USP25 enables a balanced innate immune response.

Cycles of ubiquitination and deubiquitination critically regulate growth factor-mediated activation of Akt signaling.

K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor-mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor-induced ubiquitination of Akt have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. We showed that CYLD was a DUB for Akt and suppressed growth factor-mediated ubiquitination and activation of Akt. CYLD directly removed ubiquitin moieties from Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake, and, when injected into mice, growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in determining its plasma membrane localization and activation--and further identify CYLD as a molecular switch for these processes.

BSTA promotes mTORC2-mediated phosphorylation of Akt1 to suppress expression of FoxC2 and stimulate adipocyte differentiation.

Phosphorylation and activation of Akt1 is a crucial signaling event that promotes adipogenesis. However, neither the complex multistep process that leads to activation of Akt1 through phosphorylation at Thr³°8 and Ser47³ nor the mechanism by which Akt1 stimulates adipogenesis is fully understood. We found that the BSD domain-containing signal transducer and Akt interactor (BSTA) promoted phosphorylation of Akt1 at Ser47³ in various human and murine cells, and we uncovered a function for the BSD domain in BSTA-Akt1 complex formation. The mammalian target of rapamycin complex 2 (mTORC2) facilitated the phosphorylation of BSTA and its association with Akt1, and the BSTA-Akt1 interaction promoted the association of mTORC2 with Akt1 and phosphorylation of Akt1 at Ser47³ in response to growth factor stimulation. Furthermore, analyses of bsta gene-trap murine embryonic stem cells revealed an essential function for BSTA and phosphorylation of Akt1 at Ser47³ in promoting adipocyte differentiation, which required suppression of the expression of the gene encoding the transcription factor FoxC2. These findings indicate that BSTA is a molecular switch that promotes phosphorylation of Akt1 at Ser47³ and reveal an mTORC2-BSTA-Akt1-FoxC2-mediated signaling mechanism that is critical for adipocyte differentiation.

TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis.

Transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1), a mitogen-activated protein 3 (MAP3) kinase, plays an essential role in inflammation by activating the IκB kinase (IKK)/nuclear factor κB (NF-κB) and stress kinase (p38 and c-Jun N-terminal kinase [JNK]) pathways in response to many stimuli. The tumor necrosis factor (TNF) superfamily member receptor activator of NF-κB ligand (RANKL) regulates osteoclastogenesis through its receptor, RANK, and the signaling adaptor TRAF6. Because TAK1 activation is mediated through TRAF6 in the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways, we sought to investigate the consequence of TAK1 deletion in RANKL-mediated osteoclastogenesis. We generated macrophage colony-stimulating factor (M-CSF)-derived monocytes from the bone marrow of mice with TAK1 deletion in the myeloid lineage. Unexpectedly, TAK1-deficient monocytes in culture died rapidly but could be rescued by retroviral expression of TAK1, inhibition of receptor-interacting protein 1 (RIP1) kinase activity with necrostatin-1, or simultaneous genetic deletion of TNF receptor 1 (TNFR1). Further investigation using TAK1-deficient mouse embryonic fibroblasts revealed that TNF-α-induced cell death was abrogated by the simultaneous inhibition of caspases and knockdown of RIP3, suggesting that TAK1 is an important modulator of both apoptosis and necroptosis. Moreover, TAK1-deficient monocytes rescued from programmed cell death did not form mature osteoclasts in response to RANKL, indicating that TAK1 is indispensable to RANKL-induced osteoclastogenesis. To our knowledge, we are the first to report that mice in which TAK1 has been conditionally deleted in osteoclasts develop osteopetrosis.

Deletion of TAK1 in the myeloid lineage results in the spontaneous development of myelomonocytic leukemia in mice.

Previous studies of the conditional ablation of TGF-ß activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells' increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1's role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1's role in leukemogenesis.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) play essential roles in the C-type lectin receptor signaling in response to Candida albicans infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.

The E3 ubiquitin ligase TRAF6 intercedes in starvation-induced skeletal muscle atrophy through multiple mechanisms.

Starvation, like many other catabolic conditions, induces loss of skeletal muscle mass by promoting fiber atrophy. In addition to the canonical processes, the starvation-induced response employs many distinct pathways that make it a unique atrophic program. However, in the multiplex of the underlying mechanisms, several components of starvation-induced atrophy have yet to be fully understood and their roles and interplay remain to be elucidated. Here we unveiled the role of tumor necrosis factor receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase and adaptor protein, in starvation-induced muscle atrophy. Targeted ablation of TRAF6 suppresses the expression of key regulators of atrophy, including MAFBx, MuRF1, p62, LC3B, Beclin1, Atg12, and Fn14. Ablation of TRAF6 also improved the phosphorylation of Akt and FoxO3a and inhibited the activation of 5' AMP-activated protein kinase in skeletal muscle in response to starvation. In addition, our study provides the first evidence of the involvement of endoplasmic reticulum stress and unfolding protein response pathways in starvation-induced muscle atrophy and its regulation through TRAF6. Finally, our results also identify lysine 63-linked autoubiquitination of TRAF6 as a process essential for its regulatory role in starvation-induced muscle atrophy.

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response.

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

NF-kappaB signal triggering and termination by tumor necrosis factor receptor 2.

Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425-439), which also recruits TRAF2 and activates NF-κB. In addition to that, human TNFR2 contains a conserved region (amino acids 338-379) which is responsible for TRAF2 degradation and therefore of terminating NF-κB signaling. TRAF2 degradation and the lack of NF-κB activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation.

Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.

Skeletal muscle wasting is a major human morbidity, and contributes to mortality in a variety of clinical settings, including denervation and cancer cachexia. In this study, we demonstrate that the expression level and autoubiquitination of tumor necrosis factor (α) receptor adaptor protein 6 (TRAF6), a protein involved in receptor-mediated activation of several signaling pathways, is enhanced in skeletal muscle during atrophy. Skeletal muscle-restricted depletion of TRAF6 rescues myofibril degradation and preserves muscle fiber size and strength upon denervation. TRAF6 mediates the activation of JNK1/2, p38 mitogen-activated protein kinase, adenosine monophosphate-activated protein kinase, and nuclear factor κB, and induces the expression of muscle-specific E3 ubiquitin ligases and autophagy-related molecules in skeletal muscle upon denervation. Inhibition of TRAF6 also preserves the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria in denervated muscle. Moreover, depletion of TRAF6 prevents cancer cachexia in an experimental mouse model. This study unveils a novel mechanism of skeletal muscle atrophy and suggests that TRAF6 is an important therapeutic target to prevent skeletal muscle wasting.

Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice.

The transcription factor Osterix (Osx) is required for osteoblast differentiation and bone formation during embryonic development, but it is not known whether Osx has an essential function in postnatal bone growth and in bone homeostasis. Conditional deletion of Osx at several time points postnatally revealed that Osx was essential for osteoblast differentiation and new bone formation in growing and adult bones. Additionally, inactivation of Osx in bones severely disrupted the maturation, morphology, and function of osteocytes. These findings identify Osx as having an essential role in the cell-specific genetic program of osteocytes. Interestingly, Osx inactivation also led to the massive accumulation of unresorbed calcified cartilage in a large area below the growth plate of endochondral bones. This specific area was also marked by an unanticipated almost complete lack of bone marrow cells and a marked decrease in the density and size of osteoclasts. This diminished density of osteoclasts could contribute to the lack of resorption of mineralized cartilage. In addition, we speculate that the abnormally accumulated, mainly naked cartilage represents an unfavorable substrate for osteoclasts. Our study identifies Osx as an essential multifunctional player in postnatal bone growth and homeostasis.

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3-cell-induced osteoclast formation.

PURPOSE: Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. RESULTS: Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. EXPERIMENTAL DESIGN: We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. CONCLUSION: Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

Structural basis for the lack of E2 interaction in the RING domain of TRAF2.

TRAF proteins are intracellular signal transducers for a number of immune receptor superfamilies. Specifically, TRAF2 interacts with members of the TNF receptor superfamily and connects the receptors to downstream signaling proteins. It has been assumed that TRAF2 is a ubiquitin ligase like TRAF6 and mediates K63-linked polyubiquitination of RIP1, a kinase pivotal in TNFalpha-induced NF-kappaB activation. Here we report the crystal structure of the RING and the first zinc finger domains of TRAF2. We show that the TRAF2 RING structure is very different from the known TRAF6 RING structure. The differences are multifaceted, including amino acid differences at the critical Ubc13-interacting site, local conformational differences, and a unique nine-residue insertion between the RING domain and the first zinc finger in TRAF2. These structural differences prevent TRAF2 from interacting with Ubc13 and other related E2s via steric clash and unfavorable interfaces. Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFalpha signaling and may indicate that TRAF2-associated proteins such as cIAPs may be the ubiquitin ligases for NF-kappaB signaling.

The E3 ligase TRAF6 regulates Akt ubiquitination and activation.

Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. We found that the protein kinase Akt undergoes lysine-63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.

A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

E2 interaction and dimerization in the crystal structure of TRAF6.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.

Hypoxia-associated factor, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leading to its oxygen-independent degradation.

The hypoxia-inducible factor 1alpha (HIF-1alpha) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1alpha is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1alpha in normoxia. Here, we describe a new regulator of HIF-1alpha, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1alpha leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1alpha in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1alpha levels in normoxia and hypoxia in both pVHL-competent and -deficient cells, whereas HAF knockdown increases HIF-1alpha levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2alpha is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1alpha accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1alpha-specific degradation pathway that degrades HIF-1alpha through a new, oxygen-independent mechanism.

Cthrc1 is a positive regulator of osteoblastic bone formation.

BACKGROUND: Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors. METHODOLOGY/PRINCIPAL FINDINGS: We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.