Joel Jules

Myeloid-derived suppressor cells function as novel osteoclast progenitors enhancing bone loss in breast cancer.

Enhanced bone destruction is a hallmark of various carcinomas such as breast cancer, where osteolytic bone metastasis is associated with increased morbidity and mortality. Immune cells contribute to osteolysis in cancer growth, but the factors contributing to aggressive bone destruction are not well understood. In this study, we show the importance of myeloid-derived suppressor cells (MDSC) in this process at bone metastatic sites. Because MDSC originate from the same myeloid lineage as macrophages, which are osteoclast precursors, we hypothesized that MDSC may undergo osteoclast differentiation and contribute to enhanced bone destruction and tumor growth. Using an immunocompetent mouse model of breast cancer bone metastasis, we confirmed that MDSC isolated from the tumor-bone microenvironment differentiated into functional osteoclasts both in vitro and in vivo. Mechanistic investigations revealed that nitric oxide signaling was critical for differentiation of MDSC into osteoclasts. Remarkably, osteoclast differentiation did not occur in MDSC isolated from control or tumor-bearing mice that lacked bone metastasis, signifying the essential cross-talk between tumor cells and myeloid progenitors in the bone microenvironment as a requirement for osteoclast differentiation of MDSC. Overall, our results identify a wholly new facet to the multifunctionality of MDSC in driving tumor progression, in this case as a novel osteoclast progenitor that specifically drives bone metastasis during cancer progression.

Selective targeting of RANK signaling pathways as new therapeutic strategies for osteoporosis

Importance of the field: Osteoporosis has become a worldwide health and social issue due to an aging population. Four major antiresorptive drugs (agents capable of inhibiting osteoclast formation and/or function) are currently available on the market: estrogen, selective estrogen receptor modulators (SERMs), bisphosphonates and calcitonin. These drugs either lack satisfactory efficacy or have potential to cause serious side effects. Thus, development of more efficacious and safer drugs is warranted. Areas covered in this review: The discovery of the receptor activator of NF-κB ligand (RANKL) and its two receptors, RANK and osteoprotegerin (OPG), has not only established a crucial role for the RANKL/RANK/OPG axis in osteoclast biology but also created a great opportunity to develop new drugs targeting this system for osteoporosis therapy. This review focuses on discussion of therapeutic targeting of RANK signaling. What the reader will gain: An update on the functions of RANKL and an overview of the known RANK signaling pathways in osteoclasts. A discussion of rationales for exploring RANK signaling pathways as potent and specific therapeutic targets to promote future development of better drugs for osteoporosis. Take home message: Several RANK signaling components have the potential to serve as potent and specific therapeutic targets for osteoporosis.

Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing.

BackgroundRNA interference (RNAi) was originally identified as a biological process in which short double-stranded RNA (dsRNA) suppress the expression of genes complimentary to the dsRNA. This cellular intrinsic gene silencing mechanism has subsequently been developed as a useful tool for studies of gene function. A major strategy for producing small interfering RNA (siRNA) in cultured cells involves the use of siRNA expression vectors in which a RNA polymerase III (Pol III) promoter and transcription stop signal are designed to constitute a functional siRNA expression cassette for production of siRNA. However, most of the available vectors contain only one siRNA expression cassette.ResultsIn order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed vectors containing multiple (up to six) tandem siRNA expression cassettes. Moreover, we demonstrated that these vectors can be used not only to produce different siRNA to simultaneously suppress the expression of multiple genes but also to maximize the silencing of a singe gene.ConclusionThe vectors containing multiple siRNA expression cassettes can serve as useful tools for maximizing the efficiency of gene silencing.

Molecular basis of requirement of receptor activator of nuclear factor κB signaling for interleukin 1-mediated osteoclastogenesis.

Background: Interleukin 1-mediated osteoclastogenesis requires permissive levels of RANKL or RANKL pretreatment. Results: Interleukin 1 can only activate the expression of osteoclast markers and osteoclastogenic transcription factor NFATc1 with permissive levels of RANKL or RANKL pretreatment. Conclusion: RANKL is involved in interleukin 1-mediated osteoclastogenesis by rendering osteoclast and NFATc1 genes responsive to interleukin 1. Significance: This is a novel mechanism of interleukin 1-mediated osteoclastogenesis. IL-1, a proinflammatory cytokine, is implicated in bone loss in various pathological conditions by promoting osteoclast formation, survival, and function. Although IL-1 alone can sufficiently prolong osteoclast survival and activate osteoclast function, IL-1-mediated osteoclastogenesis requires the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular basis of the dependence of IL-1-mediated osteoclastogenesis on RANKL is not fully understood. Here we show that although IL-1 cannot activate the expression of the osteoclast genes encoding matrix metalloproteinase 9, cathepsin K, tartrate-resistant acid phosphatase, and carbonic anhydrase II in bone marrow macrophages (BMMs), RANKL renders these osteoclast genes responsive to IL-1. We further demonstrate that IL-1 alone fails to induce the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a master transcriptional regulator of osteoclastogenesis), in BMMs but can up-regulate its expression in the presence of permissive levels of RANKL or with RANKL pretreatment. The RANK IVVY motif, which has been previously shown to commit BMMs to the osteoclast lineage in RANKL- and TNF α-mediated osteoclastogenesis, also plays a crucial role in IL-1-mediated osteoclastogenesis by changing the four osteoclast marker and NFATc1 genes to an IL-1-inducible state. Finally, we show that MyD88, a known critical component of the IL-1 receptor I signaling pathway, plays a crucial role in IL-1-mediated osteoclastogenesis from RANKL-primed BMMs by up-regulating the expression of the osteoclast marker and NFATc1 genes. This study reveals a novel mechanism of IL-1-mediated osteoclastogenesis and supports the promising potential of the IVVY motif to serve as a therapeutic target for inflammatory bone loss.

Cbfβ deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfβ required for skeletal development

Significance Cleidocranial dysplasia (CCD) is a hereditary human skeletal disease. Mutations in Runt-related transcription factor 2, which functions as a heterodimer with core binding factor β (Cbfβ), are found in most individuals with CCD. It has been suspected that Cbfβ may be responsible for other CCD cases. The pathogenesis of CCD and the role of Cbfβ in postnatal skeletogenesis remain unclear. There has been no animal model to study this disease. We demonstrate that ablation of Cbfβ in various skeletal cells results in severe craniofacial and skeletal dysplasia with the phenotype recapitulating clinical features of CCD. The findings from this study of Cbfβ in the skeleton provide insight into the role of Cbfβ in postnatal skeletogenesis and pathogenesis of CCD, which may assist in developing new therapies for CCD and osteoporosis. The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor β (Cbfβ) and the Runt-related transcription factor (RUNX)/Cbfβ complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfβ ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfβf/fOsterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfβ may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfβf/fCollagen, type 1, alpha 1 (Col1α1)–Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfβ results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfβ directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfβ highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfβ plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfβ heterodimeric complex in various skeletal cells. These insights into the role of Cbfβ in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.

Receptor Activator of NF-κB (RANK) Cytoplasmic IVVY535–538 Motif Plays an Essential Role in Tumor Necrosis Factor-α (TNF)-mediated Osteoclastogenesis

Tumor necrosis factor-α (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-κB (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 IVVY535–538 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.

Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases.

Chondrocyte-specific Knockout of Cbfβ Reveals the Indispensable Function of Cbfβ in Chondrocyte Maturation, Growth Plate Development and Trabecular Bone Formation in Mice

Despite years of research into bone formation, the mechanisms by which transcription factors specify growth plate development and trabecular bone formation remain unclear and the role of hypertrophic chondrocytes in trabeculae morphogenesis is controversial. To study the role of Core binding factor beta (Cbfβ) in postnatal cartilage development and endochondral bone formation, we generated chondrocyte-specific Cbfβ-deficient mice (Cbfβf/fCol2α1-Cre mice) using floxed alleles of Cbfβ (Cbfβf/f) and Cre driven by the Collagen 2α1 promoter (Col2α1-Cre). Cbfβf/fCol2α1-Cre mice evaded developmental and newborn lethality to survive to adulthood and displayed severe skeletal malformation. Cbfβf/fCol2α1-Cre mice had dwarfism, hypoplastic skeletons, defective bone mineralization, shortened limbs, shortened sternum bodies, and un-calcified occipital bones and hyoid bones. In the long bone cartilage, the resting zone was elongated, and chondrocyte proliferation and hypertrophy were impaired in Cbfβf/fCol2α1-Cre mice, which led to deformation of the growth plates. Primary spongiosa formation was delayed, diaphysis was shortened and trabecular bone formation was almost absent in the mutant mice. In addition, lamellar bone formation in the secondary spongiosa was also impaired. However, osteoclast formation in the trabecular bone was not affected. Cbfβ deficiency led to down-regulation of chondrocyte-regulating genes [i.e, patched (Ptc1), Cyclin D1 and Indian hedgehog (Ihh)] in the cartilage. Interestingly, the expression of Runx2 and Runx3 was not changed in the cartilage of the mutants. Collectively, the results revealed that Cbfβ is crucial for postnatal skeletal development and endochondral bone formation through its function in growth plate development and chondrocyte proliferation and differentiation. This study also revealed that chondrocyte maturation, mediated by Cbfβ, was critical to trabecular bone morphogenesis. Significantly, these findings provide insight into the role of Cbfβ in postnatal skeletogenesis, which may assist in the development of new therapies for osteoporosis.

Molecular Mechanisms of the Biphasic Effects of Interferon-γ on Osteoclastogenesis

Although interferon-γ (IFN-γ) potently inhibits osteoclastogenesis, the suppressive effect is significantly reduced when osteoclast precursors are pre-exposed to the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular mechanism underlying the biphasic effects of IFN-γ on osteoclastogenesis remains elusive. Here, we recapitulate the biphasic functions of IFN-γ in osteoclastogenesis in both tissue culture dishes and on bone slices. We further demonstrate that IFN-γ markedly suppresses the RANKL-induced expression of nuclear factor of activated T-cells c1 (NFATc1) in normal, but not RANKL-pretreated bone marrow macrophages (BMMs). Similarly, IFN-γ impairs the activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in normal, but not RANKL-pretreated, BMMs. These findings indicate that IFN-γ inhibits osteoclastogenesis partially by suppressing the expression of NFATc1 and the activation of the NF-κB and JNK pathways. Moreover, IFN-γ inhibits the RANKL-induced expression of osteoclast genes, but RANKL pretreatment reprograms osteoclast genes into a state in which they can no longer be suppressed by IFN-γ, indicating that IFN-γ inhibits osteoclastogenesis by blocking the expression of osteoclast genes. Finally, the IVVY(535-538) motif in the cytoplasmic domain of RANK is responsible for rendering BMMs refractory to the inhibitory effect of IFN-γ. Taken together, these findings provide important mechanistic insights into the biphasic effects of IFN-γ on osteoclastogenesis.

The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis

Background: The IVVY motif and TRAF-binding sites in the RANK cytoplasmic domain initiate distinct modes of action to promote osteoclastogenesis. Results: The ability of the IVVY motif to mediate osteoclast lineage commitment for osteoclastogenesis requires TRAF-binding sites. Conclusion: The RANK IVVY motif cooperates with TRAF-binding sites to induce osteoclastogenesis. Significance: This reveals a novel mechanism of RANK signaling in osteoclastogenesis. Receptor activator of NF-κB (RANK) activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TNF receptor-associated factors (TRAFs) via three cytoplasmic motifs (motif 1, PFQEP369–373; motif 2, PVQEET559–564; and motif 3, PVQEQG604–609) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY535–538), which is dispensable for the activation of TRAF-induced signaling pathways but essential for osteoclast lineage commitment by inducing the expression of nuclear factor of activated T-cells c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF/IL-1-mediated osteoclastogenesis requires RANK ligand assistance, and the IVVY motif is also critical for TNF/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that, although all three TRAF motifs play roles in TNF/IL-1-mediated osteoclastogenesis, motifs 2 and 3 are more potent than motif 1. Accordingly, inactivation of motifs 2 and 3 blocksTNF/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of motifs 2 and 3, similar to inactivation of the IVVY motif, abrogates the expression of nuclear factor of activated T-cells c1 and osteoclast genes in assays reflecting RANK-initiated and TNF/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provides novel insights into the molecular mechanism of RANK signaling in osteoclastogenesis.