Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Role of Sphingolipids in Multiple Myeloma Progression, Drug Resistance, and Their Potential as Therapeutic Targets.

Multiple myeloma (MM) is an incapacitating hematological malignancy characterized by accumulation of cancerous plasma cells in the bone marrow (BM) and production of an abnormal monoclonal protein (M-protein). The BM microenvironment has a key role in myeloma development by facilitating the growth of the aberrant plasma cells, which eventually interfere with the homeostasis of the bone cells, exacerbating osteolysis and inhibiting osteoblast differentiation. Recent recognition that metabolic reprograming has a major role in tumor growth and adaptation to specific changes in the microenvironmental niche have led to consideration of the role of sphingolipids and the enzymes that control their biosynthesis and degradation as critical mediators of cancer since these bioactive lipids have been directly linked to the control of cell growth, proliferation, and apoptosis, among other cellular functions. In this review, we present the recent progress of the research investigating the biological implications of sphingolipid metabolism alterations in the regulation of myeloma development and its progression from the pre-malignant stage and discuss the roles of sphingolipids in in MM migration and adhesion, survival and proliferation, as well as angiogenesis and invasion. We introduce the current knowledge regarding the role of sphingolipids as mediators of the immune response and drug-resistance in MM and tackle the new developments suggesting the manipulation of the sphingolipid network as a novel therapeutic direction for MM.

GFI1-Dependent Repression of SGPP1 Increases Multiple Myeloma Cell Survival.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

Getting to YES: The Evolution of the University of Pittsburgh Medical Center Hillman Cancer Center Youth Enjoy Science (YES) Academy.

The University of Pittsburgh Medical Center Hillman Cancer Center Academy (Hillman Academy) has the primary goal of reaching high school students from underrepresented and disadvantaged backgrounds and guiding them through a cutting-edge research and professional development experience that positions them for success in STEM. With this focus, the Hillman Academy has provided nearly 300 authentic mentored research internship opportunities to 239 students from diverse backgrounds over the past 13 years most of whom matriculated into STEM majors in higher education. These efforts have helped shape a more diverse generation of future scientists and clinicians, who will enrich these fields with their unique perspectives and lived experiences. In this paper, we describe our program and the strategies that led to its growth into a National Institutes of Health Youth Enjoy Science-funded program including our unique multi-site structure, tiered mentoring platform, multifaceted recruitment approach, professional and academic development activities, and a special highlight of a set of projects with Deaf and Hard of Hearing students. We also share student survey data from the past six years that indicate satisfaction with the program, self-perceived gains in key areas of scientific development, awareness of careers in STEM, and an increased desire to pursue advanced degrees in STEM.

A Novel Mouse Model for SNP in Steroid Receptor Co-Activator-1 Reveals Role in Bone Density and Breast Cancer Metastasis.

The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.

EZH2 Supports Osteoclast Differentiation and Bone Resorption Via Epigenetic and Cytoplasmic Targets.

Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPß-LAP and C/EBPß-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPß-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.

A Novel Sulforaphane-Regulated Gene Network in Suppression of Breast Cancer-Induced Osteolytic Bone Resorption.

Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1 Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption in vitro and in vivo.

Epigenetic-Based Mechanisms of Osteoblast Suppression in Multiple Myeloma Bone Disease.

Multiple myeloma (MM) bone disease is characterized by the development of osteolytic lesions, which cause severe complications affecting the morbidity, mortality, and treatment of myeloma patients. Myeloma tumors seeded within the bone microenvironment promote hyperactivation of osteoclasts and suppression of osteoblast differentiation. Because of this prolonged suppression of bone marrow stromal cells' (BMSCs) differentiation into functioning osteoblasts, bone lesions in patients persist even in the absence of active disease. Current antiresorptive therapy provides insufficient bone anabolic effects to reliably repair MM lesions. It has become widely accepted that myeloma-exposed BMSCs have an altered phenotype with pro-inflammatory, immune-modulatory, anti-osteogenic, and pro-adipogenic properties. In this review, we focus on the role of epigenetic-based modalities in the establishment and maintenance of myeloma-induced suppression of osteogenic commitment of BMSCs. We will focus on recent studies demonstrating the involvement of chromatin-modifying enzymes in transcriptional repression of osteogenic genes in MM-BMSCs. We will further address the epigenetic plasticity in the differentiation commitment of osteoprogenitor cells and assess the involvement of chromatin modifiers in MSC-lineage switching from osteogenic to adipogenic in the context of the inflammatory myeloma microenvironment. Lastly, we will discuss the potential of employing small molecule epigenetic inhibitors currently used in the MM research as therapeutics and bone anabolic agents in the prevention or repair of osteolytic lesions in MM. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Osteoblast suppression in multiple myeloma bone disease.

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts and suppression of BMSCs differentiation into functional osteoblasts. Although new therapies for MM have greatly improved survival, MM remains incurable for most patients. Despite the major advances in current anti-MM and anti-resorptive treatments that can significantly improve osteolytic bone lysis, many bone lesions can persist even after therapeutic remission of active disease. Bone marrow mesenchymal stem cells (BMSCs) from MM patients are phenotypically distinct from their healthy counterparts and the mechanisms associated with the long-term osteogenic suppression are largely unknown. In this review we will highlight recent results of transcriptomic profiling studies that provide new insights into the establishment and maintenance of the persistent pathological alterations in MM-BMSCs that occur in MM. We will we discuss the role of genomic instabilities and senescence in propagating the chronically suppressed state and pro-inflammatory phenotype associated with MM-BMSCs. Lastly we describe the role of epigenetic-based mechanisms in regulating osteogenic gene expression to establish and maintain the pro-longed suppression of MM-BMSC differentiation into functional OBs.

Growth factor independence 1 expression in myeloma cells enhances their growth, survival, and osteoclastogenesis.

BACKGROUND: In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown. METHODS: Human primary CD138+ and BMSC were purified from normal donors and MM patients' bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo. RESULTS: We found that Gfi1 expression is increased in patient's MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth. CONCLUSIONS: These results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.

Distinct mechanisms regulate IL1B gene transcription in lymphoid CD4 T cells and monocytes.

Interleukin 1ß is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases. The regulation of the 31 kDa proIL-1ß precursor coded by the IL1B gene has been extensively studied in myeloid cells, but not in lymphoid-derived CD4 T cells. Surprisingly, we found that some CD4 T cell subsets express higher levels of proIL-1ß than unstimulated monocytes, despite relatively low IL1B mRNA levels. We observed a significant increase in IL1B transcription and translation in CD4 T cells upon ex vivo CD3/CD28 activation, and a similar elevation in the CCR5+ effector memory population compared to CCR5- T cells in vivo. The rapid and vigorous increase in IL1B gene transcription for stimulated monocytes has previously been associated with the presence of Spi-1/PU.1 (Spi1), a myeloid-lineage transcription factor, pre-bound to the promoter. In the case of CD4 T cells, this increase occurred despite the lack of detectable Spi1 at the IL1B promoter. Additionally, we found altered epigenetic regulation of the IL1B locus in CD3/CD28-activated CD4 T cells. Unlike monocytes, activated CD4 T cells possess bivalent H3K4me3+/H3K27me3+ nucleosome marks at the IL1B promoter, reflecting low transcriptional activity. These results support a model in which the IL1B gene in CD4 T cells is transcribed from a low-activity bivalent promoter independent of Spi1. Accumulated cytoplasmic proIL-1ß may ultimately be cleaved to mature 17 kDa bioactive IL-1ß, regulating T cell polarization and pathogenic chronic inflammation.

A combined computational and experimental approach reveals the structure of a C/EBPß-Spi1 interaction required for IL1B gene transcription.

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

XRK3F2 Inhibition of p62-ZZ Domain Signaling Rescues Myeloma-Induced GFI1-Driven Epigenetic Repression of the Runx2 Gene in Pre-osteoblasts to Overcome Differentiation Suppression.

Multiple myeloma bone disease (MMBD) is characterized by non-healing lytic bone lesions that persist even after a patient has achieved a hematologic remission. We previously reported that p62 (sequestosome-1) in bone marrow stromal cells (BMSC) is critical for the formation of MM-induced signaling complexes that mediate OB suppression. Importantly, XRK3F2, an inhibitor of the p62-ZZ domain, blunted MM-induced Runx2 suppression in vitro, and induced new bone formation and remodeling in the presence of tumor in vivo. Additionally, we reported that MM cells induce the formation of repressive chromatin on the Runx2 gene in BMSC via direct binding of the transcriptional repressor GFI1, which recruits the histone modifiers, histone deacetylase 1 (HDAC1) and Enhancer of zeste homolog 2 (EZH2). In this study we investigated the mechanism by which blocking p62-ZZ domain-dependent signaling prevents MM-induced suppression of Runx2 in BMSC. XRK3F2 prevented MM-induced upregulation of Gfi1 and repression of the Runx2 gene when present in MM-preOB co-cultures. We also show that p62-ZZ-domain blocking by XRK3F2 also prevented MM conditioned media and TNF plus IL7-mediated Gfi1 mRNA upregulation and the concomitant Runx2 repression, indicating that XRK3F2's prevention of p62-ZZ domain signaling within preOB is involved in the response. Chromatin immunoprecipitation (ChIP) analyses revealed that XRK3F2 decreased MM-induced GFI1 occupancy at the Runx2-P1 promoter and prevented recruitment of HDAC1, thus preserving the transcriptionally permissive chromatin mark H3K9ac on Runx2 and allowing osteogenic differentiation. Furthermore, treatment of MM-exposed preOB with XRK3F2 after MM removal decreased GFI1 enrichment at Runx2-P1 and rescued MM-induced suppression of Runx2 mRNA and its downstream osteogenic gene targets together with increased osteogenic differentiation. Further, primary BMSC (hBMSC) from MM patients (MM-hBMSC) had little ability to increase H3K9ac on the Runx2 promoter in osteogenic conditions when compared to hBMSC from healthy donors (HD). XRK3F2 treatment enriched Runx2 gene H3K9ac levels in MM-hBMSC to the level observed in HD-hBMSC, but did not alter HD-hBMSC H3K9ac. Importantly, XRK3F2 treatment of long-term MM-hBMSC cultures rescued osteogenic differentiation and mineralization. Our data show that blocking p62-ZZ domain-dependent signaling with XRK3F2 can reverse epigenetic-based mechanisms of MM-induced Runx2 suppression and promote osteogenic differentiation.

The Role of Semaphorin 4D in Bone Remodeling and Cancer Metastasis.

Semaphorin 4D (Sema4D; CD100) is a transmembrane homodimer 150-kDa glycoprotein member of the Semaphorin family. Semaphorins were first identified as chemorepellants that guide neural axon growth. Sema4D also possesses immune regulatory activity. Recent data suggest other Sema4D functions: inactivation of platelets, stimulation of angiogenesis, and regulation of bone formation. Sema4D is a coupling factor expressed on osteoclasts that inhibits osteoblast differentiation. Blocking Sema4D may, therefore, be anabolic for bone. Sema4D and its receptor Plexin-B1 are commonly dysregulated in cancers, suggesting roles in cancer progression, invasion, tumor angiogenesis, and skeletal metastasis. This review focuses on Sema4D in bone and cancer biology and the molecular pathways involved, particularly Sema4D-Plexin-B1 signaling crosstalk between cancer cells and the bone marrow microenvironment-pertinent areas since a humanized Sema4D-neutralizing antibody is now in early phase clinical trials in cancers and neurological disorders.

EZH2 or HDAC1 Inhibition Reverses Multiple Myeloma-Induced Epigenetic Suppression of Osteoblast Differentiation.

In multiple myeloma, osteolytic lesions rarely heal because of persistent suppressed osteoblast differentiation resulting in a high fracture risk. Herein, chromatin immunoprecipitation analyses reveal that multiple myeloma cells induce repressive epigenetic histone changes at the Runx2 locus that prevent osteoblast differentiation. The most pronounced multiple myeloma-induced changes were at the Runx2-P1 promoter, converting it from a poised bivalent state to a repressed state. Previously, it was observed that multiple myeloma induces the transcription repressor GFI1 in osteoblast precursors, which correlates with decreased Runx2 expression, thus prompting detailed characterization of the multiple myeloma and TNFα-dependent GFI1 response element within the Runx2-P1 promoter. Further analyses reveal that multiple myeloma-induced GFI1 binding to Runx2 in osteoblast precursors and recruitment of the histone modifiers HDAC1, LSD1, and EZH2 is required to establish and maintain Runx2 repression in osteogenic conditions. These GFI1-mediated repressive chromatin changes persist even after removal of multiple myeloma. Ectopic GFI1 is sufficient to bind to Runx2, recruit HDAC1 and EZH2, increase H3K27me3 on the gene, and prevent osteogenic induction of endogenous Runx2 expression. Gfi1 knockdown in MC4 cells blocked multiple myeloma-induced recruitment of HDAC1 and EZH2 to Runx2, acquisition of repressive chromatin architecture, and suppression of osteoblast differentiation. Importantly, inhibition of EZH2 or HDAC1 activity in pre-osteoblasts after multiple myeloma exposure in vitro or in osteoblast precursors from patients with multiple myeloma reversed the repressive chromatin architecture at Runx2 and rescued osteoblast differentiation.Implications: This study suggests that therapeutically targeting EZH2 or HDAC1 activity may reverse the profound multiple myeloma-induced osteoblast suppression and allow repair of the lytic lesions. Mol Cancer Res; 15(4); 405-17. ©2017 AACR.

Kindlin-2 controls TGF-ß signalling and Sox9 expression to regulate chondrogenesis.

The signals that control skeletogenesis are incompletely understood. Here we show that deleting Kindlin-2 in Prx1-expressing mesenchymal progenitors in mice causes neonatal lethality, chondrodysplasia and loss of the skull vault. Kindlin-2 ablation reduces chondrocyte density by decreasing cell proliferation and increasing apoptosis, and disrupts column formation, thus impairing the formation of the primary ossification center and causing severe limb shortening. Remarkably, Kindlin-2 localizes to not only focal adhesions, but also to the nuclei of chondrocytes. Loss of Kindlin-2 reduces, while the overexpression of Kindlin-2 increases, Sox9 expression. Furthermore, the overexpression of Sox9 restores the defects in chondrogenic differentiation induced by Kindlin-2 deletion in vitro. In addition, Kindlin-2 ablation inhibits TGF-ß1-induced Smad2 phosphorylation and chondrocyte differentiation. Finally, deleting Kindlin-2 in chondrocytes directly impairs chondrocyte functions, resulting in progressive dwarfism and kyphosis in mice. These studies uncover a previously unrecognized function for Kindlin-2 and a mechanism for regulation of the chondrocyte differentiation programme and chondrogenesis.

Pathobiology of Paget's Disease of Bone.

Paget's disease of bone is characterized by highly localized areas of increased bone resorption accompanied by exuberant, but aberrant new bone formation with the primary cellular abnormality in osteoclasts. Paget's disease provides an important paradigm for understanding the molecular mechanisms regulating both osteoclast formation and osteoclast-induced osteoblast activity. Both genetic and environmental etiologies have been implicated in Paget's disease, but their relative contributions are just beginning to be defined. To date, the only gene with mutations in the coding region linked to Paget's disease is sequestosome-1 (SQSTM1), which encodes the p62 protein, and these mutations lead to elevated cytokine activation of NF-B in osteoclasts but do not induce a "pagetic osteoclast" phenotype. Further, genetic mutations linked to Paget's appear insufficient to cause Paget's disease and additional susceptibility loci or environmental factors may be required. Among the environmental factors suggested to induce Paget's disease, chronic measles (MV) infection has been the most studied. Expression of the measles virus nucleocapsid gene (MVNP) in osteoclasts induces pagetic-like osteoclasts and bone lesions in mice. Further, mice expressing both MVNP in osteoclasts and germline mutant p62 develop dramatic pagetic bone lesions that were strikingly similar to those seen in patients with Paget's disease. Thus, interactions between environmental and genetic factors appear important to the development of Paget's disease. In this article we review the mechanisms responsible for the effects of mutant p62 gene expression and MVNP on osteoclast and osteoblast activity, and how they may contribute to the development of Paget's disease of bone.

Increased IL-6 expression in osteoclasts is necessary but not sufficient for the development of Paget's disease of bone.

Measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) and mutation of the SQSTM1 (p62) gene contribute to the increased OCL activity in Paget's disease (PD). OCLs expressing MVNP display many of the features of PD OCLs. Interleukin-6 (IL-6) production is essential for the pagetic phenotype, because transgenic mice with MVNP targeted to OCLs develop pagetic OCLs and lesions, but this phenotype is absent when MVNP mice are bred to IL-6(-/-) mice. In contrast, mutant p62 expression in OCL precursors promotes receptor activator of NF-κB ligand (RANKL) hyperresponsivity and increased OCL production, but OCLs that form have normal morphology, are not hyperresponsive to 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ), nor produce elevated levels of IL-6. We previously generated p62(P394L) knock-in mice (p62KI) and found that although OCL numbers were increased, the mice did not develop pagetic lesions. However, mice expressing both MVNP and p62KI developed more exuberant pagetic lesions than mice expressing MVNP alone. To examine the role of elevated IL-6 in PD and determine if MVNP mediates its effects primarily through elevation of IL-6, we generated transgenic mice that overexpress IL-6 driven by the tartrate-resistant acid phosphatase (TRAP) promoter (TIL-6 mice) and produce IL-6 at levels comparable to MVNP mice. These were crossed with p62KI mice to determine whether IL-6 overexpression cooperates with mutant p62 to produce pagetic lesions. OCL precursors from p62KI/TIL-6 mice formed greater numbers of OCLs than either p62KI or TIL-6 OCL precursors in response to 1,25-(OH)2 D3 . Histomorphometric analysis of bones from p62KI/TIL-6 mice revealed increased OCL numbers per bone surface area compared to wild-type (WT) mice. However, micro-quantitative CT (µQCT) analysis did not reveal significant differences between p62KI/TIL-6 and WT mice, and no pagetic OCLs or lesions were detected in vivo. Thus, increased IL-6 expression in OCLs from p62KI mice contributes to increased responsivity to 1,25-(OH)2 D3 and increased OCL numbers, but is not sufficient to induce Paget's-like OCLs or bone lesions in vivo.

TBK1 mediates critical effects of measles virus nucleocapsid protein (MVNP) on pagetic osteoclast formation.

Paget's disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include increased sensitivity of osteoclast progenitors to 1,25(OH)2 D3 , receptor activator of NF-κB ligand (RANKL), and TNF-α; increased osteoclast numbers; and increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and IκB kinase-ϵ (IKKϵ) are IKK family members that complex with MVNP and activate both IRF3 and NF-κB pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKKϵ early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM owing to increased TBK1 protein stability. TBK1 overexpression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-κB, TAF12, and ATF7 proteins in several cell lines. Overexpression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2 D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation and on the expression of IL-6, a key contributor to the pagetic osteoclast phenotype.

Distinct mechanisms for induction and tolerance regulate the immediate early genes encoding interleukin 1ß and tumor necrosis factor α.

Interleukin-1ß and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPß and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.