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1.  Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism 
Bone research  2013;4:336-345.
NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2–3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.
PMCID: PMC4175530  PMID: 25264493
NF-κB; RANK; growth plate; chondrocytes; dwarfism
2.  Ubiquitin E3 ligase Wwp1 negatively regulates osteoblast function by inhibiting osteoblast differentiation and migration 
Ubiquitin E3 ligase-mediated protein degradation promotes proteasomal degradation of key positive regulators of osteoblast functions. For example, the E3 ligases, Smurf1, Itch and Wwp1, promote degradation of Runx2, JunB, and CXCR4 proteins to inhibit their functions. However, the role of E3 ligases in age-associated bone loss is unknown. We found that the expression level of Wwp1, but not Smurf1 or Itch, was significantly increased in CD45− bone marrow-derived mesenchymal stem cells (MSCs) from 6- and 12-month-old WT mice. Wwp1−/− mice developed increased bone mass as they aged, associated with increased bone formation rates and normal bone resorption parameters. Bone marrow stromal cells (BMSCs) from Wwp1−/− mice formed increased numbers and areas of alkaline phosphatase+ and Alizarin Red+ nodules and had increased migration potential towards CXCL12 gradients. Runx2, JunB and CXCR4 protein levels were significantly increased in Wwp1−/− BMSCs. Wwp1−/−BMSCs had increased amount of ubiquitinated JunB protein, but Runx2 ubiquitination was no change. Knocking-down JunB in Wwp1−/− BMSCs returned Runx2 protein levels to that in WT cells. Thus, Wwp1 negatively regulates osteoblast functions by affecting both their migration and differentiation. Mechanisms designed to decrease Wwp1 levels in BMSCs may represent a new approach to prevent the decrease in osteoblastic bone formation associated with aging.
doi:10.1002/jbmr.1938
PMCID: PMC3749248  PMID: 23553732
Wwp1; mesenchymal stem cells; osteoblasts; bone formation
3.  Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice 
Genesis (New York, N.Y. : 2000)  2013;51(9):667-675.
Summary
The troponin complex, which consists of three regulatory proteins (troponin C, troponin I and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3lacZ/+ mice are smaller than their WT littermates throughout development, but do not display any gross phenotypes. Tnnt3lacZ/lacZ embryos are smaller than heterozygotes, and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3lacZ/lacZ liver and kidney, which was not present in Tnnt3lacZ/+ or WT, but no other gross tissue abnormalities. X-gal staining for Tnnt3 promoter-driven lacZ transgene expression revealed positive staining in skeletal muscle and diapharam, and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle, and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional-inducible gene deletion approach.
doi:10.1002/dvg.22407
PMCID: PMC3787964  PMID: 23775847
Troponin; Knockout Mice; Muscle; Development
4.  Distribution and Alteration of Lymphatic Vessels in Knee Joints of Normal and Osteoarthritic Mice 
Objective
To investigate the distribution and alteration of lymphatic vessels and draining function in knee joints of normal and osteoarthritic mice.
Methods
For the mouse models of osteoarthritis (OA), we used mice with meniscal-ligamentous injury or mice with conditional knockout of the gene for cartilage transforming growth factor β (TGF β) type II receptor. The severity of cartilage loss and joint destruction was assessed histologically. Capillary and mature lymphatic vessels were identified and analyzed using double immunofluorescence staining and a whole-slide digital imaging system. Lymphatic drainage of knee joints was examined using near-infrared lymphatic imaging. Patient joint specimens obtained during total knee or hip arthroplasty were evaluated to verify the content validity of the mouse findings.
Results
Lymphatic vessels were distributed in soft tissues (mainly around the joint capsule, ligaments, fat pads, and muscles of normal knees). The number of lymphatic vessels, particularly the number of capillaries, was significantly increased in joints of mice with mild OA, while the number of mature lymphatic vessels was markedly decreased in joints of mice with severe OA. OA knees exhibited significantly decreased lymph clearance. The number of both capillary and mature lymphatic vessels was significantly decreased in the joints of patients with OA.
Conclusion
The whole-slide digital imaging system is a powerful tool, enabling the identification and assessment of lymphatic microvasculature in the entire mouse knee. Lymphatic capillaries and mature vessels are present in various soft tissues around articular spaces. Abnormalities of lymphatic vessels and draining function, including significantly reduced numbers of mature vessels and impaired clearance, are present in OA joints.
doi:10.1002/art.38278
PMCID: PMC4074307  PMID: 24574226
5.  A new osteopetrosis mutant mouse strain (ntl) with odontoma-like proliferations and lack of tooth roots 
European journal of oral sciences  2009;117(6):625-635.
A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.
doi:10.1111/j.1600-0722.2009.00690.x
PMCID: PMC4066957  PMID: 20121924
odontoma-like proliferations; osteoclast; osteopetrosis; tooth eruption; tooth root formation
6.  Delayed Fracture Healing and Increased Callus Adiposity in a C57BL/6J Murine Model of Obesity-Associated Type 2 Diabetes Mellitus 
PLoS ONE  2014;9(6):e99656.
Introduction
Impaired healing and non-union of skeletal fractures is a major public health problem, with morbidity exacerbated in patients with diabetes mellitus (DM). DM is prevalent worldwide and affects approximately 25.8 million US adults, with >90% having obesity-related type 2 DM (T2DM). While fracture healing in type 1 DM (T1DM) has been studied using animal models, an investigation into delayed healing in an animal model of T2DM has not yet been performed.
Methods
Male C57BL/6J mice at 5 weeks of age were placed on either a control lean diet or an experimental high-fat diet (HFD) for 12 weeks. A mid-diaphyseal open tibia fracture was induced at 17 weeks of age and a spinal needle was used for intra-medullary fixation. Mice were sacrificed at days 7, 10, 14, 21, 28, and 35 for micro-computed tomography (μCT), histology-based histomorphometry and molecular analyses, and biomechanical testing.
Results
HFD-fed mice displayed increased body weight and impaired glucose tolerance, both characteristic of T2DM. Compared to control mice, HFD-fed mice with tibia fractures showed significantly (p<0.001) decreased woven bone at day 28 by histomorphometry and significantly (p<0.01) decreased callus bone volume at day 21 by μCT. Interestingly, fracture calluses contained markedly increased adiposity in HFD-fed mice at days 21, 28, and 35. HFD-fed mice also showed increased PPARγ immunohistochemical staining at day 14. Finally, calluses from HFD-fed mice at day 35 showed significantly (p<0.01) reduced torsional rigidity compared to controls.
Discussion
Our murine model of T2DM demonstrated delayed fracture healing and weakened biomechanical properties, and was distinctly characterized by increased callus adiposity. This suggests altered mesenchymal stem cell fate determination with a shift to the adipocyte lineage at the expense of the osteoblast lineage. The up-regulation of PPARγ in fracture calluses of HFD-fed mice is likely involved in the proposed fate switching.
doi:10.1371/journal.pone.0099656
PMCID: PMC4049817  PMID: 24911161
7.  NOTCH inhibits osteoblast formation in inflammatory arthritis via noncanonical NF-κB 
The Journal of Clinical Investigation  2014;124(7):3200-3214.
NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TNF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-κB pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF-κB subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain–dependent (NICD-dependent) activation of an RBPjκ reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPjκ site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss.
doi:10.1172/JCI68901
PMCID: PMC4071381  PMID: 24892805
8.  The Role of Bone Marrow Edema and Lymphangiogenesis in Inflammatory-Erosive Arthritis 
A common feature of autoimmune diseases is perpetual production of macrophage, dendritic and/or osteoclast effector cells, which mediate parenchymal tissue destruction in end organs. In support of this, we have demonstrated previously that patients and mice with inflammatory-erosive arthritis have a marked increase in circulating CD11b+ precursor cells, which are primed for osteoclastogenesis, and that this increase in osteoclast precursors (OCPs) is due to systemically increased TNF production. From these data, we proposed a unifying hypothesis to explain these osteoimmunologic findings during the pathogenesis of inflammatory-erosive arthritis, which has three postulates: 1) myelopoiesis chronically induce by TNF has profound effects on the bone marrow and joint tissues that should be evident from longitudinal MRI; 2) TNF alters the chemokine/chemokine receptor axis in the bone marrow to stimulate OCP release into the blood, and 3) OCP-mediated lymphangiogenesis occurs in the end organ as a compensatory mechanism to drain the inflammation and remove by-products of joint catabolism. Here, we describe our recent experimental findings that support these hypotheses and speculate on how this information can be used as diagnostic biomarkers and tools to discover novel therapies to treat patients with inflammatory-erosive arthritis.
doi:10.1007/978-1-4419-1050-9_1
PMCID: PMC3932510  PMID: 19950010
Inflammatory Arthritis; Lymphangiogenesis; In vivo Imaging; 3D-MRI
9.  Chondrocyte β-Catenin Signaling Regulates Postnatal Bone Remodeling Through Modulation of Osteoclast Formation in a Murine Model 
Objective
To investigate whether β-catenin signaling in chondrocytes regulates osteoclastogenesis, thereby contributing to postnatal bone growth and bone remodeling.
Methods
Mice with conditional knockout (cKO) or conditional activation (cAct) of chondrocyte-specific β-catenin were generated. Changes in bone mass, osteoclast numbers, and osteoblast activity were examined. The mechanisms by which β-catenin signaling in chondrocytes regulates osteoclast formation were determined.
Results
The β-catenin cKO mice developed localized bone loss, whereas cAct mice developed a high bone mass phenotype. Histologic findings suggested that these phenotypes were caused primarily by impaired osteoclast formation, rather than impaired bone formation. Further molecular signaling analyses revealed that β-catenin signaling controlled this process by regulating the expression of the RANKL and osteoprotegerin (OPG) genes in chondrocytes. Activation of β-catenin signaling in chondrocytes suppressed Rankl gene transcription through a glucocorticoid receptor–dependent mechanism. The severe bone loss phenotype observed in β-catenin cKO mice was largely restored by treatment with human recombinant OPG or transgenic overexpression of Opg in chondrocytes.
Conclusion
β-catenin signaling in chondrocytes plays a key role in postnatal bone growth and bone remodeling through its regulation of osteoclast formation.
doi:10.1002/art.38195
PMCID: PMC3932359  PMID: 24431282
10.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
Objective
B cell depletion therapy (BCDT) ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritic flare in tumor necrosis factor transgenic (TNF-Tg) mice is associated with efferent lymph node (LN) “collapse,” triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. We examined whether BCDT efficacy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.
Methods
We developed contrast-enhancement (CE) MRI imaging, near-infrared indocyanine green (NIR-ICG) imaging, and intravital immunofluorescent imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-Tg mice. We tested to see if BCDT efficacy is associated with restoration of lymphatic draining and cell egress from arthritic joints.
Results
Unlike active lymphatics to normal and pre-arthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescent imaging demonstrated that CD11b+ monocytes/macrophages in lymphatic vessels afferent to expanding LN travel at high velocity (186 ± 37 micrometer/sec), while these cells are stationary in lymphatic vessels afferent to collapsed PLN. BCDT of flaring TNF-Tg mice significantly decreased knee synovial volume by 50% from the baseline level, and significantly increased lymphatic clearance versus placebo (p<0.05). This increased lymphatic drainage restored macrophages egress from inflamed joints without recovery of the lymphatic pulse.
Conclusion
These results support a novel mechanism in which BCDT of flaring joints lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
doi:10.1002/art.37709
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
11.  Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation 
The cytokines RANKL and TNF activate NF-κB signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-κB p100, which acts as an inhibitor, and TNF receptor–associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-κB signaling. Conditional OC-specific Traf3-KO (cKO) mice had mild osteoporosis and increased OC formation. RANKL induced TRAF3 degradation via the lysosome/autophagy system. The autophagy/lysosome inhibitor chloroquine reduced RANKL-induced OC formation and function by increasing TRAF3 expression in OCPs in vitro and in vivo. Although chloroquine had no effect on basal bone resorption, it inhibited parathyroid hormone– and ovariectomy-induced OC activation in WT, but not cKO, mice. Deletion of the transcription factor gene Relb resulted in increased TRAF3 expression in OCPs, which was associated with decreased RANKL-induced TRAF3 degradation. RelB directly increased expression of BECN1, a key autophagy regulator, by binding to its promoter. These data indicate that autophagic/lysosomal degradation of TRAF3 is an important step in RANKL-induced NF-κB activation in OCPs. Furthermore, treatments that increase TRAF3 levels in OCPs, including pharmacological inhibition of its degradation with compounds such as chloroquine, may limit bone destruction in common bone diseases.
doi:10.1172/JCI66947
PMCID: PMC3871219  PMID: 24316970
12.  Mechanisms of bone fragility in a glucocorticoid-treated mouse model of rheumatoid arthritis – implications for insufficiency fracture risk 
Arthritis and rheumatism  2012;64(11):3649-3659.
Objective
Glucocorticoid (GC) therapy is associated with increased fracture risk in rheumatoid arthritis (RA) patients. To elucidate the cause of this increased risk, we examined the effects of chronic inflammatory-erosive arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model.
Methods
Transgenic mice expressing human TNF-α-transgene (TNF-tg) with established arthritis and wild-type (WT) littermates were continually treated with GC (subcutaneous prednisolone controlled-release pellet; 5 mg/kg/day) or placebo for 14, 28 and 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of tibiae and lumbar vertebral bodies were assessed by micro-CT, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined.
Results
TNF-tg and GC treatment additively decreased mechanical strength and stiffness in both tibiae and vertebral bodies. GC treatment in the TNF-tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclastic activity.
Conclusions
Our findings indicate that the concomitant decrease in bone strength and increase in ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy without radiographic manifestation of fracture.
doi:10.1002/art.34639
PMCID: PMC3678247  PMID: 22832945
Glucocorticoid; Rheumatoid Arthritis; Bone Quality; Degree of Mineralization
13.  Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis 
PLoS ONE  2013;8(9):e73766.
Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
doi:10.1371/journal.pone.0073766
PMCID: PMC3767819  PMID: 24040061
14.  Tumor Necrosis Factor Inhibits Mesenchymal Stem Cell Differentiation into Osteoblasts Via the Ubiquitin E3 Ligase Wwp1 
Stem cells (Dayton, Ohio)  2011;29(10):1601-1610.
Patients with chronic inflammatory disorders, such as rheumatoid arthritis, often have osteoporosis due to a combination of Tumor necrosis factor-induced increased bone resorption and reduced bone formation. To test if TNF inhibits bone formation by affecting the commitment and differentiation of mesenchymal stem cells (MSCs) into osteoblasts, we examined the osteogenic potential of MSCs from TNF transgenic (TNF-Tg) mice, a model of chronic inflammatory arthritis. MSC-enriched cells were isolated from bone marrow stromal cells using negative selection with anti-CD45 antibody coated magnetic beads. The expression profile of MSC surface markers the osteogenic, chondrogenic, and adipogenic properties of CD45− cells were confirmed by FACS and cell differentiation assays. MSC-enriched CD45− cells from TNF-Tg mice formed significantly decreased numbers of fibroblast and ALP+ colonies and had a decreased expression of osteoblast marker genes. As TNF may upregulate ubiquitin ligases, which negatively regulate osteoblast differentiation, we examined the expression levels of several ubiquitin ligases and found that Wwp1 expression was significantly increased in MSC-enriched CD45− cells of TNF-Tg mice. Wwp1 knockdown rescued impaired osteoblast differentiation of TNF-Tg CD45− cells. Wwp1 promotes ubiquitination and degradation of JunB, an AP-1 transcription factor that positively regulates osteoblast differentiation. Injection of TNF into wild-type mice resulted in decreased osteoblast differentiation of MSCs and increased JunB ubiquitination, which was completely blocked in Wwp1−/− mice. Thus, Wwp1 targets JunB for ubiquitination and degradation in MSCs after chronic exposure to TNF, and inhibition of Wwp1 in MSCs could be a new mechanism to limit inflammation-mediated osteoporosis by promoting their differentiation into osteoblasts.
doi:10.1002/stem.703
PMCID: PMC3708970  PMID: 21809421
Tumor necrosis factor; Mesenchymal stem cells; osteoblasts; Wwp1; E3 ligase
16.  The ubiquitin E3 ligase WWP1 decreases CXCL12-mediated MDA231 breast cancer cell migration and bone metastasis☆ 
Bone  2012;50(4):813-823.
Advanced breast cancers preferentially metastasize to bone where cells in the bone microenvironment produce factors that enhance breast cancer cell homing and growth. Expression of the ubiquitin E3 ligase WWP1 is increased in some breast cancers, but its role in bone metastasis has not been investigated. Here, we studied the effects of WWP1 and itch, its closest family member, on breast cancer bone metastasis. First, we immunostained a multi-tumor tissue microarray and a breast cancer tissue microarray and demonstrated that WWP1 and ITCH are expressed in some of breast cancer cases. We then knocked down WWP1 or itch in MDA-MB-231 breast cancer cells using shRNA and inoculated these cells and control cells into the left ventricle of athymic nude mice. Radiographs showed that mice given shWWP1 cells had more osteolytic lesions than mice given control MDA-MB-231 cells. Histologic analysis confirmed osteolysis and showed significantly increased tumor area in bone marrow of the mice. WWP1 knockdown did not affect cell growth, survival or osteoclastogenic potential, but markedly increased cell migration toward a CXCL12 gradient in vitro. Furthermore, WWP1 knockdown significantly reduced CXCL12-induced CXCR4 lysosomal trafficking and degradation. In contrast, itch knockdown had no effect on MDA-MB-231 cell bone metastasis. Taken together, these findings demonstrate that WWP1 negatively regulates cell migration to CXCL12 by limiting CXCR4 degradation to promote breast cancer metastasis to bone and highlight the potential utility of WWP1 as a prognostic indicator for breast cancer bone metastasis.
doi:10.1016/j.bone.2011.12.022
PMCID: PMC3439807  PMID: 22266093
WWP1; Breast cancer; Bone metastasis; CXCR4; Degradation
17.  Non-canonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions 
Stem cells (Dayton, Ohio)  2012;30(4):709-718.
RelB and NF-κB2 are the main effectors of NF-κB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.
doi:10.1002/stem.1050
PMCID: PMC3602314  PMID: 22290873
Non-canonical NF-κB; RelB; hematopoietic stem cell; osteoblast; niche
18.  Osteoclast fusion and regulation by RANKL-dependent and independent factors 
World Journal of Orthopedics  2012;3(12):212-222.
Osteoclasts are the bone resorbing cells essential for bone remodeling. Osteoclasts are formed from hematopoietic progenitors in the monocyte/macrophage lineage. Osteoclastogenesis is composed of several steps including progenitor survival, differentiation to mono-nuclear pre-osteoclasts, fusion to multi-nuclear mature osteoclasts, and activation to bone resorbing osteoclasts. The regulation of osteoclastogenesis has been extensively studied, in which the receptor activator of NF-κB ligand (RANKL)-mediated signaling pathway and downstream transcription factors play essential roles. However, less is known about osteoclast fusion, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone. Several proteins that affect cell fusion have been identified. Among them, dendritic cell-specific transmembrane protein (DC-STAMP) is directly associated to osteoclast fusion in vivo. Cytokines and factors influence osteoclast fusion through regulation of DC-STAMP. Here we review the recently discovered new factors that regulate osteoclast fusion with specific focus on DC-STAMP. A better understanding of the mechanistic basis of osteoclast fusion will lead to the development of a new therapeutic strategy for bone disorders due to elevated osteoclast bone resorption. Cell-cell fusion is essential for a variety of cellular biological processes. In mammals, there is a limited number of cell types that fuse to form multinucleated cells, such as the fusion of myoblasts for the formation of skeletal muscle and the fusion of cells of the monocyte/macrophage lineage for the formation of multinucleated osteoclasts and giant cells. In most cases, cell-cell fusion is beneficial for cells by enhancing function. Myoblast fusion increases myofiber size and diameter and thereby increases contractile strength. Multinucleated osteoclasts have far more bone resorbing activity than their mono-nuclear counterparts. Multinucleated giant cells are much more efficient in the removal of implanted materials and bacteria due to chronic infection than macrophages. Therefore, they are also called foreign-body giant cells. Cell fusion is a complicated process involving cell migration, chemotaxis, cell-cell recognition and attachment, as well as changes into a fusion-competent status. All of these steps are regulated by multiple factors. In this review, we will discuss osteoclast fusion and regulation.
doi:10.5312/wjo.v3.i12.212
PMCID: PMC3557323  PMID: 23362465
Osteoclasts; Fusion; Dendritic cell-specific transmembrane protein; Receptor activator of NF-κB ligand; Bone resorption.
19.  Measuring intranodal pressure and lymph viscosity to elucidate mechanisms of arthritic flare and therapeutic outcomes 
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
doi:10.1111/j.1749-6632.2011.06237.x
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
20.  VEGF-C attenuates joint damage in chronic inflammatory arthritis by accelerating local lymphatic drainage 
Arthritis and Rheumatism  2011;63(8):2318-2328.
Objective
To investigate if enhancement of joint lymphangiogenesis by injecting VEGF-C adeno-associated virus (AAV) into joints has therapeutic efficacy in chronic inflammatory arthritis in mice.
Methods
TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF-C was cloned into an AAV expression vector to generate AAV-VEGF-C. AAV-VEGF-C or control AAV-Luc was injected into joints of TNF-Tg mice. MRI and lymphatic imaging were used during the 4-months following injection to assess changes in synovial volume and lymph flow from joint tissues to local draining lymph nodes. Joint inflammation, bone erosion and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry.
Results
Intra-articular administration of AAV-VEGF-C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in joint sections compared to AAV-Luc virus during the 4-month-period. This accompanied by reduced inflammation area, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates and was significantly improved with AAV-VEGF-C treatment.
Conclusion
Intra-articular injection of AAV-VEGF-C increased lymphangiogenesis and improved lymphatic drainage from inflamed joints, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.
doi:10.1002/art.30421
PMCID: PMC3149728  PMID: 21538325
VEGF-C; lymphatic system; lymphangiogenesis; inflammation; arthritis
21.  The expression of osteoprotegerin is required for maintaining the intervertebral disc endplate of aged mice 
Bone  2011;48(6):1362-1369.
Objective
Human chondrocytes and annulus fibrosus cells of intervertebral disc (IVD) express osteoprotegerin (OPG), but the effect of OPG on the pathogenesis of IVD degeneration remains unknown. Here we assessed the phenotype change of IVD in OPG−/− mice.
Methods
The IVDs from 12-, 20-, and 28-week-old OPG−/− mice and WT controls were subjected to histologic analyses including TRAP staining for osteoclasts, immunostaining for OPG and type I collagen protein expression, and TUNEL staining for apoptosis. The IVD tissues were also subjected to real time RT-PCR for mRNA expression of genes for osteoblast-osterix, ALP, and osteocalcin; for osteoclasts-trap, rank, mmp9 and cathepsin K, and for chondrocytes-aggrecan, mmp13 and Col10.
Results
OPG protein expresses at the cells of endplate cartilage and annulus fiborsis in IVDs of WT mice. Compared to WT mice, OPG−/− mice developed aging related cartilage loss and bony tissue appearance at the endplate. Stating from 20 weeks of age, IVDs from OPG−/− mice expressed significantly increased mmp13 and Col10 levels, which is associated with increased osteoblast number and elevated expression of osteoblast marker genes. Furthermore, TRAP+ osteoclasts were presented in the endplate cartilage of OPG−/− mice. These osteoclasts localized adjacently to and erosion into the cartilage. Increased expression of RANK, mmp9 and cathepsin k was detected in OPG−/− IVDs.
Conclusions
OPG at IVD plays an important role for maintaining the integrity of endplate cartilage during aging by preventing endplate cartilage from osteoclast-mediated resorption.
doi:10.1016/j.bone.2011.03.773
PMCID: PMC3130509  PMID: 21466864
Osteoprotegerin; intervertebral disc; cartilage endplate; osteoclast; intervertebral disc degeneration
22.  Src inhibitors in the treatment of metastatic bone disease: rationale and clinical data 
Clinical investigation  2011;1(12):1695-1706.
Src is a nonreceptor tyrosine kinase essential for the activation of osteoclasts, the cells that degrade bone. Src also regulates normal cell functions, cancer cell growth and metastasis to organs, including bone where tumor cells induce bone destruction by osteoclasts. Src inhibitors prevent bone destruction and tumor cell growth in animal models of metastatic bone disease, and some are being investigated in clinical trials, particularly in patients with prostate cancer, which has high bone metastatic potential. Here, we review how Src regulates osteoclast formation, activation and survival and the results of preclinical and clinical trials of Src inhibitors, which show some promise in inhibiting the effects of tumor cells on the skeleton.
doi:10.4155/cli.11.150
PMCID: PMC3289094  PMID: 22384312
bone metastasis; bone resorption; osteoclasts; Src tyrosine kinase
23.  CD23+/CD21hi B-cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice 
Arthritis Research & Therapy  2011;13(4):R138.
Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints. However, how arthritic flare occurs only in select joints during a systemic autoimmune disease remains an enigma. To better understand these observations, we developed longitudinal imaging outcomes of synovitis and lymphatic flow in mouse models of RA, and identified that asymmetric knee flare is associated with ipsilateral popliteal lymph node (PLN) collapse and the translocation of CD23+/CD21hi B-cells (B-in) into the paracortical sinus space of the node. In order to understand the relationship between this B-in translocation and lymph drainage from flaring joints, we tested the hypothesis that asymmetric tumor necrosis factor (TNF)-induced knee arthritis is associated with ipsilateral PLN and iliac lymph node (ILN) collapse, B-in translocation, and decreased afferent lymphatic flow.
Methods
TNF transgenic (Tg) mice with asymmetric knee arthritis were identified by contrast-enhanced (CE) magnetic resonance imaging (MRI), and PLN were phenotyped as "expanding" or "collapsed" using LNcap threshold = 30 (Arbitrary Unit (AU)). Inflammatory-erosive arthritis was confirmed by histology. Afferent lymphatic flow to PLN and ILN was quantified by near infrared imaging of injected indocyanine green (NIR-ICG). The B-in population in PLN and ILN was assessed by immunohistochemistry (IHC) and flow cytometry. Linear regression analyses of ipsilateral knee synovial volume and afferent lymphatic flow to PLN and ILN were performed.
Results
Afferent lymph flow to collapsed nodes was significantly lower (P < 0.05) than flow to expanding nodes by NIR-ICG imaging, and this occurred ipsilaterally. While both collapsed and expanding PLN and ILN had a significant increase (P < 0.05) of B-in compared to wild type (WT) and pre-arthritic TNF-Tg nodes, B-in of expanding lymph nodes (LN) resided in follicular areas while B-in of collapsed LN were present within LYVE-1+ lymphatic vessels. A significant correlation (P < 0.002) was noted in afferent lymphatic flow between ipsilateral PLN and ILN during knee synovitis.
Conclusions
Asymmetric knee arthritis in TNF-Tg mice occurs simultaneously with ipsilateral PLN and ILN collapse. This is likely due to translocation of the expanded B-in population to the lumen of the lymphatic vessels, resulting in a dramatic decrease in afferent lymphatic flow. PLN collapse phenotype can serve as a new biomarker of knee flare.
doi:10.1186/ar3452
PMCID: PMC3239381  PMID: 21884592
24.  Near infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute vs. chronic phases of arthritis in mice 
Arthritis and rheumatism  2010;62(7):1881-1889.
Objective
Development of an in vivo imaging method to assess lymphatic draining function in the K/B×N mouse model of inflammatory arthritis.
Methods
Indocyanine green (ICG), a near-infrared (NIR) fluorescent dye, was injected intradermally into the footpad of wild-type mice, the limb was illuminated with an 806 nm NIR laser, and the movement of ICG from the injection site to the draining popliteal lymph node (PLN) was recorded with a CCD camera. ICG-NIR images were analyzed to obtain 5 measures of lymphatic function across time. K/B×N arthritic mice and control non-arthritic littermates were imaged at one-month of age when acute joint inflammation commenced, and repeated at 3 months when joint inflammation became chronic. Lymphangiogenesis in PLNs was assessed by immunochemistry.
Results
ICG and its transport within lymphatic vessels were readily visualized and quantitative measures derived. During the acute phase of arthritis, the lymphatic vessels were dilated with increased ICG signal intensity and lymphatic pulses, and PLNs became fluorescent quickly. During the chronic phase, new lymphatic vessels were present near the foot. However, ICG appearance in lymphatic vessels was delayed. The size and area of PLN lymphatic sinuses progressively increased in the K/B×N mice.
Conclusion
ICG-NIR lymphatic imaging is a valuable method to assess the lymphatic draining function in mice with inflammatory arthritis. ICG-NIR imaging of K/B×N mice identified two distinct lymphatic phenotypes during the acute and chronic phase of inflammation. This technique can be used to assess new therapies for lymphatic disorders.
doi:10.1002/art.27464
PMCID: PMC2902657  PMID: 20309866
Near infrared; lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
25.  Expanded CD23+/CD21hi B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy1 
Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA), however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-tg) mouse model of RA displays a chronic-progressive disease that spreads from distal to proximal joints, and is generally considered to be adaptive immune system-independent. We have previously reported that knee arthritis in hTNF-tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging (CE-MRI). To better understand these changes, here we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from 2, 4-5, and 8-12 month old TNF-tg mice demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21hi, CD23+, IgMhi, CD1d+, activation marker-negative, polyclonal B cells which are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-tg disease and clears follicular and CD21hi, CD23+ B cells from the PLNs. Based on these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition and function of joint-draining lymph nodes.
doi:10.4049/jimmunol.0903489
PMCID: PMC2874087  PMID: 20435928
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy

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