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.
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.
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.
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.
Glucocorticoid; Rheumatoid Arthritis; Bone Quality; Degree of Mineralization
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.
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.
Tumor necrosis factor; Mesenchymal stem cells; osteoblasts; Wwp1; E3 ligase
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.
WWP1; Breast cancer; Bone metastasis; CXCR4; Degradation
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.
Non-canonical NF-κB; RelB; hematopoietic stem cell; osteoblast; niche
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.
Osteoclasts; Fusion; Dendritic cell-specific transmembrane protein; Receptor activator of NF-κB ligand; Bone resorption.
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.
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
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.
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.
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.
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.
VEGF-C; lymphatic system; lymphangiogenesis; inflammation; arthritis
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.
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.
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.
OPG at IVD plays an important role for maintaining the integrity of endplate cartilage during aging by preventing endplate cartilage from osteoclast-mediated resorption.
Osteoprotegerin; intervertebral disc; cartilage endplate; osteoclast; intervertebral disc degeneration
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.
bone metastasis; bone resorption; osteoclasts; Src tyrosine kinase
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.
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.
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.
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.
Development of an in vivo imaging method to assess lymphatic draining function in the K/B×N mouse model of inflammatory arthritis.
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.
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.
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.
Near infrared; lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
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.
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy
Ubiquitin ligase Smurf1-deficient mice develop an increased-bone-mass phenotype in an age-dependent manner. It was reported that such a bone-mass increase is related to enhanced activities of differentiated osteoblasts. Although osteoblasts are of mesenchymal stem cell (MSC) origin and MSC proliferation and differentiation can have significant impacts on bone formation, it remains largely unknown whether regulation of MSCs plays a role in the bone-mass increase of Smurf1-deficient mice. In this study we found that bone marrow mesenchymal progenitor cells from Smurf1−/− mice form significantly increased alkaline phosphatase–positive colonies, indicating roles of MSC proliferation and differentiation in bone-mass accrual of Smurf1−/− mice. Interestingly, Smurf1−/− cells have an elevated protein level of AP-1 transcription factor JunB. Biochemical experiments demonstrate that Smurf1 interacts with JunB through the PY motif and targets JunB protein for ubiquitination and proteasomal degradation. Indeed, Smurf1-deficient MSCs have higher proliferation rates, consistent with the facts that cyclin D1 mRNA and protein both are increased in Smurf1−/− cells and JunB can induce cyclinD1 promoter. Moreover, JunB overexpression induces osteoblast differentiation, shown by higher expression of osteoblast markers, and JunB knock-down not only decreases osteoblast differentiation but also restores the osteogenic potential to wild-type level in Smurf1−/− cells. In conclusion, our results suggest that Smurf1 negatively regulates MSC proliferation and differentiation by controlling JunB turnover through an ubiquitin-proteasome pathway. © 2010 American Society for Bone and Mineral Research.
MSC; ubiguitination; proteasome; JunB; osteoblasts
In contrast to rheumatoid arthritis (RA), Jaccoud arthritis (JA) joint inflammation in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the anti-osteoclastogenic cytokine interferon-alpha (IFN-α), whose transcriptome is present in SLE monocytes, may be responsible. To test this, we examined effects of IFN-α versus lupus disease on osteoclasts and erosion in the NZBxNZW F1 SLE mouse model with K/BxN serum-induced arthritis (SIA).
Elevated systemic IFN-α levels were obtained by administration of an adenoviral vector expressing IFN-α (Ad-IFN-α). SLE disease was marked by anti-dsDNA antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFN-α transcriptome. Micro-CT was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCP) and myeloid dendritic cells (mDC) in blood.
Administration of Ad-IFN-α to NZBxNZW F1 mice induced osteopetrosis. Pre-autoimmune NZBxNZW F1 mice are fully susceptible to focal erosions in the setting of SIA. However, NZBxNZW F1 mice with high anti-dsDNA antibody titers and the IFN-α transcriptome were protected against bone erosions. Ad-IFN-α pre-treatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r2=0.4720, p<0.01), which was associated with a decrease in circulating CD11b+CD11c− OCP, and a concomitant increase in CD11b+CD11c+ cells (r2=0.6330, p<0.05) that are phenotypic of mDC.
These findings suggest that IFN-α in SLE shifts monocyte development toward mDC at the expense of osteoclastogenesis thereby resulting in decreased bone erosion.
Jaccoud arthritis (JA); Lupus; Osteoclast; Interferon-alpha (IFN-α)
The effects of anti-resorptive agents (i.e. alendronate (Aln), osteoprotegerin (OPG)) on bone infection are unknown. Thus, their effects on implant-associated osteomyelitis (OM) were investigated in mice using PBS (placebo), gentamycin and etanercept (TNFR:Fc) controls. None of the drugs affected humoral immunity, angiogenesis, or chronic infection. However, the significant (p<0.05 vs. PBS) inhibition of cortical osteolysis and decreased draining lymph node size in Aln and OPG treated mice was associated with a significant (p<0.05) increase in the incidence of high-grade infections during the establishment of OM. In contrast, the high-grade infections in TNFR:Fc treated mice were associated with immunosuppression, as evidenced by the absence of granulomas and presence of Gram+ biofilm in the bone marrow. Collectively, these findings indicate that while anti-resorptive agents do not exacerbate chronic OM, they can increase the bacterial load during early infection by decreasing lymphatic drainage and preventing the removal of necrotic bone that harbors the bacteria.
NF-κB is a set of multifunctional transcriptional factors that regulate expression of genes involved in numerous normal cellular activities. They also are activated in many inflammatory and neoplastic conditions in which their expression may be stimulated by pro-inflammatory cytokines. NF-κB in turn, regulates the expression of cytokines and so can mediate autocrine, self-amplifying cycles of cytokine release and NF-κB activation leading to maintenance of inflammatory reactions beyond the initial stimulus, as seen in rheumatoid arthritis and asthma. Since discovery of the requirement of NF-κB for basal and cytokine-induced osteoclast formation in the mid-1990s, much has been learned about the role of NF-κB in bone. NF-κB has roles in skeletal development, endochondral ossification, osteoclast and osteoblast functions, and common bone diseases. NF-κB inhibitors have been developed, but none has made it to clinical trials for the treatment of common bone diseases. Here we review the roles for NF-κB in bone and in common bone diseases.
Investigation of the effect of lymphatic inhibition on joint and draining lymph node pathology during the course of arthritis progression in mice.
TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Mice received contrast enhanced MRI to obtain ankle and knee joint synovial volumes and draining popliteal lymph node (PLN) volumes before and 8 weeks after treatment with VEGFR-3 or VEGFR-2 neutralizing antibodies, or isotype IgG. The animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining PLNs prior to sacrifice. Lymphatic vessel formation and morphology of joints and PLNs were examined by histology, immunohistochemistry, and RT-PCR.
Compared to IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of PLNs, the number of lymphatic vessels in joints and PLNs, the lymphatic drainage from paws to PLNs, and the number of VEGF-C expressing CD11b+ myeloid cells in PLNs. However, it increased the synovial volumes and inflammatory area in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.
Lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.
Lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
The homologous to the E6-assosiated protein carboxyl terminus (HECT) domain E3 ubiquitin ligase Smurf1 is the first E3 ligase to be implicated in regulating bone cell function. The involvement of Smurf1 in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review highlights recent works exploring Smurf-regulated biological processes in bone cells and highlights recent discoveries surrounding the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Moreover, we discuss the relevance of targeting the HECT E3s through the development of small-molecule inhibitors as an anticancer therapeutic strategy.
Smurf; OSTEOBLAST; UBIQUITINATION; PROTEASOME DEGRADATION
The molecular classification for breast carcinomas has been used in clinical studies with a simple surrogate panel of immunohistochemistry (IHC) markers. The objective of this current project was to study the molecular classification of commonly used breast cancer cell lines by IHC analysis. Seventeen breast cancer cell lines were harvested, fixed in formalin and made into cell blocks. IHC analyses were performed on each cell block with antibodies to estrogen receptor (ER), progesterone receptor (PR), HER2, EGFR, CK5/6, Ki-67 and androgen receptor (AR). Among the 17 cell lines, MCF-7 and ZR-75-1 fell to Luminal A subtype; BT-474 to Luminal B subtype; SKBR-3, MDA-MD-435 and AU 565 to HER2 over-expression subtype; MDA-MB-231, MCF-12A, HBL 101, HS 598 T, MCF-10A, MCF-10F, BT-20, 468 and BT-483 to basal subtype. MDA-MB-453 belonged to Unclassified subtype. Since each subtype defined by this IHC-based molecular classification does show a distinct clinical outcome, attention should be paid when choosing a cell line for any study.
molecular classification; breast cancer; cell lines; immunohistochemistry
Osteoclasts are the cells that degrade bone to initiate normal bone remodeling and mediate bone loss in pathologic conditions by increasing their resorptive activity. They are derived from precursors in the myeloid/monocyte lineage that circulate in the blood after their formation in the bone marrow. These osteoclast precursors (OCPs) are attracted to sites on bone surfaces destined for resorption and fuse with one another to form the multinucleated cells that resorb calcified matrixes under the influence of osteoblastic cells in bone marrow. Recent studies have identified functions for OCPs and osteoclasts in and around bone other than bone resorption. For example, they regulate the differentiation of osteoblast precursors and the movement of hematopoietic stem cells from the bone marrow to the bloodstream; they participate in immune responses, and secrete cytokines that can affect their own functions and those of other cells in inflammatory and neoplastic processes affecting bone. Here, we review these findings, which define new roles for osteoclasts and OCPs in the growing field of osteoimmunology and in common pathologic conditions in which bone resorption is increased.
Osteoclast; cytokine; osteoblast; bone resorption; inflammatory arthritis
Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP-2 acts synergistically with β-catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross-talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3-E1 cells to investigate the effect of BMP-2 on β-catenin signaling. We found that BMP-2 stimulates Lrp5 expression and inhibits the expression of β-TrCP, the F-box E3 ligase responsible for β-catenin degradation and subsequently increases β-catenin protein levels in osteoblasts. In vitro deletion of the β-catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP-2 treatment. These findings suggest that BMP-2 may regulate osteoblast function in part through modulation of the β-catenin signaling.
BMP-2; β-CATENIN; LRP5; β-TrCP; OSTEOBLAST DIFFERENTIATION
Differentiation of mesenchymal stem cells into a particular lineage is tightly regulated, and malfunction of this regulation could lead to pathological consequences. Patients with osteoporosis have increased adipocyte accumulation, but the mechanisms involved remain to be defined. In this study, we aimed to investigate if micro-RNAs regulate mesenchymal progenitor cells and bone marrow stromal cell (BMSC) differentiation through modulation of Runx2, a key transcription factor for osteogenesis. We found that miR-204 and its homolog miR-211 were expressed in mesenchymal progenitor cell lines and BMSCs and their expression was induced during adipocyte differentiation, whereas Runx2 protein expression was suppressed. Retroviral overexpression of miR-204 or transfection of miR-204 oligo decreased Runx2 protein levels and miR-204 inhibition significantly elevated Runx2 protein levels, suggesting that miR-204 acts as an endogenous attenuator of Runx2 in mesenchymal progenitor cells and BMSCs. Mutations of putative miR-204 binding sites upregulated the Runx2 3′-UTR reporter activity, suggesting that miR-204/211 bind to Runx2 3′-UTR. Perturbation of miR-204 resulted in altered differentiation fate of mesenchymal progenitor cells and BMSCs: osteoblast differentiation was inhibited and adipocyte differentiation was promoted when miR-204 was overexpressed in these cells, whereasosteogenesis was upregulated and adipocyte formation was impaired when miR-204 was inhibited. Together, our data demonstrated that miR-204/211 act as important endogenous negative regulators of Runx2, which inhibit osteogenesis and promote adipogenesis of mesenchymal progenitor cells and BMSCs.
Mesenchymal stem cells; Runx2; miR-204; Osteoblast; Adipocyte
TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-κB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-κB pathway efficiently to process the IκB protein NF-κB p100 to NF-κB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-κB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-κB p100, and TNF-Tg mice lacking NF-κB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor–associated factor 3 (TRAF3), an adapter protein that regulates NF-κB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-κB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-κB p100 expression or inhibition of NF-κB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases.