NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis

Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway.Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis.Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli

Constitutively Active Canonical NF-κB Pathway Induces Severe Bone Loss in Mice

Physiologic osteoclastogenesis entails activation of multiple signal transduction pathways distal to the cell membrane receptor RANK. However, atypical osteoclastogenesis driven by pro-inflammatory stimuli has been described. We have reported recently a novel mechanism whereby endogenous mutational activation of the classical NF-κB pathway is sufficient to induce RANKL/RANK-independent osteoclastogenesis. Here we investigate the physiologic relevance of this phenomenon in vivo. Using a knock-in approach, the active form of IKK2, namely IKK2SSEE, was introduced into the myeloid lineage with the aid of CD11b-cre mice. Phenotypic assessment revealed that expression of IKK2SSEE in the myeloid compartment induced significant bone loss in vivo. This observation was supported by a dramatic increase in the number and size of osteoclasts in trabecular regions, elevated levels of circulating TRACP-5b, and reduced bone volume. Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway. Intriguingly, RelB and P52 were both required to mediate the osteoclastogenic effect of IKK2SSEE and co-expression of these two proteins was sufficient to recapitulate osteoclastogenesis in the absence of RANKL or IKK2SSEE. Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis. Deletion of p52 enabled more robust osteoclast formation by the active kinase. In summary, molecular activation of IKK2 may play a role in conditions of pathologic bone destruction, which may be refractory to therapeutic interventions targeting the proximal RANKL/RANK signal

Constitutively Activated NLRP3 Inflammasome Causes Inflammation and Abnormal Skeletal Development in Mice

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1β. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1β and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue “spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling

Expression of Bcl-2 and Bax in Mouse Renal Tubules during Kidney Development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated

Osteoclast Activated FoxP3+ CD8+ T-Cells Suppress Bone Resorption in vitro

BACKGROUND: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption. PRINCIPAL FINDINGS: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts. SIGNIFICANCE: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology

Rapamycin and the transcription factor C/EBPβ as a switch in osteoclast differentiation: implications for lytic bone diseases

Lytic bone diseases and in particular osteoporosis are common age-related diseases characterized by enhanced bone fragility due to loss of bone density. Increasingly, osteoporosis poses a major global health-care problem due to the growth of the elderly population. Recently, it was found that the gene regulatory transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is involved in bone metabolism. C/EBPβ occurs as different protein isoforms of variable amino terminal length, and regulation of the C/EBPβ isoform ratio balance was found to represent an important factor in osteoclast differentiation and bone homeostasis. Interestingly, adjustment of the C/EBPβ isoform ratio by the process of translational control is downstream of the mammalian target of rapamycin kinase (mTOR), a sensor of the nutritional status and a target of immunosuppressive and anticancer drugs. The findings imply that modulating the process of translational control of C/EBPβ isoform expression could represent a novel therapeutic approach in osteolytic bone diseases, including cancer and infection-induced bone loss

Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair