Yousef Abu-Amer - US grants

DESCRIPTION (adapted from the Investigator's abstract): Tumor necrosis factor alpha (TNF-alpha) is an inflammatory cytokine with osteoclastogenic and osteolytic activities that contribute to pathogenesis of bone disorders such as periodontal disease, post-menopausal osteoporosis, and arthritis. This proposal will investigate mechanisms underlying TNF-alpha stimulation of osteoclast formation from mouse bone marrow macrophages (BMM). Proposed studies are based on the premise that two TNF-alpha receptors transduce TNF-alpha signals in osteoclast precursors with opposing effects on osteoclast formation. The p55 receptor (p55r) is proposed to mediate positive effects of TNF-alpha (mainly soluble TNF-alpha) on osteoclast formation through activation of c-src kinase, subsequent phosphorylation and inactivation of the NFkB inhibitor IkBalpha, and activation of NFkB. The p75 receptor (p75r) is proposed to mediate inhibitory effects of TNF-alpha (mainly membrane TNF-alpha) on osteoclast formation. This hypothesis is supported by previous studies and preliminary data. The specific aims of the proposal are (1) to determine the mechanisms by which p55r promotes osteoclastogenesis, and (2) to determine the mechanisms by which p75r suppresses osteoclastogenesis.

DESCRIPTION (provided by applicant): Degenerative conditions of large weight bearing joints resulting from aging and/or pathologic conditions such as arthritis and osteoporosis lead to surgical intervention approaches chiefly total joint replacement (TJR). However, implant-derived wear debris occurs with time causing inflammatory responses culminating with osteolysis and failure of implants. Subsequent revision surgery of the failing joint implant, is often more difficult, and associated with increased morbidity and mortality especially for aging patients with weaker bones. Therefore, the need for effective approaches to diagnose, prevent, and/or treat complications of TJR have risen in recent years. Thus, better understanding of the processes and mechanisms underlying pathologic and osteolytic events leading to joint failure is essential to provide appropriate preventive and therapeutic countermeasures. The pathologic response to implant wear-debris constitutes a major component of this phenomenon and is under intense investigation. Recent work by several groups including ours has identified important cellular entities and secreted factors that contribute to inflammatory osteolysis. In previous work, we have shown that PMMA particles contribute to inflammatory osteolysis through stimulation of major pathways in osteoclast precursors, primarily NF-?B and MAP kinases. The former pathway requires assembly of large IKK complex encompassing IKK1, IKK2, and IKK?, also known as NEMO. We have shown recently that interfering with the NF-?B and MAPK activation pathways, through introduction of inhibitors and decoy molecules, impede PMMA-induced osteolysis in mouse models of experimental calvarial osteolysis and inflammatory arthritis. In our recent work, we found that PMMA particles directly activate the upstream transforming growth factor beta activated kinase-1 (TAK1) which is a key regulator of signal transduction cascades leading to activation of NF-?B and AP-1 factors. More importantly, we found that PMMA particles induce TAK1 binding to NEMO, RIP1, and UBC13. In addition, we show that PMMA particles induced TRAF6 binding to NEMO and lack of TRAF6 significantly attenuates NEMO ubiquitination. We further demonstrate that PMMA induction of NF-?B and MAPK is impaired in TAK1-null and NEMO mutant cells. These responses were not aided by TNF or RANKL. Altogether, these results led us to hypothesize that PMMA particles maybe inducing K63-linked ubiquitination of NEMO, RIP1, and other target proteins, events likely mediated by TRAF6, TAK1 and UBC13. Relevant to this hypothesis, it has been documented that a key mediator of LPS, IL-1, and RANKL signaling, namely TRAF6, is ubiquitin ligase. In separate studies, it was further established that a variety of upstream signals augment ubiquitination-based signaling network dominated by TAK1/TABs/RIP/NEMO/UBC complex. Thus, we propose to investigate the following specific aims: 1. Delineate the molecular steps underlying PMMA-induced regulation of NEMO. 2. Investigate if TRAF6/TAK1/NEMO POLYUBIQUITINATION events mediate PMMA-induced osteoclastogenesis. 3. Determine the effect of inhibiting POLY-UB-NEMO signaling on PMMA-induced calvarial osteolysis.

DESCRIPTION (provided by applicant): The transcription factor NF-(B is essential for osteoclastogenesis and is considered a key modulator of inflammatory responses. Activation of NF-(B entails induction of a large I(B kinase (IKK) complex that comprises IKK1, IKK2, and IKK(/NEMO. IKK2 and IKK1 activate the canonical (p50/p65) and non- canonical (p52/relB) NF-(B pathways, respectively. Recent studies implicate IKK2 and IKK1 as essential for osteoclastogenesis, yet the mechanisms underling this function, remain unclear. In addition, IKK2 is considered as a key mediator of inflammatory events induced by TNF, LPS, and other pro-inflammatory agents (through the classical NF-(B pathway), whereas less is known about the role of IKK1 is such responses. In the past few years we have investigated molecular pathways regulating osteoclast activity in osteolytic responses, such as inflammatory osteolysis and arthritic bone erosion, and unveiled critical regulatory steps modulating NF-(B activation in osteoclast precursors. In this regard, we find that disruption of NF-(B activation at multiple levels attenuates osteoclastogenesis and inflammatory bone erosion. Specifically, we have shown that administration of dominant-negative forms of I(B(, that resist phosphorylation by IKK2; and most recently inhibition of IKK assembly with NEMO, using a decoy NEMO binding domain (NBD) peptide, all are successful approaches to inhibit osteoclastogenesis and bone erosion, in vitro and in vivo. Most importantly, we have preliminary evidence that, 1) failure of RANKL-induced osteoclastogenesis by IKK1-null OCPs is rescued by re-introduction of IKK1 cDNA constructs, and that 2) tissue-specific deletion of IKK2 in OCPs (using CD11b-cre) results with skeletally deformed mice. It is evident that osteoclastogenesis is impaired in the absence of IKK1 or IKK2, however, the precise mechanism(s) underlying signaling of IKKs in basal and inflammatory osteoclastogenesis remain scarce. Moreover, the relative contribution of either protein to these responses is indefinite. It is also known that IKK1 and IKK2 share considerable sequence and domain similarities, albeit they maintain distinct and non-overlapping functions. Thus, we hypothesize that IKK1 and IKK2 differentially regulate basal and inflammatory osteoclastogenesis through the alternative and classical NF-(B activation pathways. Hence, clarifying the individual roles of IKKs in osteoclastogenesis by investigating the details of their molecular signaling in osteoclasts, utilizing germ-line and specific tissue deletions of the relevant genes, will enable us to design strategies and selective inhibitors directed against IKK1 and IKK2 that may be useful for regulating osteoclastogenesis and alleviating inflammatory osteolysis in various bone resorptive disorders. Thus, we propose to investigate the following specific aims: 1) Determine the molecular role of IKK1 and IKK2 in basal and inflammatory osteoclastogenesis. 2) Determine the mechanism by which tyrosine phosphorylation regulates the fate and activity of IKK2 in the osteoclast lineage and it's impact on osteoclastogenesis. 3) Determine the role of IKK1 and IKK2 in inflammatory osteolysis, in vivo. PUBLIC HEALTH RELEVANCE: The transcription factor NF-(B is essential for osteoclastogenesis and modulates inflammatory responses. NF-(B is activated by the serine kinases, I(B kinase (IKK)-1, IKK-2, and IKK(/NEMO. Gene deletions of IKK1 or IKK2 led to defects in osteoclasts and inflammatory responses. Using IKK1 and IKK2-null mice, we propose to investigate the molecular domain contributions of IKK1 and IKK2 to osteoclastogenesis and inflammatory osteolysis, as is the case in inflammatory arthritis. Our proposal holds promise to identify novel selective anti-osteolytic therapies.

DESCRIPTION (provided by applicant): Bone development is tightly regulated by bone forming cells, osteoblasts, and bone resorbing cells, osteoclasts. Therefore, understanding the mechanisms governing osteoclastogenesis is crucial for addressing bone loss pathologies. Differentiation of osteoclasts is governed by RANK ligand which activates several signal transduction pathways, including MAP kinases and NF-?B pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-¿-activated kinase-1 (TAK1) to the receptor RANK. TRAF6, TAK1 and other signaling partners undergo extensive post-translational modifications aimed at stabilizing RANK signaling and enabling precise regulation and execution of proper down stream signals, primarily NF-?B activation. Precise regulation of NF-?B activity is crucial to maintain normal osteoclast activity and bone homeostasis. Conversely, abnormal activity of this transcription factor causes deleterious inflammatory osteolysis. In fact, we discovered recently that constitutive activation of IKK2 is sufficient to induce RANKL-independent osteoclastogenesis in vitro. More convincingly, we reported that knock-in of constitutively active IKK2 causes severe bone loss in mice. Given that IKK2 phosphorylation and activation is governed by TAK1, a MAP kinase heavily implicated in poly-ubiquitination and stabilization of RANK-TRAF6 complexes and down-stream signaling, we decided to investigate its molecular role in osteoclastogenesis. Thus, we generated mice harboring myeloid-specific deletion of TAK1. These mice displayed all hallmarks of osteopetrosis primarily defective osteoclastogenesis. Mechanistically, we observed that Tak1-null precursors fail to generate osteoclasts. More importantly, we discovered diminished expression of key osteoclastogenic proteins including TRAF6, NEMO and Notch-NICD. This phenomenon was associated with accumulation of NUMBL, a previously described neuron protein. Consistent with these observations, we established that exogenous expression of NUMBL induces degradation of TRAF6, NEMO, NOTCH1-NICD, and inhibits osteoclastogenesis in vitro. Inhibition of NUMBL using a dominant negative PTB-phosphotyrosine-binding of NUMBL and shRNAs knockdown of NUMBL enhanced expression of TRAF6 and NEMO and did not inhibit osteoclastogenesis in wild-type cells. In addition, inhibition of NUMBL using a dominant negative PTB of NUMBL and exogenous expression of NOTCH1-NICD restored osteoclastogenesis in TAK1-null cells. Based on these observations we hypothesize that NUMBL is a repressor of osteoclastogenesis and its expression is regulated by TAK1. Deletion of TAK1 leads to accumulation of NUMBL protein which induces degradation of TRAF6, NEMO and NICD proteins, and subsequently blocks osteoclastogenesis. To test this hypothesis, we propose to investigate the following specific aims: 1) Determine the mechanism by which TAK1 regulates NUMBL expression. 2) Determine the mechanism by which TAK1 deletion regulates and impedes expression of TRAF6, NEMO and NICD. 3) Determine the effect of genetic ablation of NUMBL on the osteopetrotic phenotype of TAK1-null mice.

ABSTRACT: The transcription factor NF-kB is expressed ubiquitously in all cell types and is readily activated by numerous factors and cytokines. Baseline NF-kB activity is essential for skeletal development and physiologic cellular functions. In contrast, its exacerbated and often uncontrolled activity during inflammation leads to undesired harmful effects with major dysfunctional consequences including osteolysis. Hence, therapies targeting NF-kB have been highly pursued to combat most inflammatory diseases. Unfortunately, most available therapies are inefficient owing to lack of selectivity in such complex and ubiquitous signaling pathway wherein the essential beneficial functions of NF-kB are blocked along side the harmful effects leading to detrimental outcomes. Therefore, there is an unmet need to decode NF-kB signaling to identify specific targets that assign signal specificity and distinguish between physiologic and pathologic functions. To address this critical knowledge gap, we focused on RANKL-induced osteoclastogenesis as a proof of concept and set out to decipher the NF-kB molecular machinery and identify the signal-specific molecular signature that controls this response in osteoclast progenitors and maintains skeletal homeostasis. We hypothesize that the IKK scaffold IKK?/NEMO serves as a platform that site-specifically assembles unique signal activating or suppressing protein complexes in cell and stimulus specific manners. This hypothesis is based on recent advances implicating NEMO as a scaffold that integrates signaling molecules in response to a wide range of stimuli at lysine (K) specific sites (refer to Fig 2). These modifications include, lysine poly-ubiquitination, SUMOylation, and according to our novel finding, ISGylation; a process of attaching the ubiquitin-like protein, ISG15 (IFN-stimulated gene) to target proteins. We conduced comprehensive NEMO lysine mutational analysis and identified the NEMO K270 residue as a crucial RANKL-regulation target. Specifically, NEMO harboring K270A mutation (NEMOK270A) elicits exacerbated osteoclastogenesis. More importantly, myeloid knock-in mice of the NEMOK270A that we generated displayed severe osteopenia and osteolysis. Mechanistically, autophagy is significantly decreased in NEMOK270A BMMs. Furthermore, proteomic screen identified interferon-stimulated gene-15 (ISG15) as a potential regulator of osteoclastogenesis and autophagy. Thus, our overarching hypothesis is: RANKL-induced binding of ISG15 to NEMO at K270 is essential to restrain osteoclastogenesis by assembling a negative-feedback response. We further posit that mutating K270 hinders this regulatory process leading to reduced autophagy and uncontrolled osteoclastogenesis. Our aims are: Aim 1: Determine the mechanism by which NEMO, through its K270 site, maintains physiologic and restrains pathologic/exacerbated osteoclastogenesis. Aim 2: Determine the role of RANKL-induced ISG15 as the ubiquitin-like protein that facilitates NEMO- K270-mediated autophagy and control of physiologic osteoclastogenesis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are highly prevalent and have reached epidemic proportions in the US and worldwide. These joint diseases are characterized by inflammation, swelling (particularly in RA), pain, and limited mobility. Despite significant advances in developing anti-inflammatory therapies, biologics, and symptomatic pain relief measures, significant shortcomings in treating these diseases remain, buttressing the need for robust research to meet this urgent health predicament. A wide range of small animal models of joint disease, including RA and OA, has been developed in recent years and helped advance our understanding of disease pathology, underlying mechanisms, disease management and therapeutic intervention. However, the reproducible implementation of these models is challenging, especially in the hands of non-experts and due to scarcity of validated benchmark criteria across studies. At the same time, tests of animal behavior, sensitivity, and musculoskeletal function have demonstrated value in identifying symptoms and joint dysfunction in rodent models of arthritis. Yet, the full spectrum of creation of joint injury/disease models and evaluation of functional outcomes to achieve comprehensive analysis is rarely used by most research groups due to limited availability of essential resources. Core D will address this need by supporting model implementation and functional assessment as an integrated resource. Our ability to do so rests on the collective expertise of the Core leaders in inflammatory joint disease (Dr. Abu-Amer), post-traumatic OA (Drs. O?Keefe and Shen) and functional assessment of joint pain and dysfunction (Drs. Guilak and Setton). Notably, four of these investigators joined the WUSTL Research Community in the past few years, which has provided our Center with a unique opportunity to develop this Resource Core. Our goal is to advance current knowledge to bridge gaps in our understanding of the cellular, molecular and functional basis of joint arthritis, and to develop and evaluate new therapeutic strategies. The Core will standardize protocols and support the reproducible implementation of RA and OA models for widespread use by the Research Community. We will facilitate collaboration with Cores B and C to enable comprehensive analyses. Importantly, the Core will organize critical resources, including a facility for testing murine musculoskeletal function and behavior. We will establish a new, organized biomaterial resource to collect and store tissue and serum samples from RA and OA mouse models, which will be made available as a standard resource for histology, gene and protein screens by all investigators. Finally, the Core will provide hands-on training and enrichment program to train the next generation of joint investigators. Our Specific Aims are: Aim 1: Support the implementation and utilization of reproducible OA and RA mouse models. Aim 2: Provide measures of biomechanics, behavior, and function to assess mouse joint function. Aim 3: Establish murine OA and RA biomaterials repository. Aim 4: Provide hands-on training, outreach and enrichment.