Kyung-Hyun Park-Min, Ph.D. - US grants

DESCRIPTION (provided by applicant): Negative Regulation of Osteoclastogenesis by Inflammatory signals Rheumatoid arthritis (RA) is a chronic inflammatory disease in which immune cells and synovial fibroblasts produce pro-inflammatory cytokines, in particular TNF and IL-1, and drive an inflammatory state leading to destruction of affected joints. One important consequence of inflammation is the generation of osteoclasts, myeloid lineage cells that effectively resorb bone and thus are directly responsible for bone erosion and morbidity in RA. This application will focus on mechanisms of inhibition of the generation of osteoclasts. TLRs (Toll-like Receptors) are potent activators of inflammation and have been implicated in driving inflammatory bone resorption. However, at the same time that they activate inflammation, TLRs also induce potent homeostatic mechanisms to limit the intensity of inflammation and thus limit associated tissue damage. Disease progression is evidence of relatively ineffective feedback inhibition that is unable to restrain inflammation and bone resorption. Thus, we have initiated studies to understand the effective homeostatic regulation that occurs during physiological resolution of inflammation and quiescent phases of disease. Our overall hypothesis is that augmentation of physiological homeostatic mechanisms represents an effective approach to limiting bone resorption associated with inflammation and thus can form the basis for novel therapeutic approaches. We have found that TLR stimulation strongly suppresses osteoclastogenesis by inhibiting RANK, a key receptor required for osteoclastogenesis. The mechanism of TLR-mediated inhibition involves induction of a M-CSF receptor (c-Fms) shedding and proteolytic processing by activated TNF-alpha converting enzyme (TACE, also known as ADAM-17), potentially leading to cellular unresponsiveness to M-CSF. Cleaved c-Fms subsequently undergoes a series of proteolytic reactions that results in generation of the 50-kDa intracellular domain cleavage fragments (referred to as MICD). Interestingly, our results reveal that MICD is able to translocate into the nucleus, and expression of MICD enhances osteoclastogenesis. MICD generation is also diminished by inflammatory signals. We will use human systems that are directly relevant for RA pathogenesis as well as mouse system to test the in vivo role of MICD and TACE in inflammatory diseases. The long-term goals of this project are to: 1) understand molecular mechanisms by which c-Fms shedding and processing into MICD regulate osteoclastogenesis and the association of TACE with this process, and 2) identify the role of MICD in M-CSF signaling and differentiation of osteoclasts and macrophages. We anticipate that our studies will yield insights into homeostatic regulation that can not only be exploited for therapeutic interventions to suppress bone resorption associated with joint destruction but will also broaden understanding of the actions of M-CSF in the field of osteoimmunology. PUBLIC HEALTH RELEVANCE: The erosion of bone is a key factor in the development and exacerbation of chronic inflammatory diseases, such as rheumatoid arthritis. Osteoclasts are the specific cells that cause bone erosion, and this application explores new mechanisms whereby osteoclasts can be inhibited. By deterring the development and activation of osteoclasts in inflammatory settings, we can hinder bone erosion and therefore prevent disease development.

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which immune cells and synovial fibroblasts produce pro-inflammatory cytokines and drive an inflammatory state leading to the destruction of affected joints. Bone erosion is a diagnostic hallmark of RA and commonly precedes the development of clinical symptoms. Osteoclasts are myeloid lineage cells that effectively resorb bone and are directly responsible for bone erosion and morbidity in RA. Thus, our overall hypothesis is that a better understanding of the regulation of osteoclast differentiation and activity is likely to yield novel targets for therapies that limit pathological bone resorption. We have found that the transcription factor MYC and MYC-dependent transcriptional programs are activated by RANKL during early osteoclast differentiation. Although MYC has been implicated in osteoclastogenesis, the precise mechanisms by which MYC affects the homeostasis and function of osteoclasts remain largely unexplored. We have found that MYC is required for osteoclast differentiation and regulates the genes that are associated with metabolism and translation during osteoclastogenesis. Interestingly, both MYC and NFATc1 expression are significantly elevated in synovial osteoclast precursors (OCPs) from patients with RA that have a greater potential for differentiating into osteoclasts. OCPs are thought to reprogram their metabolism to meet the energy demands of osteoclasts, which must fuse into multinucleated cells and synthesize molecules to resorb bone. However, the contribution of metabolic pathways to osteoclast differentiation and the key molecule that regulates metabolic reprogramming are not well understood. Therefore, we hypothesize that MYC plays an important role in RANKL-induced metabolic reprogramming and MYC is one of the major contributors to generate hyperactive osteoclasts in inflammatory bone diseases by altering specific metabolic pathways. To test our hypothesis, we proposed three specific aims :1) to characterize the role of MYC in osteoclastogenesis in vivo, 2) to identify the molecular mechanisms underlying the regulation and function of MYC, and 3) to investigate mechanisms by which MYC regulates metabolic reprogramming in osteoclasts. This study will advance our understanding of the role of MYC in osteoclast differentiation, the role of metabolic reprogramming occurring during osteoclast differentiation, and the crosstalk between MYC and metabolic reprogramming during osteoclastogenesis. In addition, as therapies directly targeting MYC activation are not presently available in the clinic, identification of effector molecule(s) downstream of MYC that play important roles in osteoclast differentiation may serve as novel therapeutic targets for the treatment and prevention of pathological bone resorption. Therefore, the overall impact of this project is to yield insights that will not only broaden our understanding of the role of MYC in the field of osteoimmunology, but can also be exploited to develop therapeutic interventions to suppress bone resorption by hyperactive osteoclasts resulting from deregulated MYC expression.

Osteoclasts are large myeloid-derived multinucleated cells primarily responsible for bone resorption. Dysregulation of osteoclast differentiation can result in net bone resorption and is key to the pathophysiology of rheumatoid arthritis, osteoporosis, and lytic bone metastasis. Thus, understanding the mechanism of osteoclast differentiation is of great therapeutic importance for nearly all forms of metabolic bone disease. Our long-term goals are 1) to elucidate a role of the newly described regulatory pathway in osteoclastogenesis and arthritic bone resorption and 2) to develop an effective and specific way to treat bone loss in RA. c-FMS, a receptor for M-CSF/ IL-34, transduces essential signals for the differentiation of osteoclasts and macrophages. Aberrant expression of c-FMS (or M-CSF) has been linked to exacerbation of diseases such as inflammatory arthritis and cancers. We have identified a novel regulatory pathway of c-FMS initiated by TACE (TNF-? converting enzyme). This pathway involves the coordinated, sequential cleavage of c-FMS by TACE, ?-secretase, and calpain, which results in the generation of intracellular domain cleavage fragments (referred to as FICD). Myeloid-specific TACE-deficient mice have high bone mass with decreased osteoclast numbers and ameliorate bone destruction in TNF-? induced arthritis in TNF transgenic (tg) mice. We found that FICD generation is critical for osteoclastogenesis, as enforced expression of FICD rescues the impaired osteoclastogenesis seen in TACE deficient cells. Thus, in addition to the well known role of c-FMS to activate conventional M-CSF signaling pathways as a surface receptor, c-FMS is also processed into the FICD that traffics into the nucleus, where it functions as a positive regulator of osteoclastogenesis. The existing paradigm is that shedding of c-FMS from the cell surface is a negative event related to loss of a signaling receptor. However, our study led us to discover that TACE-mediated shedding of c-FMS provides a positive signal for osteoclastogenesis. Here, we seek to build upon our novel findings to unravel the mechanisms by which the TACE/FICD axis regulates bone resorption, with a specific focus on inflammatory arthritis. Thus, we hypothesize that :1) TACE deficiency- mediated attenuation of arthritic bone resorption is, in part, due to lack of FICD, 2) Inhibition of the TACE/FICD axis ameliorates arthritic bone resorption, and 3) FICD targets the transcriptomic program in osteoclastic bone resorption. Our specific aims are 1) to investigate the underlying mechanisms behind how the TACE/FICD axis regulates pathological bone resorption in TNF-tg mice and 2) to elucidate the mechanism by which the TACE/FICD axis regulates osteoclastogenesis by identifying the direct pathways/targets of FICD. We anticipate that a better understanding of the diverse roles of this TACE/c- FMS/FICD pathway will advance our understanding of fundamental osteoclast biology, and targeting the TACE/FICD axis may provide new ways to attenuate bone resorption in inflammatory arthritis.