Pathologic bone resorption due to osteoporosis, fragility fractures and cancers pose a significant economic burden to healthcare costs and lost wages. These osteolytic diseases share a similar pathophysiology, so understanding the mechanisms of bone resorption greatly aids the search for treatment options. The need to develop therapeutic options with minimal side effects is strong, but requires greater elucidation into the mechanisms of inflammatory bone resorption. We previously confirmed the role of pERK1/2 on the production of pro-osteoclastogenic cytokines such as RANKL, MCSF, COX-2 and IL-1b. Then, we focused on a seemingly overlooked target for inhibiting osteoclastogenesis, IL-1b, an inflammatory cytokine implicated in pathologic osteolysis. An IL-1b Converting Enzyme (ICE, or Caspase-1) inhibitor showed dramatic inhibition of osteoclastogenesis. However, Caspase-1inhibitor -/- osteoclast precursors did not demonstrate impaired osteclastogenesis, implying off-target components to this osteolysis-inhibition mechanism. Subsequently, our proteomic screening efforts yielded us Calregulin (CRG) as our osteoclastogenic-inhibition focus. Subsequent in vitro and in vivo studies met with great success as administration of recombinant human Calregulin (rhCRG) demonstrated inhibition of osteoclastogenesis in vitro in osteoclast-precursor macrophages and inhibition of LPS-induced osteolysis in vivo. rhCRG inhibited NFATc1 among several osteoclastogenic transcription factors. The new direction of this competitive renewal is to test a novel hypothesis that Calregulin inhibits osteoclastogenesis in vitro and inflammation-associated osteoclastogenesis in vivo. Therefore, our aims for this proposal employ 3 parallel Aims to determine mechanisms and therapeutic translation in vitro and in vivo.
Aim 1 will determine mechanisms by which rhCRG interferes with key osteoclastogenic pathways such as NFATc1 in vitro. We will determine whether rhCRG interferes with osteoclastogenic gene expression, calcium oscillation, NFATc1 activity, and bone resorption. In addition, we will examine whether rhCRG/NFATc1 functionally interact with 6 candidate proteins which we identified on Liquid Chromatography/Mass Spectrometry of His-CRG pulled-down proteins.
Aim 2 will determine whether CRG regulates macrophage activation in inflammatory osteolysis in vitro and in vivo. Macrophage activation of M1 or M2 plays pro- and anti-inflammatory roles, respectively. By determining the CRG's role in the polarization of M2 macrophages and inhibition of M1, we will establish an alternative mechanism.
Aim 3 will establish a therapeutic role of topical rhCRG in inflammatory osteolysis in vivo. We will induce osteolysis with clinically relevant RANKL, TNF and LPS to determine whether rhCRG can prevent or treat inflammation-associated osteoclastogenesis and bone resorption. We will measure bone protective effects of rhCRG using Cathepsin K molecular imaging, dynamic bone histomorphometry and TRAP staining. Overall impact is high in that we expect to unravel novel anti-osteoclastogenic mechanisms and therapeutic promise of rhCRG in the context of inflammatory osteolysis.
Musculoskeletal disorders are the leading cause of disability in the U.S., and they are projected to become more prevalent with the rapid growth in population of people sixty-five and older. A significant number of people will require medical intervention fo bone loss due to osteoporosis and metastatic bone cancers. While current therapeutics have their merits and employ diverse mechanisms of action, there are side effects that become evident after years of clinical application. Therefore, there exists an unmet need to develop novel and safe pharmaceutical therapies for bone loss. The identification of Calregulin as a regulator of osteoclastogenesis, a major factor in bone loss, will expand scientific knowledge and therapeutic opportunities for developing potentially safer treatments for bone loss.
|Fischer, Charla R; Mikami, Maya; Minematsu, Hiroshi et al. (2017) Calreticulin inhibits inflammation-induced osteoclastogenesis and bone resorption. J Orthop Res 35:2658-2666|
|Worthley, Daniel L; Churchill, Michael; Compton, Jocelyn T et al. (2015) Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell 160:269-84|
|Patel, Neel; Nizami, Saqib; Song, Lee et al. (2015) CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways. J Orthop Res 33:1474-86|
|Kim, Kyung Ok; Hsu, Anny C; Lee, Heon Goo et al. (2014) Proteomic identification of 14-3-3? as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells. J Orthop Res 32:848-54|
|Lee, Heon Goo; Hsu, Anny; Goto, Hana et al. (2013) Aggravation of inflammatory response by costimulation with titanium particles and mechanical perturbations in osteoblast- and macrophage-like cells. Am J Physiol Cell Physiol 304:C431-9|
|Lee, Heon Goo; Minematsu, Hiroshi; Kim, Kyung Ok et al. (2011) Actin and ERK1/2-CEBP? signaling mediates phagocytosis-induced innate immune response of osteoprogenitor cells. Biomaterials 32:9197-206|
|Minematsu, Hiroshi; Shin, Mike J; Celil Aydemir, Ayse B et al. (2011) Nuclear presence of nuclear factor of activated T cells (NFAT) c3 and c4 is required for Toll-like receptor-activated innate inflammatory response of monocytes/macrophages. Cell Signal 23:1785-93|
|Seo, Sung Wook; Lee, Daniel; Minematsu, Hiroshi et al. (2010) Targeting extracellular signal-regulated kinase (ERK) signaling has therapeutic implications for inflammatory osteolysis. Bone 46:695-702|