We will study regulation of osteoclastogenesis by calcium, focusing on a recently discovered calcium-release activated calcium channel pathway. Activation of osteoclast resorption is the final common pathway in pathologic bone destruction, and Ca2+ signaling is critical to RANKL-stimulated osteoclastogenesis. In osteoclast precursors, Ca2+ signals activate the transcriptional regulator NFATc1, which drives expression of osteoclast-specific proteins. Other effects of NFATc1 include a role, now poorly defined, in fusion of osteoclast precursors. However, the the spatial and temporal patterns of Ca2+ fluxes regulating differentiation were largely unknown. We identified the calcium release activated calcium channel Orai1 as a mediator of extracellular Ca2+ influx in osteoclast precursors in vitro. In human osteoclast differentiation in vitro, we showed that absence or inhibition of Orai1 blocked the formation of mature multinucleated osteoclasts and greatly reduced bone mineral resorption. Studies of Orai1-/- mice showed similar changes in vivo, with associated with skeletal abnormalities, confirming the importance of Orai1 in bone regulation. We hypothesize that Orai1 mediates critical Ca2+ signals that regulate osteoclast formation by controlling the fusion of precursor cells.
In Aim 1 we will define in vivo the function of Orai1 in osteoclast precursors. We will generate a mouse allowing tissue-selective deletion of Orai1 from osteoclast precursors. This will avoid pitfalls of complete Orai1 deficiency, which affects multiple cell type, leading to early mortality. We will compare the skeletal effects of a novel tissue-selective Orai1 knockout mouse to those of the global Orai1 knockout, to distinguish direct from indirect effects of Orai1 on osteoclastogenesis and skeletal regulation. We will analyze the skeletal effects of selective Orai1 deficiency in osteoclast precursors in vivo and identify abnormalities of differentiation caused by loss of Orai1 from osteoclast precursors.
In Aim 2 we will determine the mechanisms by which Orai1 calcium signals modulate osteoclast precursor gene transcription to regulate osteoclast formation, particularly cell fusion. This work will include studies defining Orai1-dependent Ca2+ fluxes in response to osteoclast differentiation signals via measurement of Ca2+ oscillations with respect to osteoclast differentiation and activity in wild type and Orai1 deficient cells. Pathway analysis will, further, define NFATc1-regulated gene expression as a function of Ca2+ activation in Orai1-deficient and WT cells, and we will identify and analyze fusion- related genes dependent on Orai1. These studies will define mechanisms through which Orai1 regulates osteoclastogenesis, which will guide the development of new strategies to control pathologic osteoclastic activity.

Public Health Relevance

We will study the mechanism by which a recently discovered calcium-release activated calcium channel, Orai1, controls differentiation of osteoclasts, the cells that destroy bone in arthritis and osteo- porosis. The work will produce an osteoclast-precursor specific Orai1-knockout mouse that will allow the function of this pathway to be studied in living animals, in isolation from other factors. Orai-1 de- pendent calcium signaling, and downstream pathways involved in differentiation and cell fusion, will be analyzed in vitro, using both mouse and human cells, to assure applicability of the work to humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
4R01AR065407-04
Application #
9116042
Study Section
Musculoskeletal Tissue Engineering Study Section (MTE)
Program Officer
Alekel, D Lee
Project Start
2013-09-16
Project End
2018-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
4
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Tantikanlayaporn, Duangrat; Tourkova, Irina L; Larrouture, Quitterie et al. (2018) Sphingosine-1-Phosphate Modulates the Effect of Estrogen in Human Osteoblasts. JBMR Plus 2:217-226
Tourkova, Irina L; Liu, Li; Sutjarit, Nareerat et al. (2017) Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes. Lab Invest 97:1072-1083
Papachristou, Nicholaos I; Blair, Harry C; Kypreos, Kyriakos E et al. (2017) High-density lipoprotein (HDL) metabolism and bone mass. J Endocrinol 233:R95-R107
Blair, Harry C; Soboloff, Jonathan; Robinson, Lisa J et al. (2016) Suppression of arthritis-induced bone erosion by a CRAC channel antagonist. RMD Open 2:e000093
Blair, Harry C; Kalyvioti, Elena; Papachristou, Nicholaos I et al. (2016) Apolipoprotein A-1 regulates osteoblast and lipoblast precursor cells in mice. Lab Invest 96:763-72
Blair, Harry C; Sepulveda, Jorge; Papachristou, Dionysios J (2016) Nature and nurture in atherosclerosis: The roles of acylcarnitine and cell membrane-fatty acid intermediates. Vascul Pharmacol 78:17-23
Tourkova, Irina L; Witt, Michelle R; Li, La et al. (2015) Follicle stimulating hormone receptor in mesenchymal stem cells integrates effects of glycoprotein reproductive hormones. Ann N Y Acad Sci 1335:100-9
Palagano, Eleonora; Blair, Harry C; Pangrazio, Alessandra et al. (2015) Buried in the Middle but Guilty: Intronic Mutations in the TCIRG1 Gene Cause Human Autosomal Recessive Osteopetrosis. J Bone Miner Res 30:1814-21
Zacherl, Janelle R; Tourkova, Irina; St Croix, Claudette M et al. (2015) Elaidate, an 18-carbon trans-monoenoic fatty acid, but not physiological fatty acids increases intracellular Zn(2+) in human macrophages. J Cell Biochem 116:524-32
Robinson, Lisa J; Mancarella, Salvatore; Songsawad, Duangrat et al. (2012) Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development. Lab Invest 92:1071-83