The initial event in glucocorticoid (GC)-induced osteonecrosis, in many cases, is death of bone-synthesizing osteoblasts. Our preliminary work shows that this osteoblast death, at least in major part, is not directly caused by the GC. Bone growth and survival is regulated by many systems, vascular endothelial growth factor (VEGF) being of critical importance. Developing osteoblasts, prominent in in high turnover bone such as femoral head trabecular bone, express the adrenocorticotropic hormone (ACTH) receptor. We discovered that osteoblasts express VEGF strongly in response to ACTH. Continuous steroid treatment reduces ACTH to very low levels. In rabbits with high-dose GC, we showed that intermittent administration of ACTH greatly reduced osteonecrosis. Our hypothesis is that ACTH is a key regulator of bone growth and survival, particularly in regions with rapid bone turnover. However, mesenchymal stem cells or osteoblasts express VEGF receptors and ACTH receptors. Further, our pilot studies show that VEGF increases growth and differentiation or osteoblasts. Because ACTH is one of several factors that regulate VEGF production in bone, systematic study is needed to determine how ACTH, VEGF, and other regulatory pathways interact in bone. Gaps in understanding include that downstream actions of ACTH in bone cells are poorly understood. The interactions of ACTH with other systems that regulate VEGF are unclear. These interactions may be mediated by inflammatory cells, hypoxia, or additional cell signals. It is not known whether ACTH synthesis occurs in bone. It is not known how ACTH action in bone varies with frequency or dose of ACTH administration.
Aim 1 will study the mechanism of response of osteoblasts to ACTH and VEGF. To assure relevancy to human disease, study will include human cells in vitro. To determine whether ACTH provides survival signals in addition to VEGF, we will study the response of osteoblasts to VEGF, with and without ACTH. Cell proliferation and matrix synthesis will be measured, as well as production of regulatory cytokines by osteoblasts. To define the VEGF response, we will make osteoblasts with VEGF receptors -1 and -2 (ft-1 and fk-1) eliminated. This will allow ACTH effects on osteoblasts to be defined in the absence of autocrine VEGF response.
Aim 2 will determine how ACTH modulates VEGF production in GC-treated bone. To determine how immune cells regulate production of VEGF by osteoblasts, we will make mixed cultures including macrophages or lymphocytes. In addition, the Scarb1 mouse model, which has elevated ACTH at normal GC and dense bone, will be studied to determine changes to bone and vasculature in vivo. We will characterize VEGF production in culture, and determine whether VEGF regulation involves specifc cytokines, in tissue cultures and in mice. To establish the effect of hypoxia, we will compare VEGF production in 7% versus 20% oxygen in human cell cultures. Analysis will include production of key cytokines as a function of oxygen tension. To determine whether ACTH synthesis occurs in bone, we will analyze pro-opiomelanocorticoid (POMC) expression and processing in osteoblasts, lymphocytes and macrophages.
Aim 3 will defne the dependency of VEGF synthesis in bone on ACTH concentration and dose interval using depot methylprednisolone acetate (MPA)-treated rabbits. Rabbits are the best in vivo model for CG-induced osteonecrosis; steroid diabetes will occur but will be controlled with insulin. Effects on VEGF production of varying IV ACTH injection, relative to depot MPA alone or no treatment, will establish concentration dependency. ACTH will be injected daily, at 8 AM, at 0.01 to 0.3 mg/kg, for 28 days. Osteonecrosis, bone turnover, serum ACTH and corticosteroids will be measured. This will establish the dose dependency of ACTH osteonecrosis suppression. To defne effect of frequency of administration on efficacy, we will compare the effects of ACTH 0.05 ?kg and 0.15 ?kg twice daily versus 0.1 or 0.3 ?kg once daily. This work will use innovative methods to defne a novel metabolic regulatory pathway in bone. It will establish new mechanisms that contribute to osteonecrosis, which may allow reduction of its occurrence.

Public Health Relevance

Glucocorticoid (GC)-induced osteonecrosis of bone is a common and miserable problem. It ecessitates hip replacement in about 50,000 people in the US annually, including in many Veterans. We discovered that a large component of osteonecrosis is not caused directly by GC, but by adrenocorticotropic hormone (ACTH). ACTH is reduced to very low levels by GC treatment. Specifcally, ACTH causes bone cells to make VEGF, a major growth and survival factor. The work here will defne the downstream actions of ACTH in human bone cells and in a mouse model, including the interactions of ACTH with other systems that regulate VEGF. Addition- ally, how ACTH action varies with time or quantity of administration will be determined using rabbits, the best animal model. This will establish new mechanisms that contribute to osteonecrosis, which may allow reduction of the severity, or prevention entirely, of a common type of osteonecrosis.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX002490-03
Application #
9275423
Study Section
Endocriniology A (ENDA)
Project Start
2014-10-01
Project End
2018-09-30
Budget Start
2016-10-01
Budget End
2017-09-30
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Veterans Health Administration
Department
Type
Independent Hospitals
DUNS #
033127569
City
Pittsburgh
State
PA
Country
United States
Zip Code
15240
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; 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
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