Background/Purpose:

Human Immunodeficiency Virus (HIV) infection devastates the immune system but also affects tissues and organs such as kidney, liver, central nervous system, heart and bone. Bone alterations have been observed in HIV disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment of patients with Tenofovir alone or in combination (as part of HAART), leads to further changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated; tenofovir-phosphate inhibits HIV-reverse transcriptase by mimicking AMP. We have recently found that Tenofovir inhibits Pannexin-1/Connexin-43-mediated ATP release from cells and decreases extracellular adenosine levels and fibrosis in murine models. As adenosine and ATP are key regulators of bone homeostasis, we determined whether Tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism.

Methods:

M-CSF/RANKL-induced osteoclast (OC) and stimulated osteoblast (OB) differentiation were studied in primary murine bone marrow culture as the number of TRAP-positive or Alizarin Red-positive cells, respectively, after challenge with Tenofovir (1nM-100µM) alone or in combination with Dipyridamole (1nM-100µM), an agent that increases extracellular adenosine by blocking cellular adenosine uptake. Pannexin-1 and Connexin-43 expression were permanently knocked down in RAW264.7cells by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. OC/OB differentiation markers were study by RT-PCR, and intracellular pathways by Western Blot.

Results:

Tenofovir produced a dose-dependent increase in OC differentiation (EC50=44.5nM) that was reversed by Dipyridamole (IC50=0.3µM). Tenofovir increases Cathepsin K and NFATc1 mRNA levels during OC differentiation, and the effect was reversed by Dipyridamole. When both Pannexin-1 and Connexin-43 were absent, Tenofovir did not increase OC number. Dipyridamole reversed the effect of Tenofovir on pERK1/2, pp38 and NFkB nuclear translocation. Tenofovir inhibits OB differentiation in a dose-dependent manner (IC₅₀=0.4µM) and treatment with Dipyridamole reversed this effect (EC₅₀=10nM). Tenofovir increases RANKL mRNA expression and decreases OPG mRNA expression during OB differentiation; these effects are reversed by Dipyridamole. We have also found alterations in beta catenin signaling pathway due to Tenofovir treatment.

Conclusion:

Tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism, a finding that suggests that treatment with agents that increase local adenosine concentrations, like Dipyridamole, might prevent bone loss due to Tenofovir treatment.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/tenofovir-a-nucleoside-analog-reverse-transcriptase-inhibitor-for-treatment-of-hiv-promotes-osteoclast-differentiation-and-decreases-osteoblast-formation-by-a-mechanism-depending-on-atp-release-and-2/