Downregulation of Gnas, Got2 and Snord32a following tenofovir exposure of primary osteoclasts

Abstract

Clinical observations have implicated the antiretroviral drug tenofovir with bone density loss during the management of HIV infection. The goal of this study was to investigate the in vitro effects of tenofovir exposure of primary osteoclasts in order to gain insights into the potential mechanisms for the drug-induced bone density loss. We hypothesized that tenofovir may alter the expression of key genes involved in osteoclast function. To test this, primary osteoclasts were exposed to physiologically relevant concentrations of the prodrug tenofovir disoproxil fumarate (TDF), then intensive microarray analysis was done to compare tenofovir-treated versus untreated cells. Specific downregulation of Gnas, Got2 and Snord32a were observed in the TDF-treated cells. The functions of these genes help to explain the basis for tenofovir-associated bone density loss. Our studies represent the first analysis of the effects of tenofovir on osteoclast gene expression and help to explain the basis of tenofovir-associated bone density loss in HIV-infected individuals.

Figure 1

Tenofovir structure and primary osteoclast cell viability following drug exposure. The structure of tenofovir (A) and tenofovir disoproxil fumarate (TDF, the prodrug of tenofovir) (B) is shown. C. Viability of primary osteoclasts following exposure to TDF. A TDF dilution series was added to primary osteoclast cultures for 72 hr, refreshing every 24h. Cell viability was determined by ATP detection. Tartrate-resistant acid phosphatase (TRAP) staining of untreated (D) and TDF-treated (E) primary osteoclasts and analysis (F) is shown.

Figure 2

Target genes, Gnas, Got2 and Snord32a, following tenofovir exposure of primary osteoclasts. A. Gnas or stimulatory G-protein alpha subunit (GαS) is a key part of the classic signal transduction pathway linking receptor-ligand interactions with the activation of adenylate cyclase and a variety of cellular responses (including the MEK-Erk signaling pathway). B. Got2 (glutamate oxaloacetate transaminase 2) is a mitochondrial enzyme involved in energy transduction, specifically amino acid metabolism as well as the urea and tricarboxylic acid cycles. C. Snord32a (small nucleolar RNA, C/D box 32A) is a member of the small nucleolar RNA (snoRNA) group of highly abundant, non-polyadenylated, non-coding transcripts. The C/D box snoRNAs are associated with methylation, and specifically guide site-specific 2′-O-methylation of RNAs, which impact gene expression.