Tenofovir treatment of primary osteoblasts alters gene expression profiles: Implications for bone mineral density loss

Abstract

There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss.

Introduction

There are currently over 30 million people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) has been very successful in the clinical management of HIV infection. Nucleoside reverse transcriptase inhibitors (NRTIs) are used in virtually all HAART regimens. The NRTIs target HIV reverse transcriptase and prevent the synthesis of viral cDNA from the genomic RNA. NRTI’s act as chain terminators of HIV DNA synthesis since NRTIs lack a 3′-hydroxyl group on the deoxyribose and following incorporation the next incoming deoxynucleotide cannot form a new 5′–3′ phosphodiester bond that is required to extend the synthesis of the DNA chain. HIV drug resistance and associated drug metabolic disorders have driven the need for the identification of new drug targets and the development of new anti-HIV drugs.

Tenofovir, 9-(R)-{2-(phosphonomethoxy)propyl}adenine or PMPA, is a nucleotide analog that was approved in 2001 for use in treating HIV infection and more recently for chronic hepatitis B infection. Tenofovir disoproxil fumarate (TDF) is the prodrug form. Tenofovir is popular and well prescribed for several reasons. First, tenofovir has improved potency as it is a nucleotide and has an abbreviated intracellular activation pathway to allow a more rapid and complete conversion to the active drug. Second, TDF has a labile lipophilic group to facilitate penetration through target cell membranes. Third, tenofovir is not known to be a substrate, inducer or inhibitor of human cytochrome P450 enzymes in vitro or in vivo. Fourth, tenofovir has high potency and an unusually durable response when used in trials of single-agent therapy intensification in highly treatment-experienced individuals. Fifth, the active metabolite, tenofovir diphosphate, exhibits a long intracellular half-life in both resting and activated peripheral blood mononuclear cells, which allows for single daily dosing.

HIV infection [1], [2], [3], [4] as well as tenofovir treatment of adults [5] have been implicated in causing osteopenia. Tenofovir-associated loss of bone mineral density has also been observed with children and adolescents [6], [7], [8]. Similar findings of reduced bone mineral density have been reported with use of tenofovir during infection of macaques with simian immunodeficiency virus [9], [10], [11].

Osteoblasts, which are derived from meschenchymal stem cells, synthesize collagen and glycoproteins that forms osteoid [12]. Osteoclasts, which are derived from hematopoietic stem cells, on the other hand, resorb bone. Bone is a dynamic tissue characterized by cycles of bone resorption and bone formation. Osteoblast differentiation is regulated by many cytokines including bone morphogenetic proteins (BMPs), which are necessary for inducing osteoblast differentiation and maintaining the differentiated state of committed osteoblasts [13], [14]; transforming growth factor-beta (TGF-β), which is a regulator of osteoblast differentiation [15]; and fibroblast growth factor (FGFs), which antagonizes the positive osteoblast differentiation effects of the Wnt signaling pathway [16]. Extensive cell signaling between osteoblasts and osteoclasts is required for maintaining a balance in the activities of osteoblasts and osteoclasts. Changes in osteoblast function can lead to reduced bone and either osteopenia or osteoporosis.

Based upon the observations of tenofovir-associated reduced bone mineral density, we sought to explore the basis for these clinical observations. In this study, we investigated whether tenofovir could perturb osteoblast gene expression profiles in a manner that could impact osteoblast function in bone formation. To do this, we initially harvested primary murine osteoblasts and exposed them to various TDF concentrations. TDF concentrations that were physiologically relevant to the clinical dosing in humans were then further used for treating primary osteoblasts followed by isolation of total RNA for use in microarray analysis. We observed a wide spectrum of gene expression changes in osteoblast genes, including many associated with osteoblast function in bone formation. Our findings show for the first time that tenofovir can result in the perturbation of osteoblast gene expression and that these changes implicate loss of osteoblast function that could lead to reduced bone mineral density.