8th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV


San Francisco, California - September 24 - 26, 2006

MODIFICATION OF TRANSCRIPTIONAL ACTIVITY AND REGULATION DRIVES OSTEOBLAST RESPONSE TO HIV AND ITS TREATMENT IN VITRO

Antiviral Therapy 2006; 11:L9 (abstract no. 11)

EJ Cotter, N Chew, WG Powderly, PP Doran and AP Malizia
UCD School of Medicine and Medical Sciences, Mater Hospital, Dublin, Ireland

This oral abstract is a combination of data presented within Posters 103 and 104 (see abstracts below).

ABSTRACT 103
Antiviral Therapy 2006; 11:L62

HIV-1 PROTEASE INHIBITORS SELECTIVELY INDUCE GENE EXPRESSION ALTERATIONS ASSOCIATED WITH REDUCED CALCIUM DEPOSITION IN PRIMARY HUMAN OSTEOBLASTS

AP Malizia, EC Cotter, WG Powderly and PP Doran
General Clinical Research Unit, Mater Misericordiae University Hospital, School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland

HIV infected patients are at increased risk of decreased bone mineral density. Some studies have implicated antiretroviral therapy as a contributor to the decreased bone mineral density seen in treated HIV-1 patients. In this study we explore the interactions between protease inhibitors (PI) and primary human osteoblast gene expression, highlighting a group of dysregulated genes which are potentially key factors in reducing bone formation. Runx2 mRNA expression, calcium deposition and alkaline phosphatase activity (ALP) activity decreased significantly in human osteoblast cultures after exposure to the PIs nelfinavir (NFV) and indinavir (IDV). Saquinavir (SQV), ritonavir (RTV), indinavir (IDV) or nelfinavir (NFV) exposure induced significant changes in genotypic expression as assessed by gene-chip microarray analysis. The altered genes from each group were compared to each other and a list of 8 upregulated and 13 downregulated genes only after NFV and IDV exposure was identified. This set includes TIMP-3 which has been previously demonstrated to be involved in osteoblast differentiation and extracellular matrix development processes. Silencing TIMP3 mRNA expression using siRNA duplexes enhanced calcium deposition and ALP activity significantly, even after exposure to NFV and IDV. Our data suggests a link between reduced osteoblastic phenotype and a group of 21 altered genes following NFV and IDV treatment, and also suggests TIMP-3 may involved in the PI induced inhibition of osteoblast function.

ABSTRACT 104
Antiviral Therapy 2006; 11:L62

HIV PROTEINS SUPPRESS OSTEOBLAST FUNCTION VIA MODULATION OF RUNX-2 AND PPARγ TRANSCRIPTION FACTOR ACTIVITY

EJ Cotter, N Chew, WG Powderly and PP Doran
UCD School of Medicine and Medical Sciences, Mater Hospital, Dublin, Ireland

AIMS: Decreased bone mineral density (BMD) has been associated with HIV infection, however the exact molecular mechanism of decreased BMD remains to be elucidated. RUNX-2 is a transcription factor that drives the development and maintenance of an osetoblastic phenotype in mesenchymal stem cells (MSCs) and osteoblasts (OBs), while PPARγ is associated with pro-adipogenic development in MSCs. In this study we have examined in vitro the effect of exposure to HIV proteins on osteoblast function and on the expression and activity of PPARγ and RUNX-2.

METHODS: An immature human osteoblast (hOB) cell line (PromoCell) was cultured and treated with HIV p55-gag, HIV gp120 and HTLV env (100 ng/ml, 24 h) (NIH Aids Reagent Program). HTLV-1 env, was used to control for non-specific viral effects. Cells were analysed for calcium deposition and alkaline phosphotase activity (ALP) using quantitative alizarin red staining and p-NNP assay respectively. Real-Time PCR with gene specific primers was used to analyse mRNA levels of RUNX-2, and PPARγ. RUNX- 2 and PPARγ activity were assessed using a commercially available assay (Active Motif).

RESULTS: Exposure of hOBs to p55-gag and gp120 significantly altered osteoblast phenotype, as evidenced by reduced calcium deposition of hOBs by 30 ±10% and 29 ±8% and a reduction in ALP activity (11 ±5% and 19 ±0.1% respectively P=0.05). Both p55-gag and gp120 significantly reduced RUNX-2 mRNA levels (60 ±14% and 35% respectively P=0.05), however no significant changes in PPARγ mRNA levels were observed. The activity of RUNX-2 was reduced by both p55-gag and gp120 (38 ±5% and 25 ±0.8% respectively P=0.05), while gp120 significantly increased PPARγ activity (179 ±50%, P=0.05).

CONCLUSION: Our findings suggest that exposure to HIV proteins decreases bone formation in osteoblasts and this change in phenotype is paralleled by alterations in RUNX-2 and PPARγ transcription factor activity in osteoblasts. Alterations of these activities may provide a molecular mechanism that contributes to HIV-associated reduction in BMD.

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2006-09-24
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