Role of NEDD8 in HIV-associated lipodystrophy

Abstract

The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy.

One hundred HIV-1-infected patients were included in the study. Using an in vitro model of adipogenesis we evaluated the effects on adipogenesis of the forced expression of NEDD8 together with efavirenz, stavudine, saquinavir, amprenavir and indinavir, belonging to the three main classes of anti-HIV medications.

We showed that NEDD8 expression level is higher in the peripheral blood of HIV patients developing lipodystrophy. Coherently, forced expression of NEDD8 in an in vitro model of adipogenesis was able to perturb expression of some key proteins involved in adipogenesis, such as C/EBPα and PPARγ, possibly acting throughout the NEDD8/p27/β-catenin pathway. Moreover, three out of five evaluated drugs were able to affect adipocyte differentiation: efavirenz, stavudine and saquinavir. Finally, we have shown that NEDD8 was expressed in the fat tissue of lipodystrophic patients, being significantly higher in the lipodystrophic patients with respect to the controls, thus further confirming the altered NEDD8 expression in the fat tissue of HIV-infected patients affected by lipodystrophy.

Taken together, our data support the hypothesis of an implication of NEDD8 through p27 and β-catenin pathways in the disruption of adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy with qualitative and quantitative differences according to diverse antiretroviral treatments. These evidences indicate the NEDD8/β-catenin/p27 pathway as a possible molecular target for prevention of lipodystrophy development in patients under HAART therapy.

Introduction

The introduction of highly active antiretroviral therapy (HAART) has significantly changed the clinical course of HIV disease, with prolonged survival and better quality of life for HIV-infected patients. However, these combination therapy regimens have been demonstrated to cause a metabolic syndrome consisting of lipodystrophy/lipoatrophy, dyslipidemia and type 2 diabetes mellitus, with an overall increased cardiovascular risk of about 1.4 cardiac events/1000 years of therapy (Barbaro, 2006; Boyd and Reiss, 2006). The pathogenetic bases of HAART-associated lipodystrophy are still poorly known. In the last few years, the attention of the researchers has been targeted to the implication of mitochondrial function and stress metabolic enzymes impairment in the development of lipodystrophy. Preliminary reports showed that the HAART regimen can affect the quantity of mitochondrial DNA even from the beginning of the therapy and that adipocyte differentiation can be consequently altered. It has also been demonstrated that different mtDNA blood levels are associated with different HAART regimens (Gougeon et al., 2004; Barragan et al., 2006). Adipocyte differentiation is largely controlled by two families of transcription factors: the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs) (Moldes et al., 2003).

C/EBPα and PPARγ are two transcription factors responsible for establishing and maintaining the adipocyte phenotype. The C/EBPα and PPARγ expressions increase dramatically during adipocyte differentiation, and in pre-adipocytes these factors are expressed in low level (Gregoire et al., 1998).

In the early phase of adipocyte differentiation, the different members of the C/EBP family form heterodimers among them and are able to bind and regulate, through a DNA-binding domain, the expression of many genes fundamental for the early steps of adipogenesis. Instead, in a second phase of adipocyte differentiation, the different members of the PPARs family form heterodimers among themselves and are able to bind and activate specific nuclear hormone receptors representative for the mature adipocyte (Gregoire et al., 1998). Interestingly, the evaluation of the expression of adipocytic differentiation markers, such as PPARγ and C/EBPα, has confirmed the impairment of the fat tissue differentiation in HIV patients (Bastard et al., 2002). Some studies also suggested that disturbances in adipose tissue gene expression are already present in untreated HIV-1-infected patients, thus indicating a role of HIV-1 itself in eliciting adipose tissue alterations that could be worsened by HAART, which ultimately leads to lipodystrophy (Giralt et al., 2006). Nevertheless, during cellular differentiation and for the correct cell homeostasis it is required that several proteins are degraded in a spatially and temporally controlled manner. Principal actors in this regulatory mechanism are ubiquitins (Crosetto et al., 2006). The ubiquitilation consists of large protein complexes, divided into different groups called ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3), also known as SCF complexes. The complexity of the ubiquitination system is further complicated by the identification of other ubiquitin-like molecules such as ubiquitin cross-reactive protein (UCRP), sentrin and NEDD8 (neural precursor cell-expressed developmentally down-regulated) (Pan et al., 2004). The activation of the SCF is regulated by the ligation of NEDD8. This process is called “neddylation” and is able to increase the ubiqutin ligase activity of E3 in part by increasing its affinity for E2. The NEDD8-ylation stimulates ubiquitylation of several key proteins such as Ikβ and p27 (Podust et al., 2000), and regulates the cytoplasmic level of β-catenin (Tateishi et al., 2001). Drawing from this background, we decided to investigate the possible involvement of NEDD8 in adipogenesis and, consequently, in HIV-related lipodystrophy.