Microarray analysis was subsequently performed to investigate the effect of antiretroviral drug combinations on gene expression profile of differentiating 3T3-L1 cells. Since the in vitro effect of antiretroviral drugs on gene expression of 3T3-L1 cells has been shown to occur mainly in the early phases of differentiation [2,7], the time point corresponding to day 3 of differentiation was chosen for microarray experiments. To analyse the effect of NRTI, 3T3-L1 cells were induced to differentiate in the presence or absence of ZDV 10 μM and 3TC 20 μM, whereas to analyse the effect of the combination of two NRTI with a PI, 3T3-L1 cells were grown in the presence of ZDV 10 μM and 3TC 20 μM, associated with either IDV 20 μM or SQV 20 μM. For each treatment protocol, the gene expression profile of drug-treated 3T3-L1 cells was compared with that of untreated (i.e., cultured in the same differentiation medium without antiretroviral drugs) 3T3-L1 cells at day 3 of differentiation. To analyse the global cellular genetic responses after the various treatments, microarray data were subjected to hierarchical clustering analysis, which showed that gene expression profiles of cells treated with the two combinations of PI–NRTI were quite similar, but markedly distinct from that of treatment with only NRTI. Moreover, drug combinations including a PI had a greater impact on cellular gene expression profile than treatment with NRTI, as suggested by the higher number of up- and down-regulated genes. In fact, when 3T3-L1 cells were treated with the combination of NRTI, a total of 58 genes were differentially regulated at 99% confidence level, whereas when cells were treated with the NRTI and IDV or SQV, the number of differentially regulated genes increased to 98 and 132, respectively. Of these genes, 44 were differently regulated and had the same expression pattern in all three treatment conditions and 26 were differently regulated in the two PI-including treatment conditions, but not in the treatment condition including only NRTI.
Of the 44 genes significantly modulated by NRTI, both alone and in combination with a PI, 32 were up-regulated by treatment, whereas 12 were inhibited (Table 2). Induced genes included transcription regulators and genes involved in signal transduction. Some of these genes showed increased expression during adipogenesis and were further enhanced by antiretroviral drugs, but others, such as Aebp-1 (adipocyte enhancer-binding protein 1) and Timp-2 (tissue inhibitor of metalloproteinase 2), were found to be repressed upon adipocyte differentiation, but markedly induced by treatment with NRTI. Aebp1 was originally characterized as a repressor of transcription of the adipocyte fatty acid binding protein gene aP2 , an important marker of adipocyte late differentiation. Aebp1 seems to correspond to aortic carboxypeptidase-like protein, i.e, a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle differentiation . Over-expression of this protein in preadipocytes has been reported to inhibit adipogenesis [10,11] and to promote preadipocyte transdifferentiation into smooth muscle-like cells , even though this finding has not been confirmed by other authors . Tissue inhibitors of metalloproteinase are a family of four secreted proteins (TIMP-1 to TIMP-4) that selectively inhibit matrix metalloproteinases. The proteolytic activity of matrix metalloproteinases has been hypothesized to play a critical role in the early step of adipocyte differentiation, as suggested by their differential regulation in adipose tissue and by the demonstration that inhibitors of matrix metalloproteinases decrease C/EBPβ expression and adipocyte differentiation .
Some genes were modulated only by NRTI treatment, but not by treatment conditions including PI. Up-regulated genes included Bid3, an important inductor of apoptosis through the regulation of mitochondrial function and caspase-3 activation , whereas inhibited genes included the transcription factor Tcfcp2l3. An inactivating mutation of the corresponding human gene TFCP2L3 was identified in a large family with an autosomal dominant form of progressive non-syndromic sensorineural hearing loss .
Of the 26 genes significantly modulated by both treatment conditions including NRTI and a PI, but not by NRTI alone, 14 were up-regulated by treatment, whereas 12 were inhibited (Table 2). In particular, treatment with PI markedly induced the transcription factor Prdm1/Blimp1. Prdm1/Blimp1 is a transcriptional repressor with Krüppel-type zinc fingers which has been demonstrated to be essential for B-cell development  and, recently, also to play a critical role in the specification of mouse primordial germ cells and in the regulation of cell fate specification and morphogenetic processes [22,23]. Other genes induced by PI included those encoding extracellular matrix proteins (i.e., laminin gamma 2 and decorin), proteases (i.e., complement component 1r), and proliferins 1–3, which were found to be repressed in differentiated adipocytes. PI also induced expression of Δ-6 fatty acid desaturase (Fad2), a key enzyme in the biosynthesis of polyunsaturated fatty acids, such as arachidonic acid and decosahexaenoic acid, which is expressed in nearly all human tissues . Transcription of Fad2 is induced by SREBP-1c and PPARα ligand activators [25,26], but suppressed by polyunsaturated fatty acids via inhibition of SREBP-1c [25,26] and by PPARγ agonists . Increased Δ-6 desaturase activity has been associated with insulin resistance . Of note is that PI dramatically induced expression of Wnt10a (wingless related MMTV integration site 10a) and repressed Lef1 (lymphoid enhancer factor 1), which are both involved in Wnt signaling pathway. Activation of the Wnt signaling pathway has been shown to inhibit the differentiation of 3T3-L1 preadipocytes by preventing the induction of C/EBPα and PPARγ , to block the development of white and brown adipose tissue and, in differentiated brown adipocytes, to promote their conversion to white adipocytes . Wnt10a encodes a secreted signaling protein and has been demonstrated to be over-expressed in preadipocytes with decreased ability to differentiate into mature brown adipocytes . The effect of Wnt signaling on adipogenesis has been shown to be mediated by both β-catenin-dependent and β-catenin-independent mechanisms . LEF1 and the other members of this family of nuclear transcription factors, in response to Wnt signals, associate with β-catenin and activate Wnt-responsive target genes . Analysis of Lef1-deficient mice indicated that LEF1 may have a function in epithelium-to-mesenchyme signaling networks. In fact, targeted inactivation of Lef1 resulted in a complete block of development of multiple ectodermal appendages, such as teeth, vibrissae, hair, and mammary glands [33,34]. Our results show that Lef1 expression is induced during adipogenesis and inhibited by PI, thus suggesting a role in adipocyte differentiation. In the Lef1-deficient mouse model, Wnt10a is expressed independently of Lef1 in the dental epithelium , in agreement with the discordant expression pattern observed in our study in adipocytes. In addition to Lef1, PI down-regulated expression of several other genes typically expressed in differentiated adipocytes and mainly involved in lipid metabolism, such as Fsp27 (fat specific gene 27), Lep (leptin), Adn (adipsin), Adipoq (adiponectin), and Pfkfb3 (inducible 6-phosphofructo-2-kinase), and already known to be inhibited by PI [2,7], as well as other genes induced during adipogenesis, but less well characterized, such as Mrap, Cd36/FAT, Hist1h4i, G0s2, and S100a8. Mrap encodes melanocortin 2 receptor accessory protein, which has been recently identified as an interacting partner of the ACTH receptor MC2R and has been supposed to have a role in the trafficking MC2R from the endoplasmic reticulum to the cell surface . MRAP was first identified as a protein that is up-regulated upon differentiation of 3T3-L1 cells into adipocytes . Interestingly, Mc2r is also up-regulated in 3T3-L1 cells during differentiation via PPARγ and mediates the lipolytic effects of ACTH . The G0/G1 switch gene (G0s2) is involved in cell cycle regulation and has a temporal pattern of expression similar to that of Mrap, being induced during adipogenesis and further increased by PPARγ agonists . CD36/FAT expression is also induced by PPARγ during adipocyte differentiation . CD36/FAT mediates the uptake and accumulation of lipids in macrophages, adipose tissue and skeletal muscle  and its deficiency has been associated with dyslipidaemia and insulin resistance [41,42]. The role of CD36 in the pathogenesis of HAART-associated dyslipidaemia has already been investigated but with opposing findings. In fact, CD36 expression in circulating monocytes of HIV-infected patients treated with antiretroviral therapy including a PI has been reported to be reduced in a study , but increased in a more recent investigation in a larger population of patients . Similarly, PI have been demonstrated to both inhibit  and induce  CD36 expression in different human cell lines in vitro. S100A8 belongs to the S100 family of calcium-binding proteins and, together with S100A9, is expressed in cells of the myeloid lineage where its is predominantly localized to the cytoplasm . The secreted S100A8/S100A9 complex specifically binds polyunsaturated fatty acids (such as arachidonic acid) in a calcium-dependent manner  and interacts with CD36/FAT to facilitate cellular uptake of fatty acids . Thus, PI could inhibit fatty acid accumulation and adipogenesis by down-regulation of both CD36/FAT and S100A8 expression. Indeed, polyunsaturated fatty acids, and in particular arachidonic acid, have been shown to stimulate adipogenesis probably by acting as PPARγ agonists . In this context, up-regulation of Fads2 (Δ6-fatty acid desaturase) by PI could be the consequence of reduced intracellular polyunsaturated fatty acids. Finally, treatment with PI significantly inhibited expression of Esr2, which encodes oestrogen receptor β (ERβ). This result is in agreement with our findings in HIV-positive patients receiving antiretroviral therapy . Our study demonstrated reduced ERβ mRNA levels in the subcutaneous adipose tissue of lipodystrophy patients, the down-regulation of ERβ expression in the adipose tissue of HIV-positive patients receiving antiretroviral therapy containing PI, and the restoration of ERβ mRNA levels after switching from PI . Thus, ERβ could represent another nuclear transcription factor involved in the cascade of events triggered by PI that lead to impairment of adipocyte differentiation and metabolism. Overall, our observations with microarray experiments are consistent with previous reports which demonstrated that PI down-regulate the expression of lipogenesis genes, such as Fsp27, Lep, Adn, Pfkfb3, and Adipoq, which were reported to be repressed both in in vitro studies [2,7,51–53] and in vivo in the subcutaneous adipose tissue from lipodystrophic HIV-infected patients [7,50,54–56]. Moreover, our microarray study identifies new genes, such as Mrap, Cd36/FAT, and S100a8, that are inhibited in vitro by PI and that are also involved in adipogenesis and adipocyte function. Our results are thus in agreement with the presence of peripheral lipoatrophy in HIV-infected patients treated with PI . Peripheral lipoatrophy, central fat accumulation, and lipomatosis are common problems in adult patients with HIV-1 infection on antiretroviral drugs. Many of the adverse metabolic effects associated with PI therapy, including hypertriglyceridaemia and insulin resistance, resemble those seen in patients with the rare congenital and acquired lipodystrophy syndromes . Thus it has been proposed that peripheral lipoatrophy may be the primary effect caused by PI therapy, which subsequently leads to other adverse effects such as insulin resistance and many other endocrine disturbances ultimately leading to fat redistribution with an increase of the visceral fat and of lipids stored also inside the muscle fibres . Therefore, the generation of the ‘hypertrophic phase’ in fat distribution must be seen as a secondary event linked to the alteration of the endocrine-metabolic milieu and the loss of the capacity by subcutaneous fat cells to store adequately the flux of free fatty acids within the cell and thus preferentially channelling these substrates towards other targets, which are the fat cells of the visceral area and other cells which get ‘fatty’, as is the case of muscle cells and hepatocytes . Although what happens in vivo should be distinguished from what may be seen in in vitro models, our findings, together with the observations by others , support the general hypothesis of a primary damage of the subcutaneous adipose cell.
To confirm microarray results, the expression of a subset of genes modulated during differentiation and in response to treatment with NRTI and PI was further investigated using quantitative RT–PCR in a time-course experiment (Fig. 2). Overall, quantitative RT–PCR evaluation confirmed results obtained with microarray analysis. Expression of Srebp-1c, C/EBPα, C/EBPβ, and Pparγ mRNA was evaluated as early marker of adipogenesis, whereas expression of Leptin represented a marker of fully differentiated adipocytes. Among these genes, only Leptin expression was shown to be significantly modulated by antiretroviral drugs at microarray analysis and Srebp-1c probes were not represented in our microarray slides. As expected, in 3T3-L1 cells, Srebp-1c, C/EBPα, C/EBPβ, and Pparγ were rapidly and transiently induced during the early phase of adipocyte differentiation, whereas Leptin mRNA levels increased in the late phase of adipogenesis. Treatment with the NRTI ZDV and 3TC did not significantly modify expression of these genes at all time points of adipocyte differentiation, even though a slight decrease of Srebp-1c and Leptin mRNA as compared with untreated control cells could be observed during early and late adipocyte differentiation, respectively. These results are in agreement with Oil Red O staining which did not show a significant inhibition of adipocyte differentiation. At variance with NRTI, the PI SQV and IDV determined a significant inhibition of Srebp-1c, C/EBPα, Pparγ, and Leptin expression, and this inhibition was more evident with SQV than with IDV (Fig. 2). PI also determined a decrease, although not significant, of C/EBPβ transcript levels. The combination of a PI with two NRTI had the same effect on gene expression as treatment with only a PI.
Among the novel genes that microarray analysis demonstrated to be modulated by antiretroviral drugs, Mrap, Fads2, Aebp1, G0s2, Cd36 and Wnt10a were selected for further investigation using quantitative RT–PCR. These genes had different expression profiles during adipogenesis, since G0s2 was transiently induced during the early phase of adipogenesis, Mrap and Cd36 were induced during the late phase of adipogenesis, Aebp1 and Wnt10a were rapidly and markedly reduced during adipogenesis, whereas Fads2 expression remained unchanged during adipocyte differentiation. RT–PCR analysis showed that treatment with NRTI significantly increased Aebp1 mRNA levels as compared to untreated control cells, but had no effect on transcript levels of the other marker genes, thus confirming microarray results. Treatment with PI determined a marked reduction of Mrap, G0s2, and Cd36 mRNA levels and an increase of Wnt10a and Fads2 expression, both at early and late phases of differentiation. Even in this case, the association of a PI with two NRTI did not seem to modify the effect observed with the single drug treatment. The milder effect of NRTI than PI on adipocyte differentiation could in part be accounted to the relatively lower concentration of NRTI than PI used in our study. Although NRTI altered the gene expression profile in differentiating adipocytes, these effects were probably not so marked to be detectable at morphological examination and Oil Red O quantification.
The time-course experiments were replicated with different combinations of anti-retroviral drugs, including the NRTI zalcitabine (ddC) 0.2 μM and stavudine (d4T) 10 μM, which have been more strongly linked to lipoatrophy , and the PI lopinavir (LPV) 10 μM. Moreover, since the effects of NRTI on mitochondria and thus their presumed toxicity is time dependent, the time-course experiment was prolonged to 16 days (Fig. 3). Also with these combinations of NRTI, no significant alterations of cell proliferation or adipogenesis were observed, even after prolonged (16 days’) treatment, whereas the number of differentiated adipocytes was reduced by LPV treatment. This effect of LPV on adipocyte differentiation was paralleled by abnormal expression of genes involved in adipogenesis (Fig. 3).
In this study, by microarray gene expression analysis, we identified genes modulated by NRTI and PI during early adipogenesis. We hypothesize that up-regulation of master transcription factors and modulation of the Wnt signaling pathway by PI could represent a key event leading to inhibition of adipocyte differentiation and down-regulation of expression of adipocyte-specific markers, such as adiponectin, leptin, MRAP, Cd36/FAT, and S100A8. With respect to PI, the effect of NRTI on adipocyte differentiation and gene expression profile was milder, even though NRTI modulated the expression of tissue inhibitors of metalloproteinases and transcription factors, such as Aebp1, which could play an important role in the determination of the adipocyte phenotype. As already demonstrated for ERβ , abnormal expression of these genes could be at the basis of HAART-associated lipodystrophy and could represent a potential target for the treatment of this syndrome.
Monia Pacenti is a recipient of a fellowship from IOV (Istituto Oncologico Veneto).
This work was supported by grants no. 40F.57 from ISS (Istituto Superiore di Sanità) and no. RSF 168/04 from Regione Veneto to Giorgio Palù, and by grant no. 2003061834_006 MIUR-PRIN to Roberto Vettor.
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