Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipodystrophy: implications for the pathogenesis of the disease
Pinti, Marcelloa; Gibellini, Laraa; Guaraldi, Giovannib; Orlando, Gabriellab; Gant, Timothy Wc; Morselli, Eugeniaa; Nasi, Milenaa; Salomoni, Paoloc; Mussini, Cristinab; Cossarizza, Andreaa
aDepartment of Biomedical Sciences, Section of General Pathology, University of Modena and Reggio Emilia School of Medicine, Italy
bInfectious Diseases Clinics, University of Modena and Reggio Emilia, Modena, Italy
cMRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, UK.
Received 27 October, 2009
Revised 14 December, 2009
Accepted 5 January, 2010
Correspondence to Andrea Cossarizza, MD, PhD, Department of Biomedical Sciences, Section of General Pathology, University of Modena and Reggio Emilia School of Medicine, via Campi 287, 41125 Modena, Italy. Tel: +39 059 2055415; fax: +39 059 2055426; e-mail: email@example.com
Background: HAART can provoke metabolic changes and body fat redistribution, resulting in lipodystrophy, a side effect significantly involving mitochondrial function. Mitochondrial DNA (mtDNA) depletion caused by nucleosidic reverse transcription inhibitors is supposed to be a crucial mechanism in the pathogenesis of mitochondrial damages.
Methods: In adipose tissue from 22 HIV-positive patients with lipodystrophy and 20 healthy controls, we analyzed gene expression by microarray analysis and real-time PCR. The most upregulated gene was further studied in the human adipocytic cell line SW872 by real-time PCR, western blot, transient transfection assays and flow cytometry.
Results: We identified 18 genes differently expressed between lipodystrophy patients and controls, and focused our attention on the nuclear-encoded mitochondrial protease LON, essential in mtDNA maintenance. In SW872 cells, treatment with stavudine (d4T) doubled LON levels, in parallel with mtDNA depletion. As d4T increased reactive oxygen species (ROS) intracellular content, we measured LON in presence of deoxyribose, which causes oxidative stress but not mtDNA depletion, and observed LON upregulation. Ethidium bromide, which markedly depletes mtDNA, did not alter LON levels. The antioxidant glutathione inhibited the increase of intracellular ROS and the increase in LON caused by d4T or deoxyribose.
Conclusion: LON upregulation was due to d4T-induced ROS production, rather than due to mtDNA depletion, and represents a response to an oxidative stress. Other mechanisms than mtDNA depletion thus exist that explain nucleosidic reverse transcription inhibitors toxicity. This observation provides a rationale for possible therapeutic interventions aimed at reducing intracellular ROS content in patients assuming HAART.
The combination of drugs belonging to different categories defined as HAART prolongs, for extremely long periods, the course of the infection with the HIV. However, HAART can cause several side effects among which lipodystrophy, a syndrome characterized by peripheral fat wasting in limbs and face; accumulation of visceral fat; cervical fatpads; breast adiposity; hyperlipidaemia; and insulin resistance are very common .
Nucleosidic inhibitors of the reverse transcriptase (NRTI), main components of HAART, display relevant toxicity for mitochondria [2–6]. They are incorporated into mitochondrial DNA (mtDNA) by DNA polymerase-γ, causing chain termination and mtDNA depletion [2,7]; this depletion was observed in muscle, liver and adipose tissue of HAART-treated HIV patients with lipodystrophy [8–11]. In turn, mtDNA depletion can cause impairment of oxidative phosphorylation, excessive lactate production and finally mitochondrial dysfunction .
Different studies [3,9,10,12–16] demonstrated an association between NRTI-containing therapeutic regimens and mitochondrial toxicity such as lactic acidaemia, hepatic impairment and changes in mtDNA content. NRTIs are mainly responsible for fat loss and, in such a process, mitochondrial impairment and dysregulation of the synthesis, maintenance and functionality of mtDNA play a central role. However, the potential role and influence of other genes than those contained in mtDNA, and especially of those that regulate mitochondrial activity, is poorly known.
In order to identify possible modifications of gene expression in fat tissue from HIV-positive patients with lipodystrophy, we performed a microarray analysis on fresh tissue from these patients, who were compared with healthy donors. After confirmation assays by real-time PCR, we found that there were profound changes in the transcription profile, with significant alterations in several genes involved in mitochondrial metabolism, mtDNA transcription and regulation. Among these, we focused our attention on the role of a protein deeply involved in mtDNA regulation, that is, the ATP-dependent LON protease. LON is preserved from bacteria to humans, and it is crucial for mtDNA replication [17–24]. Mammalian LON binds single-stranded DNA with specificity for a G-rich consensus sequence (such as TTGGTAGGGGGATGA) either in vitro or in vivo [20,22,23,25]. Furthermore, LON interacts with DNA polymerase-γ and Twinkle, in a yet not defined mechanism , and is involved in turnover of damaged proteins [26–29]. We found that in HIV-positive patients with lipodystrophy, the LON protease was significantly more expressed and, by using an in-vitro model of human adipocytes, that its upregulation is due to the oxidative stress induced by NRTI rather than to the decrease in mtDNA.
Patients and methods
A total of 22 HIV-1-infected patients receiving HAART (mean age 50 years, range 32–66; man: woman ratio 1.2: 1) and 20 seronegative healthy controls (mean age 53 years, range 27–64) who underwent plastic surgery for aesthetical reasons were enrolled in this study (Table 1). None of them was classifiable as obese, according to the BMI. Patients, all of Caucasian origin, were followed by the Metabolic Clinic of the Infectious and Tropical Diseases Department (University of Modena and Reggio Emilia, Modena, Italy). Written informed consent was obtained from each patient before study entry; experimentation guidelines of the authors' Institution were followed according to Italian laws.
HAART was given for a mean of 78 weeks (range 22–108); all patients had taken stavudine (d4T) for at least 22 weeks. Median HIV infection duration was 13 years (range 6–22), mean CD4+ T-cell level was 517 ± 153 cell/μl (range 320–886) and mean viral load was 12 174 ± 33 475 copies/ml (range <50–75 002). Lipodystrophy was diagnosed when the patient and the physician agreed on the presence of facial or limb lipoatrophy, with increased deposits of fat in the abdomen or trunk. After informed consent, tissue specimens were obtained from the abdominal zone, either for patients or healthy controls. The microarray analysis was performed in 10 out of 22 lipodystrophy patients and 10 out of 20 seronegative controls; real-time PCR confirmation was performed in all of the samples, and western blot for LON in 13 samples (eight patients and five controls).
Gene expression was first analyzed in samples from 10 lipodystrophy patients and 10 healthy controls. We analyzed the expression of 5664 genes with a microarray designed and produced by the Microarray Laboratory of the Toxicology Unit, MRC (Leicester, UK). Among genes spotted in the array, 800 encode transporters, 794 are involved in cell cycle regulation, 623 in apoptosis, 623 in transcription regulation, 467 in cell proliferation, 363 in cell adhesion, 346 in lipid and cholesterol metabolism, 300 in electron transport and 380 in immune modulation or inflammation. The genes encoding mitochondrial proteins are 167, and the genes involved in response to stress are 103. More details are available at the following web address: http://126.96.36.199/SystemsToxicology/Microarray_Databases/Databases_CloneDatabase.aspx
Cell culture and treatments
We used a human liposarcoma cell line, that is, SW872 (obtained from American Type Culture Collection, Manassas, Virginia, USA), previously characterized as a good cell model for adipocyte gene expression [30–32]. SW872 were cultured in Leibovitz's L15 medium (Invitrogen, Grand Island, New York, USA) supplemented with 10% foetal bovine serum and 50 μg/ml gentamycin (NovoPharm, Toronto, Canada) in the presence of 5% CO2 at 37°C; 5 × 105 cells/ml were plated and treated as described below for up to 7 days in Leibovitz's complete medium. Incubation media were renewed every 2 days. The production of reactive oxygen species (ROS) was analyzed after 2, 6, 14, 24, 48, 72, 96 and 144 h of treatment with d4T (up to 400 μmol/l from Sigma–Aldrich, Saint Louis, Missouri, USA); deoxyribose (Sigma–Aldrich) was used at the final concentration of 5 mmol/l, reduced glutathione (GSH; Sigma–Aldrich) at the concentration of 10 mmol/l. Hydrogen peroxide (H2O2) and menadione (Sigma–Aldrich) were used as positive controls at final concentrations of 100 and 10 μmol/l, respectively. All of the conditions were studied at least in triplicate.
RNA extractions, reverse transcription and real-time PCR
RNA was extracted from adipose specimens using Lipid RNeasy tissue kit (QIAgen, Hilden, Germany) following instructions supplied from manufacturer. RNA was reverse transcribed with Superscript III MMLV (Invitrogen) following instructions of the manufacturer, using random hexamers as primers; 1/50 of synthesized cDNA was used in each reaction of amplification, which was performed in triplicate for each gene.
The expression of several differentially expressed genes was assessed by real-time PCR using TaqMan Assays with Universal Probe Library Probe sets (Roche Biochemicals, Mannheim, Germany) following supplier instructions. Primers for these sets of reaction were designed using Roche software available on line at https://www.roche-applied-science.com/sis/rtpcr/upl/adc.jsp.
Real-time PCR for mitochondrial DNA and mitochondrial RNA
mtDNA copies per cell was quantified as described, using kits from GeneMoRe Italy Srl (Modena, Italy) . Data were expressed as mtDNA copies per cell, calculated for each sample from the ratio between mtDNA copies and nuclear DNA copies multiplied by 2. Quantification of mtRNA transcripts was performed as previously described, using kits from GeneMoRe . RNA from cells or tissue specimens was extracted and reverse transcribed as above. cDNA was used to quantify three different mitochondrial transcripts (ND1, ND6 and CYTB) and one nuclear transcript (L13) . Values are expressed as number of copies of each mtRNA normalized to number of copies of L13 calculated on the same cDNA sample.
Western blotting of LON protein
Total cellular protein (12 μg) from adipose tissue samples, or from SW872 cells incubated in the presence or absence of the above-mentioned substances, was subjected to electrophoresis in a 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated in the presence of a human LON protease rabbit antisera (a kind gift from Dr K. Suzuki, University of New Jersey, New Jersey, USA), washed and incubated with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP; Serotec, Kidlington, UK). HRP activity was detected using Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, Massachusetts, USA). The blot was developed, and the densitometric analysis was performed using ImageQuant TLv2005 software (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Detection of hydrogen peroxide
Intracellular H2O2 content was measured by 2,7-dicholorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) . Cells were stained with 2 μmol/l H2DCFDA for 30 min at 37°C, washed with phosphate buffer solution (PBS) and analyzed as described .
Detection of superoxide anions (O2−)
Cells were stained with hydroethidine (Invitrogen) at the concentration of 1 μmol/l. The probe reacts with O2− to give EtBr, which binds DNA and can be visualized by flow cytometry .
The intracellular levels of ROS were analyzed after 2, 6, 14, 24, 48, 72, 96 and 144 h of treatment with d4T 400 μmol/l and deoxyribose 5 mmol/l. H2O2 and menadione (Sigma, final concentrations 100 and 10 μmol/l, respectively) were used as positive controls of the experiments (not shown in this manuscript). Cell cultures were incubated with hydroethidine for 40 min at 37°C and centrifuged at 200g for 5 min. Cells were then resuspended in 1 ml PBS, incubated with H2DCFDA for 30 min at 37°C, centrifuged as above, resuspended in 1.5 ml PBS and analyzed by flow cytometry. All cytofluorimetric analyses were performed using a 16-parameters CyFlow ML (Partec GmbH, Münster, Germany), equipped with a blue solid-state laser, an ultraviolet mercury lamp and a red diode laser. A minimum of 10 000 cells per sample were acquired in list mode and analyzed with FlowJo 6.3 software; each experiment was repeated at least four times.
Analysis of microarray data was performed using NorTT software, developed at Microarray lab of MRC Toxicology unit by Shu-Dong Zhang and Timothy W. Gant (available at the web address http://www.le.ac.uk/mrctox/microarray_lab/Microarray_Softwares/Microarray_Softwares.htm). Briefly, this software filters and normalizes GPR data file, generated by GenePix softwares, and performs statistical t-tests for each analyzable gene. The output file generated by NorTT is a file in which are indicated the genes whose expression is significantly modified between the two analyzed groups and the percentage of false positive results. We have considered expression changes significant when P value was lower than 0.001.
Data obtained after staining with H2DCFDA represent the median of the net fluorescence value ± SD. The net fluorescence value was obtained, linearizing the fluorescence values from the logarithmic scale and subtracting the linearized median value of the blank (i.e., the sample containing cells treated in the same manner, but without any staining) from the median fluorescence value of the stained sample . This allows eliminating any influence related to autofluorescence changes.
Data regarding mtDNA quantification are reported as number of copies of mtDNA per cell, and represent the mean ± SD of three different experiments. mtRNA are reported as number of copies of mitochondrial transcript/number of copies of L13 transcript, and represent the mean ± SD of three different experiments. LON protein content has been calculated as volume of LON band/volume of control protein band, and is reported as the relative ratio in respect to the first control sample, set arbitrarily to 1.
Changes in relative expression of genes evaluated by real-time PCR, in mtDNA and mtRNA levels, and in the expression of LON protein were analyzed with GraphPad Prism 4.0. Data regarding changes in mRNA relative expression over time have been analyzed by analysis of variance for repeated measures and followed by appropriate post-hoc test. Data regarding relative protein expression and differences in mtDNA copies have been analyzed by Student's t-test. Values of P less than 0.05 were considered as statistically significant.
Analysis of the expression of mRNA in fat tissue from lipodystrophy patients
In order to confirm the well known observation regarding the decrease in mtDNA content in adipocytes from lipodystrophy patients, we tested adipose tissue of 10 lipodystrophy patients and 10 healthy controls and, as expected, we found a significant reduction of mtDNA (116 ± 35 vs. 345 ± 115 copies/cell, P < 0.05). Then, in these samples, we analyzed the expression of 5664 genes. Choosing 0.001 as the cutoff of the P value for microarray analysis, we could identify 18 genes whose expression was significantly different between lipodystrophy patients and controls (Table 2), with a fraction of 0.308 expected as false positive (corresponding to 4.63 false-positive genes). Among these genes, four (PRSS15, RAB32, CYP11B1 and POLRMT) encode proteins that localize in mitochondria and are involved in processes related to mitochondrial dynamics or in the regulation of mtDNA transcription, two (CYP11B1 and CYP2D6) are isoforms of cytochrome P450, two (PSMD12 and RBX1) encode proteasome components, two (ABCA1 and ADARB1) are involved in lipid metabolism, two (MARK3 and STK39) are kinases involved in intracellular signal transduction, two (TNFAIP1 and TRAF5) are linked to tumor necrosis factor response and one (BMPR2) is a receptor for bone morphogenetic proteins also involved in adipogenesis.
To confirm the aforementioned data, we quantified the expression of all these genes by real-time PCR analysis in the same samples and, in other tissue samples, obtained from other lipodystrophy patients and controls (for a total of 22 patients and 20 healthy donors). The genes whose mRNA expression was significantly different between patients and controls are reported in Fig. 1(a). Two (PRSS15 and RAB32) of the genes related to mitochondrial functionality showed an average increase in mRNA levels of 3.73 and 2.47-folds, respectively. The increase was significant also for STK39 and BMPR2 (Fig. 1a). A significant difference in mRNA expression was also observed for ABCA1, CYP2D6, PDE4A, PSMD12, CYP11B1, TNFAIP1 and POLMRT (not shown). On the contrary, we could not confirm the results observed with microarray analysis for RBX1, LSM1, ADARB1, TRAF5, GYG, ABCA1 and PAPOLA, whose expression was similar in patients and controls.
Expression of LON protease in lipodystrophy patients and in cell models
We focused our attention on PRSS15, a nuclear gene encoding the ATP-dependent mitochondrial protease LON, which is involved in mtDNA replication and in the turnover of mitochondrial proteins. We first confirmed the results observed with mRNA at the protein level, by performing western blot analysis of the levels of LON protein in adipose tissue. Also in this case, LON was markedly higher in lipodystrophy patients than in controls (Fig. 1b).
We designed an in-vitro model by using the fully differentiated adipocytic cell line SW872 . As the NRTI d4T has a profound impact on mitochondrial activity, significantly alters mtDNA replication  and is strongly associated with lipodystrophy [38–40], we chose this drug for inducing a damage in SW872 cells. A high dose of the drug (400 μmol/l, much more than that present in plasma of d4T-treated patients) was used because of the nature of the cells under investigation and to maximize the effect in a relatively short time. d4T significantly inhibited cell proliferation (Fig. 2a) and provoked a time-dependent decrease in mtDNA content (Fig. 2b). We also observed a parallel, significant reduction in three representative mitochondrial transcripts (CYTB, ND1 and ND6), whose levels halved by 6 days of d4T treatment (data not shown).
By using real-time PCR, we analyzed LON mRNA levels in cells treated with d4T, and found that LON transcription was doubled after 24 h of treatment; mRNA levels showed lower increases after 72 and 144 h of treatment in a way that did not seem to be directly correlated with the number of copies of mtDNA (Fig. 2c).
LON levels are not influenced by decrease in mitochondrial DNA content
To evaluate whether LON mRNA levels were directly influenced by mtDNA levels, we analyzed its levels in cells treated with ethidium bromide (EtBr), which depletes mtDNA . Treating SW872 cells for up to 96 h with 50 ng/ml EtBr caused a relevant mtDNA decrease, which was well evident even after 48 h (Fig. 2d). The same treatment did not cause a parallel modification in LON mRNA levels (Fig. 2e).
Not only does d4T deplete mtDNA but it can also increase intracellular levels of ROS . Thus, we asked whether ROS could play a role in LON increase. By using flow cytometry, we analyzed ROS levels in the presence of d4T, EtBr or deoxyribose, which depletes cells of GSH and causes oxidative stress without affecting mtDNA replication [42,43]. We found that, in contrast with EtBr, both d4T and deoxyribose caused a significant increase of H2O2 at different times of culture; when the antioxidant GSH was added to cultures, such increase was abolished (Fig. 3a and b). Similar results, including the protection by GSH, were obtained by analyzing the levels of O2− (Fig. 3b). By using real-time PCR, we found that the levels of LON mRNA increased after 24 h of treatment with either d4T or deoxyribose (Fig. 3c). The presence of GSH inhibited the increase in LON mRNA caused by d4T and deoxyribose. Finally, the quantification of the levels of LON protein by western blot confirmed the results we observed with mRNA (Fig. 3d).
Profound changes in gene expression occur in adipose tissue from HIV-positive patients with lipodystrophy, and we found that several genes involved in mitochondrial function are significantly upregulated. In some cases, such an increase could likely represent a compensatory mechanism. This could be the case of POLRMT, which is the sole responsible for mtDNA transcription, and whose higher expression could represent an effort to maintain a normal level of mtRNAs despite the progressive reduction of mtDNA. However, previous data obtained in our laboratory indicate that a constant reduction of mtRNA, which parallel mtDNA depletion, can be observed in adipose tissue from lipodystrophy patients, as well as in cell lines treated with NRTIs [33,37], suggesting that this possible compensatory mechanism is not sufficient to maintain mitochondrial functionality.
PRSS15 gene, encoding for mitochondrial protease LON, was significantly upregulated in adipose tissue from lipodystrophy patients. A possible explanation could be that LON is essential for maintaining mtDNA copies, and its higher expression represents a compensatory mechanism in response to mtDNA depletion. Because of difficulty to identify a significant number of male patients undergoing plastic surgery, a certain lack of equivalence exists between the two groups as far as sex and HIV status are concerned, which theoretically could affect results. For this reason, a more accurate analysis of the effects of drugs on LON expression has been performed in a well defined and controlled cellular model. The results obtained on SW872 cells likely exclude the presence of a direct mechanism connecting mtDNA depletion with LON upregulation. On the contrary, LON appears to be sensitive to intracellular ROS levels, in agreement with the idea that LON can be considered a ‘stress protein’, able to respond to various agents, including heating, serum deprivation and high levels of H2O2 . Thus, it is possible to hypothesize that higher levels of LON are required to eliminate an excess of mitochondrial-oxidized proteins, which are the main target of LON proteolytic activity , as well as for protecting mtDNA from ROS-induced damage. A study  has shown that the capability of LON to bind mtDNA is significantly modified in the presence of oxidative stress; this observation is in agreement with the hypothesis that LON could effectively protect mtDNA.
As HIV has a relevant responsibility in provoking the onset of a proinflammatory milieu, the hypothesis that the upregulation of LON is caused by the direct or indirect action of the virus cannot be definitively ruled out. However, this hypothesis is quite unlikely, as samples we have analyzed came from patients with a long history of HAART treatment, which have reduced HIV viral load to undetectable levels in 14 out of 22 patients. Furthermore, adipose tissue is not one of the major sites of infection by HIV. Further studies in patients who are HIV-infected naive for HAART or HIV-infected under HAART previous to lipodystrophy development will be useful to clarify this point.
Preliminary data indicate that the ROS-induced upregulation of LON was mainly due to an increase in the promoter activity (Pinti et al., manuscript submitted). We found that the region encompassing nt −623/+1 is essential for upregulating LON in the presence of oxidative stress, and that the deletion of this region abrogates the response to ROS in adipocytes. This was observed either in adipocytic (SW872) or in hepatocytic (HepG2) cell lines, suggesting that its upregulation is likely a nontissue-restricted phenomenon.
For several years, the attention of different research groups, including ours, has been largely focused on damages induced by antiretroviral drugs on mtDNA and on the consequence of its depletion [46,47]. Our data show that oxidative stress caused by NRTIs can play an important role in the pathogenesis of lipodystrophy, and that part of the response (i.e., changes of gene expression that can be observed in adipose tissue) is not due to mtDNA depletion per se but to the response to ROS increase.
LON is considered a crucial component of mitochondrial ‘quality control’ system and represents one of the most important components for removing selectively nonassembled and misfolded proteins (reviewed in ). Contrasting data exist regarding the expression of LON during oxidative stress of different type and strength, and in different animal or cell models [44,48]. Here, we show that LON expression is increased either in vitro or ex vivo in the presence of a mild but continuous oxidative stress. Unpublished data on the regulation of LON promoter strongly support this view (Pinti et al., manuscript submitted). Such upregulation of LON is in perfect agreement with the high turnover of damaged proteins caused by ROS increase. However, the limited LON expression increase, we observed, could not be sufficient to compensate such damages. The accumulation of oxidized mitochondrial proteins, typically observed with physiological aging or in age-related disorders, could occur at higher rate in lipodystrophy, and could significantly contribute to the pathogenesis of such a side effect of antiretroviral therapy.
This paper was partially supported by VI Programma Nazionale di Ricerca sull'AIDS 2006 (Istituto Superiore di Sanita', Rome, Italy), grants No. 30G.62 and 40G.62 to A.C., and by a grant from MIUR - PRIN 2008 (Project: ‘Regulation of Lon protease expression in response to oxidative damage to mitochondria’) to A.C.
M.P., L.G., T.W.G., E.M. and M.N. performed research; G.G., G.O. and C.M. selected patients and collected clinical samples and data; M.P. and T.W.G. analyzed data; P.S and A.C. designed research; M.P, P.S. and A.C. wrote the paper.
There are no conflicts of interest.
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AIDS; HAART; HIV; lipodystrophy; LON; mitochondria; mitochondrial DNA
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