HIV-1 Expression Induces Tubular Cell G2/M Arrest and Apoptosis

Abstract

Human renal biopsy studies suggest the presence of HIV-1 and associated signs of injury in renal tubular epithelial cells. Because renal epithelial cells lack conventional HIV-1 receptors, the modus operandi of HIV-1 in the induction of tubular cell injury remains a mystery. In the present study, we evaluated the role of HIV-1 gene expression in human proximal tubular cell apoptosis and cell cycle progression. HIV-1- or vector-transduced cells were assayed for cellular injury and cell cycle defect. HIV-1-transduced cells showed the progressive loss of viability in a time-dependent manner. Similarly, HIV-1-transduced cells showed greater apoptosis when compared with vector-transduced cells. A higher number of HIV-1 expressing cells showed cell cycle arrest at G2/M phase and enhanced tubular cell expression of phospho-p53(ser15), phospho-cdc-2(Tyr 15), and phospho-chk-2 (Thr 68). These findings suggest that in addition to the activation of apoptotic pathway, HIV-1-induced G2/M arrest may also contribute to tubular cell injury.

Keywords:

• HIV-1
• tubular cells
• cell cycle
• apoptosis

Human immunodeficiency virus-associated nephropathy (HIVAN) is one of the clinical manifestations of AIDS and the single most common cause of chronic renal disease in HIV-1 seropositive patients.[1–3] HIVAN is characterized by proteinuria with rapidly developing azotemia and histologically by focal glomerulosclerosis (often a collapsing type) and microcystic dilatation of tubules. Other lesions that are not part of HIVAN are seen in some HIV infected patients, including immunoglobulin A nephropathy, immune complex glomerulonephritis, membranous nephropathy, and proliferative glomerulopathies.[1–4] The molecular mechanisms involved in these non-HIVAN renal lesions are expected to be different as these renal lesions bear no resemblance to the histological features seen in HIVAN.

For a long time, it was not clear whether the pathogenesis of HIVAN was due to HIV infection in the renal cells or due to an indirect effect of the systemically dysregulated immune system. Studies designed to address this issue have shown that HIVAN can occur at any point during AIDS progression with no apparent correlation with either viral burden or CD4+ T cell number.[5] Using elegant experimental approaches, Bruggeman et al. demonstrated that the expression of the HIV-transgene in renal cells was necessary and sufficient for the development of HIVAN.[6] These observations suggest that a direct effect of HIV-1 expression is necessary and sufficient for the development of HIVAN in human patients.

Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIVAN.[7–9] Using in situ hybridization, Marras et al. have shown HIV-1 gag and nef mRNA in renal epithelial cells of HIVAN patients.[10] The same group showed that phylogenetic analysis of the DNA sequence from infected renal epithelial cells as well as the corresponding sequences from peripheral blood mononuclear cells of the same patients reveal evidence of tissue-specific viral evolution.[10] These results suggest not only the existence of a renal viral reservoir but also a localized replication of HIV-1 in the kidney.

In the past, various investigators reported that HIV-1 induces tubular cell apoptosis and necrosis in in vitro studies.[9],[11] However, in these studies, the entry of HIV-1 into tubular cells has not been reported. Because renal tubular cells do not express conventional HIV-1 receptors such as CD4, CCR5, and CXCR4, it was likely that this effect of HIV-1 on tubular cells might be related to some extrinsic interactions.[10],[12],[13] Ray et al. infected primary tubular cells with certain strains of HIV-1.[14] These investigators showed the occurrence of degenerative changes in both infected and uninfected tubular cells. However, the occurrence of apoptosis was not studied in these studies. It was also not clear whether these tubular degenerative changes were the result of entry of HIV-1 or related to extrinsic effects of HIV-1. Thus, it may be important to evaluate the effects of HIV-1 on tubular cells after it becomes part of their cellular genome. In present study, we have studied the effect of HIV-1 expression on tubular cell injury.

Tubular cells can undergo several cell fates in response to injury, including proliferation, de-differentiation, hypertrophy, senescence, apoptosis, or necrosis.[15],[16] The regulation of these responses takes place at the level of the cell cycle, coordinated by positive regulators-cyclins and cyclin-dependent kinases and negative regulators-cyclins and cyclin-dependent kinase inhibitors. In the present study, we studied the effect of HIV-1 expression on tubular cell cycle progression.

MATERIALS AND METHODS

Preparation of Pseudotype Viruses

A gag/pol deleted HIV-1 construct, pNL4-3:ΔG/P-EGFP, which contains enhanced green fluorescent protein (EGFP) in gag open reading frame, was used to infect the cells, and a lentiviral vector pHR-CMV-IRES2-EGFP-ΔB was used as a control. The gag/pol and VSV.G envelope genes were provided in trans using pCMV R8.91 and pMD.G plasmids, respectively. Infectious viral supernatants were produced by transient transfection of 293T cells using Effectin transfection reagent (QIAGEN Inc., Valencia, California, USA) according to the manufacturer's instructions. Viral supernatants were collected at 48 and 72 hrs post-transfection. Harvested supernatants were cleared by centrifugation at 2,000 rpm (828 × g) followed by filtration through 0.45 μM filter. Viral suspensions were frozen at −80°C for storage. The viral stocks were titrated by infecting Hela cells, followed by flow cytometric analysis of cells that were positive for the reporter molecule, GFP. Viral titers were calculated with the following equation:

where F is the frequency of GFP-positive cells found by flow cytometry, C₀ is the total number of target cells at the time of infection, V is the volume of inoculum, and D is the virus dilution factor. Viral stocks ranging from 105 to 106 GFP expressing units/mL were obtained.

Cell Culture and Viral Transduction

HK-2 cells (obtained from American Type Culture Collection, Manassas, Virginia, USA) were grown in Keratinocyte-SFM media (Invitrogen, Carlsbad, California, USA) supplemented with epidermal growth factor (5 ng/mL) and bovine pituitary extract (40 μg/mL). The cells were grown at 37°C in 5% CO₂ and replenished with fresh media every third day. HK-2 cells were subcultured by washing twice with 1 × phosphate buffered saline and harvested by trypsinization with 0.05% trypsin (GIBCO). The viral stock was used to transduce HK-2 cells at an MOI of 1.0 in the presence of 10 μg/mL polybreneor mock infected with control supernatant. The virus inoculum was allowed to absorb for 3 h at 37°C and then replaced with fresh medium.

Determination of Cell Injury

HIV-1 transduced HK-2 cells were harvested and suspended in 0.2% Trypan blue. Cells were counted using a hemocytometer and assessed for the trypan blue exclusion. Cells that were blue were scored as injured cells.

Analysis of Apoptosis

FACS analysis was performed to identify the cells undergoing apoptosis. The cells were transduced with HIV-1 and vector alone. After incubation period of 24, 48, and 72 h, cells were harvested and stained with Annexin-V and 7-AAD using Annexin V:PE Apoptosis Detection Kit I (BD Pharmingen, San Diego, California, USA) as the manufacturer's instructions. In brief, cells were harvested and washed three times with cold PBS. Cells were washed twice with cold PBS and then resuspended in 1 × binding buffer at a concentration of 1 × 10⁶ cells/mL. Cells (1 × 10⁵) were added with 5 μl of Annexin V-PE and 5 μl of 7-AAD followed by gentle shaking and incubation for 15 min at RT (25°C) in the dark. Cells were added with 400 μl (1×) binding buffer and analyzed for apoptosis by flow cytometry gating on the positive cells for EGFP. Data were analyzed by CELL Quest program (BD Pharmingen).

Measurement of Cell Death by Cell Death ELISA

Cell death was determined using a Cell Death Detection ELISA^PLUS kit (Roche Applied Science, Mannheim, Germany). 2.5 × 10³ HK-2 cells were seeded into each well of a 96-well plate and transduced with viral stocks as mentioned above. After 48 and 72 h, samples were collected, and ELISA was performed according to the manufacturer's instruction.

TUNEL Assay

Detection of DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed by using a kit (R&D Systems, Inc., Minneapolis, Minnesota, USA). An equal number of the cells were grown on the coverslips in a six well plate and transduced with the virus expressing HIV-1 or empty vector. Cells were fixed in 3.7% formaldehyde to prevent the loss of low molecular weight DNA fragments and permeabilized using cytonin to make the DNA accessible to labeling enzyme. Endogenous peroxidase activity was quenched using hydrogen peroxide. Biotinylated nucleotides were incorporated into the 3′-OH ends of the DNA fragments by Terminal deoxynucleotidyl Transferase (TdT). The biotinylated nucleotides were detected by using streptavidin-horseradish peroxidase conjugate followed by the substrate diaminobenzidine (DAB). After rinsing with PBS, cells were counterstained using methyl green and examined under bright light microscope.

Flow Cytometry Analysis of Cell Cycle Stage

To determine the stage of cell cycle, cells were transduced with HIV-1 and vector alone. FACS analysis was performed 24, 48, and 72 h post-transduction, gating on the positive cells for EGFP. Cells were washed twice and 1 × 10⁶ cells were resuspended in 70% ethanol followed by 1 h fixation in formalin at 4°C. Fixed cells were washed twice with PBS and resuspended in 0.5 mL/10⁶ cells propidium iodide/RNase staining buffer (BD Pharmingen) in the dark to analyze their DNA content. GFP-positive cells were gated, and the cellular DNA content in fixed cells was then assessed with a FACS flow cytometer. Data were analyzed by CELL Quest program (BD Pharmingen).

Protein Extraction and Western Blot Analysis

HK-2 cells were transduced with HIV-1 and vector alone as described earlier. At the end of the incubation period, cells were washed three times with PBS, scraped into a modified RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 0.1% SDS, 10 μl/mL of protease inhibitor cocktail, and 100 μg/mL of PMSF) and transferred to a microcentrifuge tube. The cell lysates were centrifuged at 15,000 g for 30 min at 4°C. The supernatant was analyzed for total protein content. One hundred micrograms of protein was heated at 100°C for 5 min and separated on a 12% PAGE gel. The protein bands were electrotransferred to a nitrocellulose membrane in transfer buffer containing 48 mM Tris-Cl, 39 mM glycine, and 20% methanol at 4°C overnight. Nonspecific binding to the membrane was blocked for 1 h at room temperature with blocking buffer (5% fat free milk in 1× PBS with 0.1% Tween 20). The membrane was then incubated for 16 h at 4°C with primary antibodies (mouse monoclonal anti-Phospho-p53 (Ser15), rabbit polyclonal anti-phospho-Chk-2 (Thr68), and rabbit polyclonal anti-Phospho-cdc-2 (Tyr15) antibody, Cell Signaling Technology Inc., Danvers, Massachusetts, USA) in blocking buffer, followed by incubation for 1 h at room temperature with the appropriate secondary antibody in blocking buffer. Signals were visualized using chemiluminescence reagent (Perkin Elmer Lab., Inc., Boston, Massachusetts, USA), exposing the membrane to x-ray film (Eastman Kodak, Rochester, New York, USA). To determine loading, blots were stripped and reprobed for β-actin. Quantitative densitometry was performed on the identified bands by using a computer-based measurement system (IS-1000 Digital Imaging System, Alpha Innotech, San Leandro, California, USA).

Statistical Analysis

Statistical analysis was performed using GraphPad-Instat software. A Newman-Keuls multiple comparison test was used, and p values were calculated.

RESULTS

Time Course Effect of HIV-1 on Renal Proximal Tubular Epithelial Cell Injury

To determine the effect of HIV-1 expression on viability of tubular cells, mock-, HIV-1-, or vector-transduced tubular cells were stained with Trypan blue at variable time periods (12, 24, 36, 48, 60, and 72 hrs). As shown in Figure 1, mock- and vector-transduced cells did not show any alteration in viability of tubular cells. On the other hand, cells with HIV-1 expression showed time-dependent effect on tubular cells injury (see Figure 1).

Figure 1. Time course effect of HIV-1 on renal proximal tubular epithelial cell injury. Equal numbers of HIV-1-, vector alone,- or mock-transduced tubular cells were incubated in media for variable time periods (12, 24, 36, 48, 60, and 72 hrs). Subsequently, cellular injury was assessed by Trypan blue exclusion technique. Results (means ± SD) are from four series of experiments each carried out in triplicate. *p < 0.001 compared with respective mock- and vector- transduced cells.

HIV-1 Expression Induces Apoptosis in Tubular Cells

To study the effect of HIV-1 expression on tubular cell apoptosis, cells transduced with HIV-1 or vector were stained with annexin-V after 24, 48, and 72 h of transduction. Subsequently, apoptosis assay was carried out by flow cytometry. Vector- and HIV-1- expressing cells were gated for GFP-positive cells. HIV-1 expressing tubular cells showed an approximate two-fold increase in apoptosed cell numbers when compared with vector expressing cells (see Figure 2A). HIV-1 expression promoted tubular cell apoptosis at all time points (see Figure 2B).

Figure 2. Tubular cell expression of HIV-1 promotes apoptosis. Equal numbers of tubular cells were transduced with either HIV-1 or vector alone. After 24, 48, and 72 hours of transduction, cells were stained with annexin-V and then studied for apoptosis by flow cytometry. Vector and HIV-1 expressing HK-2 cells were gated for GFP positive cells. (A) Annexin V-stained cells; marker M1 indicates apoptosed cells. (B) Cumulative data showing percentage of apoptosed cells. Results (means ± SD) are from three sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vectors; **p < 0.05 compared with respective vectors.

To confirm the effect of HIV-1 expression on tubular cell apoptosis, in parallel sets of experiments, cells were transduced with either HIV-1 or vector. After the incubation period of 48 and 72 hours, the occurrence of apoptosis was assayed by cell death ELISA. HIV-1-expressing tubular cells showed an approximate two-fold increase in apoptosed cell numbers when compared with vector-expressing cells (see Figure 3).

Figure 3. Effect of HIV-1 expression on cell death. Equal number of cells (2.5 × 10⁴ cells/well) were seeded in a 96-well plate and incubated overnight at 37°C. Cells were transduced with either HIV-1 or vector alone. Cell death ELISA was carried out at 48 and 72 h post-transduction in quadruplicate wells. The results (mean ± SD) are from three sets of experiments plotted for each group of cells. *p < 0.001 compared with respective vector controls.

To further confirm the effect of HIV-1 on tubular cell apoptosis, tubular cells were treated under above-mentioned conditions for 48 and 72 hours and then assayed for apoptosis by TUNEL method. Representative micrographs of vector-transduced and HIV-1-transduced tubular cells (at 48 and 72 h) are shown in Figure 4 (vector-transduced cells, Figures 4A and 4B; HIV-1-transduced cells, 4C and 4D). HIV-1-expressing cells showed an approximate 5- (48 h) and 8-fold (72 h) increase in apoptosed cells (see Figure 4).

Figure 4. Effect of HIV-1 expression on tubular cell apoptosis. Equal number of HK-2 cells were grown on poly-L lysine coated glass coverslips. After incubation of 48 and 72 hours, cells were assayed for apoptosis by TUNEL assay. The upper panel shows representative micrographs of vector-transduced cells at (A) 48 and (B) 72 hours of incubation. The lower panel shows representative micrographs of HIV-1 transduced cells at (C) 48 and (D) 72 hours of incubation (×200). Apoptosis cells with dark brown nuclei are indicated by arrows. Cumulative data of apoptosed cells in vector- and HIV-1- transduced cells are shown in the bar diagram. Results (means ± SD) are from four sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vector; **p < 0.001 compared with respective vector and HIV (48 h).

Tubular Cell HIV-1 Expression Induces Cell Cycle Arrest at G2/M Phase

To determine the effect of HIV-1 expression on progression of cell cycle, HK2 cells were transduced with either HIV-1 or vector alone. After 24, 48, and 72 h of transduction, cells were stained with propidium iodide and examined for cell cycle stage by flow cytometry. Vector and HIV-1 expressing HK-2 cells were gated for GFP positive cells. HIV-1 expressing cells showed a two- to three-fold increase in G2/M phase of cell cycles at different time points when compared with vector-transduced cells (see Figure 5). The effect of HIV-1 expression on G2/M arrest was maximal at 72 hours.

Figure 5. Tubular cell HIV-1 expression induces cell cycle arrest in G2/M phase. Equal numbers of cells were transduced with either HIV or vector alone. After 24, 48, and 72 hours of transduction, cells were stained with propidium iodide and examined for cell cycle stage by flow cytometry. Vector and HIV-1 expressing HK-2 cells were gated for GFP positive cells.(A) Histograms showing cell cycle stage of GFP positive cells. (B) Cumulative data showing percentage of cells in G2/M phase. Results (means ± SD) are from three sets of experiments each carried out in triplicate. *p < 0.001 compared with respective vector- and HIV-1-transduced cells at 24 and 48h.

Effect of Tubular Cell Expression of HIV-1 on the Activation of Cell Cycle/Checkpoint Signaling Pathways

To evaluate the effect of tubular cell expression of HIV-1 on the activation of cell cycle signaling pathways, HK-2 cells were transduced with either HIV-1 or vector alone followed by incubation for 72 hrs. Subsequently, cells were prepared for Western blots and probed for Phospho-P53 (Ser 15), Phospho-cdc-2 (Tyr 15), and phospho-chk-2 (Thr 68). Tubular cells expressing HIV-1 showed higher expression of phospho-p53 when compared with cells transduced with vector alone (see Figure 6A). Similarly, HIV-1 transduced cells showed an increase in expression of phospho-Chk2 when compared with cells transduced with vector alone (see Figure 6B). Moreover, HIV-1 expressing cells showed enhanced expression of phospho-cdc2 when compared with vector expressing tubular cells (see Figure 6C).

Figure 6. Effect of HIV-1 expression on tubular cell cycle/checkpoint signaling pathways. Equal numbers of HK-2 cells were transduced with either HIV-1 or vector alone, followed by incubation for 72 hours. Subsequently, cells were harvested, proteins were extracted, and Western blots were prepared and probed for phospho-P53 (Ser 15), phospho-cdc-2 (Tyr 15), and phospho-chk-2 (Thr 68). Three sets of experiments were carried out. (A) The upper panel shows a representative gel of tubular cell expression of phospho-P53 (Ser 15) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-P53 and actin. *p < 0.01 compared with vector. (B) The upper panel shows a representative gel of tubular cell expression of phospho-Chk2 (Thr 68) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-Chk2 and actin. *p < 0.05 compared with vector. (C) The upper panel shows a representative gel of tubular cell expression of phospho-cdc-2 (Tyr 15) under HIV-1 and vector transduction states. The middle panel shows tubular cell content of actin under similar conditions. The lower panel shows cumulative data on densitometric analysis of the ratio of phospho-cdc-2 and actin. *p < 0.05 compared with vector.

DISCUSSION

In the present study, HIV-1 expressing tubular cells showed an accelerated rate of apoptosis. This proapoptotic effect of HIV-1 was associated with the arrest of the tubular cell cycle at G2/M phase. Moreover, tubular cells expressing HIV-1 showed the activation of phospho-p53, phospho-Chk2, and phospho-cdc2. These findings suggest that HIV-1 expression in tubular cells may be contributing to cellular injury through arrest at G2/M phase via activation of phospho-p53, phospho-Chk2, and phospho-cdc-2.

The timely progression through the cell division cycle ensures the correct transmission of genetic information from a cell to its daughters. A quiescent cell on stimulation leaves a resting phase (G0) and enters the gap 1 (G1) phase, prior to DNA synthesis (S), followed by a second gap (G2) phase and cell mitosis (M). Cell cycle checkpoints maintain the order and fidelity of cell-cycle events in response to replicative stress and DNA strand breaks.[17],[18]. The transition from G2 into M is regulated by the serine-threonine cdk 1. The activation of cdk 1 requires the association of their positive subunit, cyclin B, and the phosphorylation of Thr161 by Cdk-activating kinase. These cdk-cyclin complexes are also negatively regulated by phosphorylation at Tyr15, which is catalyzed by inhibitory protein kinases.[17],[18] During G2/M transition, dephosphorylation of Tyr15 by cdc25 protein phosphatase cdc25B triggers cdk1 (cdc2)-cyclin B activation. During the DNA-damage response, the activation of ATM/ATR and downstream checkpoint kinase, CHK2, leads to the phosphorylation of all cdc25 phosphatases, which creates binding sites of 14-3-3 proteins and sequestration of the phosphatases away from cdk1 (cdc2)-cyclin B.[19–24] These molecular events provoke G2M cell cycle arrest. For example, in fission yeast, entry into mitosis is regulated by the phosphorylation status of tyrosine 15 (Tyr15) on cdc2, which is phosphorylated by Wee1 and Mik1 kinases during G2 and rapidly dephosphorylated by cdc25 phosphatase to trigger entry into mitosis.[25] These cell cycle regulatory elements are targets of DNA damage and DNA replication checkpoints, two regulatory pathways that induce cell cycle arrest.[26] In the present study, tubular cell HIV-1 expression was associated with phosphorylation of Tyr25 and CHK2. These findings indicate that DNA damage induced by HIV-1 triggers phosphorylation of Tyr15 and DNA replication checkpoint kinase2. These events led to G2/M arrest in tubular cells.

P53 attenuates the activity of cyclinB1 promoter or promotes the transcriptional activation of the sequestering protein 14-3-3.[27] It has also been reported that p53-regulated GADD45 protein associates and inhibits cdk1 (cdc2)-cyclin B kinase activity in response to DNA damage.[28] In the present study, HIV-1 enhanced p53 phosphorylation of tubular cells. Thus, it appears that HIV-1-induced phosphorylation may have also contributed to tubular cells G2/M arrest.

The p53-mediated pathway has been extensively studied in context to apoptosis.[29–35] At the level of transcription, multiple p53 target genes, especially those of Bcl-2 family members, play crucial roles in promoting apoptosis.[29–30] The Bax gene was one of the first p53-dependent apoptotic targets to be described.[29],[30] Subsequently, other Bcl-2 family members, including BH3-only members Bid, Puma, and Noxa have been described.[31–33] Others have reported that p53 itself targets mitochondria and activates Bax and Bak.[34],[35] It is likely that HIV-1-induced p53 phosphorylation might have also contributed to tubular cell apoptosis.

Previously, many investigators have reported the effect of HIV-1 on tubular cell injury.[9],[11] Because tubular cells do not carry the known conventional HIV-1 receptors, it was not clear whether the reported effect of HIV-1 happened through extrinsic interaction of HIV-1 with tubular cells or occurred as a result of entry of HIV-1 into these cells.[12],[13] Ray et al. showed HIV-1 entry into tubular cells; however, they did not comment on occurrence of apoptosis.[14] The present study was designed to evaluate the effect of HIV-1 entry into tubular cells. We studied only those cells in which HIV-1 entry (GFP +ve) has occurred. Thus, the present study confirms that HIV-1 entry into tubular cells promotes apoptosis.

Interestingly, tubular cells expressing HIV-1 showed G2/M arrest only after 72 hours of transduction. Therefore, it appears that occurrence of HIV-1-induced tubular cell injury at later time periods is mediated through cell cycle dysregulation. Thus, HIV-1 may induce tubular cell injury through multiple pathways.

ACKNOWLEDGMENTS

This study was supported from Grant RO1 DA12111 from the National Institutes of Health, Bethesda, Md. There is no conflict of interest in carrying out these studies.