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Originally published online as doi:10.1189/jlb.0405194 on November 10, 2005

Published online before print November 10, 2005
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(Journal of Leukocyte Biology. 2006;79:192-201.)
© 2006 by Society for Leukocyte Biology

HIV-1 TAT represses transcription of the bone morphogenic protein receptor-2 in U937 monocytic cells

Robert L. Caldwell*,{dagger},1, Radhika Gadipatti{ddagger}, Kirk B. Lane{ddagger} and Virginia L. Shepherd*,{dagger},2

Departments of
* Pathology and
{ddagger} Medicine, Vanderbilt University School of Medicine, and
{dagger} Department of Veterans Affairs Medical Center, Nashville, Tennessee

2 Correspondence: VA Medical Center/Research Service, 1310 24th Ave., South, Nashville, TN 37212. E-mail: Virginia.l.shepherd{at}vanderbilt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bone morphogenetic protein receptor-2 (BMPR2) is a member of the transforming growth factor-ß receptor family and is expressed on the surface of several cell types including endothelial cells and macrophages. Recently, a cause for familial primary pulmonary hypertension (FPPH) has been identified as mutations in the gene encoding BMPR2. Three forms of pulmonary hypertension (PH) exist, including PPH, FPPH, and PH secondary to other etiologies (sporadic PH) such as drug abuse and human immunodeficiency virus (HIV) infection. It is interesting that these subtypes are histologically indistinguishable. The macrophage is a key target cell for HIV-1, significantly altering macrophage cell function upon infection. HIV-1 trans-activator of transcription (Tat), an immediate-early product of the HIV-1 lifecycle, plays an important role in mediating HIV-induced modulation of host cell function. Our laboratory has previously shown that Tat represses mannose receptor transcription in macrophages. In the current study, we examined activity from the BMPR2 promoter in the macrophage cell line U937 and potential regulation by Tat. Transfection of U937 cells with BMPR2 promoter-reporter constructs revealed dose-dependent repression of BMPR2 promoter activity in the presence of Tat. Experiments using truncations of the BMPR2 promoter localized Tat-mediated repression to the first 208 bases of the promoter. Decreased BMPR2 transcription resulted in altered downstream signaling. Similar to mothers against decapentaplegics (SMAD) phosphorylation and SMAD6 expression, in response to BMP2 treatment, were down-regulated after Tat treatment. Finally, HIV-1 infection and treatment with Tat protein of the U937 human monocytic cell line resulted in a decreased, endogenous BMPR2 transcript copy number.

Key Words: monocytes/macrophages • AIDS • cell-surface molecules • gene regulation • lung


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus type 1 (HIV-1) infection of the host includes a cascade of events that optimizes viral replication while evading the ensuing immune response. HIV-1, or trans-activator of transcription (Tat), is a 101- to 72-amino acid protein, expressed early in the virus lifecycle [1 ]. Functionally, Tat is an essential, potent stimulator of HIV-1 transcription from the viral promoter [1 , 2 ] as well as suppressor of the immune response through interaction with host transcription factors [3 , 4 ]. HIV-1 transcription is up-regulated significantly by the interaction of Tat with host nuclear proteins such as cyclin T1, facilitating enhanced elongation of the viral transcript [5 ]. Structurally, Tat possesses the canonical transactivator characteristics including a nucleic acid-binding domain and activation domain. Full-length Tat protein is encoded by two exons, resulting in an 86- or 101-amino acid product, depending on the viral isolate [2 ]. Later, when HIV-1 infection becomes less efficient, synthesis of the truncated, one-exon product is observed. However, both Tat proteins up-regulate HIV-1 transcription.

Besides its essential role in trans-activating HIV-1 transcription, Tat also regulates host cell functions, primarily at the level of transcription. In addition to Tat expression in infected cells, Tat protein can be released from infected cells and act as a soluble cell modulator [6 ], thus expanding its role to infected as well as neighboring, noninfected cells. A number of studies have reported a variety of physiological effects of Tat including inhibition of antigen-induced T cell proliferation [7 ], increased proliferation of renal glomerular epithelial cells [8 ], regulation of apoptosis in T cells [9 ], dysregulation of cytokine expression [10 11 12 ], up-regulation of HIV coreceptors [13 , 14 ], and promotion of chemotaxis [15 ] and microvascular invasion [16 , 17 ]. As macrophages represent an infectable cell population, they serve as producers and/or targets of Tat action during HIV infection. In a recent report, our laboratory demonstrated that decreased expression of the mannose receptor, a pattern recognition receptor expressed on macrophages [18 ], was mediated in part by inhibition of mannose receptor transcription by endogenous and exogenous Tat [19 ].

Another project within our group focuses on the role of the bone morphogenetic protein receptor-2 (BMPR2) in familial primary pulmonary hypertension (FPPH). Previous studies have shown that a number of heterogeneous mutations in the gene encoding BMPR2 lead to FPPH [20 21 22 ]. Although considerable work has focused primarily on the role of BMPR2 in development, specifically in ectopic bone, tooth, and cartilage formation [23 24 25 26 ], recent studies have begun to examine the role of BMPR2 in the adult. For example, reports have shown that BMP treatment of pulmonary artery smooth muscle cells (PASMC) from normal lungs results in decreased cell proliferation [27 ]. PASMC from patients with FPPH exhibited abnormal growth responses to BMP treatment, suggesting that normal BMP signaling through wild-type BMPR2 may regulate cell growth. In addition, BMP treatment of osteoblasts has recently been reported to induce cyclooxygenase-2 transcription [28 ].

Different forms of pulmonary hypertension (PH) exist, including FPPH and PH, secondary to other etiologies {sporadic PH (SPH) [29 ]}. Etiologies associated with SPH include collagen vascular disease, exposure to drugs, and HIV-1 infection {HIV-related PH (HRPH) [30 ]}. It is interesting that the FPPH and HRPH subtypes are histologically indistinguishable [30 , 31 ]. The development of HRPH reduces the probability of survival by half as compared with HIV-infected individuals without HRPH [32 ]. As a result of the histopathological identity of FPPH and HRPH, we postulated that modulation of host gene transcription by HIV infection might mimic FPPH by genetically altering expression of BMPR2. As the HIV Tat protein is the major transcriptional regulator of host gene expression during HIV infection [33 ], we investigated the effect of endogenous and exogenous Tat on BMPR2 transcription and function in monocytic cells.

BMPR2 is a member of the transforming growth factor-ß receptor (TGF-ßR) superfamily and is the receptor mediating BMP signaling [34 ]. Downstream signaling following binding of BMPs involves the phosphorylation of membrane-bound, receptor-regulated similar to mothers against decapentaplegic (SMAD)1, -5, and -8. BMPR2 maintains a constitutively active serine/threonine kinase domain in the cytoplasmic tail of the receptor, which phosphorylates a second member of this family, BMPR1, following interaction with BMP ligands [35 ]. After phosphorylation by activated BMPR1, SMAD1, -5, and -8 are phosphorylated and individually bind the cytosolic SMAD4. The heterodimer then translocates to the nucleus, associates with the host transcription machinery, or binds specific gene promoters directly [36 37 38 39 ] and initiates transcription from BMP-responsive promoters, including the inhibitory SMAD6 [35 ]. After its transcription and translation, SMAD6 competes with SMAD1, -5, and -8 for SMAD4 and the kinase domain of BMPR1, repressing the BMPR2-SMAD signaling cascade [40 41 42 ].

Identical disease pathologies between HRPH and FPPH, the ability of HIV-1 Tat to repress macrophage gene transcription, and the recent discovery that mutations in BMPR2 result in FPPH prompted us to investigate whether Tat regulates transcription of BMPR2. In the current report, we present evidence that HIV-1 Tat specifically represses transcription of BMPR2 in macrophages and that Tat-mediated repression is localized to the first –208 base pairs (bp) of the BMPR2 promoter. We also show that decreased phosphorylation of receptor-associated SMAD1, -5, and -8 as well as decreased transcription and expression of SMAD6 are downstream, biological consequences associated with decreased expression of BMPR2. Finally, we demonstrate that HIV-1-infected U937 cells express decreased levels of an endogenous BMPR2 transcript.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reporter plasmids and expression vectors
The human BMPR2 promoter was cloned by polymerase chain reaction (PCR) amplification of a genomic fragment delineated by primers corresponding to positions –1834 to –1814 for the sense primer and +4 to +23 for the antisense primer. The resulting fragment was cloned into pGEM-T EASY® (Promega, Madison, WI). The clone was confirmed by sequencing and comparison with the 5' region associated with the human BMPRII locus (National Center for Biotechnology Information, Acct. #AC064836). The promoter was subcloned using primers corresponding to the desired 5' and 3' ends. The primers were further modified to include a KpnI site on the 5' primers and a SacI site to facilitate insertion into the luciferase reporter plasmid pGL3E (Promega). Following PCR amplification, promoter amplicons were digested with KpnI and SacI, and the resulting fragment was cloned into similarly digested pGL3E. The promoter-reporter constructs were amplified by passage through DH5{alpha} Escherichia coli and confirmed by DNA sequencing.

Cloning of the mannose receptor promoter driving luciferase (MRp-Luc) has been described previously [19 ]. Tat expression vector and the HIV-1 long-terminal repeat (LTR)-Luc reporter construct were kind gifts from Dr. Eric Verdin (Gladstone Institute, University of California, San Francisco). pGL3-Luc, cytomegalovirus (CMV)-Renilla-luciferase (RL), and CMV-Luc constructs were purchased from Promega. The BMP-responsive element (BRE)-Luc construct was a gift from Dr. Peter ten Dijke (Netherlands Cancer Institute, Amsterdam). The murine SMAD6 promoter reporter construct was obtained from Dr. Mitsuyasu Kato (University of Tsukuba, Japan). The HXB2 HIV-1 Pol expression construct and HIV-1 (Ada strain) virus stock were kind gifts from Dr. Paul Spearman (Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN).

Tat protein preparation
The 86-amino acid isoform of the Tat protein from the National Institutes of Health AIDS Reagent Repository was reconstituted in phosphate-buffered saline (PBS) containing 1 mg/ml bovine serum albumin (BSA) and 0.1 mM dithiothreitol and deaerated by bubbling with helium. The protein was stored at –80°C in the dark.

Transfection of U937 monocytic cells
Human U937 monocytes were purchased from American Tissue Culture Collection (Manassas, VA) and maintained in RPMI (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS; Mediatech, Herndon, VA) and antibiotics and were transfected as described previously [19 ]. Briefly, U937 cells (6x106) were suspended in Dulbecco’s modified Eagle’s medium (DMEM; 6 ml) and seeded at 1 ml per well in a six-well plate (Falcon, Becton Dickinson, San Jose, CA). For each transfection, DMEM (145 µl) was aliquoted into a 5-ml round-bottom tube (Falcon). Reporter plasmid (3 µg), effector plasmid, or empty vector (1 µg) and RL (1 µg) were added. Superfect (5 µl, Qiagen, Valencia, CA) was aliquoted into each tube, and the mixture was allowed to incubate at room temperature for 10 min. The transfection mixture was then added to each well, 0.5 ml complete media was added to the transfection reactions, and the reaction mixtures were incubated at 37°C, 5% CO2, for 24 h. The cells were then lysed, and luciferase activity was measured using the dual luciferase assay kit (Promega), per the manufacturer’s instructions. Resulting light emission was quantified using a luminometer.

BRE-Luc and SMAD6-Luc assays
Determination of luciferase activity from the BRE-Luc construct in the presence of Tat and/or BMP was accomplished by transfection of U937 cells with BRE-Luc (2 µg). U937 cells were transfected as described above in RPMI medium with 0.1% FBS and antibiotics. BMP2 (50 ng/ml, a gift from Dr. Anthony. Celeste, Wyeth-Genetics Institute, Cambridge, MA) was added to the cells 8 h after addition of the transfection reaction. Cells were collected 24 h after incubation, and luciferase activity was determined as described. Decreased luciferase expression from the SMAD6-Luc activity in the presence of Tat and/or BMP was determined by resuspension of U937 cells in RPMI with 0.1% FBS and antibiotics followed by transient transfection with SMAD6-Luc (2 µg) plus Tat expression plasmid or empty vector (2 µg). Twenty-four hours later, BMP2 (50 ng/ml) or PBS was added, and the cells were then assayed for luciferase activity after incubation for an additional 24 h.

Immunoblot analysis for phosphorylated SMAD (P-SMAD)1, -5, and -8 and SMAD6
U937 cells (2x106) were pelleted and resuspended in RPMI containing 0.1% FBS with antibiotics and plated into a six-well culture dish (Corning, NY). Cells were treated for 24 h with Tat protein (50 ng/ml) or PBS. Cells were then treated with BMP2 (50 ng/ml) or PBS for 15 min, as previously reported [43 ]. After incubation, cells were collected, washed, and lysed in immunoprecipitation buffer [20 mM Tris (pH 7.75) containing 1% Triton X-100, 0.5% deoxycholate, 0.15 M NaCl, 0.02% sodium azide, 0.34 trypsin inhibitory units of aprotonin/ml, and phosphatase inhibitors (Sigma Chemical Co., St. Louis, MO); 10 µl/ml lysate]. Cell lysate proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, which was washed briefly with Tris-buffered saline/0.1% Tween to remove excess salt and blocked in 5% BSA (1 h). Anti-P-SMAD1, -5, and -8 (Cell Signaling Technology, Beverly, MA) were added to the blocking solution at a 1:1000 concentration. The blot was then incubated overnight at 4°C and was washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000). P-SMAD proteins were visualized by incubation of the blot in 0.2 M Tris-HCl (pH 8.5), 2.5 mM luminol, 0.4 mM p-coumaric acid, and 0.0002% H2O2, followed by exposure of X-OMAT film (Kodak, Rochester, NY). To normalize for protein loading, the blot was stripped with NaOH (200 mM) and reprobed for human transferrin receptor (TF-R; Zymed, San Fransisco, CA). Densitometry was performed to quantify P-SMAD intensity using the UN-SCAN-it digitizing system.

Detection of SMAD6 protein in the presence of Tat and incubation with BMP2 were performed similarly to immunoblotting for P-SMAD with the following modifications: After 24 h incubation with Tat (50 ng/ml) or PBS, U937 cells were incubated with BMP2 (50 ng/ml) or PBS for 24 h as described previously [44 ]. Cells were washed and lysed, and cellular proteins were separated by SDS-PAGE as described above. After transfer, the blot was washed and blocked in 5% milk (1 h). Anti-SMAD6 antibody (Zymed) was added to the blocking solution at a 1:1000 concentration and incubated with the blot at 4°C overnight. The blot was washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000), and bands were visualized as described above. Densitometry was performed on a nonspecific band common to all samples to normalize for protein loading using the UN-SCAN-it gel digitizing system.

Reverse transcription (RT) real-time, quantitative polymerase chain reaction (PCR)
To examine the effect of infection of cells on BMPR2 transcription, U937 cells (6x106) were resuspended in RPMI containing 0.1% FBS and antibiotics. Cells were infected with 600 ng wild-type Ada strain HIV-1 or mock-infected. After overnight infection, cells were washed and resuspended in fresh medium (0.1% FBS, RPMI, and antibiotics). To examine the effect of treatment of cells with exogenous Tat, U937 cells (2x106) were pelleted and resuspended in RPMI containing 0.1% FBS with antibiotics and plated into a six-well culture dish. Cells were treated for 24 h with Tat protein (50 ng/ml) or vehicle. After 24 h, infected cells or cells treated with Tat were collected and lysed with TRIzol reagent (1 ml, Invitrogen, Carlsbad, CA). Total RNA was purified by the Qiagen RNeasy purification system as described by the manufacturer. Total RNA collected from HIV-1-infected U937 cells was reverse-transcribed to cDNA as follows: Total RNA (1 µg) was incubated with 5x buffer (4 µl, Promega), 100 mM dithiothreitol (2 µl, Promega), 10 mM deoxy-unspecified nucleoside 5'-triphosphate (2 µl, Boehringer Mannheim, Germany), RNase inhibitors (0.8 µl, Invitrogen), random primer (1 µl, Pharmacia, Uppsala, Sweden), and Moloney murine leukemia virus (1 µl, Promega). RNase-free water was added for a total volume of 20 µl. The RT reaction was mixed, briefly spun, and incubated at 37°C for 1 h. Real-time PCR was performed by first making a stock solution using the following concentrations per reaction: water (10.75 µl), mastermix (10.75 µl, Qiagen), SYBR Green (0.25 µl, Qiagen), BSA (0.25 µl), forward primer (0.5 µl), reverse primer (0.5 µl), and 100 mM MgCl2 (1 µl). The solution was briefly mixed and aliquoted into a real-time PCR tube (24 µl stock solution per reaction), followed by the addition of template cDNA or standard (1 µl). Amplification of BMPR2 cDNA was normalized to {gamma}-actin cDNA. Primers for BMPR2 amplification were as follows (Integrated DNA Technologies, Coralville, IA): forward, 5'-CTG GAC AGC AGG ACT TCA CA-3'; reverse, 5'-CTT GGG CCC TAT GTG TCA CT-3'. Primers for {gamma}-actin were: forward, 5'-TCC TGT GGC ATC CAC GAA ACT-3'; reverse, 5'-GAA GCA TTT GCG GTG GAC GAT-3'. To monitor HIV-1 infection of U937 cells, the highly conserved HIV-1 Pol gene was amplified using the following primers: forward, 5'-GGG CCT GAA AAT CCA TAC AA-3'; reverse, 5'-CCT TTC CAT CCT TGT GGA AG-3'. Standard curves for BMPR2 and HIV-1 Pol were derived from BMPR2 and Pol expression constructs, and {gamma}-actin standard curves were derived from genomic DNA. Real-time PCR reactions were performed using the Cepheid Smart Cycler (Sunnydale, CA), per the manufacturer’s instructions.

Statistical analysis
Statistical significance was determined using t-tests to compare two data sets or ANOVA to determine significance between multiple data sets (InStat software, GraphPad, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIV Tat represses transcription of BMPR2
Previous studies have shown that Tat represses transcription of the mannose receptor in macrophages [19 ], as well as major histocompatibility complex (MHC) classes I and II in T lymphocytes [3 , 45 46 47 48 49 ]. To determine if Tat represses BMPR2 transcription in macrophages, a 1725-bp BMPR2 (–1725) promoter driving the luciferase reporter gene was transiently cotransfected with Tat expression vector or empty vector into the human U937 monocytic cell line. We observed a greater than 50% reduction in BMPR2 promoter activity in the presence of Tat (Fig. 1A ). Transcription from the CMV promoter was unaffected by Tat, and Tat significantly enhanced transcription from the HIV promoter or LTR, as reported [1 , 2 ]. An approximate tenfold induction from the LTR was observed when the amount of Tat expression vector was increased (data not shown). Tat also repressed transcription from the mannose receptor promoter as described previously [19 ]. When the increasing Tat expression vector was cotransfected with the BMPR2 promoter reporter construct, a dose-dependent repression of BMPR2 promoter activity was determined, with a maximum inhibition of 80% with 2 µg Tat vector (Fig. 1B) . Tat protein expression in these transfection experiments was verified by Western blotting as described previously [19 ].



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  		Figure 1. HIV-1 Tat represses BMPR2 promoter activity in U937 cells. (A) U937 monocytes were transiently cotransfected with a BMPR2 promoter reporter construct (3 µg) plus Tat expression plasmid or empty vector (1 µg) and RL (1 µg) to control for transfection efficiency. As positive controls, the MRp-Luc (MR) or the LTR-Luc (HIV LTR) was transfected in place of BMPR2-Luc. CMV-Luc (CMV; 3 µg) was cotransfected with Tat expression plasmid (solid bars) or empty plasmid (1 µg) as a negative control (open bars). Firefly luciferase and RL activities were measured using the dual luciferase assay system (Promega) according to the manufacturer’s instructions. Data are expressed as the firefly luciferase activity for each sample normalized to the RL activity and are the mean of triplicates ± SD. This experiment is representative of five separate experiments. *, P < 0.05. (B) Increasing amounts of Tat expression vector were cotransfected with BMPR2-Luc (2 µg). The total amount of DNA was kept constant by addition of empty vector. Results are expressed as the firefly luciferase activity for each sample normalized to the RL activity and are expressed as the percentage of control cells with empty vector alone. This experiment is representative of five separate experiments performed in triplicate.

 
HIV-1 Tat-mediated repression from the BMPR2 promoter is localized to the first 208 bp
To localize the region in the BMPR2 promoter where Tat represses BMPR2 promoter activity, truncations of the BMPR2 promoter (–1725, –998, –487, –208) were cloned into the pGL3 reporter plasmid and transiently transfected in the presence or absence of the Tat expression vector. As shown in Figure 2 , inhibition of promoter activity was ~50% for the –1725, –998, and –487 constructs and a slightly greater level of inhibition (39%) for the –208 construct. However, the Tat-mediated inhibition was not significantly different between the –1725-bp and the –208-bp promoter-luciferase vector.



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  		Figure 2. HIV-1 Tat-mediated repression from the BMPR2 promoter is localized to the first 208 bp. U937 cells were transiently transfected with truncations of the BMPR2 promoter constructs (–1725, –998, –487, –208) driving the luciferase reporter gene (3 µg) with Tat expression plasmid or empty vector (1 µg) as described in Materials and Methods. Cells were collected 24 h post-transfection, and luciferase activity was measured with a luminometer. Results are expressed as the firefly luciferase activity for each sample normalized to the RL activity and are expressed as the percentage of control cells with empty vector alone. These data are representative of four separate experiments.

 
Tat decreases activity from a BRE after BMP treatment
BMP binding to BMPR2 ultimately results in the phosphorylation of SMAD1, -5, and -8 and their physical association with SMAD4 in smooth muscle cells [50 ], osteoblasts [43 ], and epithelial cells [36 , 51 ]. This complex then migrates to the nucleus to bind transcription factors and/or promoters of specific genes. To determine if repression of BMPR2 transcription results in a reduction in downstream signaling following BMP ligation, a BRE-Luc was transfected into the U937 monocytic cell line. This construct has been shown previously to interact specifically with P-SMAD1 and -5 [52 ]. As shown in Figure 3A , when cells expressing BRE-Luc were treated with BMP2 (50 ng/ml), an eightfold increase in luciferase expression was observed. There was no change in luciferase activity from the BRE-Luc construct when Tat was coexpressed in the absence of BMP. When Tat was coexpressed in the presence of BMP2, we observed a significant decrease (50%) in BRE-Luc activity. These data suggest that Tat-mediated repression of BMPR2 results in decreased activation of SMAD1 and -5.



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  		Figure 3. Tat decreases activity from a BRE after BMP treatment. (A) The BRE-Luc construct (2 µg) was transiently cotransfected with empty vector or Tat expression vector (2 µg) and RL (1 µg). After incubation of cells and DNA for 8 h, BMP2 (50 ng/ml) was added to the indicated wells for 16 h. Cells were collected and lysed, and luciferase activity was measured. Results are firefly luciferase activity for each sample normalized to RL activity and are expressed as the percentage of luciferase activity in control cells transfected with empty vector. Experiments were performed in triplicate in two separate experiments, and results are the mean ± SD. *, P < 0.001, for BRE-Luc plus BMP2 versus BRE-Luc minus BMP2 and for BRE-Luc plus BMP2 versus BRE-Luc plus BMP2 plus Tat. (B) U937 cells (2x106) were suspended in RPMI containing 0.1% FBS with antibiotics (2 ml). Cells were incubated with Tat protein (50 ng/ml) or PBS for 24 h. Cells were stimulated with BMP2 (50 ng/ml) or PBS for 20 min, followed by lysis of cells and separation of proteins by SDS-PAGE. P-SMAD1, -5, and -8 were detected by Western analysis. The blot was stripped and probed for the TF-R to normalize SMAD levels. Densitometry was performed to quantify SMAD phosphorylation intensity and TF-R using UN-SCAN-it gel digitizing system. P-SMAD levels normalized to TF-R levels are shown below the immunoblot results. This experiment is representative of two separate experiments.

 
Tat-mediated repression of BMPR2 transcription decreases SMAD phosphorylation
To confirm our hypothesis that Tat-mediated repression of BMPR2 decreases SMAD activation through decreased phosphorylation, U937 cells were incubated with Tat protein (50 ng/ml) or PBS for 24 h. Cells were treated with BMP2 (50 ng/ml) or PBS for 20 min, and SMAD phosphorylation levels were determined via immunoblot analysis. We observed an increase in SMAD phosphorylation in BMP2-treated cells, and following treatment with BMP2 in the presence of Tat, phosphorylation of SMAD proteins was decreased (Fig. 3B) . Densitometric analysis was performed on the bands for P-SMAD and normalized to levels of TF-R, a protein that is not regulated by Tat. Results of this analysis showed an ~2.3-fold increase in SMAD phosphorylation induced by BMP2, with almost complete inhibition of the BMP2 response by the addition of Tat.

Tat represses activity from the SMAD6 promoter after BMP treatment
BMP interaction with BMPR2 ultimately initiates transcription of SMAD6 via binding of phosphorylated SMAD1 and SMAD5 to the SMAD6 promoter [35 ]. We hypothesized that Tat-mediated inhibition of BMPR2 transcription would result in decreased SMAD6 transcription. To test this hypothesis, the murine SMAD6 promoter driving a luciferase reporter gene was transiently transfected into U937 cells with Tat expression vector or empty vector. Cells were also treated with BMP2 (50 ng/ml) or PBS. As shown in Figure 4A , treatment of cells expressing SMAD6-Luc with BMP2 resulted in increased luciferase activity, and inclusion of Tat resulted in inhibition of the BMP2-mediated enhancement of SMAD6-Luc activity. There was no effect of Tat alone on SMAD6 promoter activity.



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  		Figure 4. BMP2-induced activity from the SMAD6 promoter and SMAD6 protein levels are decreased by pretreatment with Tat. (A) SMAD6-Luc (2 µg) was cotransfected in the presence or absence of Tat expression vector (2 µg) plus RL to control for transfection efficiency (1 µg). After incubation of the transfection mixture with cells (12 h), indicated wells were treated with BMP2 (50 ng/ml). Luciferase activity was measured post-BMP2 treatment (12 h). Results are the mean ± SD of triplicate determinations of firefly luciferase activity normalized to RL activity and are expressed as the percentage of control cells (Con) with empty vector alone. This experiment is representative of two separate experiments. *, P < 0.001, comparing SMAD6-Luc minus BMP2 versus SMAD6 plus BMP2; **, P < 0.001, comparing SMAD6-Luc plus Tat minus BMP2 versus SMAD6-Luc plus Tat plus BMP2. (B) U937 cells (2x106) were suspended in RPMI, 0.1% FBS with antibiotics (2 ml), and incubated with Tat protein (50 ng/ml) or PBS for 24 h. Cells were then stimulated with BMP2 (50 ng/ml) or PBS for 24 h. The cells were lysed, and proteins separate on SDS-PAGE gel for Western blotting analysis for SMAD6 protein levels. After detection of SMAD6 protein via chemiluminesence, normalization was performed using a nonspecific band, which remained constant and seen on all blots, followed by densitometry as described above. This experiment is representative of two separate experiments.

 
Tat-mediated repression of endogenous BMPR2 transcription decreases SMAD6 protein levels
To support our findings that Tat-mediated repression of BMPR2 transcription decreases SMAD6 transcription, we examined SMAD6 protein levels in the presence of BMP2, with or without Tat protein. U937 cells were treated for 24 h with Tat protein (50 ng/ml) or PBS followed by incubation with BMP2 (50 ng/ml) or PBS for 24 h. Western blotting was performed to examine SMAD6 protein levels. As shown in Figure 4B , we observed a 2.2-fold increase in SMAD6 protein levels when cells were treated with BMP2, and this increase was blocked when cells were coincubated with Tat protein. These data support our findings that HIV-1 Tat represses transcription of BMPR2, resulting in decreased downstream effects of BMPR2 signaling.

HIV-1 infection or treatment with exogenous Tat represses transcription of endogenous BMPR2 message in U937 cells
Studies presented above demonstrate that Tat mediates repression of BMPR2 transcription using transient transfection of a monocytic cell line with a construct containing the luciferase reporter gene driven by the BMPR2 promoter. To determine the effect of HIV-1 infection on macrophage expression of endogenous BMPR2 transcript, U937 cells were infected with a macrophage-tropic, wild-type HIV-1 strain (Ada, 100 ng/106 cells) or mock-infected, and BMPR2 transcript levels were determined by real-time PCR. Total RNA was collected from HIV- or mock-infected cells, purified, and reverse-transcribed as described in Materials and Methods. Probes specific for BMPR2 were used to amplify BMPR2 mRNA levels, and reactions were normalized to {gamma}-actin. As shown in Figure 5 , BMPR2 message was reduced by 50% in infected cells compared with mock-infected cells. To monitor for HIV-1 infection, the highly conserved HIV-1 Pol gene was amplified from cDNA obtained from HIV- or mock-infected U937 cells. Pol was amplified from the cDNA of HIV-infected cells and was not amplified in the mock-infected cells (data not shown). To determine if treatment with exogenous Tat would also reduce levels of BMPR2 mRNA, cells were treated for 24 h with 50 ng/ml Tat (+Tat) or vehicle alone (control). RNA was isolated and reverse-transcribed as described in Materials and Methods, and BMRP2 transcript levels were determined as above. As shown in Figure 5B , transcript levels were reduced by 79%.



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  		Figure 5. HIV-1 infection and treatment with Tat protein repress transcription of endogenous BMPR2 message in U937 cells. (A) U937 cells (6x106) were resuspended in RPMI with 0.1% FBS and antibiotics (2 ml) and incubated with macrophage-topic, wild-type HIV-1 (600 ng) or media. After overnight infection, cells were washed and resuspended in RPMI, 0.1% FBS with antibiotics (2 ml). After 24 h, cells were pelleted, and total RNA was isolated as described in Materials and Methods. Total RNA was reverse-transcribed, and target cDNA was amplified as described in Materials and Methods. Loading was normalized by amplification of the {gamma}-actin gene using genomic DNA for the standard curve. Infection of U937 cells was monitored by amplification of the highly conserved Pol gene (data not shown). These data are representative of three separate experiments and are expressed as the mean percentage of BMPR2 relative abundance from the infected cells as compared with mock-infected cells ± SD. *, P < 0.05. (B) U937 cells (2x106) were resuspended in RPMI with 0.1% FBS and antibiotics (2 ml) and incubated with Tat protein (50 ng/ml) or vehicle. After 24 h, cells were pelleted and RT-PCR performed as described in A. These data are from three separate experiments ± SD. *, P < 0.0001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HIV-1 infection of macrophages regulates a variety of host cell functions such as cytokine production [12 ] and apoptotic signaling [53 ]. Several HIV-1 proteins have been shown to be involved in this regulation, resulting in altered host cell protein expression at various levels, including post-translational trafficking of targeted proteins and transcriptional up- or down-regulation of specific genes [33 , 54 , 55 ]. Numerous studies have demonstrated that HIV-1 Tat, a potent transactivator of HIV-1 transcription in both macrophages and T cells, modulates transcription from the promoters of several host genes involved in the immune response. Expression of the mannose receptor [19 ], MHC class I [46 47 48 49 ], MHC class II [3 ], and ß2-microglobulin [45 ] is down-regulated, resulting in decreased pathogen clearance and antigen presentation associated with these molecules. In the current study, we have extended these studies to Tat-mediated regulation of another macrophage surface receptor, BMPR2, which is a member of the TGF-ßR superfamily. Using transient transfection assays of the BMPR2 promoter driving the luciferase reporter gene, we have shown that Tat expression in U937 cells reduced BMPR2 promoter-luciferase activity by greater than 50%. Regulation was localized to a region in the first 208 bp of the BMPR2 promoter and resulted in altered downstream signaling following ligand-receptor interaction. In addition, infection of U937 cells with a macrophage-tropic virus and treatment with recombinant Tat protein decreased endogenous BMPR2 transcript levels, suggesting that this down-regulation may occur in the setting of HIV infection.

BMPR2 is a surface molecule found on a variety of developing and adult mammalian tissues, but little is known about the function of this protein in the adult. BMPs are the natural ligands for BMPR2 and are found in most developing tissues, appearing to be principally involved in bone and cartilage formation [56 ]. Most information about BMPR2 function has come from studies in the developing embryo. BMP4 and -7 colocalize in the developing lung buds, and studies examining normal and targeted misexpression of BMP4 have shown that BMP4 plays a role in embryonic lung morphogenesis [57 ]. Ligation of BMPs by BMPR2 induces phosphorylation of SMAD1, -5, and -8, followed by increased transcription of the inhibitory SMAD6. In the current study, we have shown that Tat not only reduces BMPR2 transcription but blocks ligand-induced signal transduction by reducing SMAD activation. A block in this pathway could have additional consequences on other pathways requiring these signaling molecules. Intracellular signaling through the SMAD pathway is initiated by BMP and TGF-ß1. After binding to the respective receptors, signaling is initiated by two different subsets of receptor-bound SMADs. However, signaling from both subsets intersects at SMAD4, which facilitates nuclear translocation of BMP and TGF-ß1 signaling. For example, SMAD2 and -3 are phosphorylated by TGF-ß1 signaling through its receptor and converge on cytoplasmic SMAD4 in a similar manner to BMPR2 signaling [24 , 58 ]. Thus, events initiated by TGF-ß1 and BMP signaling compete for a limited pool of SMAD4. Perturbations of BMP signaling as a result of altered or decreased BMPR2 expression might tilt SMAD4 use toward the TGF-ß1 signaling cascade.

In the current study, we have demonstrated that Tat-mediated repression of BMPR2 transcription localizes to the first 208 bp, but the mechanism involved has not yet been delineated. Tat enhancement of the rate of HIV transcriptional elongation is dependent on the interaction of Tat with a variety of host proteins including the core RNA polymerase II [59 , 60 ], TATA-binding protein associated factor (TAFII)55 [61 ], TFIIH [62 ], cyclin-dependent protein kinase 7 [63 ], SP1 [64 ], nuclear factor of activated T cells (NFAT)1 [65 ], and cyclin T1 [66 ]. It has been suggested that this Tat–host protein binding results in regulation of HIV transactivation and at the same time, pulls these factors away from specific host genes, altering transcription from these promoters. For example, Tat represses MHC class I transcription but does not bind directly to the promoter DNA [48 ]. Inhibition appears to proceed via a direct interaction of Tat with TAFII250, a component of the general transcription factor TFIID, which is a core protein in the transcriptional initiation complex. Tat down-regulates MHC class II transcription by binding to cyclin T1, preventing the interaction of cyclin T1 with the class II transactivator CIITA [3 ]. Tat interacts directly with NFAT1 in T cells, increasing NFAT1-driven transcription, leading to up-regulation of cytokines such as interleukin (IL)-2 [65 ]. Preliminary studies from our laboratory suggest that regulation of BMPR2 as well as the mannose receptor might involve a mechanism similar to MHC class II regulation, whereby Tat binding of cyclin T prevents transcriptional initiation from these promoters (R. L. Caldwell, V. L. Shepherd, manuscript in preparation).

In the current study, we have shown that Tat, expressed intracellularly in U937 cells and added extracellularly, was capable of repressing BMPR2 transcription and function. Zauli et al. [67 ] reported in 1995 that Tat can be released from infected cells and act as a soluble stimulator of cell proliferation. In addition, Tat has been detected in the serum of HIV-1-infected patients at levels of 1–3 ng/ml [68 ]. Tat protein includes a basic domain, which facilitates its movement across membranes and entry into the nucleus when added exogenously to cells. Effects of exogenous Tat on T cells include up-regulation of IL-2 [69 ], IL-4 [70 ], IL-6 [70 ], and c-Fos [71 ] and down-regulation of ß2-microglobulin [5 ] and MHC class II molecules [3 ]. In macrophages, exogenous Tat transcriptionally represses mannose receptor expression [19 ] and increases expression of tumor necrosis factor {alpha} (TNF-{alpha}) [10 ] and IL-6 [12 ]. These studies expand the role for this HIV-derived protein to infected and noninfected cells, greatly expanding the potential for Tat to modulate gene expression in the infected host and contribute to a wide variety of pathologies.

The role of Tat-mediated repression of BMPR2 transcription and function in the context of HIV-infected individuals is not yet understood. It is compelling to speculate that Tat-mediated repression of BMPR2 transcription might result in HRPH, mimicking a disease that is caused by frameshift or mis-sense mutations in the BMPR2 gene, resulting in decreased BMPR2 function and/or expression. The recent finding that mutations in the BMPR2 gene can be identified in a significant percentage of patients with FPPH and SPH suggests that signaling via this receptor plays a critical role in the maintenance of normal pulmonary vascular physiology. Forty-six different germ-line mutations in the gene encoding BMPR2 have been identified, including mis-sense and frameshift mutations [56 ], which result in the loss of expression or inactivation of the receptor, but no link has been found as to how these BMPR2 mutations give rise to PH. However, the finding that arterial remodeling involves hypertrophy and proliferation of endothelial cells, smooth muscle cells, and intimal cells [56 ] and that BMP2 inhibits growth of aortic vascular smooth muscle cells in vitro [72 , 73 ] suggests that altered BMPR2 might lead to uncontrolled proliferation.

In the current study, we found that Tat down-regulates BMPR2 expression and signaling by ~50%. Reports on FPPH have suggested that this level of decreased BMPR2 expression can lead to altered lung physiology. However, we do not know if Tat-mediated reduction of BMPR2 is linked directly to the development of HRPH in HIV-infected individuals. In addition, recent studies have suggested that reductions of less than 50% in BMPR2 expression might not result in PH conditions and that mutations in other genes might be required for the PH phenotype to be expressed. For example, mutations in the TGF-ßRII gene are found in tumors [74 ] and atherosclerotic lesions [75 ]. Reduced expression of the proapoptotic Bax gene in endothelial cells from plexiform lesions in patients with SPH could result in inhibition of apoptotic cascades [76 ]. As with SPH, Tat may reduce expression of other genes in concert with BMPR2 to reach the stage of HRPH. For example, Tat up-regulates the antiapoptotic Bcl-2 gene and the cytokines TNF-{alpha}, IL-1, IL-2, monocyte chemoattractant protein-1, and TGF-ß1 in macrophages. Regulation of these proteins could contribute to optimal conditions for productive HIV-1 infection and survival and also cripple the immune response to HIV and other invading pathogens. From the profound effects on the immune system to secondary complications such as HRPH, the potential regulation of other genes during HIV infection is warranted to fully understand the significance of Tat in normal host cell function.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Department of Veterans’ Affairs (V. L. S.). K. B. L. and V. L. S. contributed equally to this study. We are grateful to Dr. Rasul Abdolrasulnia (Department of Pathology, Vanderbilt University School of Medicine) and Tom Blackwell (Department of Pulmonary Medicine, Vanderbilt University School of Medicine) for their technical assistance, to Dr. Paul Spearman (Department of Pediatrics, Vanderbilt University School of Medicine) for use of his BL-3 facilities for our HIV-1 infection studies, and to the Skin Disease Research Center Molecular Genetics Core for their assistance with the real-time PCR experiments.


   FOOTNOTES
 
1 Current address: Vanderbilt Orthopaedic Institute, Division of Musculoskeletal Oncology, Vanderbilt University, Nashville, TN 37232-8774. Back

Received April 14, 2005; revised September 9, 2005; accepted September 10, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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