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The Journal of Immunology, 2004, 172: 7642-7646.
Copyright © 2004 by The American Association of Immunologists

Rac1 and Toll-IL-1 Receptor Domain-Containing Adapter Protein Mediate Toll-Like Receptor 4 Induction of HIV-Long Terminal Repeat1

Ozlem Equils2, Zeynep Madak3, Chunrin Liu, Kathrin S. Michelsen, Yonca Bulut and Daning Lu

Division of Pediatric Infectious Diseases, Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center and University of California School of Medicine, Los Angeles, CA 90048


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-{kappa}B activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human immunodeficiency virus-1-infected patients are frequently coinfected with multiple opportunistic and pathogenic microorganisms that lead to increased HIV-1 replication (1, 2). The presence of an opportunistic infection is an independent predictor of HIV disease progression (3); however, the molecular mechanisms involved in this process are not clearly known.

HIV-1 replication is regulated through HIV-long terminal repeat (HIV-LTR),4 and although HIV-LTR alone can serve as its own promoter, early mRNA transcription relies primarily on binding of cellular transcription factors, including NF-{kappa}B, to the LTR (4, 5, 6, 7). The predominant complex binding to the LTR-{kappa}B sites in activated cells is NF-{kappa}B p50-p65 heterodimer. In unstimulated cells, NF-{kappa}B is restricted to the cytoplasm through its interaction with inhibitor proteins of the I{kappa}B family. Activation of NF-{kappa}B occurs through phosphorylation and proteolysis of the I{kappa}B inhibitor, with subsequent translocation of active NF-{kappa}B into the nucleus, where it can bind to its cognate binding sites (8, 9). Regulation of NF-{kappa}B activation is also dependent on the inducible phosphorylation and trans-activation activity of p65 (10).

We have recently shown that Gram-negative bacterial LPS induces HIV-LTR trans-activation via innate immune system receptor, Toll-like receptor 4 (TLR4) and IL-1R-associated signaling molecules (i.e., myeloid differentiation protein (MyD88), IL-1R-activated kinase (IRAK), TNF receptor-associated factor-6 (TRAF6), and NF-{kappa}B-inducing kinase, which lead to I{kappa}B{alpha} phosphorylation and NF-{kappa}B activation (11).

Rac1 has recently been shown to induce NF-{kappa}B activation through both I{kappa}B{alpha} phosphorylation (12) and phosphatidylinositol 3-kinase-regulated p65 trans-activation (13). We have previously shown that chemical inhibition of phosphatidylinositol 3-kinase partially blocked the LPS-induced HIV-LTR trans-activation (11). Arbibe and colleagues (14) have shown that Rac1 played role in TLR2-mediated NF-{kappa}B activation. Currently, the role of Rac1 in LPS/TLR4-induced HIV-LTR trans-activation is unknown.

In the present study we demonstrate that LPS stimulation of human dermal microvascular endothelial cells activates Rac1, and that coexpression of dominant negative (DN) Rac1 partially blocks the LPS-induced HIV-LTR trans-activation. Furthermore, expression of a constitutively active Rac1 construct induces HIV-LTR trans-activation in HMEC. We also show that constitutively active Rac1 induces HIV-LTR trans-activation through the recently identified adapter molecule, Toll-IL-1R (TIR) domain-containing adapter protein (TIRAP). TIRAP was shown to mediate TLR1, TLR2, TLR4, and TLR6, but not TLR3, TLR5, TLR7, or TLR9 signaling (15, 16) and was suggested to provide signaling specificity to TLRs. Our observations are important in understanding the parallel signaling mechanisms involved in microbial Ag activation of HIV replication and may potentially help develop novel therapeutic agents targeting cellular molecules to block HIV replication during opportunistic infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

The immortalized human dermal microvessel endothelial cells (HMEC) were obtained from Dr. Candal (Centers for Disease Control and Prevention, Atlanta, GA) (17). HMEC were cultured in MCDB-131 medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, and 100 µg/ml penicillin and streptomycin in 24-well plates. The cells were routinely used between passages 10 and 14 as described previously (18). Highly purified, phenol-water-extracted, and protein-free (<0.0008% protein) Escherichia coli LPS, prepared according to the method described by McIntire et al. (19), was obtained from S. N. Vogel (Uniformed Services University, Bethesda, MD).

Plasmids

The HIV-LTRwt-luciferase vector has been previously described (20). Briefly, it carries U2+R regions of the HIV-LTR (LAI strain) from nucleoside –644 (Xhol) to +78 (HindIII). The pCMV-{beta}-galactosidase vector was used as previously described (18). DN-Rac1 (Rac1N17) cDNA was obtained from Dr. Broek (University of Southern California, Los Angeles, CA). Constitutively active Rac1 (Rac1V12) cDNA was obtained from Upstate Biotechnology (Lake Placid, NY). Flag-tagged TIRAP cDNA was obtained from R. Medzhitov (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT). The DN cDNA construct of MyD88 has been characterized and described previously (21).

Adenovirus expression vectors

Recombinant adenoviral vectors expressing either green fluorescent protein (Ad.CMVGFP) or a constitutively active mutant of Rac1 (Rac1V12) were obtained from J. F. Engelhardt (University of Iowa, Iowa City, IA) (22, 23). Briefly, recombinant adenoviruses were generated in 293 cells according to a procedure described by Anderson et al. (23). Adenoviral stocks were prepared as previously described (24) and were stored in 10 mM Tris with 20% glycerol at –80°C. The functional titers of adenoviral stocks were determined by plaque titrating on 293 cells. Typically one multiplicity of infection (MOI) was equal to 2 x 105 particle/PFU.

Tissue culture and infection

HMEC were plated at a concentration of 50,000 cells/well in 24-well plates and were cultured in MCDB-131 with 10% serum overnight. Adenoviral infections were performed for 2 h at 37°C, in MCDB-131 without FBS. After infections, an equal volume of MCDB-131 with 20% serum was placed on the wells, and the infections were continued for a total of 24 h. Most studies used various MOI to test recombinant adenoviral vectors. Cell death was assessed by measuring the lactate dehydrogenase activity in the supernatants according to the manufacturer’s protocol (Roche, Indianapolis, IN).

Transfection of HMEC

After 2 h of adenovirus infection, HMEC were cotransfected with reporter genes pCMV-{beta}-galactosidase (0.1 µg) and HIV-LTR-luciferase (0.1 µg) constructs using FuGene 6 transfection reagent (Roche) following the manufacturer’s instructions as reported previously (25). Cells were transfected overnight and then stimulated for 6 h with LPS suspended in growth medium. Cells were then lysed, and luciferase activity was measured with a Promega kit (Madison, WI) and a luminometer. {beta}-Galactosidase activity was determined by colorimetric method as described previously (18). Ninety-five percent of the HMEC was infected with the adenovirus vector.

Rac1 activation assay

In the inactive state, Rac-GDP is unable to associate with p21-activated kinase 1 (PAK-1). Activation of Rac1 leads to exchange of GDP for GTP; activated GTP-Rac1 then associates and activates PAK-1 (26). GST fusion proteins corresponding to the p21-binding domain of human PAK-1 (PBD; residues 67–150) were used to precipitate the activated Rac-GTP from cell lysates using a Rac1 activation assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. Briefly, confluent HMEC were stimulated with 50 ng/ml LPS and incubated at 37°C for various periods of time. Cells were then harvested into lysis buffer (20 mM HEPES (pH 7.4), 0.5% Nonidet P-40, 10 mM MgCl2, 10 mM {beta}-glycerophosphate, 10% glycerol, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) at the various time points by scraping. The same amount of total protein from clarified lysate was incubated with GST-PBD to precipitate GTP-bound Rac1 according to the manufacturer’s instructions (Upstate Biotechnology). Precipitated GTP-bound Rac1 was resolved on a 4–20% SDS-PAGE and immunoblotted using a Rac1 mAb (Upstate Biotechnology). Six percent of the cell lysate was also resolved in a 4–20% SDS-PAGE and immunoblotted to measure the total amount of Rac1. The nonspecific ~31-kDa band seen in the Western blot was reported by Upstate Biotechnology to be an uncharacterized protein cross-reacting with the Ab.

Statistics

In transfection experiments data shown are the mean ± SD of three or more experiments and are reported as a percentage of LPS-stimulated HIV-LTR promoter activity. Student’s t test was used to assess statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS stimulation of HMEC activates Rac1

Rac1 has been known to play a role in microbial Ag-induced innate immune responses. In RAW cells and umbilical cord endothelial cells, Rac1 mediates Gram-negative bacterial LPS (TLR4 ligand)-induced TNF-{alpha} (27) and IL-8 production (28). Recently, Rac1 has also been shown to mediate Gram-positive Staphylococcus aureus (TLR2 ligand) activation of NF-{kappa}B in THP-1 human monocytic cell lines and TLR2-transfected 293 cells (14). We have previously shown that in the HMEC system, stimulation of TLR4 with LPS leads to HIV-LTR trans-activation and viral replication (11). To assess whether LPS stimulation of HMEC activates Rac1, confluent cells were stimulated with LPS (50 ng/ml) for different periods of time (0, 5, 15, and 30 min), and Rac1 activation was assessed by immunoprecipitating GTP-bound Rac1 using GST-PBD and Western blotting for Rac1. LPS-induced a rapid and transient Rac1 activation, starting at 5 min after stimulation and peaking at 15 min (Fig. 1). As Rac1 plays a key role in cell movement and phagocytosis, the transient nature of LPS-induced Rac1 activation may be an innate mechanism of the cell to direct its movement to the target pathogen. These results confirm that LPS stimulation of HMEC leads to Rac1 activation.



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  		FIGURE 1. LPS stimulation of HMEC activates Rac1. HMEC were stimulated with 50 ng/ml LPS for various periods of time. Activated Rac1 was immunoprecipitated with GST-PBD beads, and Western blotting was performed using anti-Rac1-antibody according to the manufacturer’s directions. GTP-{gamma} sulfate is provided as the positive control in the kit. The data shown are representative of at least three independent experiments.

 
Rac1 modulates LPS-induced HIV-LTR trans-activation

Next, we assessed the role of Rac1 in LPS-induced HIV-LTR trans-activation by cotransfecting HMEC with HIV-LTR-luciferase and {beta}-galactosidase reporter vectors and either with dominant negative Rac1 (RacN17) or empty vector constructs at various concentrations before LPS stimulation. Cells were lysed after 5 h of stimulation, and luciferase activity was measured. The results were normalized for transfection efficiency using a colorimetric galactosidase assay as described previously (18). We observed that expression of Rac1N17 inhibited LPS-induced HIV-LTR trans-activation in a dose-dependent manner; and the reduction at highest concentration of Rac1N17 (1 µg) was ~50% (Fig. 2). At this concentration, Rac1N17 construct did not induce cell death, as assessed by lactate dehydrogenase release (data not shown). These data suggest that Rac1 is involved in LPS-induced HIV-LTR trans-activation.



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  		FIGURE 2. DN-Rac1 (Rac1N17) blocks LPS-induced HIV-LTR trans-activation in a dose-dependent manner. HMEC were transiently cotransfected with {beta}-galactosidase (0.1 µg), HIV-LTR-luciferase (0.1 µg) constructs, and either DN-Rac1 (Rac1N17) cDNA or empty vector pcDNA3 as described in Materials and Methods. Cells were then stimulated with LPS for 5 h, and HIV-LTR activation was determined by luciferase assay using a luminometer. A {beta}-galactosidase colorimetric assay was performed to normalize for transfection efficiency. LPS-induced luciferase activity was considered 100%, and the results are expressed as the percent luciferase activity relative to the LPS-induced luciferase activity. The results shown are from three or more independent experiments and are reported as the mean ± SD.

 
Constitutively active Rac1 induces HIV-LTR trans-activation

To further define the involvement of Rac1 in LPS-induced HIV-LTR trans-activation, we assessed whether the expression of a constitutively active form of Rac1 (Rac1V12) could mimic the effect of LPS treatment and lead to enhanced HIV-LTR trans-activation. As shown in Fig. 3, infection of HMEC with adenovirus vector expressing Rac1V12 for 20 h led to a dose-dependent increase in HIV-LTR luciferase activity compared with adeno-GFP control. Rac1V12 at MOI 200 and 400 led to 50 and 100% increases in HIV-LTR activation, respectively, above that induced by LPS (50 ng/ml for 5 h). Importantly, Rac1V12-induced HIV-LTR trans-activation was achieved in the absence of LPS stimulation, and infection of HMEC with adeno-GFP did not increase luciferase activity (data not shown). These observations together with those observed by the DN-Rac1 experiments suggest that Rac1 participates in the signaling pathways involved in the induction of HIV-LTR trans-activation.



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  		FIGURE 3. Infection of HMEC with adenovirus vector expressing constitutively active Rac1 (Rac1V12) induces HIV-LTR trans-activation in a dose-dependent manner. HMEC were infected with adenovirus vector containing constitutively active Rac1 (Rac1V12) construct as described in Materials and Methods and then cotransfected with HIV-LTR luciferase (0.1 µg) and {beta}-galactosidase (0.1 µg) cDNA using FuGene6 overnight. Cells infected with adeno-GFP were included as a control (data not shown). HIV-LTR activation was determined by luciferase assay using a luminometer. A {beta}-galactosidase assay was performed to normalize for transfection efficiency. In parallel, the cells were stimulated with LPS (50 ng/ml) for 5 h, and LPS-induced luciferase activity was measured using a luminometer. LPS-induced HIV-LTR trans-activation in the absence of Rac1V12 expression was considered 100%, and the results are expressed as the percent luciferase activity relative to the LPS-induced luciferase activity. The data shown are the mean ± SD of three or more independent experiments.

 
TIRAP, but not MyD88, mediates Rac1 activation of HIV-LTR

LPS stimulation of TLR4 has been known to induce both MyD88-dependent and MyD88-independent signaling cascades to activate NF-{kappa}B (29), and TIRAP has recently been suggested to control activation of MyD88-independent signaling pathways downstream of TLR4 (16). However, this was recently disputed by Yamamoto et al. (15), who suggested that TIRAP had a crucial role in the MyD88-dependent signaling pathway shared by TLR2 and TLR4. Rac1 has recently been shown to associate with the IL-1R complex via interactions with both MyD88 and the IL-1R accessory protein, and a pathway emanating from MyD88 and involving IRAK1, TRAF-6, and Rac1 was shown to be involved in the trans-activation of gene expression by the p65 subunit of NF-{kappa}B in response to IL-1 (30). To examine the roles of MyD88 and TIRAP in Rac1-induced HIV-LTR trans-activation we cotransfected HMEC with HIV-LTR-luciferase, Rac1V12, and {beta}-galactosidase cDNA and empty vector pcDNA3, DN-TIRAP, or DN-MyD88 constructs at various concentrations using FuGene 6 overnight. The cells were then stimulated with LPS (50 ng/ml) or medium for 5 h, and HIV-LTR activation was assessed by luciferase assay. As expected, both DN-TIRAP (Fig. 4A) and DN-MyD88 (Fig. 4B) blocked LPS-induced HIV-LTR trans-activation; however, only the expression of DN-TIRAP blocked the constitutively active Rac1 (Rac1V12)-induced HIV-LTR activation (Fig. 4). Similar to the findings of Jefferies et al. (30), we observed that DN-Myd88 did not block Rac1V12-induced HIV-LTR, which suggests that Rac1 may be downstream of MyD88. We propose that both MyD88 and TIRAP play roles in HIV-LTR trans-activation, and Rac1-induced HIV-LTR trans-activation is TIRAP dependent.



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  		FIGURE 4. A, LPS-induced HIV-LTR trans-activation is blocked by DN-TIRAP, and TIRAP is involved in Rac1-activation of HIV-LTR. HMEC transiently expressing, Rac1V12 cDNA, HIV-LTR-luciferase, and {beta}-galactosidase cDNA were cotransfected with either DN-TIRAP or empty vector using FuGene6, and HIV-LTR activation was determined by luciferase assay using a luminometer. {beta}-Galactosidase colorimetric assay was performed to normalize for transfection efficiency. LPS-induced luciferase activity was expressed as 100%, and Rac1V12-induced luciferase activity was expressed as a percentage of LPS-induced luciferase activity. Cell death was assessed under the microscope. The data shown are the mean ± SD of three or more independent experiments. *, p < 0.05. B, Rac1 activation of HIV-LTR is MyD88 independent. HMEC transiently expressing, Rac1V12, HIV-LTR-luciferase, and {beta}-galactosidase cDNA were cotransfected with either DN-MyD88 cDNA (0.5 µg) or empty vector, and HIV-LTR activation was determined by luciferase assay using a luminometer. A {beta}-galactosidase colorimetric assay was performed to normalize for transfection efficiency. Cell death was assessed under the microscope. LPS-induced luciferase activity was expressed as 100%, and Rac1V12-induced luciferase activity was expressed as a percentage of LPS-induced luciferase activity. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HIV infection is characterized by progressive immune dysfunction, leading to AIDS and opportunistic infections by a wide variety of microorganisms, which is an independent risk factor for death in the HIV-infected population (31). The innate immune activation-driven HIV replication may in part explain the aggressive course of HIV infection in individuals coinfected with opportunistic organisms, such as those in sub-Saharan Africa, where coinfections are common and often continuous (32, 33). Currently the molecular mechanisms involved in enhanced HIV replication during opportunistic infections are not well understood. Therefore, delineating the molecular mechanisms that regulate activation of latent HIV is of great clinical significance to develop novel modalities to control HIV replication during opportunistic infections.

We have shown that LPS interaction with TLR4 induces a signaling cascade, including MyD88, IRAK1, and TRAF6, which leads to NF-{kappa}B activation and HIV-LTR trans-activation (11), and that costimulation of TLR4 with either TLR2 or TLR9 induces HIV replication in an additive manner (34). In this study we show that in addition to the IL-1R signaling cascade, Rac1 and TIRAP also play roles in HIV-LTR trans-activation.

The Rho family of small GTPase, Rac1, is known to regulate critical cellular functions, such as cell growth, apoptosis, cytoskeleton organization, and development (35, 36). Rac1 is also implicated in different aspects of host defense against bacteria, including leukocyte chemotaxis, pathogen phagocytosis, production of oxygen radicals, and activation of multiple stress response cascades (35, 36, 37, 38, 39, 40, 41). Currently, the information on the role of Rac1 in HIV replication is limited to the potentiation effect of Rac1 on HIV negative factor-induced HIV replication and HIV disease progression in infected cells (42). In addition, CD28-dependent activation of HIV-1 transcription has recently been shown to require GTPase activity of Rac1 in T lymphocytes (43).

Rac1 has recently been shown to be a part of the IL-1R complex and TLR2 (14) and associates with MyD88, IRAK1, and TRAF6 to mediate p65 and NF-{kappa}B trans-activation (30). Currently there are no data on TLR4 activation of Rac1 and its role in HIV replication. Our data suggest that LPS stimulation of TLR4 induces Rac1, which may, in turn, lead to HIV-LTR trans-activation.

TLR signaling is initiated by the TIR domain, which recruits cellular adapter proteins that contain TIR domains. Four such adapter proteins have been discovered to date, and have been named MyD88, Mal (MyD88 adapter-like; also known as TIRAP (TIR domain-containing adapter protein)), TIR domain-containing adapter inducing IFN-{beta} (44), and TIR domain-containing adapter inducing IFN-{beta}-related adaptor molecule (45). The adapter proteins are suggested to confer specificity to TLR signaling; however, the molecules involved in TIRAP signaling have yet to be identified. Rac1 has been shown to associate with Myd88 (30). Our data suggest that Rac1 is also involved in TIRAP signaling; however, immunoprecipitation experiments did not show association of Rac1 with TIRAP (data not shown). This may also be due to unstable nature of Rac1-TIRAP association upon activation of cells. Another possibility is that TIRAP acts downstream of Rac1 to induce HIV-LTR trans-activation.

Our results emphasize the role of Rho GTPase Rac1 and TIRAP in HIV-LTR trans-activation. These results suggest that novel therapeutic agents targeting Rac1 or Rac1-activated signaling molecules may potentially be developed to control HIV-1 replication.


   Footnotes
 
1 This work was supported by National Institutes of Health Child Health Research Center Grant P30HD34610 (to O.E.), Howard Hughes Prime Award, and National Institutes of Health Grant KO8AI51216 (to O.E.). Back

2 Address correspondence and reprint requests to Dr. Ozlem Equils, Department of Pediatrics, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 4220, Los Angeles, CA 90048. E-mail address: ozlem.equils{at}cshs.org Back

3 Current address: University of California, 600 16th Street, San Francisco, CA 94107. Back

4 Abbreviations used in this paper: LTR, long terminal repeat; DN, dominant negative; EC, endothelial cells; GFP, green fluorescent protein; HMEC, human dermal microvessel endothelial cells; IRAK, IL-1R-activated kinase; MOI, multiplicity of infection; MyD88, myeloid differentiation protein; PAK-1, p21-activated kinase 1; PBD, p21-binding domain of human PAK-1; TIR, Toll-IL-1R; TIRAP, TIR domain-containing adapter protein; TLR, Toll-like receptor; TRAF, TNF receptor-associated factor. Back

Received for publication November 5, 2003. Accepted for publication April 14, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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