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Originally published online as doi:10.1189/jlb.0805429 on December 5, 2005

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(Journal of Leukocyte Biology. 2006;79:388-396.)
© 2006 by Society for Leukocyte Biology

Cyclin T1 but not cyclin T2a is induced by a post-transcriptional mechanism in PAMP-activated monocyte-derived macrophages

Li-Ying Liou, Richard E. Haaland, Christine H. Herrmann and Andrew P. Rice1

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas

1 Correspondence: Baylor College of Medicine, Department of Molecular Virology and Microbiology, One Baylor Plaza, Houston, TX 77030. E-mail: arice{at}bcm.tmc.edu


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ABSTRACT
 
Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin-dependent kinase 9 as the catalytic subunit and different cyclin subunits—cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P-TEFb subunit is highly regulated in monocyte-derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome-mediated proteolysis. Cyclin T1 can be reinduced by pathogen-associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P-TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P-TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome-mediated proteolysis. Significantly, the cyclin T2a subunit of P-TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate-limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome-mediated proteolysis.

Key Words: P-TEFb • Cdk9 • HIV Tat • transcription • proteasome


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INTRODUCTION
 
Monocytes originate from myeloid progenitor cells in the bone marrow and then circulate in peripheral blood and begin to differentiate to macrophages upon penetrating through blood vessels and migrating to peripheral tissues [1 , 2 ]. Macrophages play important roles in innate and adaptive immunity with their phagocytic activity and antigen-presenting ability, respectively. As part of the innate immune response, macrophages are one of the first lines of defense against pathogens. Upon sensing a pathogen, macrophages become activated and secrete cytokines and chemokines which can interfere with replication of the pathogen and stimulate an adaptive immune response. Toll-like receptors (TLRs) on the surface of macrophages play a critical role in sensing and responding to pathogens (reviewed in ref. [3 ]). TLRs recognize conserved features of pathogens, pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) and peptidoglycan (PGN). Upon recognition of a PAMP, TLRs transduce signals to macrophages, which lead to a broad cellular response, including the synthesis of cytokines, chemokines, and nitric oxide. Cytokines secreted by activated macrophages serve as autocrine or paracrine factors to further expand the state of macrophage activation and to instruct effector cells of the immune system.

Despite the ability to counter infection, macrophages are targets of many infectious agents, such as human immunodeficiency virus (HIV), dengue virus, Salmonella, Leishmania major, and Mycobacterium tuberculosis [4 5 6 7 ]. In the case of HIV, although infected macrophages contribute only a small amount to the total viral load in vivo, they play a critical role in viral pathogenesis. Infected macrophages secrete chemokines which recruit and promote the infection of CD4+ T lymphocytes, thereby leading to high levels of virus production [8 ]. HIV infection of CD4+ T cells and macrophages is dependent on the activation and maturation state of the cells [9 10 11 ]. Activated CD4+ T cells are more susceptible to infection than resting T cells, and macrophages are more susceptible to infection than monocytes. In monocytes, restrictions to HIV-1 replication are likely to be a result of limiting levels of cellular factors needed at various stages of the replication cycle. Entry of HIV-1 into monocytes is limited by low levels of CC chemokine receptor 5 (CCR5), the major coreceptor used by HIV-1 to enter macrophages. Upon macrophage differentiation, CCR5 is up-regulated, and this results in efficient viral entry [12 ]. Our recent results have indicated that the expression level of cyclin T1, the cellular cofactor for the viral Tat protein, is also limiting in monocytes [13 , 14 ].

Replication of the HIV-1 provirus requires the viral Tat protein, which activates RNA polymerase II transcriptional elongation of the integrated provirus by binding to a cellular protein kinase which is composed of cyclin-dependent kinase 9 (Cdk9) and cyclin T1 (reviewed in refs. [15 16 17 18 ]). Cdk9 and cyclin T1 are components of a general RNA polymerase II elongation activator, positive transcription elongation factor b (P-TEFb). Multiple P-TEFb complexes exist in mammalian cells, which contain Cdk9 associated with different cyclin regulatory subunits, cyclin T1, T2a, T2b, or K [19 ]. Cyclin T2a and T2b are isoforms of the same gene and are generated by differential splicing. P-TEFb complexes activate transcriptional elongation through the hyperphosphorylation of the carboxyl terminal domain of RNA polymerase II, as well as the phosphorylation of negative factors which act to limit polymerase elongation [20 21 22 ]. Tat makes direct protein–protein contact with cyclin T1 and can therefore only interact with the cyclin T1-containing P-TEFb complex [23 , 24 ]. The Tat/Cdk9/cyclin T1 complex is targeted to the RNA polymerase II complex by binding to the transactivation-responsive RNA (TAR) element which forms at the 5' end of nascent viral transcripts.

A significant portion of P-TEFb complexes associates with a small nuclear RNA (snRNA), known as 7SK, and the HEXIM1 or HEXIM2 proteins [25 26 27 28 ]. P-TEFb kinase activity in vitro is reduced when it is associated with 7SK snRNA and HEXIM proteins, and it has been proposed that 7SK snRNA and HEXIM proteins function to negatively regulate P-TEFb function. However, although siRNA depletion of 7SK in HeLa cells can activate reporter plasmids, the depletion has no effect on expression from the HIV-1 long-terminal repeat following infection or the c-myc and myeloid cell leukemia-1 genes, three transcription units known to be highly dependent on P-TEFb function [29 ]. The siRNA depletion of 7SK does induce apoptosis after relatively extended culture times, indicating that 7SK has an essential function, but the relation of this function to P-TEFb and transcriptional elongation remains to be determined.

In circulating monocytes isolated from healthy blood donors, Cdk9 is typically expressed at high levels, and cyclin T1 is expressed at a low level [14 ]. Upon differentiation to macrophages in vitro, cyclin T1 protein expression is up-regulated within the first few days of culture by a post-transcriptional mechanism. We previously made the unexpected observation that cyclin T1 expression is shut off after 1–2 weeks in culture by a mechanism that involves proteasome-mediated proteolysis, which may be mediated by the proline, glutamic acid, serine, and threonine (PEST) sequence at its carboxyl-terminus [30 ]. Cyclin T1 expression can be re-induced by PAMPs after it is shut off in late, differentiated macrophages, and HIV-1 infection can also re-induce cyclin T1 in late-differentiated macrophages. The induction of cyclin T1 by PAMPs or HIV-1 infection indicates that up-regulation of cyclin T1 is a component of the innate immune response.

The expression pattern of cyclin T2a, T2b, and K during macrophage differentiation and activation is a significant issue, as these additional regulatory subunits of P-TEFb might be capable of compensating for loss of cyclin T1 expression. In our previous studies, we were unable to examine the expression pattern of these cyclins as a result of the lack of adequate antisera. In this study, we developed conditions to measure cyclin T2a in immunoblots and observed that expression of this P-TEFb subunit remains constant during differentiation and is not induced by PAMPs. We have also observed that the 7SK snRNA and the HEXIM1 protein are rapidly up-regulated as monocytes differentiate to macrophages in vitro. Late in differentiation, HEXIM1 protein expression is shut off, but 7SK snRNA levels remain high. We also investigated mechanisms involved in PAMP re-induction of cyclin T1. Our data indicate that PAMPs induce cyclin T1 with relatively slow kinetics through a post-transcriptional mechanism which appears to involve the inhibition of proteasome-mediated proteolysis. Our results indicate that cyclin T1 expression is highly regulated in macrophages, and its shut-off appears to be compensated by other P-TEFb cyclin subunits, and this is likely to have significance for gene expression in innate immune responses.


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MATERIALS AND METHODS
 
Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy HIV-seronegative blood donors (obtained from the Gulf Coast Regional Blood Center, Houston, TX) by centrifugation in Isolymph (Gallard/Schlesinger, Plainview, NY) density gradient as described previously [14 ]. Monocytes were isolated from PBMCs by adherence of 2.5 x 106 PBMCs/ml to plastic cell culture dishes (Sarstedt, Germany) at 37°C for 1 h in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 1% human serum (Sigma Chemical Co., St. Louis, MO), penicillin, and streptomycin (Invitrogen Life Technologies). Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Adherent cells were then incubated with RPMI 1640 containing 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) for another 2 h. After additional washes with prewarmed PBS, cells were cultured in RPMI 1640 containing 10% FBS and antibiotics described above, plus granulocyte macrophage-colony stimulating factor (10 U/ml, R&D Systems, Minneapolis, MN). A detailed protocol is at http://www.bcm.edu/molvir/RiceHerrmann.

Monocyte-derived macrophage (MDM) treatment
MDMs were treated for the times indicated in text with PAMPs at the following final concentrations: LPS (from Salmonella typhosa, 100 ng/ml, Sigma Chemical Co.), lipoteichoic acid (LTA; from Staphylococcus aureus, 10 µg/ml, Sigma Chemical Co.), PGN (from S. aureus, 10 µg/ml, Sigma Chemical Co.), double-stranded RNA poly (I:C; 50 µg/ml, Sigma Chemical Co.), and cytosine-phosphate-guanine DNA (2 µM, 5'-phosphorothioate-TCG TCG TTT TGT CGT TTT GTC GTT, Invitrogen Life Technologies). For MDMs incubated with LPS (100 ng/ml) for 1 or 4 h, LPS was washed out with prewarmed PBS twice, and cells were cultured in RPMI 1640 containing 10% FBS and antibiotics at 37°C. For some experiments, culture supernatants were collected as conditioned media and applied to MDMs derived from another donor as indicated in the text. For tumor necrosis factor {alpha} (TNF-{alpha}) assays, culture supernatants were collected at the indicated times, and TNF-{alpha} levels were measured by enzyme-linked immunosorbent assay (ELISA; BioSource, Camarillo, CA) following the manufacturer’s protocol. Proteasome inhibitor, MG101 (LLnL), was purchased from Sigma Chemical Co. and was used at a final concentration of 50 µM for 30 min.

HIV infection
Pseudotyped HIV-1 was generated and concentrated as described previously [30 ]. Briefly, 293T cells were transfected with a plasmid expressing vesicular stomatitis virus glycoprotein (VSV-G), a plasmid expressing HIV-1 Tat, and a proviral plasmid of HIV-green fluorescent protein (GFP; nef, vpr, vpu, vif, env-deleted) or HIV-1 BRU3 strain (env-deleted) by calcium phosphate coprecipitation. Viral stock was prepared by centrifugation at 23,000 rpm for 2.5 h in an SW28 rotor from culture supernatants collected 3 days post-transfection [31 ]. The viral pellet was resuspended with PBS, and the viral titer for HIV-Ires-GFP was determined to be ~108 infection units/ml after concentration, as estimated with infections in 293T cells.

Infection of MDMs was performed with 0.1 ml concentrated virus in the presence of 2.5 ml fresh medium and 6 µg/ml polybrene for 8 h. The estimated multiplicity of infection was ~15, inferred from the titers of HIV-Ires-GFP stock. Infected cells were determined for intracellular p24, measured by flow cytometry analysis using phycoerythrin-conjugated monoclonal antibody (KC57-RD1, Beckman Coulter, Fullerton, CA).

Immunoblots
Cell extracts were prepared as described previously [32 ]. Briefly, cells were incubated in lysis buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 5 mM dithiothreitol] containing protease inhibitors (2 µg/ml aprotinin, 1 µg/ml leupeptin, and 2.5 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined by a Bio-Rad protein assay (Bio-Rad, Hercules, CA), and 20 µg total protein was loaded onto sodium dodecyl sulfate-9% polyacrylamide gels. Immunoblots were performed using enhanced chemiluminescence (Pierce, Rockford, IL) for detection, according to the manufacturer’s protocol. Rabbit anti-Cdk9 (Santa Cruz Biotechnology, CA), anti-HEXIM-1 (a generous gift from Olivier Bensaude, Laboratoire de Régulation de l’Expression Génétique, Paris, France), and anti-actin (Sigma Chemical Co.) antibodies were used at a dilution of 1:5000; goat anti-cyclin T1 antibody (Santa Cruz Biotechnology) and goat-anti cyclin T2a (Santa Cruz Biotechnology) were used at a dilution of 1:1000. Horseradish peroxidase-conjugated goat anti-rabbit and donkey anti-goat immunoglobulin G (Santa Cruz Biotechnology) were used at a dilution of 1:5000.

Northern blots
To detect 7SK snRNA, cells were washed twice with PBS and lysed in lysis buffer as described above containing protease inhibitor cocktail (Sigma Chemical Co.) and RNase inhibitor (Invitrogen Life Technologies). Cell extracts were split into separate tubes for immunoblots or Northern blots. RNA was isolated from cell extracts using TRIzol reagent (Invitrogen Life Technologies). RNA was separated on 10% urea-polyacrylamide gels, and Northern blot hybridization was performed as described previously using the ULTRAHyb Northern blot kit (Ambion, Austin, TX) and a 5' end-labeled oligonucleotide probe for 7SK snRNA (5'-CCTCCTCTATCGGGGATGGTCGTCC-3') [33 ].

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) as described previously [14 ]. Briefly, MDMs at the indicated times were harvested, and RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instruction. RT was performed at 37°C for 1 h in a total volume of 50 µl containing 1 µg RNA, random hexanucleotide primer (Invitrogen Life Technologies), 5 mM deoxy-unspecified nucleoside 5'-triphosphates, and Omniscript RT (Qiagen, Valencia, CA). Real-time PCR was carried out simultaneously for cyclin T1, Cdk9, and ß-actin in 96-well optical reaction plates in triplicate. All primers were designed using Beacon Designer 2.0 (Premier Biosoft, Palo Alto, CA), and the amplification products are examined by gel electrophoresis or melt-curve analysis using the MyIQ system to verify specificity of products. Each PCR reaction contained 1x SYBR Green Supermix (Bio-Rad), 2 µl cDNA, and 400 nM final concentration of each primer in a 25-µl reaction. PCR reaction was performed using a Bio-Rad iCycler with a 3-min hot-start, followed by 40 cycles of 15 s at 95°C, 1 min annealing, and amplification at 55°C. The threshold cycle value was determined using the MyIQ software program (Bio-Rad). The RNA levels of cyclin T1 or TNF-{alpha} were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA, and the fold change was calculated by comparing the normalized RNA levels in PAMP-treated MDMs with mock-treated ones.


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RESULTS
 
HEXIM1 and 7SK but not cyclin T2a are induced following macrophage differentiation
Previously, we observed a large increase in the association of 7SK with P-TEFb when quiescent peripheral blood lymphocytes (PBLs) are activated in vitro, a condition in which transcription is globally up-regulated [29 , 33 ]. To further investigate whether 7SK and HEXIM1 might regulate P-TEFb regulation during macrophage differentiation, we examined the expression patterns of 7SK and the HEXIM1 protein. Although the HEXIM2 protein also associates with P-TEFb, its expression level is generally much lower than HEXIM1 (refs. [28 , 34 ] and our unpublished data). Monocytes were isolated from a healthy blood donor and cultured for up to 19 days under conditions that allow for differentiation into macrophages. Cell extracts were prepared, and protein levels of cyclin T1, Cdk9, and HEXIM1 were determined by immunoblot analysis, in which ß-actin was used as an internal control. Total RNA was also prepared and examined for 7SK levels by Northern blot hybridization. Cyclin T1 was expressed at a low level in monocytes isolated from this donor, and cyclin T1 and Cdk9 proteins were up-regulated at Day 4, consistent with previous results [14 ]. 7SK and HEXIM1 were also present at low levels in Day 0 monocytes and were induced strongly at Day 4 (Fig. 1A ). We observed similar inductions of 7SK and HEXIM1 following differentiation of monocytes from multiple blood donors (data not shown). The induction of 7SK and HEXIM1 expression is typical for many genes, as freshly isolated monocytes are quiescent cells that carry out only low levels of transcription and other metabolic processes, and upon differentiation, many aspects of gene expression are activated, including transcription of hundreds of genes [35 ]. The increases in gene expression during monocyte differentiation, and the associated increase in expression of 7SK and HEXIM1 are similar to their induction when quiescent PBLs are activated [29 , 33 ].


Figure 1
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  		Figure 1. Expression of HEXIM1, cyclin T1, cyclin T2a, and 7SK snRNA in MDMs. (A) Human monocytes were isolated and cultured for the indicated days under conditions that allow differentiation to macrophages. Cell extracts were prepared at the indicated days, and the expression levels of HEXIM1, cyclin T1, cyclin T2a, and Cdk9 were examined by immunoblots. Cellular RNA was also isolated, and the levels of 7SK snRNA were determined by a Northern blot. (B) MDM cultures were treated with LPS at Day 20 and incubated for 48 h before preparation of cell extracts at 22; MG101 was added for 2 h prior to preparation of cell extract at Day 22. Protein expression was determined by immunoblots. (C) MDM cultures were allowed to differentiate until Day 15 and infected with HIV-GFP or HIV-BRU3. Cell extracts were prepared 3 days later (Day 18), and protein expression was determined by immunoblots. (D) Extracts were prepared from MDM cultures, which were incubated for 4, 11, or 14 days (D) as indicated, and cultures were treated with LPS or LTA for 48 h or MG101 for 2 h prior to preparation of extracts. Protein expression was determined by immunoblots.

Although cyclin T1 is induced early during macrophage differentiation, we have repeatedly observed that cyclin T1 expression is shut off at later stages of macrophage differentiation, typically after macrophages have been maintained for 7–10 days in culture under these conditions [14 ]. In addition, we have observed that Cdk9 expression usually declines several days subsequent to the shut-off of cyclin T1 [36 ], and cyclin T1 and Cdk9 can be re-induced following shut-off by treatment with LPS or other PAMPs, as well as with proteasome inhibitors [30 , 36 ]. We therefore examined regulation of HEXIM1 and 7SK in late-differentiated macrophages, which were cultured up 19 days (Fig. 1A , right). HEXIM1 expression was found to be shut off subsequent to that of cyclin T1, but its decline was more rapid than that of Cdk9. Unlike cyclin T1, Cdk9, and HEXIM1 proteins, the level of 7SK remained constant throughout the time course of differentiation (Fig. 1A) .

To determine if LPS or proteasome inhibitors could re-induce HEXIM1 levels after shut-off, macrophages were treated on Day 20 with LPS for 48 h or on Day 22 with the proteasome inhibitor MG101 for 2 h, and the levels of HEXIM1, cyclin T1, Cdk9, and 7SK were examined (Fig. 1B) . LPS and MG101 treatment restored cyclin T1, Cdk9, and HEXIM1 protein expression to high levels but had no effect on 7SK. These results indicate that HEXIM1 is regulated similarly to cyclin T1 and Cdk9 late in macrophage differentiation.

Previously, we reported that HIV-1 infection re-induces cyclin T1 expression in late-differentiated macrophages [30 ]. To determine whether HEXIM1 can be induced by HIV-1 infection of macrophages, MDMs were infected with HIV-1 pseudotyped with VSV-G at Day 15 of differentiation and were analyzed for protein expression at Day 18 in an immunoblot (Fig. 1C) . In the experiment shown in Figure 1C , expression of cyclin T1 and HEXIM1 was shut off in Day 18 macrophages, and Cdk9 was not. This is consistent with the kinetics of shut-off shown in Figure 1A , in which shut-off of cyclin T1 and HEXIM1 precedes that of Cdk9. Pseudotyped HIV-1 infection led to the induction of cyclin T1 but not Cdk9. It is interesting that HEXIM1 was also induced by the infection of pesudotyped HIV-1, suggesting that HEXIM1 is regulated similarly to cyclin T1.

Because of the importance of P-TEFb to general RNA polymerase II elongation, it was unexpected to observe that expression of cyclin T1 but not Cdk9 was extinguished late in differentiation [14 ]. It is possible that the other cyclin subunits of Cdk9 might compensate for the loss of cyclin T1 and thereby maintain P-TEFb function. In our previous studies, we were unable to measure expression levels of these alternative cyclins (T2a, T2b, and K) as a result of lack of adequate antisera and conditions for immunoblots. However, we have developed conditions to examine cyclin T2a in immunoblots. Extracts were prepared from MDM cultures from two donors, which were incubated for 4, 11, and 14 days, with and without LPS or LTA or a proteasome inhibitor (Fig. 1D) . The immunoblot analyses indicate that in contrast to cyclin T1, cyclin T2a expression remains relatively stable during MDM differentiation. LPS or LTA activation and a proteasome inhibitor also had no effect on cyclin T2a expression. These results suggest that the stable expression of cyclin T2a may maintain the level of P-TEFb function that is likely to be essential late in differentiation.

Cyclin T1 induction is a late response in PAMP-activated MDMs
We were interested in determining whether cyclin T1 induction was an early or late response in PAMP-activated MDMs. MDMs were therefore treated with LPS, LTA, PGN, and poly (I:C) for times ranging from 0.5 to 48 h. Protein levels of cyclin T1, Cdk9, and ß-actin were analyzed in immunoblots (Fig. 2A ). Cdk9 expression was not yet shut off in the MDM cultures shown in Figure 2A , and it remained a fairly constant level after PAMPs stimulation. In contrast, cyclin T1 expression was shut off in the MDM cultures, and its induction was not detectable at 0.5 or 4 h after LPS treatment (Fig. 2A , lanes 2 and 3). However, cyclin T1 expression was induced by 20 h of treatment and remained a high level at 48 h of treatment (lanes 4 and 5). Other PAMPs used to activate MDMs [LTA, PGN, and poly (I:C)] showed a similar kinetics to LPS of cyclin T1 induction (Fig. 2A , lanes 6–14). In additional independent experiments, LPS, LTA, PGN, and poly (I:C) did not induce cyclin T1 at 0.5 or 4 h of stimulation but did so at 20–48 h (data not shown). The relatively slow induction of cyclin T1 by PAMPs suggests that its up-regulation may not be linked directly to the TLR4 signaling pathway but may be a secondary response to PAMP activation. To verify that MDMs were capable of an early response to PAMPs, the levels of secreted TNF-{alpha} in culture supernatants were measured by ELISA (Fig. 2B) . TNF-{alpha} induction was detected 2 h after PAMPs treatment, and TNF-{alpha} levels continued to increase up to 4 h after PAMPs treatment. This kinetics of TNF-{alpha} induction shown in Figure 2B is consistent with previous studies of LPS-stimulated MDMs [6 , 37 ].


Figure 2
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  		Figure 2. Induction of cyclin T1 is not an early response to PAMPs activation. (A) MDM cultures were treated with LPS, LTA, PGN, and poly (I:C) for times ranging from 0.5 to 48 h as indicated. Cell extracts were prepared, and expression levels of indicated proteins were determined in immunoblots. (B) Cultured supernatants from A were collected, and TNF-{alpha} was assayed by ELISA. Similar results were observed for three independent donors.

Cyclin T1 is induced by intracellular factors activated by PAMPs
The duration of LPS stimulation of macrophages can influence the cellular response [35 , 38 ]. Therefore, we examined whether relatively long incubations with LPS were an optimal condition to induce cyclin T1 expression. Duplicate cultures of late-differentiated MDMs were treated with LPS for 1 or 4 h, washed to remove LPS, and then incubated with LPS-free medium to 24 or 48 h. Cell extracts prepared to measure cyclin T1 and Cdk9 expression levels in immunoblots. Cyclin T1 was induced at 24 h in the MDMs exposed to LPS for 1 h or 4 h, and exposure for 4 h showed a significantly stronger induction (Fig. 3A , lanes 2 and 3 and 6 and 7). In addition, MDM cultures treated continuously with LPS for 20 or 24 h showed an induction of cyclin T1, which was similar to the level induced by a 1-h treatment but less than a 4-h treatment (lanes 1–3, 6 and 7, and 11–14). These results suggest that prolonged activation by LPS may stimulate a negative-feedback mechanism that reduces the magnification of cyclin T1 induction.


Figure 3
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  		Figure 3. Conditioned medium from LPS-activated MDMs does not induce cyclin T1 in late-differentiated MDMs. (A) MDM cultures were treated with LPS for the times indicated in the top row. After treatment, cells were washed with PBS to remove LPS and incubated with LPS-free medium for 24 or 48 h, upon which time extracts were prepared (times indicated in the second row). Protein expression levels are determined by immunoblots. (B) Conditioned media were collected at 24 h after treatment with 1 or 4 h to LPS. Separate cultures of late-differentiated MDMs were treated with conditioned media for 24 h, and protein expression levels were examined by immunolots.

The slow induction of cyclin T1 by PAMPs raises the possibility that a factor secreted from activated cells may act in a paracrine manner to up-regulate cyclin T1. To investigate this issue, we examined conditioned medium from LPS-activated MDMs for an activity that could induce cyclin T1 in late-differentiated MDM cultures. Conditioned media from MDM cultures treated with LPS for 1 or 4 h were used to treat a separate late-differentiated culture in which cyclin T1 but not Cdk9 has been shut off (Fig. 3B) . No significant induction of cyclin T1 was observed by conditioned media, suggesting an intracellular factor rather than a secreted molecule is involved in the induction of cyclin T1.

Induction of cyclin T1 by PAMPs is involved in a post-transcriptional regulation
To investigate mechanisms involved in the induction of cyclin T1, we used real-time RT-PCR assays to examine whether cyclin T1 mRNA is up-regulated in PAMP-stimulated MDMs. TNF-{alpha} mRNA was used as a positive control in these assays, as TNF-{alpha} mRNA is known to be up-regulated following PAMP activation [39 40 41 42 ]. In a pilot experiment, TNF-{alpha} RNA was found to increase by 4 h after LPS treatment (data not shown), in agreement with previous studies [6 , 43 ]. Therefore, RNA was isolated from MDMs after 4 h of treatment for assays for TNF-{alpha} RNA, and RNA was isolated 20 h after treatment for assays for cyclin T1 RNA, as cyclin T1 protein induction is high at this time (see Fig. 2A ). TNF-{alpha} and cyclin T1 RNAs were normalized to GAPDH, whose level is unchanged by PAMP activation. TNF-{alpha} RNA levels increased between ten- and 40-fold following PAMPs activation (Fig. 4A ). In contrast, cyclin T1 RNA levels remained constant after 20 h of incubation with PAMPs (Fig. 4B) , a time in which cyclin T1 protein is induced (see Fig. 2A ). We obtained similar results for cyclin T1 RNA levels at 4 h after PAMP stimulation (data not shown). These results indicate that the induction of cyclin T1 protein expression by PAMPs does not involve an increase in RNA levels and must therefore involve a post-transcriptional mechanism.


Figure 4
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  		Figure 4. Induction of cyclin T1 by PAMPs involves a post-transcriptional mechanism. Total RNA was isolated from MDMs after 4 h of treatment for TNF-{alpha} RNA assays (A) or after 20 h of PAMP treatment for cyclin T1 RNA assays (B). Real-time RT-PCR assays performed to analyze cyclin T1, TNF-{alpha}, and GAPDH RNA. The fold-change of cyclin T1 and TNF-{alpha} RNA represents RNA levels normalized to GAPDH RNA in PAMP-stimulated MDMs relative to those in untreated cells.

LPS treatment of MDMs appears to inhibit proteasome-mediated proteolysis of cyclin T1
We reported previously that cyclin T1 can be re-induced by a brief treatment with proteasome inhibitors, indicating that the shut-off of cyclin T1 expression in late-differentiated MDMs is a result of active proteolysis [30 ]. We were interested in investigating whether PAMP induction of cyclin T1 might involve a block to proteolysis. We therefore treated late-differentiated MDMs with LPS, the proteasome inhibitor MG101, or a combination of the two agents. If LPS and MG101 induce cyclin T1 through an action at different rate-limiting steps, the combination of the two would show a greater induction than either agent alone. Conversely, if LPS and MG101 were acting at the same rate-limiting step, the induction by the combination would be similar to that of each agent alone. As shown in Figure 5A , treatment with MG101 alone induced cyclin T1 expression in two donors (compare lanes 1 and 2 with 11 and 12). When MDMs were incubated with LPS alone for times ranging from 0.5 to 48 h, cyclin T1 was also induced as expected (lanes 7–10). When MDMs were incubated with LPS for these time periods and then treated with MG101 for 30 min, cyclin T1 was not "super-induced" (lanes 3–6 and 13–15). Rather, the combination of LPS and MG101 led to an induction similar to that of either agent alone. We performed a similar experiment with LTA and MG101 and observed that the level of cyclin T1 induction by either agent alone was similar to the level by the combination of the two agents (Fig. 5B) . These data suggest that LPS and LTA are likely to induce cyclin T1 by acting at the same rate-limiting step as that of MG101. It therefore appears likely that PAMPs induce cyclin T1 in late-differentiated MDMs by blocking proteasome-mediated proteolysis.


Figure 5
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  		Figure 5. Induction of cyclin T1 by LPS is through a mechanism inhibiting the action of proteasome. MDM cultures were stimulated with LPS (A) or LTA (B) for indicated times; 50 µM MG101 was added where indicated to cultures 30 min prior to addition of PAMPs. Extracts were prepared, and protein levels were determined by immunoblots. Two independent donors are shown for LPS and LTA (with and without MG101 treatment).


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DISCUSSION
 
Given its role as a subunit of the general RNA polymerase II elongation factor P-TEFb, the expression level of cyclin T1 in macrophages has important consequences for gene expression and innate immune functions. In addition, as cyclin T1 is the target of the HIV-1 Tat protein, the expression level of cyclin T1 is a critical determinant in the amount of virus produced in HIV-infected macrophages. Our previous studies have shown that cyclin T1 is highly regulated during the course of macrophage differentiation and activation [14 ]. In this study, we have extended this analysis by examining 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and have been proposed to negatively regulate its activity [25 26 27 28 ]. We found that 7SK and HEXIM1 are present at only low levels in freshly isolated monocytes, and like cyclin T1, their expression is induced during the course of differentiation (Fig. 1) . Late in differentiation, 7SK expression remains high, and HEXIM1 is shut off subsequent to that of cyclin T1 by proteasome-mediated proteolysis. The expression pattern of 7SK and HEXIM1 in monocytes and early-differentiated MDMs is similar to that in resting and activated PBLs [29 , 33 ]. 7SK and HEXIM1 expression levels are low in quiescent monocytes and PBLs, and they are strongly up-regulated by monocyte differentiation and PBL activation, two conditions in which there is a global increase in RNA polymerase II transcription.

Given the importance of P-TEFb to expression of RNA polymerase II-transcribed genes, it was unexpected to observe that cyclin T1 expression is extinguished late in macrophage differentiation [14 ]. In this study, however, we observed that expression of cyclin T2a, another cyclin that can associate with Cdk9 and constitute P-TEFb activity, is stable throughout differentiation. It is likely that the stable expression of cyclin T2a is therefore able to maintain P-TEFb function throughout differentiation and compensate for the loss of cyclin T1. The functional consequences of the specific induction of cyclin T1 by macrophage activation are unknown, but it may lead to the increased expression of a subset of P-TEFb-dependent genes that are important for the innate immune response.

PAMP activation of macrophages results in a broad cellular response that plays a critical role in innate immunity. Transcriptional profiling studies in LPS-stimulated macrophages have grouped the inducible genes into four classes: immediate-early (induced within 30 min), early (30 min–2 h), middle (2–7 h), and late (>7 h) [44 ]. The kinetics of induction of cyclin T1 by LPS and other PAMPs places it in the late gene class (Fig. 2A) . This relatively slow induction suggests that cyclin T1 is unlikely to be a direct response to PAMP signaling through TLRs, but its up-regulation may be a secondary response dependent on the synthesis of a PAMP-induced protein. A number of cytokines are known to be induced by LPS and are therefore logical candidates for such a protein, such as the immediate-early genes TNF-{alpha}, interleukin (IL)-1, and IL-6 and the late-induced gene cytokines transforming growth factor-ß and IL-10. However, we were unable to detect an activity in conditioned medium from PAMP-activated MDMs, which could induce cyclin T1 (see Fig. 3B ). Although it is a negative result, our observation suggests that the putative factor responsible for the PAMP-induction of cyclin T1 is not a soluble molecule.

The shut-off of cyclin T1 in late-differentiated MDM is a result of a proteasome-mediated process (ref. [30 ] and Fig. 5 ). The PEST sequence in the carboxyl terminus of cyclin T1 likely confers sensitivity to proteolysis in MDMs, as PEST sequences in G1 cyclins are known to confer ubiquitylation to these cyclins and their subsequent degradation in proteasomes [24 , 45 ]. It is notable that cyclin T2a (or T2b) does not contain a PEST sequence at its carboxyl terminus. We have been unable to detect direct ubiquitylation of cyclin T1 in macrophage extracts by immunoblot analyses. In addition, we used immunoprecipitation to purify hemagglutinin (HA)-tagged cyclin T1 from 293T cells; we incubated this purified cyclin T1 with late-differentiated macrophage extracts under conditions that allow in vitro ubiquitylation. However, we were unable to detect any ubiquitylation of HA-cyclin T1, although we were able to detect high levels of ubiquitylation of the positive control cyclin E (unpublished result). Although subject to the limited conclusions that can be inferred from negative results, these experiments do raise the possibility that cyclin T1 may be degraded in a proteasome-dependent manner, which does not involve its direct poly-ubiquitylation. There are several examples of proteins, which can be degraded by a proteasome-dependent mechanism and do not involve the direct ubiquitylation of the protein, such as ornithine decarboxylase, c-Jun, calmodulin, troponin C, the cyclin-dependent kinase inhibitor p21, and p53 [46 47 48 49 50 ].

It is noteworthy that proteasome inhibitors have no effect on the expression level of cyclin T1 in activated PBLs and several transformed cell lines, suggesting that the proteasome targeting of cyclin T1 is specific to MDMs and perhaps a limited number of other cell types [30 ]. It is possible that late-differentiated MDMs specifically express an E3 ligase activity or another type of factor, which has an implication in proteasome function. PAMP activation of MDMs may lead to a loss of expression of such factors, leading to the restoration of cyclin T1 protein expression. Comprehensive transcriptional profiling by DNA microarray methodology of monocytes and late-differentiated MDMs with and without PAMP treatment may identify candidate genes involved in this process. It will be important to identify the factor(s) responsible for the proteasome-mediated proteolysis of cyclin T1 in macrophages, as this will enable the investigation of the mechanism whereby cyclin T1 is shut off late in differentiation as well as the investigation of how this process is controlled in PAMP-activated MDMs.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institutes of Health AI45374 (A. P. R.) and AI42558 (C. H. H.). We thank Wendong Yu for help with real-time PCR assays and Dr. Olivier Bensaude for antibodies.

Received August 2, 2005; revised September 21, 2005; accepted October 10, 2005.


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