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(The FASEB Journal. 2001;15:483-491.)
© 2001 FASEB

HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway

GIORGIO ZAULI*1, DANIELA MILANI{dagger}, PRISCO MIRANDOLA*,{dagger}, MERI MAZZONI{dagger}, PAOLA SECCHIERO{dagger}, SEBASTIANO MISCIA* and SILVANO CAPITANI{dagger}

* Institute of Normal Morphology, ‘G. d’Annunzio’ University of Chieti; 66100 Chieti, Italy; and
{dagger} Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, 44100 Ferrara, Italy

1Correspondence: Institute of Normal Morphology, ‘G. D’Annunzio’ University of Chieti; via dei Vestini 6, 66100 Chieti, Italy. E-mail: g.zauli{at}morpho.unich.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The addition of low concentrations (0.1–1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60–120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24–48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.—Zauli, G., Milani, D., Mirandola, P., Mazzoni, M., Secchiero, P., Miscia, S., Capitani, S. HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway.


Key Words: extracellular Tat • signal transduction • cAMP • PC12 • HIV-1-associated dementia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE HUMAN IMMUNODEFICIENCY virus type 1 (HIV-1) Tat protein plays an essential role in viral gene expression and replication, mainly through a direct interaction with the trans-activation-responsive RNA element, located within the long terminal repeat of viral genome. Tat is translated from a multiply spliced mRNA containing two coding exons. The first exon encodes 72 amino acids and is relatively conserved; the second exon is less well conserved and varies considerably in length from 14 to 31 amino acids (1) .

Tat is actively released by HIV-1-infected cells (2 , 3) and, when used at concentrations of 0.1 µM or higher, induces massive and rapid neuronal cell death (4 5 6 7 8 9 10 11 12 13) . Since microglial cells and astrocytes are infected with HIV-1 but neurons are not (14) , it has been hypothesized that HIV-1 Tat protein locally released by infected cells may produce neuronal dysfunction and/or loss. In this respect, Tat protein is expressed in cells obtained from the central nervous system of patients affected by encephalitic AIDS (14) . It has also been shown that, at concentrations of 0.1–10 nM, Tat protein activates various signal transduction pathways in neurons, including phosphatidylinositol 3 kinase (PI-3K) (15 , 16) , ERK/MAPK (17) , and the cyclic adenosine monophosphate (cAMP) -dependent protein kinase A (PKA) pathway (18) .

The cyclic nucleotide response element binding protein (CREB) is a 43–46 kDa nuclear transcription factor that recognizes the highly conserved sequence known as cAMP-responsive element (CRE): 5'-TGACGTCA-3' (19) . CREB activation results from post-translational modifications, such as phosphorylation of serine-133 (Ser-133) in situ by PKA or calmodulin kinase, following increases in intracellular cAMP or Ca2+ levels, respectively (20 , 21) . More recently it has been shown that the activation of the ERK/MAPK pathway can also induce CREB phosphorylation on Ser-133 (22) . Phosphorylation of CREB at Ser-133 represents a critical step for trans-activation of CRE-dependent gene promoters (19) . Whereas the expression of the CREB gene is constitutive at low basal levels in many tissues (23) , CREB mRNA and protein are synthesized cyclically at high levels in the testis (24) . Moreover, CREB expression depends mainly on the CRE elements present in its promoter (25) , suggesting that autoregulation may affect the transcriptional expression of the CREB gene.

Since it has been clearly established that CREB plays a central role in promoting the survival/function of neuronal cells in response to neurotrophic factors (22 , 26 , 27) , the aim of this study was to investigate whether HIV-1 Tat protein affects the Ser-133 phosphorylation levels and expression of CREB in neurons. We chose as a model system the rat PC12 neuronal cell line, which has been shown to reproduce many of the biological responses to Tat protein observed in vivo or in cultured primary neurons (4 5 6 7 8 9 10 11 12 13 14 15 16 17 18) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and treatment of the cells
Synthetic (86 amino acid, derived from the HIV-1 T cell line-tropic BH10 strain; Technogen, Caserta, Italy) Tat protein was dissolved in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA, fraction V Chon; Sigma Chemicals, St. Louis, Mo.) and stocked at -70°C before use. Forskolin (Sigma), H89, SB203580, IBMX, LY294002, and wortmannin (Calbiochem, La Jolla, Calif.) were prepared in DMSO and stored at -70°C.

Rat pheochromocytoma PC12 cells were obtained from American Type Culture Collection (Rockville, Md.) and cultured in D-MEM (Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% horse serum (HS, Gibco) plus 5% fetal calf serum (FCS, Gibco). PC12 cells were serum-starved in D-MEM plus 0.1% FCS for 40 h before treatment with Tat protein (0.1–1 nM corresponding to 1–10 ng/ml), forskolin (10 µM), either alone or in combination, for 5–120 min. Equivalent volumes of PBS containing 0.1% BSA were used as negative control. In blocking experiments performed with pharmacological inhibitors, PC12 cells were pretreated for 1 h at 37°C with H89 (1 µM), SB203580 (10 µM), IBMX (0.1 mM), LY294002 (10 µM), wortmannin (0.1 µM), or equal volumes of DMSO.

Western blotting
For analysis of Ser-133 CREB, total CREB, tyrosine phosphorylated and total AKT and ß-tubulin proteins, Western blot was performed on ~10 x 106 cells per experimental point. Cell pellets were supplemented at 4°C with a lysis buffer containing 1% deoxycholate, 1 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (without Triton X-100) for 10 min. Cell lysates were sonicated and either immediately processed by Western blot or centrifuged at 100,000 g for 1 h at 4°C. In the latter case, the supernatants were discharged and the pellets, containing the nuclei, were solubilized in 100 µl of lysis buffer containing 0.1% Triton X-100. Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad, Richmond, Calif.) according to the manufacturer’s protocol. Equivalent (100 µg) amounts of proteins per sample were subjected to electrophoresis on a 10% sodium dodecyl sulfate-acrylamide gel. The gel was then electroblotted onto a nitrocellulose membrane; equal loading of protein in each lane was confirmed by brief staining of the blot with 0.1% Ponceau S, followed by destaining prior to reacting with the specific antibodies. Blotted membranes were blocked for 30 min in a 3% suspension of dried skimmed milk in PBS and incubated overnight at 4°C with 1) a rabbit polyclonal anti-CREB serum, 2) a rabbit serum directed against the phosphorylated Ser-133 form of CREB (both from Upstate Biotechnology Inc., Lake Placid, N.Y.; 1:1000 dilution), 3) a monoclonal anti-tyrosine phosphorylated AKT, 4) a monoclonal anti-AKT (both from PharMingen/Transduction Laboratories, San Diego, Calif.; dilution 1:250), or 5) a monoclonal anti-ß-tubulin (Sigma; dilution 1:500). Filters were washed and incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (to detect Ser-133 phosphorylated and whole CREB, 1:1500 dilution, Sigma) or anti-mouse IgG (to detect tyrosine phosphorylated or whole AKT or ß-tubulin, 1:1500 dilution, Sigma) in 0.1% BSA. Specific reactions were revealed with the ECL Western blotting detection reagent (Amersham Corp., Arlington Heights, Ill.).

Assay of cAMP
The intracellular cAMP levels were measured using the cAMP assay kit (Amersham) according to the manufacturer instructions. Briefly, after 40 h of starvation in D-MEM + 0.1% FCS, 100 mm plates of PC12 cells were treated with synthetic Tat (1 nM), BSA solution (0.1%) or forskolin (10 µM) for up to 120 min. The incubation was stopped by the addition of the lysis buffer provided by the manufacturer. The intracellular cAMP content of the samples was measured by ELISA and expressed as pM/100 µg of proteins.

Plasmids and transfection experiments
The following plasmids, wild-type -1264 pCREB-CAT, -1264°CRE 1 + 2 mut pCREB-CAT, mutated in the CRE sites, and poCAT empty vector (25) , were a generous gift of Dr. Habener (Massachusetts General Hospital, Harvard Medical School, Boston, Mass.).

Transient transfection experiments were performed using the PerFect transfection kit reagent (Invitrogen, Carlsbad, Calif.) following manufacturer’s instructions. Briefly, semi-confluent T35 culture flasks of PC12 were transfected with 5–10 µg of plasmid DNA. Thirty-six hours post-transfection, cells were treated with synthetic Tat (0.1–10 nM), forskolin (10 µM) used alone, or in various combinations. Twelve hours after treatment, cell lysates were assayed for chloramphenicol acetyl transferase (CAT) activity, using volumes of extract corresponding to equal protein amounts.

Detection of apoptosis
Apoptosis was evaluated in PC12 cells by using the TdT-mediated D-UTP-biotin nick end labeling (TUNEL) technique to monitor the DNA fragmentation in situ, following a previously described procedure (28) .

Statistical analysis
Statistical analysis was performed using the two-tailed Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rapid Ser-133 phosphorylation of CREB induced by Tat protein in PC12 neuronal cells
In the first group of experiments, we investigated whether extracellular Tat modulates the phosphorylation levels of the transcription factor CREB in PC12 neuronal cells. For this purpose, PC12 cells were serum-starved in order to lower the high levels of endogenous Ser-133 CREB phosphorylation observed in cells cultured with 10% HS + 5% FCS (not shown); 40 h after serum starvation in D-MEM + 0.1% FCS, PC12 cells were treated with low concentrations of synthetic Tat protein (01–1 nM) for up to 60 min. As a positive control of CREB Ser-133 phosphorylation, we used forskolin (10 µM), a known activator of adenylate cyclase (23) . PC12 nuclear homogenates were subjected to Western blot analysis with anti-Ser-133 phosphorylated CREB and anti-CREB (Fig. 1 ) polyclonal sera, followed by densitometric analysis. The time course of Tat-mediated CREB phosphorylation in PC12 cells revealed that extracellular Tat maximally increased the levels of Ser-133 CREB phosphorylation between 15 and 30 min (Fig. 1 and Table 1 ). At these time points, no significant variations in the total amount of nuclear CREB levels were noticed using an anti-CREB serum (Fig. 1) .



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  		Figure 1. Short-term effect of Tat on CREB Ser-133 phosphorylation in PC12 neuronal cells. Levels of Ser-133 phosphorylated CREB (P-CREB) and total CREB were evaluated by Western blot on enriched nuclear fractions obtained from PC12 cells, serum starved for 40 h, and treated for the time indicated (min) with Tat (1 nM) or forskolin (10 µM). Densitometric analysis of the bands is reported in arbitrary units (a.u.). A representative of four separate experiments is shown.


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  		Table 1. Densitometric analysis of P-CREB protein expression in PC12 cells treated with forskolin or Tat in the absence or presence of various pharmacological inhibitorsa

Bimodal changes of the intracellular cAMP levels in Tat-treated PC12 cells
Among several pathways that can induce CREB phosphorylation on Ser-133 in neuronal cells (20 21 22 23 , 26 , 27 , 29) , a major role is played by PKA. Thus, to ascertain whether PKA was involved in Tat-mediated CREB phosphorylation in PC12 cells, we used the pharmacological compounds H89, a specific inhibitor for the PKA pathway, and SB203580, an inhibitor of the p38 MAPK pathway, used as control. Western blot analysis of PC12 nuclear cell lysates showed that H89, but not SB203580, significantly (P<0.05) inhibited the Tat-mediated Ser-133 CREB phosphorylation (Fig. 2 and Table 1 ). This suggested that activation of the cAMP/PKA pathway was required for the Tat-induced CREB phosphorylation in these cells.



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  		Figure 2. Effect of a PKA pharmacological inhibitor on the short-term Tat-induced CREB Ser-133 phosphorylation in PC12 cells. Western blot was performed on enriched nuclear fractions obtained from PC12 cells treated with BSA (0.1%) or Tat protein (1 nM) for the times indicated (min) in the presence of H89 (1 µM) or SB203580 (10 µM). Densitometric analysis of the bands is reported in arbitrary units (a.u.). A representative of three separate experiments is shown.

To confirm this hypothesis, the intracellular levels of cAMP were analyzed after treatment with Tat, used alone (Fig. 3A ) or in combination with forskolin (Fig. 3B ). Consistent with a role for the cAMP/PKA pathway in inducing CREB phosphorylation in PC12 cells, Tat stimulated a rapid (peak at 5 min) elevation of intracellular cAMP over the basal levels observed in control (BSA-treated) cells (Fig. 3A ). At later time points, the intracellular levels of cAMP in Tat-treated cells progressively declined and were significantly (P<0.01, at 60–120 min) lower than those found in BSA-treated control cells (Fig. 3A ).



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  		Figure 3. Effect of Tat on the intracellular levels of cAMP in PC12 cells. The amount of intracellular cAMP was analyzed in total cell extracts obtained from PC12 cells treated for the time indicated (min) with Tat (1 nM) (A) or Tat (1 nM) plus forskolin (10 µM) (B). C) Cells were supplemented for 30 min with IBMX (0.1 mM) and then treated as in panel B. The data are expressed relative to cells treated with BSA (0.1 ng/ml, Control), set at 100. Data represent the means ± standard deviations of three (B, C) to six (A) independent experiments performed in duplicate.

To further investigate this biphasic effect of Tat on the intracellular levels of cAMP, particularly the secondary inhibitory phase, experiments were performed by treating PC12 cells with a combination of Tat plus forskolin. As expected, treatment of PC12 cells with forskolin induced a rapid and persistent elevation of intracellular cAMP (Fig. 3B ). The simultaneous addition of Tat and forskolin to PC12 cells resulted in an additive (P<0.05) effect of the two agonists on the cAMP levels at early time points (5 min). On the other hand, at later time points (60–120 min) the intracellular levels of cAMP were significantly (P<0.01) lower in cells treated with Tat + forskolin than in cells treated with forskolin alone (Fig. 3B ). Moreover, pretreatment of PC12 cells with IBMX, a broad inhibitor of cyclic nucleoside phosphodiesterases (PDE), completely abrogated the ability of Tat to decrease the levels of cAMP in PC12 cells costimulated with forskolin (Fig. 3C ). Taken together, these findings suggest that the decline of intracellular cAMP levels observed in Tat-treated cells was mediated by PDE activity.

In parallel, aliquots of the same samples examined for intracellular cAMP were subjected to Western blot to analyze the levels of CREB Ser-133 phosphorylation (Fig. 4 ). At 5–10 min, the combination of Tat plus forskolin showed an additive effect (P<0.05) on CREB Ser-133 phosphorylation, as demonstrated by the densitometric analysis of the Western blot bands (Fig. 4 and Table 1 ). At 60–120 min, the same combination induced levels of CREB Ser-133 phosphorylation significantly (P<0.05) lower than forskolin alone (Fig. 4 and Table 1 ). These changes in Ser-133 phosphorylation took place in the absence of significant modifications of total nuclear CREB (Fig. 4) . Changes in CREB Ser-133 phosphorylation levels closely paralleled those in intracellular cAMP (Fig. 3A , B ). In this and other experiments, a doublet of immunoreactive proteins instead of a single band was sometimes observed. The faster migrating band likely represents a different CREB isoform (21 , 26) . Due to the proximity of the two phosphorylated bands, stripping, and reprobing with the anti-CREB serum did not allow us to discriminate which band represented CREB. Densitometric analysis was then calculated considering an area comprising both phosphoproteins.



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  		Figure 4. Effect of combination of Tat and forskolin on short-term CREB Ser-133 phosphorylation in PC12 cells. Levels of Ser-133 phosphorylated CREB (P-CREB) and total CREB were evaluated by Western blot on enriched nuclear fractions obtained from PC12 cells treated with BSA (0.1%), Tat (1 nM), forskolin (10 µM), Tat (1 nM) plus forskolin (10 µM) for the time indicated (min). Densitometric analysis of the bands is reported in arbitrary units (a.u.). A representative of four separate experiments is shown.

Tat stimulates PDE through a PI-3K/AKT pathway
We next investigated how extracellular Tat induced the PDE activity responsible for the secondary decrease in the intracellular cAMP levels and in CREB Ser-133 phosphorylation. We took advantage of the fact that it had been shown that some PDE isoforms can lie downstream the serine-threonine kinase AKT (30 31 32) , which in turn is activated by PI 3-K (33 , 34) . Having previously demonstrated that extracellular Tat activates PI 3-K in PC12 cells (15) through p125FAK tyrosine kinase (16) , we next asked whether Tat also activates AKT in these cells and whether AKT may be an intermediate in Tat-induced stimulation of PDE. As shown in Fig. 5A , synthetic Tat (0.1–1 nM) rapidly stimulated AKT. Moreover, pretreatment of PC12 cells with two unrelated pharmacological inhibitors of PI 3-K (10 µM LY294002 and 0.1 µM wortmannin) before Tat addition significantly increased CREB Ser-133 phosphorylation in PC12 cells with respect to cells treated with vehicle containing DMSO (Fig. 5B and Table 1 ). These data strongly suggested that Tat promoted PDE activity through a PI 3-K/AKT-dependent pathway.



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  		Figure 5. A) Short-term effect of Tat on AKT tyrosine phosphorylation in PC12 neuronal cells. Levels of tyrosine-phosphorylated AKT and total AKT were evaluated by Western blot whole cell lysates of PC12 cells, serum starved for 40 h, and treated for the time indicated (min) with Tat (1 nM). B) Effect of the PI 3-K/AKT pharmacological inhibitors on the short-term Tat-induced CREB Ser-133 phosphorylation in PC12 cells. Western blot was performed on enriched nuclear fractions obtained from PC12 cells treated with BSA (0.1%) or Tat protein (1 nM) for the times indicated (min) in the presence of vehicle containing DMSO, LY 294002 (10 µM), or wortmannin (0.1 µM). A, B) A representative of four separate experiments is shown. Densitometric analysis of the bands is reported in arbitrary units (a.u.).

Inhibition of CREB promoter activity and CREB protein expression after prolonged treatment of PC12 cells with Tat
It has been shown that steady-state levels of CREB are extremely sensitive to variations of intracellular cAMP, due to the presence of cAMP response elements in its promoter (25) . Thus, in the next group of experiments we investigated whether the ability of extracellular Tat to modulate the levels of intracellular cAMP and CREB Ser-133 phosphorylation could affect CREB transcription. PC12 cells were transfected with a plasmid containing the intact CREB promoter (wild-type -1264 pCREB-CAT) and a CREB promoter mutated in the CRE sites (-1264°CRE 1+2 mut pCREB-CAT), fused to CAT (25) . The basal transcriptional activity of wild-type pCREB-CAT was low but clearly detectable in BSA-treated PC12 cells, and was significantly (P<0.05) reduced by the addition of Tat for the last 12 h of culture (Fig. 6A ). On the other hand, no CAT activity was detected when PC12 cells were transfected with either mut pCREB-CAT or poCAT empty vector (data not shown). This suggested that the Tat-mediated activation of PDE (Fig. 3A ) was able to down-modulate the CREB promoter activity in PC12 cells.



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  		Figure 6. Effect of Tat on basal (A) and forskolin-stimulated (B, C) transcriptional activity of wild-type pCREB-CAT reporter plasmid. A) PC12 cells were transfected with pCREB-CAT reporter construct and treated with different concentrations of Tat protein. The data are expressed relative to activity in cells treated with BSA alone, set at 100. B) PC12 cells were transfected with pCREB-CAT reporter construct and treated simultaneously with forskolin (10 µM) plus Tat (0.1–10 nM). C) PC12 cells were transfected with pCREB-CAT reporter construct, then treated with forskolin (10 µM) plus Tat (1 nM) added 1 h before or after forskolin. A—C) CAT assay was performed 12 h after treatments. The data are expressed relative to activity in cells treated with forskolin alone, set at 100. Data are expressed as means ± SD of three to five independent experiments.

This hypothesis was corroborated in next experiments, in which PC12 cells were transfected with pCREB-CAT and then treated with forskolin plus Tat (Fig. 6B ). As expected, due to the ability of forskolin to elevate the cAMP levels (Fig. 3B ), when pCREB-CAT transfected PC12 cells were treated with forskolin, a pronounced increase of CREB promoter activity was noticed. The addition of Tat resulted in a dose-dependent inhibition of the forskolin-induced pCREB-CAT activity with respect to cells treated with forskolin plus BSA (Fig. 6B ). This inhibitory effect of Tat on forskolin-induced pCREB-CAT activation was also consistently observed when transfected PC12 cells were treated with Tat 1 h before adding forskolin (Fig. 6C ). On the other hand, no significant variations were noticed when Tat was added 1 h after forskolin (Fig. 6C ). The transcriptional activity of mut pCREB-CAT was also undetectable in cultures stimulated with forskolin used alone or in combination with Tat, confirming that the CRE sites played a central role in driving CREB transcription (data not shown).

To verify the relevance of Tat-induced down-modulation of CREB promoter, in the next group of experiments we analyzed the total amount of CREB protein in PC12 cells after prolonged Tat treatment (Fig. 7 ). For this purpose, cell lysates obtained from whole PC12 cells and nuclei were analyzed for the content of CREB and its levels of phosphorylation on Ser-133 after 24–48 h from the addition in culture of 1 nM Tat. As shown in Fig. 7 and Table 2 , a significant decline in both Ser-133-phosphorylated CREB and whole CREB protein was reproducibly observed between 24 and 48 h. This decrease could not be ascribed to a nonspecific loss of proteins since equal loading of proteins in each lane was confirmed by staining the blot with Ponceau (not shown) and by densitometric analysis of ß-tubulin (Table 2) . The possibility that the loss in CREB protein expression was due to a nonspecific cytotoxicity of Tat was ruled out by an analysis of apoptosis at different time points in Tat- and BSA-treated cells (Table 3 ). In fact, in four separate experiments the percentage of apoptosis, analyzed by using the TUNEL technique, was lower (P<0.05) in PC12 cells treated with Tat then in cells treated with BSA (0.1%) up to 24 h post-treatment, whereas no significant differences were observed after 48 h (Table 3) . At later time points (72–96 h post-treatment), however, a moderate but significant (P<0.01) increase of apoptosis was noticed in Tat-treated with respect to BSA-treated cultures. Thus, the decrease of CREB protein expression anticipated the loss in cell viability.



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  		Figure 7. Prolonged effect of Tat on CREB Ser-133 phosphorylation and CREB expression in PC12 neuronal cells. Levels of Ser-133 phosphorylated CREB (P-CREB) and total CREB were evaluated by Western blot on both whole cell lysates (cells) and enriched nuclear fractions (nuclei) obtained from PC12 cells, serum starved for 40 h, and treated for the time indicated (hours) with Tat (1 nM). Densitometric analysis of the bands is reported in arbitrary units (a.u.). A representative of four separate experiments is shown.


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  		Table 2. Densitometric analysis of P-CREB, total CREB and ß-tubulin in PC12 cells treated with Tat (1 nM) for up to 48 ha


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  		Table 3. Percentage of apoptosis in serum-starved PC12 cells evaluated by the TUNEL technique at various time points after Tat or BSA additiona


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Approximately 25% of HIV-1 infected individuals will develop HIV-1-associated dementia, and HIV-1 infection is now the leading cause of dementia in children and young adults, in whom other kinds of dementia are uncommon (35) . HIV-1-associated dementia complex manifests in severely immune-compromised patients and involves cognitive, behavioral, and motor dysfunction. This unusual slowly progressive illness is thought to be due to HIV-1 invasion of the central nervous system. With the development of highly active anti-retroviral therapy, it is now recognized that symptoms of HIV-1 dementia are reversible at least in some patients (36) . However, these beneficial effects may not be long-lasting due to development of drug resistance. Therefore, to effectively treat or prevent HIV-1 dementia, an understanding of its pathogenesis is essential.

At the neuropathological level, a range of abnormalities has been reported in persons with HIV-1 dementia, including extensive loss of neurons within certain regions of the brain (37 , 38) and high levels of neuronal apoptosis (39 , 40) . The induction of neuronal dysfunction is believed to occur as a result of the production and release of a number of neurotoxic factors, which include HIV-1 Tat protein, that can be released locally by infected microglial cells or across the blood–brain barrier (41) . Detectable levels of Tat protein have been demonstrated to be present in the sera of HIV-1-seropositive patients (42) , and Tat can easily across the blood–brain barrier (43) . Thus, it is possible that also systemic Tat may reach the central nervous system. How extracellular Tat elicits its biological effects on neuronal cell survival/growth is still incompletely understood. In fact, Tat protein can be taken up by intact neurons and localizes to the nucleus quite rapidly both in vitro (4) and in vivo (44) . In addition, Tat interacts with a variety of surface receptors, including receptors for integrins (45) , members of the vascular endothelial growth factor receptor family (46) , and receptors for chemokines (47) .

Previous studies have shown that at micromolar concentrations, Tat can be neurotoxic by inducing rapid and massive apoptotic cell death of neuronal cells both in vitro and in vivo (4 5 6 7 8 9 10 11 12 13) . These high concentrations of Tat cause neurotoxicity by direct neuronal depolarization (9) , by increasing levels of intracellular calcium (8 , 11) , and by activating excitatory amino acid receptors (6 , 10) .

In this study, we have demonstrated for the first time that synthetic extracellular Tat also shows subtler, but potentially noxious effects on neuronal cell survival at concentrations as low as 0.1–1 nM. In fact, after an initial rapid induction of CREB phosphorylation on Ser-133, Tat induced a secondary, prolonged down-regulation of CREB expression. These changes in CREB phosphorylation and expression anticipated and correlated with the bimodal effect of low Tat concentrations on PC12 cell viability: early protection from apoptosis induced by serum withdrawal (up to 24 h), followed by secondary increase of apoptosis (72–96 h) with respect to BSA-treated cells.

CREB is encoded by a gene that contains at least 11 exons spread over more than 40 kilobases of DNA located on the long arm of human chromosome 2 (48 , 49) . CREB is the prototype of the CREB/CREM/ATF superfamily of bZIP or leucine zipper transcription factors, including similar factors with a basic DNA binding domain that recognize the CRE motif. Due to its essential role for neuronal cell survival (26 , 27) and as a mediator of long-term memory (50) , the Tat-induced down-regulation of CREB expression is expected to have profound detrimental effects on neuronal cell survival and/or function. What is particularly remarkable is that these effects were obtained at relatively low Tat concentrations.

In considering the possible levels of Tat that may be present in vivo, concentrations of 0.01–0.1 nM of Tat in the sera of HIV-1-infected individuals have been reported (42) . Tat can be expected to be present at higher concentrations in tissues with active HIV-1 replication or in proximity to cells with productive HIV-1 infection, such as in regions containing inflammatory infiltrates and productively infected microglia and macrophages (51) . Therefore, although Tat within the brain has never been measured, it is not unreasonable to expect that concentrations of around 0.1–1 nM may be present in close proximity to virus-positive microglia or brain macrophages. However, in our opinion the neurotoxic effects associated with higher (µM) concentrations of Tat (4 5 6 7 8 9 10 11 12 13) are very unlikely to occur in vivo in the brain of HIV-1 infected individuals.

Although we cannot exclude that the down-regulation of CREB requires internalization and nuclear localization of extracellular Tat protein (4) , our data strongly suggest that Tat down-modulates CREB gene expression, altering the intracellular levels of cAMP. In fact, even though the early and rapid elevation of cAMP is likely due to the activation of adenylate cyclase, the signal transduction pathway involved in the secondary CREB down-regulation requires activation of a PI 3-K/AKT/PDE pathway. It has previously been shown that the steady state, intracellular levels of cAMP are controlled predominantly by PDE (52) . It is noteworthy that CREB expression is extremely sensitive to variations of the intracellular cAMP levels due to the presence of critical CRE elements in its promoter (25) .

Although this study was performed using a neuronal cell line in vitro and there may be important differences with in vivo systems, our data strengthen the notion that Tat may be an important cause of neurocognitive dysfunction in HIV-1-seropositive individuals. Moreover, we have provided a molecular mechanism to explain how low concentrations of extracellular Tat can impair the survival/function of neuronal cells by decreasing the levels of cAMP and the expression of CREB protein. Several authors, including ourselves, have previously proposed and shown the potential usefulness of a Tat vaccination to slow or even arrest the progression toward AIDS in both nonhuman primates and human beings infected with simian immunodeficiency virus or HIV-1, respectively (3 , 53 54 55 56) . If our present findings were confirmed with in vivo studies, they might suggest that a vaccination of HIV-1-seropositive individuals with Tat protein would be beneficial to counteract the occurrence of neurological abnormalities, frequently observed in these patients.


   ACKNOWLEDGMENTS
 
This work was supported by AIDS project from the Italian Ministry of Health, Italy and by local funds of the ‘G. D’Annunzio’ University of Chieti.

Received for publication June 1, 2000. Revision received August 2, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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