- Top of page
- Materials and methods
The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.
The human immunodeficiency virus (HIV) encodes the highly conserved transcriptional transactivator Tat, which is expressed early in the viral life cycle and plays a pivotal role in viral replication as well as in the progression to overt acquired immune deficiciency syndrome (AIDS) (Cullen, 1993; Jones & Peterlin, 1994). Tat, endogenously expressed by HIV-1-infected cells, exerts its function after binding to a specific RNA target (Dingwall et al, 1989). In particular, Tat protein binds to the transactivation response (TAR) RNA stem-loop located from +1 to +60 in the 5′ of the long-terminal repeat (LTR) viral promoter inducing a massive viral RNA transcription (Feng & Holland, 1988; Marciniak & Sharp, 1991; Cullen, 1993). In addition, endogenous Tat is able to elicit an effective increase of transcriptional RNA viral elongation (Feng & Holland, 1988; Marciniak & Sharp, 1991; Cullen, 1993).
Interestingly, Tat can also induce biological effects in various cell types in a cytokine-like fashion. In fact, it has been shown that Tat can be actively released and detected in the culture supernatants of both acutely HIV-1-infected and tat-transfected cells (Frankel & Pabo, 1988; Ensoli et al, 1993). Moreover, soluble Tat can be rapidly taken up by uninfected cells in which it localizes in both the cytoplasm and nuclear compartments (Frankel & Pabo, 1988; Mann & Frankel, 1991). We, and other authors, have previously demonstrated that, in its extracellular form, Tat protein affects the survival/proliferation of CD4+ T lymphocytes by a paracrine/autocrine loop (Zauli et al, 1995; Zauli & Gibellini, 1996). While Tat inhibits cell proliferation at high (μmol/l-nmol/l) concentrations (Li et al, 1995; Westendorp et al, 1995; Zauli et al, 1996; McCloskey et al, 1997), at lower (pmol/l) physiological concentrations Tat promotes the survival (Gibellini et al, 1995; Zauli et al, 1995) and the proliferation of quiescent CD4+ T cells, in cooperation with anti-CD3 and anti-CD28 monoclonal antibodies (Zauli et al, 1996; Secchiero et al, 2000).
In this context, we have previously demonstrated that tat gene endogenously expressed in both lymphoid and monocytic cells upregulates the expression of c-fos (Gibellini et al, 1997), a proto-oncogene, which constitutes the transcription factor AP−1 in association with c-jun. It should be noticed, however, that endogenous tat increased the expression of c-fos only when cells were cultured in the presence of high [15% fetal calf serum (FCS)] serum concentrations and were stimulated with phytohaemagglutinin or phorbol esters.
The aim of this study was to ascertain whether low physiological concentrations of extracellular Tat protein, which have been detected in the body fluids of HIV-1-seropositive individuals (Westendorp et al, 1995), were able to modulate c-fos expression. We also investigated which region of the c-fos promoter was responsive to extracellular Tat and whether the Tat-mediated induction of c-fos led to the formation of the AP-1 complex. This is particularly relevant as AP-1 plays a crucial role in the activation/proliferation of various cell types, including T lymphocytes (Sassone-Corsi et al, 1988; Angel & Karin, 1991; Karin et al, 1997).
- Top of page
- Materials and methods
Several studies have convincingly demonstrated that Tat protein has a profound impact on the survival/function of both HIV-1-infected and uninfected lymphoid CD4+ T cells, through its ability to be secreted by HIV-1-infected cells (Ensoli et al, 1993; Zauli et al, 1995). This intriguing cytokine-like accomplishment indicates that Tat can act in an autocrine/paracrine fashion (Ensoli et al, 1993; Zauli et al, 1995).
Opposite effects of lymphoid cell survival/growth have been demonstrated in the presence of low (pmol/l) (Gibellini et al, 1995; Zauli et al, 1995) and high (μmol/l-nmol/l) (Li et al, 1995; Westendorp et al, 1995; Zauli et al, 1996) concentrations of extracellular Tat, indicating that the vast array of biological responses observed in the presence of this regulatory HIV-1 protein is concentration dependent. In this respect, Tat has been detected in body fluids at subnanomolar concentrations, which suggests that picomolar concentrations of Tat probably reflect the physiopathological concentrations present in vivo at the sites of an active HIV-1 replication (Westendorp et al, 1995). Moreover, tat-transgenic mice studies showed an increased proliferative activity, leading to oncogenic transformation in lymphoid tissues, which further suggests that the main Tat biological effect in vivo is a proliferative one (Vellutini et al, 1995; Altavilla et al, 1999).
To further investigate how low concentrations of extracellular Tat affect lymphoid CD4+ T-cell function, in this study we demonstrated for the first time that synthetic extracellular Tat induces c-fos expression in low serum-starved Jurkat cells. The c-fos proto-oncogene represents a key factor of the AP-1 transcription factor complex, which stimulates several gene promoters by binding to the TRE sequences (Angel & Karin, 1991; Karin et al, 1997). Of note, the Tat-mediated induction of c-Fos protein resulted in the functional activation of AP-1, as demonstrated in CAT assay experiments. The ability of extracellular Tat to activate AP-1 is of primary importance to explain the upregulation of interleukin 2 (IL-2) production observed in lymphoid T cells treated with both intracellular or extracellular Tat (Westendorp et al, 1994; Ott et al, 1997).
By using promoter deletion mutants, we also demonstrated that the two main c-fos promoter elements responsive to extracellular Tat are the CRE and SRE domains located at −60 and −323, respectively, even though the SIE element (located at −340) also contributes to the Tat-mediated activation.
It should be noted that these data obtained with extracellular Tat are significantly different from those obtained with endogenously expressed tat (Gibellini et al, 1997). In fact, we have previously shown that the activation of c-fos promoter in response to endogenous tat gene in the same (Jurkat) cell line (i) requires the presence of high serum concentration (> 10% FCS) and phytohaemagglutinin, and (ii) is mediated by the SRE element, but not the SIE and CRE elements. Our present and previous findings suggest that intracellular and extracellular Tat may select different targets on the c-fos promoter, depending on the activation state of the cells as well as on the presence of additional stimuli. In this respect, it is also noteworthy that the c-fos promoter shows a very complex regulation (Van Straaten et al, 1983), undergoing a post-induction downregulation in response to c-Fos protein itself, which negatively regulates the SRE site of its own promoter. Interestingly, the CRE element (−60) of the c-fos promoter is not affected by c-Fos protein. Taken together, these findings suggest a dual role of endogenous and extracellular Tat in the c-fos promoter regulation in CD4+ T cells.
We have also demonstrated that, among the various intracellular signal transduction pathways activated by Tat protein (Borgatti et al, 1997; Li et al, 1997; Gibellini et al, 1998; Kumar et al, 1998), the ERK/MAPK pathway, which leads to CREB Ser133 phosphorylation (Gibellini et al, 1998), plays an important role in mediating c-fos promoter activity by extracellular Tat. However, it is conceivable that additional pathways are involved in c-fos activation, probably acting at the level of the SIE element.
In conclusion, the ability of extracellular Tat to activate c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to AIDS and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells (Gallo, 1999).