Pentoxifylline and Other Protein Kinase C Inhibitors Down-Regulate HIV-LTR NF-κB Induced Gene Expression
This investigation deals with the molecular mechanism of anti-human immunodeficiency virus type 1 (HIV-1) action of pentoxifylline (PTX) [1-(5′-oxohexyl)-3,7-dimethylxanthine] a drug widely used for the treatment of conditions involving defective regional microcirculation.
Materials and Methods
The inhibition by PTX of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA)-mediated activation by phorbol ester (PMA) and tumor necrosis factor alpha (TNF-α) of HIV-1-LTR-regulated reporter gene expression was studied in human CD4+ T lymphocytes (Jurkat) and human embryo kidney cells (293-27-2). A protein kinase C is involved in activation of NF-κB in whole cells, identified by using inhibitors specific for PKC- or PKA-catalyzed NF-κB activation in whole cell and cell-free systems.
PTX inhibited PKC- or PKA-catalyzed activation of NF-κB in cytoplasmic extracts from unstimulated Jurkat or 293-27-2 cells, but not interaction of preactivated NF-κB with its motifs. Calphostin C, a specific inhibitor of PKC, inhibited NF-κB activation and HIV-1 LTR-driven reporter gene expression in both PMA- and TNF-α-treated cells. In contrast, although H88 specifically inhibited PKA activity in the cell-free extract, it did not affect NF-κB action in PMA- or TNF-α-treated cells.
The mechanism of inhibitory action of PTX on virus replication and NF-κB-induced trans-activation of HIV-1 gene expression has been elucidated as due to blocking PKC-dependent PMA- or TNF-α-induced activation of NF-κB in Jurkat and 293-27-2 cells. Other protein kinase inhibitors may be useful in down regulating transcription of HIV-1 provirus and thereby virus replication in HIV-infected patients.
Human immunodeficiency virus type 1 (HIV-1) is implicated in the deadly disease acquired immune deficiency syndrome (AIDS). Identification and characterization of agents that block its replication are currently being investigated intensively. Provirus transcription is regulated by interactions of several cellular and viral trans-acting proteins, including NF-κB, with specific sequences in the long terminal repeat (LTR) of HIV-1 (1). We have reported that treatment of cells with pentoxifylline (PTX) [1-(5-oxohexyl)-3,7-dimethylxanthine] inhibits HIV-1 replication (2). PTX is also widely used for the treatment of different human peripheral blood disorders. The drug down-regulates HIV-1 LTR-driven gene expression in stimulated cells through its action upon the two nuclear factor kappa B (NF-κB) motifs in the LTR (3). The antiviral action of PTX may be due to inhibition of any one or more of at least six steps of trans-activation by NF-κB in whole cells: (1) activation of a protein kinase; (2) activation of NF-κB by kinase action upon the inactive cytoplasmic NF-κB-IκB complex; (3) NF-κB release from phosphorylated IκB; (4) translocation of NF-κB into the nucleus; (5) its interaction with the two NF-κB motifs in the HIV-1 LTR; and (6) bound NF-κB stimulation of promoter function, the end result of these steps.
NF-κB is present in most cells as an inactive complex that is activated by diverse agents such as cytokines TNF-α and interleukin-1 (IL-1) (4,5), phorbol ester (PMA), cAMP, protein synthesis inhibitors, lipopolysaccharides, deoxycholate, and formamide (6, 7, 8, 9). NF-κB is also activated by double-stranded RNA (10) and the HTLV protein tax-1 (11). Activation of NF-κB is dependent on the phosphorylation of IκB, an inhibitor complexed to NF-κB in the cytoplasm (7,12, 13, 14). The reaction is catalyzed by various protein kinases in different cell types (15). Mitogen-like phorbol ester (PMA)-induced activation is mediated by protein kinase C (PKC), as is induction by some cytokines such as TNF-α (16, 17, 18, 19). Meichle et al. (20) suggested a PKC-independent physiological activation of NF-κB, and cell-specific differences in PKC-dependent activation of NF-κB are reported (21). IL-1-induced activation of NF-κB in certain cells was mediated by cAMP-dependent protein kinase A (PKA) (9,22). The involvement of Raf-1 kinase in the activation of NF-κB was also reported (23).
The inhibitors of protein kinases used in most of the above studies have overlapping actions on both PKC and PKA. Contradictory results were obtained with the frequently used inhibitors H7 [1-(5-isoquinoline-sulfonyl)-2-methylpipirazine hydrochloride] (24, 25, 26, 27, 28), H8 (29), and staurosporine (20). Effects of H7 were also dependent on the commercial source (30). H7 may be a mixture of two isomers; some preparations have a higher concentration of one or the other (31). Other protein kinase inhibitors were recently described that have more specific and selective inhibitory activity against PKA and PKC. Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is a selective inhibitor of PKC with a reported IC50 of 0.05 µM, whereas the IC50 of this drug for PKA is about 50 µM (32,33). Calphostin C inhibits protein kinase C by competing for the binding site of diacylglycerol and PMA, and its action is light-dependent (34). Qatsha et al. (35) reported that Gö 6976, a nonglycosidic indolocarbazole and a selective inhibitor of PKC (IC50 0.02 µM) is an antagonist of HIV-1 induction from latent/low-level-producing reservoir cells in vivo. Chelerythrine is another newly identified selective PKC inhibitor (36). Its IC50 for PKC is 0.66 µM and IC50 for PKA is 170 µM. In contrast, H88 [N-(2-cinnamylaminoethyl)-5-isoquinolinesulfonamide], a derivative of H89 (37), is a selective inhibitor of PKA with IC50 of 0.36 µM whereas IC50 of this drug for PKC is 75 µM.
In this investigation we have attempted to identify the site or sites of PTX action in the pathway of NF-κB-mediated trans-activation of HIV-1 LTR. Initially we examined the influence of PTX on protein kinase-catalyzed cytoplasmic activation of NF-κB and its interaction with the motifs, the two events (steps 2 and 5) of this multistep NF-κB-mediated trans-activation process. In a cell-free system, PKC and PKA catalyze activation of NF-κB. We characterized two specific inhibitors (calphostin C and H88) and a nonspecific inhibitor (H7) and then used them to define the PMA- and TNF-α-induced activation pathway of NF-κB in the intact cells as dependent on PKC which is inhibited by PTX and by calphostin C, another PKC-specific inhibitor.
Materials and Methods
Jurkat CD4+ T lymphocytes were maintained in RPMI 1640 (GIBCO/BRL, Gaithersburg, MD, U.S.A.) tissue culture medium supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2 and 95% air. 293-27-2 is a human embryo kidney cell line in which the pNAZ expression plasmid (38,39) carrying the HIV-1 LTR fused to a bacterial lacZ gene was stably integrated into the chromosome. This cell line, obtained from Dr. L. A. Herzenberg of Stanford University, was maintained in complete Dulbecco’s modified Eagle’s medium (DMEM, GIBCO/BRL) supplemented with 10% bovine calf serum (BCS) and L-glutamine in 5% CO2 and 95% air at 37°C.
Pentoxifylline (PTX) was a gift from Hoechst-Roussel Pharmaceuticals, Inc. (Somerville, NJ, U.S.A.). Protein Kinase C (PKC) purified from rat brain was obtained from Promega Biotech (Madison, WI, U.S.A.). This PKC consists of α, β, and γ isoenzymes and is free of other kinases. cAMP-dependent protein kinase A (PKA), 8-bromo-cAMP, dithiothreitol (DTT), phenyl methyl sulfonyl fluoride (PMSF), phosphatidyl serine, 4-B-phorbol-12-B-myristate acetate (PMA), and ATP were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Tumor necrosis factor-α (TNF-α) was from Endogen Inc. (Boston, MA, U.S.A.). Calphostin C was from Kamiya Biochemical Co. (Thousand Oaks, CA, U.S.A.), and H88 and H7 were from Seikagaku America, Inc. (Rockville, MD, U.S.A.). The complementary strands of the oligonucleotide carrying the wild-type NF-κB motif (5′-TCGACAGGGACTTTCCGAGAG-3′) and the mutated NF-κB motif (TCGACACTC ACTT…) were custom synthesized in the core facilities of the Dana-Farber Cancer Institute.
Fractionation of Cytoplasmic Extracts
Jurkat and 293-27-2 cells were grown in their respective tissue culture media under the above-described conditions. Cytoplasmic extracts were prepared according to the method of Dignam et al. (40) and fractionated by 0–40% saturation with ammonium sulfate as described by Ghosh and Baltimore (16). The ammonium sulfate precipitate containing the NF-κB-IκB complex was dissolved in buffer A (Tris-HCl, pH 7.5, 20 mM, KCl 100 mM, EDTA 0.5 mM, DTT 1 mM, PMSF 0.5 mM, and 10% glycerol) and dialyzed against a large volume of buffer A. A protease inhibitor cocktail (16) was added to the dialyzed samples, and they were stored at −80°C. Protein content was determined according to the method described by Bradford (41).
Cell-Free NF-κB Activation System
Protein kinase C-catalyzed activation of NF-κB was carried out in a reaction mixture of 5 µl total volume containing 1–2 µg of ammonium sulfate-precipitated cytoplasmic proteins from unstimulated cells, 100 mM KCl, 30 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 0.5 mM CaCl2, a lipid mix (10 µM PMA, 0.28 mg/ml of phosphatidyl serine, 0.3% Triton X-100), and 200 µM ATP in the absence or presence of specified amounts of purified PKC. The reaction was carried out at room temperature for 30 min. The cAMP-dependent PKA-catalyzed activation of NF-κB was conducted in a reaction volume of 5 µl containing 1–2 µg of ammonium sulfate-precipitated cytoplasmic proteins from unstimulated cells, 25 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 75 µM ATP, and 1 mM 8-bromo-cAMP in the absence or presence of purified cAMP-dependent PKA. The reaction was carried out at room temperature for 30 min. The activation reaction mediated by either kinases was analyzed by electrophoretic mobility shift assay (EMSA) using a 32P-labeled oligonucleotide carrying the wild-type NF-κB motif under conditions described previously (3). The level of active NF-κB produced in the cell-free reaction mixture was quantitated by densitometric scanning of the specific autoradiographic signals as described previously (3).
Whole Cell Experiments
To identify the specific kinase that participates in the pathway of PMA- or TNF-α-induced activation of NF-κB in whole cells, stable expression of HIV-1 LTR-driven reporter lacZ gene in 293-27-2 and transient expression of chloramphenicol acetyl transferase (CAT) gene in Jurkat cells was determined. Cells were treated with specific inducers and kinase inhibitors as described in the figure legends. In the case of 293-27-2 cells in which HIV-1 LTR-lacZ fusion gene construct was stably integrated, both NF-κB binding (by EMSA) and its influence on the reporter gene expression (by β-gal activity) were measured in the nuclear and cytoplasmic extracts of treated cells as previously described (3). Unstimulated or stimulated (with PMA or TNF-α) Jurkat cells were transiently transfected with pHIV-1 LTR-CAT and treated with specific inhibitors (either calphostin C or H88) under conditions described in the figure legends. CAT activity in cell extracts was determined as described previously (3).
PKC- and PKA-Dependent Activation of NF-κB in a Cell-Free System
A cell-free system was described for the activation catalyzed by purified PKC of NF-κB by phosphorylation of IκB in the cytoplasmic inactive NF-κB-IκB complex and release of active NF-κB (9,16). In this investigation we have established a similar system with a partially purified cytoplasmic NF-κB-IκB complex from unstimulated Jurkat (T lymphoid) or 293-27-2 (human embryo kidney) cells. PKC or cAMP-dependent protein kinase (PKA) was added, to activate NF-κB as measured by electrophoretic mobility shift assay (EMSA), by its specific retardation of a 32P-labeled oligonucleotide carrying the NF-κB motif (3). Since our goal is to quantitate the level of released active NF-κB from the cytoplasmic inactive NF-κB-IκB complex, we reasoned that measurement of free NF-κB binding with its motif to be a more direct assay of the activation step than measurement of the phosphorylated IκB, the other product of the reaction.
Effects of PTX on Activation of NF-κB by PKC or PKA in a Cell-Free System
To identify sites of this inhibitory action of PTX in the multistep activation pathway, we initially utilized the PKC/PKA-catalyzed cell-free NF-κB activation system. A concentration-dependent inhibition by PTX of activation of NF-κB catalyzed by externally added purified PKC and PKA (Fig. 3C) to extracts of unstimulated cells was observed (IC50 0.4–0.5 mM). Similar patterns of inhibition of PKC/PKA-catalyzed activation of NF-κB were observed with extracts from both Jurkat (Fig. 3, upper panels) and 293-27-2 (Fig. 3, lower panels) cells (Fig. 3C). This kinase inhibition by PTX is consistent with blocking of PKC/PKA activity by other established specific kinase inhibitors.
These two sets of data, in addition to the previously reported results with whole cells (2,3,42) suggest that at least one primary site for inhibition by PTX of HIV-1 LTR-mediated gene activation and virus replication is on the protein kinase-dependent activation of NF-κB.
Effects of Other Kinase Inhibitors upon NF-κB Activation
PTX inhibited both PKC- and PKA-catalyzed activation of NF-κB in the cell-free system. But these results do not specify which of the two kinases inhibited by PTX is involved in the PMA- or TNF-α-mediated activation in whole cells. Therefore, properties of some established relatively specific protein kinase inhibitors were first tested for their effects in the cell-free NF-κB activation system. They were then used to characterize specific kinase involvement in activation of NF-κB in whole cells.
H88 is reported to be a specific inhibitor of cAMP-dependent protein kinase A (PKA) (37). This differential inhibitory property of the drug was examined on the PKC- and PKA-catalyzed NF-κB activation in the cell-free system. Figure 5B demonstrates a concentration-dependent inhibition by H88 of PKA-catalyzed release of active NF-κB (IC50 about 0.1 µM). No significant inhibition of PKC-catalyzed activation of NF-κB by H88 at more than 500-fold higher concentrations was detected, demonstrating that H88 is a specific inhibitor of PKA-catalyzed NF-κB activation in this system (Fig. 5B).
H7 has an overlapping inhibition of both the protein kinases. The IC50’s of the compound for PKC and PKA have been reported to be 6 and 3 µM, respectively (43,44). In the cell-free NF-κB activation system, H7 inhibited both PKC and PKA, although it showed a preferentially greater inhibitory influence on PKC (Fig. 5C). A low-level inhibition of PKA with H7 was consistently observed in five independent experiments, but the results were variable. A typical result is presented in Fig. 5C. The IC50 of H7 for PKA or its specificity for PKA or PKC could not be conclusively determined from the results of these experiments.
TNF-α- and PMA-Induced NF-κB Activation Pathway in Whole Cells
Active NF-κB in the nuclear extracts of PMA- or TNF-α-treated 293-27-2 is elevated as determined from the increased binding of the factor with its motif (3). A simultaneous elevation of HIV-1 LTR-driven reporter gene product, β-galactosidase activity, in the same PMA- or TNF-α-treated cells was also detected. PTX inhibited these increased activities. Since PTX inhibited both PKC- and PKA-catalyzed activation of NF-κB in the cell-free system it remains unclear which kinase in whole cells is inhibited. The kinase involved was further identified by treating intact 293-27-2 with an activator in the presence of the inhibitors of PKC or PKA whose specificity had been characterized in the cell-free NF-κB activation system.
The possibility that a PKA-catalyzed pathway is also involved in the activation with either PMA or TNF-α of NF-κB in whole cells was examined both in 293-27-2 and Jurkat cells by using the specific PKA inhibitor H88 (as shown in Fig. 5B). In 293-27-2 cells NF-κB interaction with its motifs and HIV-1 LTR-driven reporter gene expression were measured in PMA (Fig. 7, upper panels)- and TNF-α (Fig. 7, lower panels)-stimulated cells. H88 did not significantly influence either of these parameters of NF-κB action in these cells. The low level of H88-mediated inhibition observed was not concentration-dependent. Similarly, as shown in Fig. 6, H88 did not influence HIV-1 LTR-driven CAT activity in PMA- or TNF-α-stimulated Jurkat cells transiently transfected with pHIV-1 LTR-CAT plasmid. These results substantiate the conclusion of the experiments with calphostin C, that the PMA- or TNF-α-mediated activation of NF-κB was catalyzed primarily by PKC in both the cell types used in this investigation.
Similar to results found with H7 in the cell-free system, in whole cells this compound exhibited inconsistent and low-level inhibition of both kinases at overlapping inhibitory concentrations (data not shown). Therefore, H7 was not useful in further elucidating the pathway of PMA or TNF-α stimulation in whole cells.
PTX, a widely used drug for the treatment of peripheral blood disorders in humans, inhibits HIV-1 LTR activation and viral replication by blocking the trans-activation by NF-κB (3). In these previous experiments with whole cells, the exact site of action of the drug in the multistep NF-κB trans-activation process was not identified. To elucidate this question, several steps of the NF-κB trans-activation process were analyzed by a combination of experiments conducted with a cell-free system and in whole cells. The cell-free system allowed us to identify one site of action of PTX as the kinase-mediated activation of NF-κB. These results, however, do not rule out the possibility that PTX may inhibit additional events in the multistep pathway of NF-κB-mediated activation of HIV-1 LTR. Recognizing that any cell-free system is subject to uncertainties of altered conditions in extracts versus in vivo, we studied the specific event of protein kinase mediated NF-κB activation both in cell-free system and in whole cells and obtained consistent results in both of these working systems.
The kinases involved in activation of NF-κB in whole cells have been studied by other investigators using less specific inhibitors at overlapping inhibitory concentrations (see Introduction). Our results suggest that H7 preferentially inhibited PKC over PKA in a cell-free system and in whole cells. But on the basis of these experiments the activation pathway of NF-κB by PKC versus PKA could not be conclusively established. Results obtained with this inhibitor suffer in general from low specificity and potential inhibition of yet other kinases of which there are a very large number.
The conclusions reached here were obtained by using two relatively specific protein kinase inhibitors, calphostin C and H88, which showed stringent specificity on one or the other kinase as shown by their inhibitions in extracts of the NF-κB-IκB complex as substrate. The specificity of inhibitory action of calphostin C observed in a cell-free system, the drug’s inhibition of both NF-κB binding to its motif, and HIV-1 promoter function in whole cells suggest that the critical step of the physiological activation by TNF-α is a PKC-catalyzed release of active NF-κB. This conclusion is further corroborated by results with the PKA-specific inhibitor H88, which did not influence PMA- or TNF-α-induced activation of NF-κB and HIV-1 LTR-driven gene expression, suggesting that PKA does not play a major role in this process, at least in the two cell types used in this investigation. H88, however specifically inhibited PKA in a cell-free system, showing that the compound is active. That a low, concentration-independent inhibition of reporter gene activity was detected (Fig. 7), and that H88 at higher concentrations was cytotoxic (data not shown) suggest that H88 was taken up by these cells. Inhibition of the activation of NF-κB mediated via a kinase other than PKC such as Raf-1 in whole cells, is not eliminated. However, the pattern of inhibition of PKC by PTX is similar to that observed with a structurally dissimilar compound, calphostin C, which is a specific inhibitor of this enzyme, thereby validating this conclusion that the inhibitory influence of PTX is on PKC in whole cells. Recent results of Ratner and Bandres (45) on the suppressive action of the viral protein nef in T cells, which is also mediated by down regulation of NF-κB action via a PKC-dependent mechanism, further substantiate the role of PKC in NF-κB activation and regulation of viral gene expression.
PTX is unlikely to prevent dissociation of phosphorylated IκB from NF-κB because specificity of such a mechanism would not be expected to show the similar results observed with structurally different kinase inhibitors. Inhibition of NF-κB transport into the nucleus is possible but is not involved in the cell-free system. PTX was shown not to interfere directly with the binding of active NF-κB to its motif.
As expected, PMA-induced activation of NF-κB is catalyzed by PKC. The novel aspect of these results is the role of PKC in the physiologically more relevant activation by TNF-α. PTX can inhibit PKC-catalyzed activation of NF-κB, and thus may in part provide a mechanism for the previously reported anti-NF-κB and anti-HIV-1 action of PTX (2,3).
We have consistently observed a lack of strict correlation between effects on NF-κB interaction with its motif versus HIV-1 promoter-driven reporter gene expression (3). The inhibitory influence of PKC-specific calphostin C on NF-κB interaction with its motifs was about 80%, as compared with an inhibition of HIV-1 promoter function of only 30-50% (Fig. 7). A noninhibitable basal level of NF-κB in PMA- or TNF-α-treated cells might limit maximal inhibition. This is further corroborated by our observation that the basal NF-κB binding activity and HIV-1 LTR-driven reporter gene activity in unstimulated cells could not be completely eliminated by either of these PKC inhibitors. An alternative possibility is that an HIV-1 promoter activation pathway independent of NF-κB might be involved. However, mutations in the NF-κB motifs eliminated most of the HIV-1 promoter activity, suggesting that NF-κB-mediated activation of HIV-1 LTR is the major pathway in these cells (3).
The PKC inhibitors may also act on virus replication at other sites and activations of other trans-activators. Possible additional interventions of PTX at steps upstream of NF-κB activation by kinases was not eliminated. Diaz-Meco et al. (46) reported the involvement of a dominant negative protein kinase C subspecies in the activation of NF-κB. The mechanism of the inhibitory action of PTX on PKC and the role of different subspecies of this enzyme was not addressed in this investigation.
The results of this study support the potential of kinase C inhibitors as anti-HIV-1 drugs. The synthetic compound Gö 6976, a specific inhibitor of PKC, inhibits HIV-1 induction with little cell toxicity and without the development of resistance (35). PTX and calphostin C as well as this compound could down regulate HIV-1 gene expression and viral replication with little toxicity to cells. PKC-mediated phosphorylation of many cellular key compounds plays a major role in signal transduction. Thus inhibition of such an important cellular function will be deleterious to the cells. However, they might be used in lower noncytotoxic concentrations in combination with other agents such as Tat (trans-activator of transcription) inhibitors, in light of our recent observation on the cooperative action of the Tatinhibitor Ro24-7429 and the NF-κB inhibitor PTX to down-regulate viral gene expression (42).
The authors thank Dr. L. A. Herzenberg of Standard University for providing the 293-27-2 cells. This research was supported by grants from Hoechst-Roussel Pharmaceuticals Inc., Somerville, NJ, U.S.A., and by a Center for AIDS Research Award to the Dana-Farber Cancer Institute. B.J.D. is a Clinical Oncology Career Development awardee of the American Cancer Society.
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