Glutathione Depletion Associated with the HIV-1 TAT Protein Mediates the Extracellular Appearance of Acidic Fibroblast Growth Factor

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 351, No. 1, March 1, pp. 17–26, 1998 Article No. BB970566

Glutathione Depletion Associated with the HIV-1 TAT Protein Mediates the Extracellular Appearance of Acidic Fibroblast Growth Factor1 Susan R. Opalenik,* Qiang Ding,* Susan R. Mallery,† and John A. Thompson*,‡,2 *Department of Surgery and ‡Department of Biochemistry/Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294; and †Department of Oral Maxillofacial Surgery and Pathology, College of Dentistry and Medicine, Ohio State University, Columbus, Ohio 43210

Received September 2, 1997, and in revised form December 8, 1997

Primary murine embryonic fibroblasts transfected with HIV-1 TAT demonstrated decreased levels of high energy phosphates (ATP, GTP, UTP/CTP), adenine nucleotides (ATP, ADP, AMP), and both NAD//NADH redox pairs, resulting in a substantial loss of redox poise. A greater than 50% decrease in intracellular reduced glutathione (GSH) concentration was accompanied by the extracellular appearance of acidic fibroblast growth factor (FGF-1). Addition of either N-acetyl-Lcysteine or glutathione ester (GSE), but not L-2-oxothiazolidine 4-carboxylate, partially restored intracellular GSH levels and resulted in loss of extracellular FGF-1. Treatment of FGF-1-transduced cells with buthionine sulfoximine (BSO) resulted in a time- and dose-dependent decrease in total cellular GSH concentration that was accompanied by the extracellular appearance of FGF-1. Inclusion of GSE during BSO treatment eliminated the extracellular appearance of FGF1. BSO treatment of cells transfected with a mutant form of FGF-1, in which all three cysteine residues were replaced with serines, also decreased total cellular GSH concentration but failed to induce the extracellular appearance of FGF-1. Collectively, these results suggest that HIV-1 TAT induces a condition of oxidative stress, which mediates cellular secretion of FGF-1, an observation relevant to the pathophysiologic development and progression of AIDS-associated Kaposi’s sarcoma. q 1998 Academic Press

Key Words: HIV-1 TAT; FGF-1; oxidative stress; glutathione.

AIDS-associated Kaposi’s sarcoma (AIDS-KS)3 is a highly vascularized lesion composed of recruited fibroblasts, inflammatory, and endothelial cells (24, 26, 39, 53), involving cutaneous as well as visceral tissues (24, 26). Mounting experimental evidence suggests that KS arises from the altered expression of inflammatory mediators, which results in a shift from a temporary, physiologic response to the uncontrolled cellular proliferation and angiogenesis associated with KS. Indeed, cells derived from AIDS-KS lesions demonstrate increased levels of both mRNA and extracellular protein encoding several growth-promoting factors (4, 8, 19, 42, 70), including acidic fibroblast growth factor (FGF-1). The human immunodeficiency virus-1 (HIV-1) TAT protein, a potent transactivator of viral gene transcription (11, 20, 58, 59), has been investigated for its ability to act as a selective transcriptional regulator of eukaryotic genes. Numerous in vitro studies (9, 21, 32, 40, 41, 46, 48, 65, 71, 74, 75) have demonstrated TAT to modulate expression of known inflammatory response genes, including FGF-1 (43). Following absorptive uptake (37) and prior to nuclear translocation (18), extracellular TAT can stimulate the growth of cells (32, 40), including those derived from human AIDS-KS lesions (16,

1

This work was supported by NIH Grants HL48457 (S.R.M.), HL448491 (J.A.T.), and HL45990 (J.A.T.). Predoctoral fellowship support was provided by the Helen Keller Research Foundation (Q.D.). 2 To whom correspondence should be addressed at 752 Lyons-Harrison Research Bldg., University of Alabama at Birmingham, 701 South 19th St., Birmingham, AL 35294. Fax: (205) 975-7549. E-mail: [email protected].

3 Abbreviations used: AIDS, acquired immunodeficiency syndrome; KS, Kaposi’s sarcoma; HIV-1, human immunodeficiency virus-1; FGF-1, acidic fibroblast growth factor; GSH, reduced glutathione; NAC, N-acetyl-L-cysteine; GSE, glutathione ester; OTC, L-2-oxothiazolidine 4-carboxylate; BSO, buthionine sulfoximine; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HBSS, Hank’s balanced salt solution.

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0003-9861/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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18). Furthermore, the introduction of the TAT gene into the germ line of mice under the control of the HIV LTR has yielded the appearance of dermal lesions that histologically resemble KS (5, 68). Collectively, these observations support the suggestion that HIV-1-infected inflammatory cells may release a polypeptide mitogen (TAT) in vivo which initiates a localized biological cascade of molecular events resulting in the subsequent disregulated production of growth factors and cytokines relevant to the initiation and perpetuation of the angiopathologic KS lesion (16). However, the mechanisms whereby TAT transactivates expression and extracellular presentation of these factors have not been elucidated fully. Previous studies from this laboratory have demonstrated the ability of TAT, following stable transfection of primary murine fibroblasts, to induce a transformed phenotype accompanied by the extracellular appearance of FGF-1 (43). Secretion of FGF-1, which lacks a classical signal peptide sequence, is particularly relevant to AIDS-KS pathophysiology since this growth factor serves as a potent mitogen for mesoderm- and neuroectoderm-derived cells in vitro (7, 22) and functions as a hormonal inducer of angiogenesis and neurogenesis in vivo (66, 67, 69). It is relatively well established that the biological activity of FGF-1 involves productive heparin sulfate proteoglycan-dependent cell surface interactions with specific high affinity receptors (43, 57). Under normal physiologic conditions, intracellularly sequestered FGF-1 does not appear to be available to mediate its full biological potential (57). Consequently, mechanisms whereby this cytosolic growth factor gains access to the extracellular environment become fundamental for understanding the regulation of FGF-1 biology. Besides HIV-1 TAT expression (43), recent studies also have demonstrated cellular secretion of FGF-1 in response to heat shock (28, 29) and serum starvation (57). Since heat shock and serum starvation responses overlap those induced by oxidative stress (30, 31, 60, 73), a condition recognized to be associated with most pathophysiologic conditions, including HIV-1 infection (61), the role of TAT as an inducer of oxidant stress was examined in vitro. We now report that HIV-1 TAT expression in primary murine fibroblasts results in depletion of reduced glutathione (GSH), the most abundant nonprotein thiol of the cell and the major nonenzymatic antioxidant defense system. In addition, several lines of evidence provide a first indication that this form of oxidant stress induces the secretion of FGF-1. These observations provide mechanistic insight relevant not only to regulation of FGF-1 biology but also to the development and progression of AIDS-KS. MATERIALS AND METHODS Cell culture. Primary murine fibroblasts were isolated and genetically engineered to express biologically active forms of the full-

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length (101 amino acids) HIV-1 TAT protein (from ARV-2), human FGF-1 (amino acids 21–154), or a mutant of FGF-1 (amino acids 21– 154), in which the three cysteine residues were replaced with serines (cys-free FGF-1), as previously described (43, 57, 64). Murine fibroblasts expressing either b-galactosidase (b-gal) or neomycin phosphotransferase (NPT) served as controls (43, 57). RT-PCR, Western, in situ immunohistochemical, heparin-binding, DNA synthesis, enzymatic activity, and transient transfection techniques were used to confirm transcription, translation, localization, and biologic functionality of individual transgenes (43, 57). Fibroblasts were maintained using DMEM (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated FBS (Hyclone Laboratories) and 100 units/100 mg penicillin/streptomycin (Life Technologies, Inc.). Where indicated, cells were incubated (16 h) in media (Hank’s balanced salt solution, HBSS) containing defined concentrations of buthionine sulfoximine (BSO), N-acetyl-L-cysteine (NAC), glutathione ester (GSE), or L-2oxothiazolidine 4-carboxylate (OTC), each from Sigma Chemical Co. Population doublings were monitored and only early, stabilized passages were used for these studies. Viability (trypan exclusion) of each cell population under defined conditions was determined by hemacytometer in duplicate, using five separate measurements per condition. Release of preincorporated [814 C]adenine (New England Nuclear) also was utilized as a measure of cytotoxicity. Cells were cultured (4 h) under normal conditions with 4 mM [8-14C]adenine and washed twice with fresh media prior to initiation of described treatments. The percentage of total [8-14C]adenine released into the media was determined as described (44). The proliferation index of individual cell populations was obtained by labeling (4 h) with 10 mM bromodeoxyuridine (BrdU, Sigma) and in situ immunohistochemical analysis (43, 57). Determination of cellular bioenergetic status and redox poise. Cells were seeded (1 1 104/cm2) and allowed to attach (16 h) under normal growth conditions. Cultures were washed (PBS), fed (72 h) DMEM containing 10% (v/v) FBS, harvested (0.25% trypsin, 0.5 M EDTA), counted, washed twice with PBS, and resuspended at a concentration of 4 1 106 cells in 500 ml of PBS. Cellular extractions were conducted essentially as described (36). Cellular levels of nucleotides and nucleosides were determined by high performance liquid chromatography (HPLC) on two independently prepared samples (25). Adenine nicotinamide nucleotides and respective nucleosides were separated on a Partisil 10 SAX column (Whatman) using a phosphate and pH gradient. Buffer A consisted of 0.01 M H3PO4 , pH 2.65; buffer B was 0.75 M KH2PO4 , pH 4.5, with detection at 254 nm. On many of the cellular samples, the UTP and CTP peaks coeluted. Therefore, the levels of these high-energy phosphates were expressed as the sum of total UTP plus CTP. Determination of cellular reduced glutathione (GSH) concentration. Cells were seeded, grown, and harvested as described (36). Three, 1-ml aliquots (4 1 106 cells/ml PBS) were centrifuged (2 min, 10,000 rpm, 47C) for each cell type. Cell pellets were resuspended in 125 ml 15% (w/v) trichloroacetic acid (TCA), incubated on ice for 10 min, and centrifuged (20 min, 14,000 rpm, 47C). Supernatants were transferred to fresh ice-cold microcentrifuge tubes. Cell pellets were reextracted with an additional 125 ml 15% (w/v) TCA and respective supernatants combined. Cell pellets were retained for protein determination using the Bradford assay (Pierce Immunochemicals). To neutralize the TCA, 130 ml of 4 M K2HPO4 (47C) was added to the combined supernatants. Samples were maintained on ice and analyzed within 2 h of extraction. Cellular levels of GSH were determined enzymatically (1). Individual supernatants (50 ml) were added to a cuvette containing 500 ml of 50 mM KH2PO4 , 50 mM KH2PO4 , and 2 mM CDNB (Sigma), pH 7.0, and the baseline absorbance at 340 nm was recorded (Perkin– Elmer–Lamda 3B). Glutathione transferase (Sigma) was added (0.5 unit), the samples were mixed, incubated at 257C for 15 min, and the absorbance at 340 nm was recorded. GSH concentrations were determined by comparison with a GSH (Sigma) eight point standard curve prepared concurrently.

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TAT-MEDIATED GSH DEPLETION AND FGF-1 SECRETION TABLE I

HPLC Nucleotide Analyses of Control- and TAT-Transfected Primary Murine Fibroblastsa Nucleotide

Control

TAT

AMP ADP ATP Total (AMP / ADP / ATP) NAD/ NADH Total (NAD/ / NADH) NADP/ NADPH Total (NADP/ / NADPH) GTP UTP / CTP Total (ATP / GTP / UTP / CTP) Energy charge Redox poise

0.59 { 0.04 2.17 { 0.03 41.86 { 0.78 44.62 { 0.86 9.28 { 0.31 6.79 { 0.13 16.07 { 0.44 0.18 { 0.01 0.17 { 0.00 0.35 { 0.02 9.67 { 0.18 16.49 { 0.32 78.02 { 1.28 0.96 0.74

0.31 { 0.09 1.65 { 0.17 33.27 { 1.51 35.23 { 1.77 7.96 { 0.32 3.69 { 0.22 11.64 { 0.54 0.37 { 0.04 0.38 { 0.04 0.75 { 0.08 6.34 { 0.31 11.04 { 0.49 50.65 { 2.31 0.97 0.49

Energy Charge Å a

[ATP] / 1/2 [ADP] [ATP] / [ADP] / [AMP]

Redox Poise Å

[NAD(P)H] [NAD(P)/]

All numbers are presented as nmol/mg { SD from triplicate analysis of two independently prepared samples.

Western analysis. Total proteins were extracted from individual cell populations and conditioned media using heparin-Sepharose (Pharmacia) chromatography as previously described (43, 57). Affinity-extracted proteins were fractioned either under reducing or nonreducing conditions using routine 15% (w/v) SDS–PAGE or 15% (w/ v) limited SDS–PAGE, respectively (43, 57). Gels were transferred electrophoretically to polyvinylidene difluoride membranes (Immobilon-P, Millipore) and incubated with an affinity-purified polyclonal antibody (1 mg/ml) against FGF-1 (55). As a control for FGF-1 specificity, the polyclonal antibody was preincubated (16 h, 47C) with a 100-fold molar excess of recombinant protein (23), a process that completely blocked immunoblot staining. Membranes were probed with HRP-conjugated goat anti-rabbit serum (1:25,000; Kirkegaard and Perry Laboratories) and incubated with chemiluminescent substrates for development (43, 57). Recombinant monomeric FGF-1 (23) or high molecular weight FGF-1 complexes generated by Cu2/ oxidation (15) served as semiquantitative immunoreactive markers for individual Western analyses. Statistics. Data were compiled and analyzed with the assistance of the StatView (Abacus Concepts, Inc.) statistical software package. The student’s paired t test was used for comparing data obtained from experimental cell populations to that of appropriately matched controls.

RESULTS

In contrast to control transfectants, primary murine fibroblasts engineered to express HIV-1 TAT demonstrated both aggressive growth behavior and spindlelike morphology characterized by a tendency toward foci formation, increased cell motility, and a general loss of anchorage-dependent and contact-inhibited growth. These observations were consistent with previous results (43). HPLC nucleotide analyses were conducted to determine the bioenergetic status of control and TAT transfectants maintained under normal culture conditions (Table I). Several similar findings were

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observed: (a) ATP, due to its recognized role as the primary cellular energy reserve, was the main triphosphate in both cell populations; (b) both control- and TAT-transfected cells possessed a high energy charge (ú0.96), indicative of ongoing, active oxidative metabolism; and (c) NAD//NADH represented the primary nicotinamide redox pair in both cultures. While similarities among the nucleotide profiles were apparent, distinct differences of other components between the two culture groups emerged. Relative to control cells, TAT-transfected fibroblasts contained substantially decreased levels of each high energy phosphate (ATP, GTP, UTP/CTP), each adenine nucleotide (ATP, ADP, AMP), and both reduced and oxidized nicotinamide nucleotides. Differences in the ratio of reduced and oxidized forms of nicotinamide-containing nucleotides (redox poise) demonstrated that control cells exhibited a more reduced status (0.74) in comparison to a more oxidized environment observed with TAT-transfected cells (0.49). Measurement of cellular GSH concentration revealed significant (Põ0.05) differences between control and TAT-transfected cell populations (Fig. 1). Under normal growth conditions, control cells exhibited a GSH concentration (48.42 { 2.97 nmol/mg protein) comparable to that previously reported for dermal fibroblasts (36). In contrast, TAT-transfected fibroblasts demonstrated a greater than 50% decrease in cellular GSH concentration (24.45 { 6.57 nmol/mg protein), an observation consistent with the more oxidized environment demonstrated by the HPLC nucleotide analyses. TAT-transfected cells maintained 16 h in Hank’s balanced salt solution (HBSS) also demonstrated signifi-

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FIG. 1. Analysis of major nonenzymatic antioxidant defense system in transfected murine fibroblasts: intracellular levels of total reduced glutathione were determined in both control (black) and TAT-transfected (white) primary embryonic murine fibroblasts. Values are expressed as nmol GSH/mg protein { standard deviations.

cantly (Põ0.05) decreased levels (20.08 { 2.96 nmol/ mg protein) of GSH compared to controls (37.53 { 1.22). Addition of 5 mM OTC every 4 h to the culture media (HBSS) did not result in a significant change in total GSH concentration in the TAT cells (22.00 { 0.67 nmol/ mg protein). However, addition of either 5 mM NAC every 4 h or 1 mM GSE every 2 h to the HBSS during the 16-h incubation resulted in a greater than 44% increase in cellular GSH concentration (28.90 { 1.68 and 29.43 { 1.66 nmol/mg protein, respectively). Previous results (43) demonstrated that TAT-transfected murine fibroblasts, maintained in DMEM and 10% (v/v) FBS, induced the secretion of FGF-1 as latent, high molecular mass complexes requiring reducing agents to activate full biological activity. The ability to monitor the extracellular appearance of the intrinsic growth factor under defined small-scale culture conditions was related to the observation that TAT induced increased levels of endogenous, murine FGF-1 mRNA and protein. In studies reported here, Western analysis of affinity-extracted media (HBSS) conditioned (16 h) by TAT-transfected cells also demonstrated the extracellular appearance of high molecular mass complexes of FGF-1. Treatment of these structures with the reducing agent DTT (0.1 M) and heat (907C, 10 min) generated the appearance of native FGF-1 migrating as a single band with a representative molecular mass (Fig. 2, lane 1). Treatment of the high molecular mass FGF1 complexes with 10 mM GSE or 50 mM each of either NAC or OTC had no apparent effect on electrophoretic mobility (data not shown). Addition of either 5 mM NAC every 4 h or 1 mM GSE every 2 h to the HBSS during the 16-h incubation resulted in the loss of extra-

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cellular FGF-1 (Fig. 2; lanes 2 and 4, respectively). In contrast, media (HBSS) conditioned (16 h) by TATtransfected cells treated with 5 mM OTC every 4 h demonstrated similar levels of extracellular FGF-1 (Fig. 2; lane 3). For more detailed analytical measurements, growth kinetics were evaluated by determining the proliferation index (BrdU incorporation) of TAT-transfected cells maintained (16 h) under defined culture conditions. The percentage of TAT-transfected cells labeled in HBSS (50–51%) was slightly higher than that previously (43) observed (40–43%) when cells were maintained in DMEM supplemented with 10% (v/v) FBS. Treatment of TAT-transfected cells maintained in HBSS with NAC and GSE resulted in approximately a 50% decrease in the labeling index (25–27%). In addition, treatment with these agents for 16 h resulted in partial restoration of the control phenotype, characterized by increased attachment, spreading, and cobblestone appearance of individual cells (Fig. 3). The morphological response to these treatments was dependent on constant presentation of the GSH supplementing agents as evidenced by the eventual reversion of the TAT-transfected cells to their characteristic transformed phenotype within 24 h. In contrast, treatment of TAT-transfected cells maintained (16 h) in HBSS with OTC had no apparent effect on either proliferation or phenotype. In contrast to TAT transfectants, normal murine fibroblasts express relatively low levels (4 ng/106 cells) of endogenous FGF-1 (57), suggesting that large scale cell culture would be required to study trafficking of the growth factor. To overcome this limitation, retroviral and eukaryotic expression vectors were designed to deliver human cDNA sequences, which would direct constitutive overexpression of intracellular FGF-1 (and mutant) as a biologically active protein. This approach should permit evaluation of growth factor partitioning in normal diploid fibroblasts under defined, small-scale culture conditions. The truncated form of the growth factor was chosen to allow discrimination between the

FIG. 2. Western analysis of medium conditioned by TAT-transfected cells: Heparin-affinity extracted proteins harvested from 16-h conditioned medium of TAT-transfected murine fibroblasts (5.0 1 107 cells/lane) were fractionated (15%, w/v, reducing SDS–PAGE) and analyzed using the affinity-purified antibody against FGF-1. Media contained either HBSS alone (lane 1) or HBSS supplemented with NAC (lane 2), OTC (lane 3), or GSE (lane 4). Recombinant preparations of FGF-110154 (0.2 mg) served as a positive control (lane 5).

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FIG. 3. In situ analysis of transfected murine fibroblasts: The phenotype of individual transfected cell populations (9 1 104/cm2) were maintained (16 h) under defined culture conditions and examined by phase-contrast microscopy (magnification, 1100). A; Control transfectants in HBSS; B; TAT transfectants in HBSS; and C; TAT transfectants in HBSS supplemented every 2 h with 1 mM GSE.

full-length endogenous murine and the transferred human gene product (57). Primary murine fibroblasts either transduced (retrovirally mediated) with FGF-1 (57) or transfected with cys-free FGF-1 (64) maintained both a constant, nontransformed cellular phenotype and normal, anchorage-dependent growth behavior. Western blot analysis (Fig. 4A; lane 2) demonstrated steady-state levels (approximately 40 ng/106 cells) of the single-copy, intracellular FGF-1 transgene that were consistent with previous observations (57). Compared to FGF-1-transductants, cys-free FGF-1-transfected cells demonstrated 8- to 10-fold higher steadystate levels (approximately 360 ng/106 cells) of the intracellular transgene (Fig. 4A; lane 3). Under these experimental conditions (analysis of 1–5 1 106 cells), Western blot analyses failed to detect endogenous murine FGF-1, an observation predicting 20 ng of FGF-1 to be below the limits of immunodetection. FGF-1 and cys-free FGF-1, affinity-extracted from the intracellular compartment of corresponding cells, displayed mitogenic behavior in a DNA synthesis assay (43, 57) similar to that obtained with recombinant FGF-1 isolated from Escherichia coli (23). Correlation of this biologic data with immunoblot analysis determined that these intracellular forms of FGF-1 retained greater than 90% of their mitogenic potential. To correlate the decreased cellular GSH concentration observed in TAT-transfected cells with secretion of FGF-1 by oxidant stress, GSH synthesis in FGF-1transduced and cys-free FGF-1-transfected cells was inhibited with BSO, a specific inhibitor of g-glutamylcysteine synthetase (49). Treatment of FGF-1 transductants (Fig. 5) and cys-free FGF-1 transfectants demonstrated a time- and dose-dependent decrease in total cellular GSH concentration. Compared to untreated cells (37.53 { 2.45 nmol/mg protein), FGF-1-transduced fibroblasts demonstrated an 81.9% decrease in total cellular GSH concentration (6.80 { 1.45 nmol/mg protein) following 16 h of treatment with 100 mM BSO in HBSS. This observation confirms that these cells have a relatively fast turnover rate of endogenous GSH

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and depend on de novo synthesis to restore intracellular levels. When maintained in the absence or presence of varying BSO concentrations at the indicated time points, both FGF-1-transduced and cys-free FGF-1transfected cells released minimal levels of preloaded [8-14C]adenine into the extracellular compartment. Normalization of [8-14C]adenine counts to total cell number determined that less than 1.0% of these cells experienced cell death/damage. Assessment of trypan blue exclusion correlated well with this assay and confirmed the absence of significant cell death/damage (õ2%) for either of these individual cell populations throughout the time course of study. Western blot analysis of media (HBSS) conditioned (16 h) by FGF-1-transduced cells failed to demonstrate detectable levels of extracellular growth factor (Fig. 4B; lane 2). Addition of 100 mM BSO to the culture media during a 16-h incubation resulted in the extracellular appearance of FGF-1 migrating as multiple high molecular mass bands (Fig. 4B; lane 3). Treatment of the heparin-extracted, high molecular mass FGF-1 complexes with the reducing agent DTT (0.1 M) and heat (907C, 10 min) generated the appearance of FGF-1 migrating as a single band with a representative molecular mass (Fig. 4B; lane 4). The absence of full-length, intrinsic murine FGF-1 in the conditioned media (Fig. 4B) was both consistent with Western analysis of the intracellular compartment (Fig. 4A) and reflective of levels below detectable limits. Addition of 1 mM GSE every 2 h to the culture media (HBSS, 100 mM BSO) during the 16-h incubation resulted in the loss of extracellular FGF-1 as determined by Western analysis following 15% (w/v) SDS–PAGE under reducing (0.1 M DTT, 907C) conditions (Fig. 4B; lane 5). In contrast to FGF-1-transduced cells, media (HBSS) conditioned (16 h) by cys-free FGF-1 transfectants, either in the absence or presence of 100 mM BSO, failed to demonstrate the extracellular appearance of the cys-free FGF-1 transgene as determined by Western analysis following 15% (w/v) SDS–PAGE under reducing conditions (Fig. 4B; lanes 6 and 7, respectively).

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DISCUSSION

AIDS-associated KS is the most common HIV-related malignancy with an incidence of 20–30% (10). The reported absence of HIV genomic sequences in AIDS-KS biopsies (13, 54) and the prevalence of nonepidemic forms of KS suggests an indirect involvement of the virus in KS pathogenesis. Experimental evidence indicates that the HIV-1 TAT gene product, either released from acutely infected inflammatory cells or delivered as a recombinant protein, modulates the expression of various eukaryotic genes and promotes growth of responding cells in vitro (9, 17, 18, 21, 32, 40, 41, 43, 46– 48, 56, 65, 71, 74, 75). Therefore, TAT may function as

FIG. 4. Western analysis of individual cell populations under defined culture conditions: A; Heparin-affinity extracted proteins harvested from total cellular extracts of cells either transduced with FGF-1 (lane 2; 5 1 106 cells/lane) or transfected with cys-free FGF1 (lane 3; 1 1 106 cells/lane) were fractionated (15%, w/v, reducing SDS–PAGE) and analyzed using the affinity-purified polyclonal antibody against FGF-1. Recombinant preparations of FGF-121-154 (0.2 mg; lane 1) and FGF-11-154 (0.1 mg; lane 4) served as positive controls. B; Heparin-affinity extracted proteins (1 1 107 cells/lane) harvested from 16-h conditioned medium (HBSS) of either FGF-1-transduced (lanes 2 to 5) or cys-free FGF-1 cells (lanes 6 and 7) were fractionated (15%, w/v, limited, nonreducing SDS–PAGE) and analyzed using the affinity-purified polyclonal antibody against FGF-1. The inclusion (/) of DTT and heat (907C) to the sample buffer and either BSO or GSE to the medium are indicated. Recombinant preparations (0.2 mg) of FGF-121-154 in the absence (lane 1) or presence (lane 8) of Cu/2 oxidation served as positive controls.

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FIG. 5. Kinetics of BSO-induced inhibition of GSH synthesis. Intracellular GSH levels (nmol/mg protein) were analyzed in FGF-1-transduced cells (4 1 106) at different time points (h) following treatment with varying doses (mM) of BSO. Each bar represents the average value { standard deviation following triplicate analysis of three independent experiments.

a link between HIV infection and convergent biological pathways responsible for the pathogenesis of KS. Oxidative stress has been suggested to be a common biologic process participating in many disease states, including those mediated by inflammation (38), HIV1 infection (6, 61), and AIDS-KS (33, 61). Previous studies have demonstrated that HIV patients, HIVinfected lymphocytes, and cultures obtained from AIDS-KS biopsies display intracellular biochemical characteristics indicative of ongoing oxidative stress (38, 61) and a decreased capacity for cytoprotection against oxidant challenge (36). An association between TAT expression and oxidant stress was first indicated with stably transfected HeLa cells, which demonstrated significant decreases in MnSOD mRNA and enzyme activity (21) as well as intracellular levels of reduced glutathione (72). Several results obtained from the experimental models described here provide additional insight into the pathophysiological consequences of intrinsic TAT expression beyond that previously reported. Compared to control cells, TAT-transfected fibroblasts demonstrated: (i) a normal high energy charge; (ii) decreased levels of high energy phosphates, adenine nucleotides, and NAD//NADH pairs; (iii) attenuated redox poise; and (iv) significantly reduced concentrations of the antioxidant tripeptide, glutathione. Collectively, these observations provide additional in vitro evidence that constitutive expression of TAT induces oxidant stress following attenuation of the major nonenzymatic antioxidant defense system within responding cells. Furthermore, the ability of TAT to induce cellular depletion of reduced glutathione is relevant to the pathogenesis of AIDS since HIV-infected patients (3, 12, 14, 27, 50, 52, 62), HIV-infected cell cultures (3, 51, 63), and AIDS-KS-derived cells (36) all demonstrate decreased concentrations of reduced glutathione. It appears unlikely that TAT-induced depletion of reduced glutathione is related directly to the observed aggressive growth behavior exhibited by these trans-

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fected cells (43). Typically, cell cycle progression of both fibroblasts and endothelial cells in culture are associated with increased levels of GSH and ATP (34, 35). Even though TAT-transfected cells maintained a high-energy charge, they contained markedly lower concentrations of ATP and had become more glycolytic. This observation is consistent with the biochemistry of many malignant cells, whereby neoplasia results in a shift from aerobic to anaerobic metabolic pathways (45). In the absence of adequate ATP production, glutathione synthesis also would be limited since generation of 1 mol of GSH requires 2 mol of ATP (Fig. 6A). Furthermore, the recycling of GSH from its oxidized disulfide form (GSSG) by glutathione reductase may be compromised in TAT-transfected cells (Fig. 6B). This process requires reducing equivalents from nicotinamide containing moieties [NAD(P)H], which are limited in TAT-transfected cells. Consequently, the observed depletion of GSH in TAT-transfected cells may reflect alterations in both the synthesis and recycling pathways. As expected, glutathione monoester, which is readily transported into cells and processed to glutathione (2), partially restored GSH concentrations in TAT-transfected cells. The inability of OTC to replenish glutathione levels in TAT transfectants would be consistent with altered GSH formation. Following cellular uptake, OTC is converted into cysteine and subsequent production of GSH is dependent upon intact synthesis machinery (49), which may be impaired by the observed limited supply of intrinsic ATP. In contrast, treatment of TAT-transfected cells with NAC resulted in an increase in GSH concentrations. NAC is distinct from OTC in that, besides providing cysteine for GSH synthesis, it also functions as a free radical scavenger to restore intracellular GSH in a thiol-dependent manner (51). The differential effects of these agents further

FIG. 6. Glutathione synthesis pathway. Overall enzymatic production of intracellular reduced glutathione (GSH) includes both biosynthesis (A) and regeneration (B) pathways involving corresponding enzymes and cofactors. Buthionine sulfoximine (BSO), a specific inhibitor of g-glutamylcysteine synthetase (X), was used experimentally to prevent biosynthesis of GSH.

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suggest that TAT-induced perturbations of the intracellular environment favor the formation of oxidized species by a thiol-dependent process. In response to heat shock (28), serum starvation (57), and HIV-1 TAT expression (43), FGF-1 is released from cells as multiple, high molecular mass complexes requiring reduction of oxidized cysteine residues to restore full mitogenic potential. These observations coupled with the specific involvement of FGF-1 cysteine residues during temperature-induced release of this growth factor (29), suggest a common FGF-1 secretory pathway responding to biologic stress. Since serum deprivation and hypothermic responses overlap those of oxidative stress (30, 31, 60, 73), we previously suggested that the intracellular redox state may play a role in the FGF-1 secretion pathway (43, 57). Indeed, in studies described here, constitutive expression of TAT significantly decreased both intracellular redox poise and concentrations of GSH, observations which coincided with the extracellular appearance of FGF-1. Exposure of TAT-transfected cells to GSE and NAC partially restored GSH levels, decreased their growth advantage (proliferation index), partially reverted their transformed phenotype, and prevented the extracellular appearance of detectable FGF-1. In contrast, treatment with OTC had no effect on GSH concentration, growth, phenotype, or levels of extracellular FGF-1. Unlike NAC, OTC will not function as a free radical scavenger since its sulfur is masked (49) and not available as a free thiol. It is unlikely that GSE and NAC treatments merely reduced the extracellular FGF-1 complexes thereby decreasing steady state levels following receptor-mediated uptake. This potential would be inconsistent with the observed reduction in growth rate and partial reversion of phenotype. Moreover, GSE and NAC, even at higher concentrations than those added to the culture media, were unable to reduce these high molecular mass complexes. A more likely interpretation of these collective observations is that in response to oxidative stress induced by HIV-1 TAT, FGF1 is secreted in a thiol-dependent manner. Additional evidence for this potential was established in primary murine fibroblasts constitutively expressing intrinsic FGF-1. BSO-depletion of intracellular GSH in FGF-1-transduced cells resulted in the extracellular appearance of FGF-1 as noncovalent, high molecular mass complexes that readily reduced (DTT) to an immunoreactive product with a representative molecular mass. Assessment of trypan blue exclusion and [8-14C]adenine release suggested that sublethal cell injury did not contribute to the release of FGF-1. Whereas the exact identity of these extracellular, high molecular mass FGF-1 complexes are not known, this result is consistent with previous observations in response to heat shock (28), serum starvation (57), and HIV-1 TAT expression (43). The major species of FGF-

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1 complexes detected in the immunoblot assay corresponded well with the reported molecular mass representative of the FGF-1 homodimer induced by copper oxidation of intrinsic cysteine residues (15). The ability of GSE to prevent the extracellular appearance of FGF1 induced by BSO treatment of FGF-1 transductants is consistent with observations in TAT-transfected cells and provides further evidence for oxidant stress as a mediator of FGF-1 secretion. The failure of cys-free FGF-1-transfected cells to secrete the transgene in response to BSO treatment is not the consequence of differential levels of gene expression, since cys-free FGF-1 transfectants expressed 8- to 10-fold more recombinant protein compared to FGF-1 transductants. Rather, this observation is more consistent with the suggestion (29) that the unconventional FGF-1 secretion pathway involves oxidation of intrinsic cysteine residues. Collectively, these results predict that HIV-1 TAT induces a condition of oxidative stress (e.g., GSH depletion) which mediates the extracellular appearance of FGF1. Whether TAT-induced, FGF-1 secretion functions in vivo as a mechanism relevant to the pathophysiologic development and progression of AIDS-Kaposi’s sarcoma remains the focus of ongoing efforts.

9.

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ACKNOWLEDGMENTS The authors thank Dr. LeeAnn MacMillan-Crow for helpful suggestions, Xiofen Liu for expert technical assistance, and Marlis Richardson for secretarial support. We also thank Dr. Francesca Tarantini (American Red Cross) for the cys-free FGF-1 cDNA construct. These studies were performed in partial fulfillment of the requirement for the Ph.D. degree from the Departments of Medical Genetics (S.R.O.) and Molecular Pathology (Q.D.) at the University of Alabama at Birmingham.

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