Inhibition of Protein Geranylgeranylation Causes a Superinduction of Nitric-oxide Synthase-2 by Interleukin-1beta in Vascular Smooth Muscle Cells*

(Received for publication, January 29, 1997, and in revised form, March 13, 1997)

Jonathan D. Finder Dagger , Jennifer L. Litz Dagger , Michelle A. Blaskovich §, Terence F. McGuire §, Yimin Qian , Andrew D. Hamilton , Paul Davies § and Saïd M. Sebti §par

From the Departments of § Pharmacology, Dagger  Pediatrics, and  Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Recently, we have designed farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298) that selectively block protein farnesylation and geranylgeranylation, respectively. In this study, we describe the opposing effects of these inhibitors on interleukin-1beta (IL-1beta )-stimulated induction of nitric-oxide synthase-2 (NOS-2) in rat pulmonary artery smooth muscle cells (RPASMC) and rat hepatocytes. Pretreatment of cells with GGTI-298 caused a superinduction of NOS-2 by IL-1beta . RPASMC treated with GGTI-298 (10 µM) prior to IL-1beta (10 ng/ml) expressed levels of NOS-2 protein five times higher than those exposed to IL-1beta alone. This superinduction of NOS-2 protein by pretreatment with GGTI-298 resulted in nitrite concentrations in the medium that were 5-fold higher at 10 ng/ml IL-1beta and 10-fold higher at 1 ng/ml IL-1beta . Furthermore, NOS-2 mRNA levels in RPASMC were also increased 6- and 14-fold (at 10 and 1 ng/ml IL-1beta , respectively) when the cells were pretreated with GGTI-298. In contrast, treatment of cells with the inhibitor of protein farnesylation, FTI-277 (10 µM), blocked IL-1beta -induced NOS-2 expression at mRNA and protein levels. Pretreatment with lovastatin, an inhibitor of protein prenylation, resulted in superinduction of NOS-2. This superinduction was reversed by geranylgeraniol, but not by farnesol, further confirming that inhibition of geranylgeranylation, not farnesylation, is responsible for enhanced NOS-2 expression. The results demonstrate that a farnesylated protein(s) mediates IL-1beta induction of NOS-2, whereas a geranylgeranylated protein(s) represses this induction.


INTRODUCTION

Nitric oxide (NO) is a radical with important physiologic effects that include modulation of vasoreactivity, prevention of thrombosis, and neurotransmission (1). NO is the product of an enzymatic reaction catalyzed by nitric-oxide synthase (NOS).1 Mammalian cells express three isozymes of NOS, neuronal (NOS-1), endothelial (NOS-3), and inducible (NOS-2) (reviewed in Ref. 2). NOS-1 and NOS-3 are constitutively active, whereas NOS-2 is not present unless the cell is triggered to produce it. The induction of NOS-2 in cells exposed to cytokines and lipopolysaccharides is implicated in the pathophysiology of shock (3).

IL-1beta is an important cytokine that is released early in inflammation and shock (4). As a single agent, IL-1beta is capable of inducing NOS-2 at levels comparable to those induced by a mixture of cytokines and lipopolysaccharides (5). IL-1beta achieves its effects through signaling pathways that have not been fully defined (6), but that appear to include the mitogen-activated protein (MAP) kinase pathway since IL-1beta activates MAP kinase itself (7-10), at least in some systems, via the kinase (MAP kinase kinase) immediately preceding it (10-12). This effect on MAP kinase kinase implicates the small GTP-binding protein Ras since this triggers activation of the pathway by interacting with Raf, a serine/threonine kinase lying immediately upstream of MAP kinase kinase (13). IL-1beta is also capable of activating stress-activated protein kinase (14, 15) and p38 kinase (16), two kinases that occupy positions in pathways distinct from MAP kinase, but that are also potentially under the control of small G proteins such as Ras, Rho, and Rac (17).

Modulating the function of these small G proteins has recently become possible with inhibitors of prenyltransferases, the enzymes responsible for attaching prenyl lipids to the proteins as the first and most critical step in post-translational processing (18). The enzymes are farnesyltransferase (FTase) (19, 20), which attaches the 15-carbon farnesyl, and geranylgeranyltransferase I (GGTase I) (21, 22), which attaches the 20-carbon geranylgeranyl. Among proteins that are farnesylated are Ha-Ras and nuclear lamins; among those that are geranylgeranylated are Rap1A, RhoA, and Rac1. Some proteins that can be both farnesylated and geranylgeranylated include RhoB and Ki-Ras 4B (23). Because farnesylation and/or geranylgeranylation of small G proteins is critical for their function, we (24-28) and others (29-34) have designed potent and highly selective inhibitors of FTase. More recently, we have designed GGTase I inhibitors (35-37). FTase inhibitors have been shown to block the processing of oncogenic Ras, to reverse transformation, to induce the accumulation in the cytoplasm of inactive Ras-Raf complexes, and to inhibit murine and human tumor growth in vivo (24-34). Furthermore, FTase inhibitors are not toxic in vivo (25, 30), are less effective at inhibiting normal cell growth (29, 31), and do not inhibit growth factor (epidermal growth factor or platelet-derived growth factor) activation of MAP kinase (37-39). Much less is known about GGTase I inhibitors, but recently, we have shown that these agents antagonize the signaling of oncogenic Ki-Ras 4B (35), decrease levels of tyrosine phosphorylation of the platelet-derived growth factor receptor (37), and block fibroblasts in the G1 phase of the cell cycle (36). Very little is known about the effects of FTase and GGTase I inhibitors in cells other than fibroblasts. Recently, Singh et al. (40) showed that an inhibitor of FTase blocked IL-1beta -stimulated induction of NOS-2 and activation of MAP kinase in cardiac myocytes. Here we demonstrate that an inhibitor of GGTase I causes superinduction of IL-1beta -stimulated NOS-2 in smooth muscle cells and hepatocytes.


EXPERIMENTAL PROCEDURES

Cell Culture

The primary culture of rat pulmonary artery smooth muscle cells (RPASMC) has been described in detail previously (5). Briefly, explants of pulmonary artery were obtained by dissection following pentobarbital euthanasia of male Harlan Sprague Dawley rats and placed intimal surface down in plastic flasks containing growth medium (1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium, 10% fetal bovine serum, and penicillin/streptomycin). Cells were allowed to migrate out of the explants for 5-7 days before removal of the explants and subsequent subpassaging by trypsinization. For the studies reported here, cells were trypsinized, replated at ~106 cells/75-cm2 flask, and grown under standard conditions of 37 °C, 5% CO2, and 100% humidity. Cells were used through the fifth passage.

Hepatocytes were isolated from male Harlan Sprague Dawley rats using the in situ collagenase perfusion technique of Geller et al. (41). After isolation, hepatocytes were plated onto gelatin-coated Petri dishes (Corning Inc.) and grown in Williams' medium E (Life Technologies, Inc.) with the addition of L-arginine (0.50 mM), insulin (1 µM), HEPES (15 mM), L-glutamine, penicillin/streptomycin, and 10% low endotoxin fetal calf serum. Cells were incubated in growth medium for 24 h, and then serum-free Williams' medium E with the above additives was used for all experimental conditions.

Induction of NOS-2

This was achieved by treating cells with recombinant human IL-1beta (R&D Systems, Minneapolis, MN) at the concentrations indicated. After 24 h, the medium was harvested for determination of nitrite concentration, and the cells for assessment of NOS-2 RNA and protein levels.

Treatment of Cells with GGTI-298 and FTI-277

RPASMC were grown to 50% confluence (generally, 24-48 h after plating). Fresh medium was added containing either FTI-277 or GGTI-298 at the concentrations indicated. The cells were allowed to grow with inhibitors for 24 h. Stimulation of cells with IL-1beta took place in basal medium (Dulbecco's modified Eagle's medium and 0.1% bovine serum albumin) with the addition of FTI-277 or GGTI-298 where indicated.

Measurement of Nitrite

To quantify the amount of NO released from cells, nitrite, its stable product in aqueous solution, was measured by the Griess reaction. Nitrite concentration was measured by mixing an aliquot of cell supernatant with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) and incubating at room temperature for 10 min. The absorbance at 550 nm was measured, and nitrite concentration was determined using sodium nitrite as a standard.

Western Blotting for NOS-2

Cell monolayers were lysed in ice-cold buffer containing 50 mM Tris (pH 8.0), 110 mM NaCl, 5 mM EDTA, 1% Triton X-100, and the protease inhibitors antipain, pepstatin, leupeptin, chymostatin, and phenylmethylsulfonyl fluoride, and the lysate was transferred to a conical tube. Protein concentration was determined using the Bradford assay (Bio-Rad). Whole cell lysate was boiled in Laemmli buffer, and 100 µg of protein/lane was loaded on a 7.5% SDS-polyacrylamide gel and separated electrophoretically. Proteins were transferred to a nitrocellulose membrane overnight at 90 mA in a Bio-Rad Trans-Blot cell. For immunoblotting, the membrane was blocked with 10% nonfat dry milk in PBS for 1 h. The primary antibody was a polyclonal rabbit anti-murine macrophage NOS-2 (Transduction Laboratories, Lexington, KY) diluted 1:1000 in 1% nonfat dry milk and applied for 2 h. After washing three times in PBS containing 0.1% Tween 20 and 1% nonfat dry milk, the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Sigma) was applied at 1:5000 dilution. The blot was washed in PBS containing 0.3% Tween 20 and 1% nonfat dry milk three times over 30 min, and positive immunoreactivity was visualized using enhanced chemiluminescence reagents (ECL, Amersham International, Buckinghamshire, United Kingdom) and by exposure to photographic film.

Determination of Ras/Rap1A Processing

Cell lysates (50 µg/lane) were electrophoresed on a 12.5% SDS-polyacrylamide gel and then transferred to nitrocellulose as described above. The membrane was blocked in PBS-T (PBS with 0.1% Tween 20) containing 5% nonfat dry milk. After washing three times in PBS-T, the membrane was probed with Y13-238, an anti-Ras antibody, at 50 µg/ml in PBS-T containing 3% nonfat dry milk for 1 h at room temperature. The blot was washed as described above and then incubated with the secondary antibody (peroxidase-conjugated goat anti-rat IgG) at a dilution of 1:1000. Positive immunoreactivity was visualized using ECL. Processing of Rap1A was assessed in the same manner described for Ras except that the membrane was probed with anti-Rap1A antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and the positive antibody reactions were visualized using peroxidase-conjugated goat anti-rabbit IgG and ECL.

Isolation of RNA and Northern Analysis

Total RNA was extracted using the method described by Chomczynski and Sacchi (42). Aliquots containing 20 µg of total RNA underwent electrophoresis on 1% agarose gel containing 3% formaldehyde. RNA was transferred to a GeneScreen membrane (DuPont NEN) and UV-cross-linked. Membranes were hybridized overnight at 42 °C with a cDNA probe to murine macrophage NOS-2 (1-2 × 106 cpm/ml) labeled with [32P]dCTP (specific activity of 3000 Ci/mM; DuPont NEN) by random priming (Boehringer Mannheim). The hybridized filters were washed at 53 °C and analyzed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).


RESULTS AND DISCUSSION

FTI-277 and GGTI-298 Inhibit Selectively the Processing of Ras and Rap1A, Respectively, in RPASMC

Recently, we have designed and synthesized small organic molecules that mimic the carboxyl-terminal tetrapeptide CAAX of proteins that are farnesylated or geranylgeranylated. These peptidomimetics are potent and selective inhibitors of FTase and GGTase I in vitro, respectively. The peptidomimetic FTI-276 is 100-fold more selective for FTase over GGTase I (IC50 = 0.5 and 50 nM, respectively) (24), whereas GGTI-297 is more selective for GGTase I over FTase (IC50 = 50 and 200 nM, respectively) (36, 37). In this study, we treated RPASMC with the methyl ester prodrugs of FTI-276 and GGTI-297, FTI-277 and GGTI-298, which enter the cell more easily (24, 36). Fig. 1A shows that treatment of RPASMC with FTI-277 (0-10 µM), as described under "Experimental Procedures," resulted in a dose-dependent inhibition of Ras processing, with no effect on the processing of the geranylgeranylated protein Rap1A. In contrast, treatment of RPASMC with GGTI-298 (0-10 µM) resulted in inhibition of Rap1A processing, with no effect on Ras processing. These results demonstrate that FTI-277 and GGTI-298 are potent inhibitors of FTase and GGTase I in smooth muscle cells and are therefore efficacious in inhibiting cellular processing of farnesylated and geranylgeranylated proteins, respectively.


Fig. 1. Effect of FTI-277 and GGTI-298 on processing of Ras and Rap1A (A) and on IL-1beta -stimulated nitrite formation (B). A, cells were treated on each of 2 successive days with either FTI-277 or GGTI-298 and then harvested and subjected to Western analysis to demonstrate inhibition of processing by a band shift from the processed (p) to the unprocessed (u) protein. In lane 1, cells were untreated; in lanes 2-5, cells were treated with FTI-277 at 0.1, 1, 5, and 10 µM, respectively; and in lanes 6-9, cells were treated with GGTI-298 at the same concentrations. B, cells were pretreated for 24 h with FTI-277 (10 µM) or GGTI-298 (10 µM) or were left untreated. Fresh medium with or without FTI-277 or GGTI-298 and containing IL-1beta at 0, 1, 3, or 10 ng/ml was then added. After a further 24 h, the medium was harvested and assayed for nitrites. The curves show the response to IL-1beta of untreated cells (bullet ) or cells treated with FTI-277 (triangle ) or GGTI-298 (down-triangle). The data are representative of two (A) and four (B) independent experiments.
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Inhibition of Protein Geranylgeranylation Causes Superinduction of NOS-2 Protein and Nitrite Levels by IL-1beta in RPASMC and Rat Hepatocytes

To determine the role of geranylgeranylated proteins in the signal transduction pathways that mediate IL-1beta induction of NOS-2, we treated RPASMC and rat hepatocytes with the GGTase I inhibitor GGTI-298 prior to treatment with IL-1beta as described under "Experimental Procedures." Fig. 1B shows that in the absence of inhibitors, IL-1beta (0-10 ng/ml) induced a concentration-dependent, but modest, increase in the medium levels of nitrite from 1 µM basal levels to 12 µM after treatment with 10 ng/ml IL-1beta . Pretreatment with GGTI-298 (10 µM) caused a dramatic increase in IL-1beta -induced nitrite formation (from 4 to 52 µM). The medium of cells treated with 1 ng/ml IL-1beta alone accumulated nitrite levels of 4 µM, whereas the medium of cells treated with GGTI-298 prior to IL-1beta accumulated levels of nitrites that were more than 10-fold higher (42 µM) (Fig. 1B). In the absence of IL-1beta , GGTI-298 increased basal levels of NOS-2 to levels comparable to those obtained by stimulation of cells with 1 ng/ml IL-1beta alone. In contrast to the effects of GGTI-298, treatment of cells with FTI-277 inhibited IL-1beta -stimulated nitrite production (Fig. 1B).

The dramatic increase in the level of nitrite brought about by GGTI-298 could be due to direct activation of NOS-2 enzymatic activity or superinduction of NOS-2 protein. To determine the effects of GGTI-298 on NOS-2 protein levels, RPASMC were treated with GGTI-298 for 24 h and then stimulated with IL-1beta for a further 24 h as described under "Experimental Procedures." The cells were harvested, and the lysates were analyzed by Western blotting using a specific anti-NOS-2 antibody. Fig. 2A shows that in the absence of GGTI-298, significant induction of NOS-2 protein levels in RPASMC occurred only at 10 ng/ml IL-1beta , whereas in the presence of inhibitor, NOS-2 was induced at concentrations as low as 1 ng/ml. In RPASMC treated with 10 ng/ml IL-1beta , GGTI-298 enhanced the ability of IL-1beta to induce NOS-2 protein by 5-fold. A similar effect was demonstrated in hepatocytes (Fig. 2B).


Fig. 2. Effect of GGTI-298 on IL-1beta -stimulated expression of NOS-2 protein levels in RPASMC (A) and hepatocytes (B). Cells were treated with and without GGTI-298 in the manner described in the legend to Fig. 1B. Following 24 h of IL-1beta stimulation, cells were harvested, and NOS-2 protein levels were determined by Western analysis. The data are representative of four (A) and two (B) independent experiments.
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We next used an alternative method to demonstrate the involvement of geranylgeranylated proteins in the regulation of NOS-2 induction by IL-1beta . Recently, we have shown that treatment of NIH-3T3 cells with lovastatin inhibited the processing of both farnesylated and geranylgeranylated proteins, with a much more pronounced effect on geranylgeranylated proteins, but that treatment of these cells with a combination of lovastatin and either geranylgeraniol or farnesol restored protein geranylgeranylation or farnesylation, respectively (43). Applying this strategy, we observed that RPASMC treated with lovastatin alone were much more responsive to IL-1beta in inducing NOS-2 (Fig. 3). This lovastatin-mediated superinduction of IL-1beta -stimulated NOS-2 was reversed by geranylgeraniol, but not by farnesol. Lovastatin inhibition of the processing of Ras and of the geranylgeranylated protein Rap1A in RPASMC was reversed with farnesol and geranylgeraniol, respectively (Fig. 3). The above results confirmed that inhibition of protein geranylgeranylation is responsible for the hypersensitivity of RPASMC to IL-1beta induction of NOS-2. The results also suggest that inhibition of protein farnesylation is not involved in the superinduction of NOS-2 by IL-1beta .


Fig. 3. Effect of lovastatin supplemented with either farnesol or geranylgeraniol on IL-1beta -stimulated expression of NOS-2 protein in RPASMC. Cells were pretreated with lovastatin (20 µM) alone or with supplementary farnesol (30 µM) or geranylgeraniol (30 µM) for 24 h. Fresh medium under the same treatment conditions with and without IL-1beta (10 ng/ml) was then added. After a further 24 h, cells were harvested and subjected to Western analysis to demonstrate NOS-2 protein expression (A) and effects on processing of Ras (B) and Rap1A (C). Inhibition of processing is indicated by a shift from a lower processed (P) band to an unprocessed (U) band. In lanes 2-5, cells were stimulated with IL-1beta ; treatment conditions were as follows: no treatment (lanes 1 and 2), lovastatin (lane 3), lovastatin + geranylgeraniol (lane 4), and lovastatin + farnesol (lane 5). The data are representative of two independent experiments.
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FTI-277 Blocks IL-1beta Induction of NOS-2 in RPASMC and Rat Hepatocytes

The above results demonstrated that inhibition of protein geranylgeranylation causes a superinduction of NOS-2 by IL-1beta . We next determined the consequences of inhibiting protein farnesylation. RPASMC were pretreated for 24 h with FTI-277 prior to a 24-h treatment with various concentrations of IL-1beta (0-10 ng/ml). The cells were then harvested, and the levels of NOS-2 protein as well as the cumulative levels of nitrites in the cell-conditioned medium were determined as described under "Experimental Procedures." Fig. 4A shows that pretreatment of RPASMC with FTI-277 (10 µM) blocked IL-1beta induction of NOS-2 protein. Hepatocytes were treated similarly, but with slightly higher IL-1beta levels (0-50 ng/ml). Fig. 4B shows a similar effect in the hepatocytes, although the effect was not as complete.


Fig. 4. Effect of FTI-277 on IL-1beta -stimulated expression of NOS-2 protein. RPASMC (A) and hepatocytes (B) were treated with and without FTI-277 (10 µM) as described in the legend to Fig. 2 and stimulated with IL-1beta for 24 h at the concentrations shown. Cell lysates were then immunoblotted to determine the levels of NOS-2 protein as described under "Experimental Procedures." The data are representative of three (A) and two (B) independent experiments.
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GGTI-298 Enhances, whereas FTI-277 Blocks, the Ability of IL-1beta to Induce NOS-2 mRNA

We next determined whether the effects of GGTI-298 and FTI-277 on NOS-2 protein and nitrite levels were due to alterations at the mRNA level. RPASMC were treated with vehicle, IL-1beta , FTI-277, GGTI-298, and IL-1beta with either FTI-277 or GGTI-298, and the levels of NOS-2 mRNA were determined by Northern blotting as described under "Experimental Procedures." Stimulation of cells with IL-1beta showed a modest induction of NOS-2 mRNA (Fig. 5, lanes 1-3), whereas treatment of cells with FTI-277 or GGTI-298 alone had no detectable effect (lanes 4 and 7). Treatment of cells with FTI-277 blocked the ability of IL-1beta to induce NOS-2 mRNA (compare lanes 1-3 with lanes 4-6). In contrast, GGTI-298 enhanced the ability of IL-1beta to induce NOS-2 by 6- and 14-fold at 10 and 1 ng/ml IL-1beta , respectively (compare lanes 1-3 with lanes 7-9). Cyclophilin mRNA levels were not affected by the treatments and were similar in all lanes of Fig. 5.


Fig. 5. Effects of GGTI-298 and FTI-277 on IL-1beta induction of NOS-2 mRNA. RPASMC were treated with and without GGTI-298 or FTI-277 and stimulated with IL-1beta . Total RNA was isolated, and the levels of NOS-2 and cyclophilin mRNAs were determined by Northern blot analysis as described under "Experimental Procedures." Lane 1, vehicle; lanes 2 and 3, IL-1beta (1 and 10 ng/ml, respectively); lanes 4-6, FTI-277 (10 µM) and IL-1beta (0, 1, and 10 ng/ml, respectively); lanes 7-9, GGTI-298 (10 µM) and IL-1beta (0, 1, and 10 ng/ml, respectively). Data are representation of three independent experiments.
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The data presented in this report clearly demonstrate that IL-1beta induction of NOS-2 in RPASMC and rat hepatocytes is under the tight control of farnesylated and geranylgeranylated proteins. Whereas inhibition of protein farnesylation blocked NOS-2 expression, inhibition of protein geranylgeranylation had the opposite effect of dramatically enhancing NOS-2 expression. The results suggest that a farnesylated protein mediates IL-1beta induction of NOS-2, whereas a geranylgeranylated protein represses this induction. A potential candidate for mediating IL-1beta induction of NOS-2 is the Ras protein. This suggestion is consistent with the recent report by Singh et al. (40) that showed that IL-1beta activates the MAP kinase pathway and induces NOS-2 in cardiac myocytes and that this can be blocked by the FTase inhibitor BZA-5B. Therefore, Ras proteins that are farnesylated and are known to activate the MAP kinase pathway are good candidates for mediating IL-1beta induction of NOS-2. Among geranylgeranylated proteins that can serve to antagonize IL-1beta induction of NOS-2 are the small G proteins Rho and Rac. Although the involvement of these proteins in growth factor signaling is now well documented, their role in cytokine signaling is less clear. It is plausible that cytokines such as IL-1beta could activate both the Ras/MAP kinase (7-10) and the Rho/Rac/stress-activated protein kinase (14, 15) pathways. Integration of the MAP kinase and stress-activated protein kinase pathways in smooth muscle cells and hepatocytes would result in the net outcome of induction of NOS-2 expression. Inhibition of the stress-activated protein kinase pathway that depends on the geranylgeranylated RhoA and Rac proteins would tip the equilibrium in the direction of releasing the repressing pathways and superinducing NOS-2. On the other hand, inhibition of the MAP kinase pathways that depend on farnesylated proteins such as Ras would block NOS-2 induction. Regardless of the mechanism by which inhibition of protein geranylgeranylation causes superinduction of NOS-2, the consequences of this novel finding are of great therapeutic potential. We are presently evaluating the potential of GGTI-298 to reverse in animal models intimal hyperplasia associated with restenosis and atherosclerosis. Our ultimate goal is to prevent local hyperplasia that compromises the success of angioplasty and surgical bypass for obstructive vascular lesions.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA-67771 (to S. M. S.) and HD-28836-04 (to J. D. F.) and by a grant-in-aid from the American Heart Association, Pennsylvania Chapter (to P. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Drug Discovery Program, H. Lee Moffitt Cancer Center and Dept. of Biochemistry and Molecular Biology, University of South Florida, 12902 Magnolia Dr., Tampa, FL 33612. Tel.: 813-979-6734; Fax: 813-979-6748.
1   The abbreviations used are: NOS, nitric-oxide synthase; IL-1beta , interleukin-1beta ; MAP, mitogen-activated protein; FTase, farnesyltransferase; GGTase I, geranylgeranyltransferase I; RPASMC, rat pulmonary artery smooth muscle cells; PBS, phosphate-buffered saline.

ACKNOWLEDGEMENTS

We thank Dr. Timothy Billiar and Debbie Williams for providing the hepatocyte cultures.


REFERENCES

  1. Schmidt, H. H. H. W., and Walter, U. (1994) Cell 78, 919-925 [Medline] [Order article via Infotrieve]
  2. Morris, S. M., Jr., and Billiar, T. R. (1994) Am. J. Physiol. 26, E829-E839
  3. Szabo, C. (1995) New Horizons 3, 2-32 [Medline] [Order article via Infotrieve]
  4. Dinarello, C. (1994) FASEB J. 8, 1314-1325 [Abstract/Free Full Text]
  5. Nakayama, D. K., Geller, D. A., Lowenstein, C. J., Chern, H. D., Davies, P., Pitt, B. R., Simmons, R. L., and Billiar, T. R. (1992) Am. J. Respir. Cell Mol. Biol. 7, 471-476 [Medline] [Order article via Infotrieve]
  6. O'Neill, L. A. J. (1995) Biochim. Biophys. Acta 1266, 31-44 [CrossRef][Medline] [Order article via Infotrieve]
  7. Ahlers, A., Belka, C., Gestel, M., Lamping, N., Scott, C., Herrmann, F., and Brach, M. A. (1994) Mol. Pharmacol. 46, 1077-1083 [Abstract]
  8. Bird, T. A., Sleath, P., deRoos, P. C., Dower, S. K., and Virca, G. D. (1991) J. Biol. Chem. 266, 22661-22670 [Abstract/Free Full Text]
  9. Gould, G. W., Cuenda, A., Thomson, F. J., and Cohen, P. (1995) Biochem. J. 311, 735-738 [Medline] [Order article via Infotrieve]
  10. Guy, G. R., Chau, S. P., Wong, N. S., Ng, S. B., and Tan, Y. H. (1991) J. Biol. Chem. 266, 14343-14352 [Abstract/Free Full Text]
  11. Guesdon, F., Freshney, N., Waller, R. J., Rawlinson, L., and Saklatvala, J. (1993) J. Biol. Chem. 268, 4236-4243 [Abstract/Free Full Text]
  12. Saklatvala, J., Rawlinson, L. M., Marshall, C. J., and Kracht, M. (1993) FEBS Lett. 334, 189-192 [CrossRef][Medline] [Order article via Infotrieve]
  13. Hallberg, B., Rayter, S. I., and Downward, J. (1994) J. Biol. Chem. 269, 3913-3916 [Abstract/Free Full Text]
  14. Bird, T. A., Kyriakis, J. M., Tyshler, L., Gayle, M., Milne, A., and Virca, G. D. (1994) J. Biol. Chem. 269, 31836-31844 [Abstract/Free Full Text]
  15. Kracht, M., Truong, O., Totty, N. F., Shiroo, M., and Saklatvala, J. (1994) J. Exp. Med. 180, 2017-2026 [Abstract]
  16. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., and Saklatvala, J. (1994) Cell 78, 1039-1049 [Medline] [Order article via Infotrieve]
  17. Marshall, M. S. (1995) FASEB J. 9, 1311-1318 [Abstract/Free Full Text]
  18. Gutierrez, L., Magee, A., Marshall, C., and Hancock, J. (1989) EMBO J. 8, 1093-1098 [Abstract]
  19. Reiss, Y., Goldstein, J., Seabra, M., Casey, P., and Brown, M. (1990) Cell 62, 81-88 [Medline] [Order article via Infotrieve]
  20. Manne, V., Roberts, D., Tobin, A., O'Rourke, E., De Vigilio, M., Meyers, C., Ahmed, N., Kurz, B., Resh, M., Kung, H.-F., and Barbacid, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7541-7545 [Abstract]
  21. Moomaw, J., and Casey, P. (1992) J. Biol. Chem. 267, 17438-17443 [Abstract/Free Full Text]
  22. James, G., Goldstein, J., and Brown, M. (1995) J. Biol. Chem. 270, 6221-6226 [Abstract/Free Full Text]
  23. Casey, P. J., Moomaw, J. F., Zhang, F. L., Higgins, Y. B., and Thissen, J. A. (1994) Recent Prog. Horm. Res. 49, 215-238 [Medline] [Order article via Infotrieve]
  24. Lerner, E. C., Qian, Y., Blaskovich, M. A., Fossum, R. D., Vogt, A., Sun, J., Cox, A. D., Der, C. J., Hamilton, A. D., and Sebti, S. M. (1995) J. Biol. Chem. 270, 26802-26806 [Abstract/Free Full Text]
  25. Sun, J., Qian, Y., Hamilton, A. D., and Sebti, S. M. (1995) Cancer Res. 55, 4243-4247 [Abstract]
  26. Vogt, A., Qian, Y., Blaskovich, M. A., Fossum, R. D., Hamilton, A. D., and Sebti, S. M. (1995) J. Biol. Chem. 270, 660-664 [Abstract/Free Full Text]
  27. Qian, Y., Vogt, A., Sebti, S. M., and Hamilton, A. D. (1996) J. Med. Chem. 39, 217-223 [CrossRef][Medline] [Order article via Infotrieve]
  28. Bernhard, E. S., Kao, G., Cox, A. D., Sebti, S. M., Hamilton, A. D., Muschel, R. J., and McKenna, W. G. (1996) Cancer Res. 56, 1727-1730 [Abstract]
  29. Kohl, N. E., Mosser, S. D., deSolms, S. J., Giuliani, E. A., Pompliano, D. L., Graham, S. L., Smith, R. L., Scolnick, E. M., Oliff, A., and Gibbs, J. B. (1993) Science 260, 1934-1937 [Medline] [Order article via Infotrieve]
  30. Kohl, N. E., Omer, C. A., Conner, M. W., Anthony, N. J., Davide, J. P., deSolms, S. J., Giuliani, E. A., Gomez, R. P., Graham, S. L., Hamilton, K., Handt, L. K., Hartman, G. D., Koblan, K. S., Kral, A. M., Miller, P. J., Mosser, S. D., O'Neill, T. J., Rands, E., Schaber, M. D., Gibbs, J. B., and Oliff, A. (1995) Nat. Med. 1, 792-796 [Medline] [Order article via Infotrieve]
  31. James, G. L., Goldstein, J. L., Brown, M. S., Rawson, T. E., Somers, T. C., McDowell, R. S., Crowley, C. W., Lucas, B. K., Levinson, A. D., and Marsters, J. C., Jr. (1993) Science 260, 1937-1942 [Medline] [Order article via Infotrieve]
  32. Garcia, A. M., Rowell, C., Ackermann, K., Kowalczyk, J. J., and Lewis, M. D. (1993) J. Biol. Chem. 268, 18415-18418 [Abstract/Free Full Text]
  33. Patel, D. V., Gordon, E. M., Schmidt, R. J., Weller, H. N., Young, M. G., Zahler, R., Barbacid, M., Carboni, J. M., Gullo-Brown, J. L., Hunihan, L., Ricca, C., Robinson, S., Seizinger, B. R., Tuomari, A. V., and Manne, V. (1995) J. Med. Chem. 38, 435-442 [Medline] [Order article via Infotrieve]
  34. Bishop, W. R., Bond, R., Petrin, J., Wang, L., Patton, R., Doll, R., Njoroge, G., Catino, J., Schwartz, J., Windsor, W., Syto, R., Carr, D., James, L., and Kirschmeier, P. (1995) J. Biol. Chem. 270, 30611-30618 [Abstract/Free Full Text]
  35. Lerner, E. C., Qian, Y., Hamilton, A. D., and Sebti, S. M. (1995) J. Biol. Chem. 270, 26770-26773 [Abstract/Free Full Text]
  36. Vogt, A., Qian, Y., McGuire, T. F., Hamilton, A. D., and Sebti, S. M. (1996) Oncogene 13, 1991-1999 [Medline] [Order article via Infotrieve]
  37. McGuire, T. F., Qian, Y., Hamilton, A. D., and Sebti, S. M. (1996) J. Biol. Chem. 271, 27402-27407 [Abstract/Free Full Text]
  38. McGuire, T. F., Qian, Y., Blaskovich, M. A., Fossum, R. D., Sun, J., Marlowe, T., Corey, S. J., Wathen, S. P., Vogt, A., Hamilton, A. D., and Sebti, S. M. (1995) Biochem. Biophys. Res. Commun. 214, 295-303 [CrossRef][Medline] [Order article via Infotrieve]
  39. James, G. L., Brown, M. S., Cobb, M. H., and Goldstein, J. L. (1994) J. Biol. Chem. 269, 27705-27714 [Abstract/Free Full Text]
  40. Singh, K., Balligand, J. L., Fischer, T. A., Smith, T. W., and Kelly, R. A. (1996) J. Biol. Chem. 271, 1111-1117 [Abstract/Free Full Text]
  41. Geller, D. A., Nussler, A. K., DiSilvio, M., Lowenstein, C. J., Shapiro, R. A., Wang, S. C., Simmons, R. L., and Billiar, T. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 522-526 [Abstract]
  42. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  43. McGuire, T. M., and Sebti, S. M. (1997) Oncogene 14, 305-312 [CrossRef][Medline] [Order article via Infotrieve]

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