Biol Res 35: 169-176, 2002

Caveolin-1-mediated post-transcriptional regulation of
inducible nitric oxide synthase in human colon
carcinoma cells

EMANUELA FELLEY-BOSCO¹, FLORENT BENDER² AND ANDREW F.G. QUEST³

¹ Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland
² University of Pennsylvania, School of Dental Medicine, Department of Microbiology, 218, Levy Building, 4010 Locust Street, Philadelphia, PA 19104, USA
³ Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027,Santiago 7, CHILE

ABSTRACT

Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO) is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS) for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2+-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue), might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in humans.

Key terms: inducible nitric oxide synthase, proteosome_mediated proteolysis, nitroxysomes, cytokines, caveolae, caveolin-1

REACTIVE OXYGEN SPECIES IN CELLULAR SIGNALING

More recent developments in cell biology have lead to the perception that reactive oxygen species (ROS), including superoxide (O₂^-), nitric oxide (NO) and hydrogen peroxide (H₂O₂), may be employed not only as cellular tools of destruction, but also to serve as mediators of cellular signal transduction. For example, the increase in O₂^--derived H₂O₂ by epidermal growth factor receptor is found to inactivate protein-tyrosine phosphatase 1B by modifying the cysteine residue located at catalytic moiety (Lee et al., 1998a). Thus, H₂O₂ production indirectly regulates tyrosine phosphorylation levels on a large number of proteins implicated in signal transduction (Finkel, 2000). Alternatively, NO produced by endothelial cells is of central importance in tonus regulation of the vasculature, where it elevates cGMP levels in smooth muscle cells by binding to guanylate cyclase (Moncada et al., 1991). However, NO also reacts with O₂^- to form peroxynitrite, a powerful nitrating and hydroxylating reagent that exerts potent cytotoxic effects. For example, exposure of cells to NO together with O₂^- leads to mutation in the p53 tumor suppressor gene contributing to an endogenous mechanism of genomic alteration (Souici et al., 2000). Given the potential cytotoxicity of ROS, the need for stringent mechanims controlling their production is apparent. Here, we shall focus the discussion on regulation of nitric oxide synthases. Given the general emphasis of this symposium on the importance of complex formation and compartmentalization in cellular signaling, we will specifically highlight here the recent, unexpected discovery of a post-transcriptional mechanism controlling inducible nitric oxide synthase (iNOS) protein and activity levels that involves a detergent-insoluble membrane compartment in caveolin-1 expressing colon cancer cells.

NOS enzymes

NO is endogenously produced by NO synthases (NOS), a family of enzymes which currently includes three different isoenzymes in mammals (Griffith and Stuehr, 1995). iNOS is an inducible isoenzyme (iNOS) now known to be expressed in a wide variety of cell types. This isoenzyme may be distinguished from endothelial (eNOS) and neuronal NOS (nNOS), the other family members, by at least two criteria: first, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to eNOS or nNOS, is widely considered to occur at the transcriptional level only; second, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca²⁺-controlled NO production by the eNOS or nNOS (Marletta, 1994; Nathan and Xie, 1994; Forstermann et al., 1995). Due to this property, iNOS expression and activity have also been linked to a number of human pathologies and particularly cancer, since increased levels of NOS expression and/or activity have been observed in human breast (Thomsen et al., 1995), central nervous system (Cobbs et al., 1995), and colon tumors (Ambs et al., 1998a).

Cellular distribution of NOS enzymes

Within cells, NOS enzymes are generally evenly distributed between cytosol and membrane fractions. In the case of eNOS, presence at the membrane is linked to modification by palmitic acid residues, which not only target to the plasma membrane in general, but are also held responsible for protein accumulation in detergent-insoluble membrane rafts (Shaul et al., 1996). These membrane microdomains are rich in glycosphingolipids and cholesterol, which contribute to their insolubility in Triton X-100 at 4°C (Anderson, 1998; Okamoto et al., 1998), a property that has facilitated their isolation and biochemical characterization. Interestingly, such approaches revealed that these fractions of the plasma membrane contain a large number of signaling molecules, including small G-proteins, heterotrimeric G-proteins, src family kinases, PKCs and the aforementioned NOS (Anderson, 1998; Okamoto et al., 1998). A structural feature of these membrane fractions is that they may also contain caveolin-1 in which case they are detectable as flask-shaped plasma membrane invaginations of 50-100 nm, referred to as caveolae (Anderson, 1998; see also Bender et al., 2002). Caveolin-1 co-immunoprecipitates with eNOS in cultured bovine endothelial cells (Feron et al., 1996; Garcia-Cardena et al., 1996) and inhibits both eNOS and nNOS activity in vivo as well as in vitro via interaction with the NOS caveolin-binding motif (Garcia-Cardena et al., 1997; Ju et al., 1997; Michel et al., 1997; Rizzo et al., 1998).

The caveolin-1 scaffolding domain binding motif is conserved in all NOS isoenzymes

All caveolin proteins, including caveolin-1 possess a short cytosolic domain called the caveolin scaffolding domain, which is involved in the formation of caveolin oligomers. This region also mediates interactions with a variety of proteins, including heterotrimeric G-proteins, Src family tyrosine kinases and eNOS. These different signaling proteins, on the other hand, contain a caveolin binding motif (FxxxxFxxF, or FxFxxxxF, where F is aromatic amino acid Trp, Phe, or Tyr, Fig.1) and association with caveolin-1 leads to inhibition of their activity (Okamoto et al., 1998). This caveolin-binding sequence is conserved in all NOS sequences published to date in Gen-Bank including iNOS (Fig.1) and is located in the arginine binding site, close to the glutamate residue that forms a hydrogen bond with the guanidino group of arginine, the substrate of NOS (Crane et al., 1997; Crane et al., 1998). Although the caveolin-binding motif is also present in iNOS, data concerning the possibility that iNOS may interact with caveolins is highly controversial (Garcia-Cardena et al., 1997; Michel et al., 1997). This possibility is particularly interesting in the context of tumor biology, since caveolin-1 is suggested to function as a tumor suppressor gene in human breast, lung and colorectal cancer (Lee et al., 1998b; Racine et al., 1999; Bender et al., 2000). The hypothesis that caveolin-1 might function as a tumor suppressor by blocking iNOS function was rather appealing, since down-regulation of caveolin-1 would be expected to promote uncontrolled iNOS activity, genotoxic damage and tumor development.

In one set of experiments using extracts from the human colon cancer cell lines HT29 and DLD-1 treated with cytokines to induce iNOS expression and different glutathione-S-transferase (GST) _ caveolin-1 fusion proteins, we sought to determine whether caveolin-1 bound to iNOS, to study what the consequences of such an interaction were for iNOS and to identify the caveolin-1 sequence elements involved. In a second set of experiments, the same cell lines stably transfected with a plasmid permitting IPTG inducible expression of caveolin-1 were employed to determine whether and how caveolin-1 might modulate cellular iNOS levels.

Fig. 1. Schematic representation of NOS domains and localization of the caveolin binding domain. The caveolin-binding sequence (FxxxxFxxF, or FxFxxxxF, where F is aromatic amino acid Trp, Phe, or Tyr) is conserved in all NOS sequences.

In vitro binding of iNOS to GST-caveolin-1 fusion proteins is lost upon deletion of a region adjacent to the scaffolding domain

As stated above, caveolin-1 is known to interact directly with eNOS and nNOS. To determine whether this is also the case for iNOS, segments of caveolin-1 including residues 1-31, 1-101, 1-134, or 1-178 (full-length) were expressed as GST-fusion proteins in E. coli and purified by affinity chromatography on glutathione (GSH)-agarose. GST or GST-caveolin-1 fusion proteins bound to GSH-agarose beads were used for in vitro binding assays with cytosol from HT29 cells where iNOS expression was stimulated by the addition of IL-6, IFNg and IL-1ß (Felley-Bosco et al., 2000). Specific binding of iNOS was detected with the GST-caveolin-1 fusion proteins containing segments 1-134 and 1-178, while iNOS binding to either GST alone, GST-cav (1-31) or GST-cav (1-101) was either low or not detectable. Thus, despite also the presence of a highly conserved scaffolding domain binding motif in iNOS (Fig.1), binding detected by this assay required the caveolin-1 element 101-134 residing just COOH-terminal of the caveolin-1 scaffolding domain (Felley-Bosco et al., 2000). These observations do not rule out a role for the scaffolding domain in contributing to caveolin-1-iNOS interactions, but certainly suggest that this is not the only element of caveolin-1 relevant to the interaction. Interestingly, a recent study has shown that the homologous region of caveolin-3 contains a WW-like domain that is implicated in the regulation of dystrophin degradation via the proteosome pathway (Sotgia et al., 2000). Currently, mutational studies are underway to determine precisely the role of the putative WW-domain of caveolin-1 and the scaffolding domain in mediating association of caveolin-1 with iNOS as well as degradation of iNOS via the proteosome pathway (Espinoza & Quest, unpublished results).

Fig. 2. Half-life of iNOS is decreased by 30% in caveolin-1 transfected cells. Pulse chase experiments were performed 15h after cytokine stimulation. Mock (dashed line) and caveolin-1 (full line) transfected HT-29 cells were labeled for 1h in the presence of 35[S]-methionine as described in previous studies. Cells were collected at the end of labeling period (100% of labeling) or medium was changed and cells were incubated for additional 3 and 6h. iNOS was immunoprecipitated in cell homogenates and resolved on 7.5% SDS-PAGE. Dry gels were analyzed using Biorad Molecular Imager FX. Values shown are representative of 2 experiments.

Caveolin-1 expression in colon cancer cells promotes degradation of iNOS via the proteosome pathway

HT29 and DLD1 derived lines expressing exogenous IPTG-inducible caveolin-1 were employed to study the consequences of enhanced caveolin-1 expression in the human carcinoma lines HT29 and DLD-1 where caveolin-1 levels are low (Bender et al., 2000) and iNOS expression can be stimulated by the addition of a cytokine cocktail (see above). In the absence of cytokines, iNOS is not expressed in HT29 cells; however, upon addition of cytokines, iNOS protein increased, reaching a maximum within 15h, and then remained at that level for at least an additional 9h. In the presence of exogenous caveolin-1, iNOS protein levels are substantially reduced at all time points and basal levels of caveolin-1 expression, observed in transfected HT29 and DLD-1 cells in the absence of IPTG, are already sufficient to observe this effect. In this context, it is important to note that the basal caveolin-1 levels in transfected colon carcinoma cells are not higher than the levels detected in normal colon mucosa samples obtained from patients (Bender et al., 2000). Reduced levels of iNOS protein are paralleled by a similar loss in iNOS activity as measured in two different assays, corroborating the notion that iNOS protein levels are decisive in this respect. Pulse-chase experiments, analyzing [³⁵S] methionine-labeled iNOS in immunoprecipitates revealed that the half-life of the iNOS in immunoprecipitates was reduced by 30%, from 7 to 5h in the HT-29 cav-1 transfected clone 13, as compared with mock-transfected HT29 cells (Fig.2). Interestingly, Northern analysis of mRNA from HT29 cells examined 15h after cytokine stimulation revealed that caveolin-1 presence does not alter iNOS mRNA levels, suggesting that caveolin-1 controls iNOS protein levels in HT29 cells by a post-transcriptional mechanism.

Caveolin-1 and a small fraction of iNOS (about 1%) co-fractionated on sucrose gradients in the presence of Triton X-100, suggesting that an interaction in situ between the two proteins is most likely to occur in detergent-insoluble, caveolae-like membrane fractions. Interestingly, a proteolytic degradation product of iNOS was detected exclusively in those fractions using specific antibodies.

To identify the protease(s) involved in caveolin-1-induced iNOS down-regulation, several inhibitors were added 15 h after cytokine stimulation. Presence of either the calpain and proteasome inhibitor N-acetyl-Leu-Leu-Norleucinal (ALLN; Rock et al., 1994) or the proteasome inhibitor lactacystin (Fenteany et al., 1995) increased iNOS levels observed after stimulation of both HT29 and DLD1 cells. Addition of (2S, 3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester (E-64D) a specific calpain inhibitor (McGowan et al., 1989), had no effect. Thus, caveolin-1 is suggested to promote iNOS degradation by a proteasome-dependent pathway (Felley-Bosco et al., 2000). Consistent with this interpretation, components of the proteasome pathway have been detected together with degradation of iNOS in detergent-insoluble caveolin-1 containing fractions (Fig.3). Furthermore, caveolin-1 has recently been identified in a two-hybrid screen as a binding partner for the proteasome subunit JAB-1 (Cao et al., 2002).

Fig. 3. Proteasome components are present in the detergent insoluble fraction. Homogenates, nuclear pellets, Triton X-100 soluble and detergent insoluble proteins (DIGs) from caveolin-1 transfected HT-29 cells were prepared in duplicate as described (Doucey et al., 2001). Extracts were separated by SDS-PAGE, transferred to membranes and incubated with anti-iNOS, anti-caveolin-1, anti-fyn, anti-actin, anti PKCa as described (Felley-Bosco et al., 2000) or with the PW8205 antibody (Affinity, UK) which recognizes proteasome components. Migration positions of molecular weight marker proteins are indicated to the left of each panel in kDa.

Perspectives: caveolin-mediated control of iNOS in "nitroxysomes" and their role in human disease

Direct interactions between caveolin-1 and iNOS in HT29 cells occur most likely in the detergent-insoluble membrane fraction, involve only a small fraction of iNOS molecules at any given time point and promote iNOS degradation. This fraction may be present at the plasma membrane and/or the trans-Golgi network where lipids initially acquire the property of detergent-insolubility during membrane biogenesis. The existence of an interaction between iNOS and caveolin-3 has been inferred from co-immunoprecipitation experiments using mouse skeletal muscle extracts (Gath et al., 1999). There, iNOS binding to caveolin-3 was proposed to promote membrane association. However, expression of caveolin-1 in HT29/cav cells did not appear to promote iNOS presence in caveolae-like fractions, although it cannot be excluded that such an increase was masked by the overall decline in iNOS protein levels.

In this context, it is interesting to note that transfected eNOS co-localizes with the Golgi marker mannosidase II in HEK 293 cells (Sessa et al., 1995). Also, in peritoneal macrophages, iNOS is detected in a perinuclear region on the trans side of the trans-Golgi network and in 50 to 80 nm vesicles near the cell periphery called "nitroxysomes " (Vodovotz et al., 1995). Thus, caveolin-1 may play a crucial role in the control of cellular iNOS function both by directing iNOS to "nitroxysomes" where iNOS would be active, as well as by initiating steps to eliminate iNOS (Fig. 4).

Fig. 4. Schematic illustration of the potential relationship between caveolin-1 and inducible nitric oxide synthase (iNOS) in cells. iNOS is depicted as being evenly distributed in the cells between membranes and cytosol. A small amount of iNOS is found in caveolae like-fractions which are morphologically detectable as coated invaginations of the plasma membrane in the presence of caveolin-1 (see highlighted region of plasma membrane). These membrane microdomains are rich in glycosphingolipids, cholesterol and glycosylphosphatidylinositol (GPI)-anchored surface proteins (see box with enlarged view and also Figure 1 in Bender et al., 2002; this issue). These may be related to a previously described population of vesicles detected near the cell periphery called «nitroxysomes". Caveolin-1 is suggested to control cellular iNOS function both by directing iNOS to such « nitroxysomes « where iNOS would be active, as well as by initiating steps to eliminate iNOS via the proteasome pathway (see box with enlarged view).

Altered iNOS turnover as a consequence of reduced caveolin-1 expression may be particularly significant in cells with mutations in the p53 tumor suppressor protein, as is the case for both HT29 (homozygous for codon 273 HIS) and DLD-1 (codon 241 mutation C->T) cells. As a consequence, iNOS gene transcription cannot be controlled via p53-dependent mechanisms in these cells (Forrester et al., 1996). Interestingly, caveolin-1 has recently been described as a major p53-induced gene in colon cancer cells (Yu et al., 1999). Thus, p53 appears to control iNOS expression directly at the transcriptional level and also indirectly via caveolin-1 at the post-translational level. Alternatively, caveolin-1 expression is likely to be regulated by dietary intake, since caveolin-1 mRNA levels are upregulated by free cholesterol and down-regulated by oxysterols (Fielding et al., 1997). Given that endogenously produced NO accelerates tumor growth (Jenkins et al., 1995; Ambs et al., 1998b), these observations in conjunction with our results suggest that human diet may contribute as an epigenetic factor towards colon tumor development and progression via modulation of caveolin-1 levels. Down-regulation of caveolin-1 is expected to promote uncontrolled iNOS activity, genotoxic damage and tumor development in humans.

ACKNOWLEDGEMENTS

Work discussed here was supported by the Swiss National Science Foundation Grant SNSF 3100-050888, the Swiss Cancer League Grant SCL 636-2-1998, Chilean National Science Foundation FONDECYT regular grant awards #1990893 and #1020585, as well as FONDAP 15010006 (to A.F.G.Q) and Swiss National Science Foundation Grant SNSF 3100-49662 (to E.F.B).

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Corresponding author: Andrew F.G. Quest, Ph.D. Laboratory of Cellular Communication Program of Cell and Molecular Biology, ICBM, Faculty of Medicine, University of Chile, Independencia 1027, Santiago, CHILE. Fax/phone 56-2-7382015. e-mail: aquest@machi.med.uchile.cl

Received: May 17, 2002. In revised form: June 07, 2002. Accepted: July 14, 2002