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n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon ¹

DAVID W. L. MA, JEONGMIN SEO, LAURIE A. DAVIDSON^,, EVELYN S. CALLAWAY, YANG-YI FAN, JOANNE R. LUPTON^, and ROBERT S. CHAPKIN^,,2

Faculty of Nutrition and
Center for Environmental and Rural Health, Texas A&M University, College Station, Texas, USA

² Correspondence: Molecular and Cell Biology Section, Faculty of Nutrition, 442 Kleberg Center, TAMU 2471, Texas A&M University, College Station, TX 77843-2471, USA. E-mail: r-chapkin{at}tamu.edu

SPECIFIC AIMS

Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, altering their functionality and subsequent downstream signaling. We investigated the in vivo ability of n-3 polyunsaturated fatty acids (PUFA), a chemoprotective class of membrane lipid-modifying dietary constituents, to modulate caveolae lipid environment and protein microlocalization and function in the colon. We began the present study by comparatively characterizing colonic caveolae-enriched membranes isolated from in vivo vs. in vitro model systems.

PRINCIPAL FINDINGS

1. In vivo characterization of colonic caveolae: differential caveolae localization of H- vs. K-Ras
Biochemical isolation of a colonic caveolae-enriched membrane fraction using density gradients from in vitro (immortalized young adult mouse colon cell line, YAMC) and in vivo (mouse and rat) sources was verified by caveolin-1 enrichment and clathrin depletion. Independently, the presence of morphologically distinct caveolae in colonocytes was confirmed by electron microscopy. Colonic caveolae lipid composition was characterized by significantly higher levels of cholesterol (µg/mg protein), sphingomyelin (mol %), and phosphatidylserine (mol %) and reduced phosphatidylcholine (mol %) compared with other membrane fractions. We report the marked enrichment of H-Ras but not K-Ras in colonic caveolae both in vitro and in vivo, providing the first in vivo evidence supporting the current model of differential microlocalization of Ras isoforms established using in vitro cell culture models exclusively.

2. Effect of n-3 PUFA on mouse colonic caveolae lipid composition
To examine the ability of n-3 PUFA to alter colonic caveolae lipid composition in vivo, mice were fed n-6 (control) or n-3 PUFA-enriched diets for 2 wk. The n-6 PUFA diet was characterized by a high content of linoleic acid, 18:2 (n-6) (54.1%) (wt %); the n-3 PUFA diet was enriched in eicosapentaenoic acid, 20:5 (n-3) (13%) and docosahexaenoic acid, 22:6 (n-3) (8.9%).

Distribution of individual phospholipid classes as well as total phospholipid content did not differ between animals fed n-6 or n-3 PUFA. After n-3 PUFA feeding, cholesterol content in caveolae was significantly (P<0.05) reduced by 46% compared with n-6 PUFA-fed animals (n=3–4, 15 mice pooled per analysis) although total cellular and plasma membrane cholesterol levels were not affected. Our data show that the fatty acyl composition of caveolae-enriched membranes are significantly altered by n-3 PUFA feeding, as indicated by marked incorporation of n-3 PUFA into phospholipids, resulting in a significant decrease in the n-6/n-3 ratio.

3. Diet alters caveolin-1 localization in mouse colon
In view of the interdependency between cholesterol and caveolin-1, we determined whether the n-3 PUFA-induced cholesterol depletion in colonic caveolae was accompanied by alterations in caveolin-1 localization. n-3 PUFA feeding significantly (P<0.05) reduced caveolin-1 in colonic caveolae by 53% relative to n-6 PUFA-fed animals (n=8, 15 mice pooled/analysis). Caveolin-1 protein expression in total cell lysates was unaffected by diet, indicating that n-3 PUFA-induced alteration is not mediated at the protein expression level. These results demonstrate that dietary n-3 PUFA alter colonic caveolae microenvironment not only by modifying membrane lipid composition, but also by displacing the major structural protein, caveolin-1.

4. Diet alters subdomain distribution of H-Ras but not K-Ras in mouse colonocytes
H-Ras signaling, unlike K-Ras, is highly sensitive to perturbations of cholesterol-rich microdomains by cholesterol depletion or dominant negative caveolin since it requires access to lipid rafts and caveolae for proper activation. We examined whether n-3 PUFA-induced modification of the caveolae microenvironment could affect Ras localization in mouse colonic caveolae. The level of H-Ras in the caveolae fraction was lower in n-3 PUFA-fed animals (74±15 ng/mg protein) by 45% relative to n-6 PUFA-fed animals (134±29 ng/mg protein) (Fig. 1 ) (n=3–4, 15 mice pooled/analysis). The decrease in caveolar H-Ras in n-3 PUFA-fed animals was not due to an effect on protein expression, since total cellular levels of H-Ras were unaffected by diet. There was no effect on K-Ras localization in all membrane fractions, consistent with the previously reported insensitivity of K-Ras localization to microdomain perturbations.

View larger version (15K): [in this window] [in a new window]   Figure 1. H- and K-Ras localization in colonic caveolae in n-6 and n-3 PUFA-enriched membranes. C57BL/6 mice were fed n-6 (control) or n-3 PUFA-enriched diets for 2 wk. Colons were collected and processed for immunoblotting. A) H-Ras and B) K-Ras localization was quantified as the mass of Ras protein/mg of protein (mean±SE, n=3–4) in the plasma membrane, intermediate, and caveolae membrane fractions using recombinant H- and K-Ras standards. Each analysis is pooled from 15 mice. *Significant difference between diets for a given fraction (P<0.05). Log transformed values were used for statistical analysis; untransformed values are shown.

5. Diet alters eNOS but not clathrin localization
To confirm that H-Ras displacement from caveolae by n-3 PUFA is not due to a global decompartmentalization of plasma membrane domains, localization of clathrin, a non-caveolae protein, was determined. The level of clathrin was significantly lower (P<0.05) in the caveolae fraction relative to the cell lysate in n-3 and n-6 PUFA-fed animals. Clathrin localization was unaffected by diet, indicating that n-3 PUFA selectively alters H-Ras microlocalization. To further address whether the modulatory effect of n-3 PUFA is confined to H-Ras or perhaps encompasses other signaling proteins in caveolae, we show that localization of endothelial nitric oxide synthase (eNOS), a dually acylated protein involved in vasoregulation, was also significantly reduced by 52% after n-3 PUFA treatment (n=6, 15 mice pooled/analysis). This reduction was not due to a change in protein expression. These data suggest that n-3 PUFA-induced modification of caveolae microenvironment may selectively influence the compartmentalization of caveolae-resident proteins.

6. n-3 PUFA suppress EGF-stimulated activation of colonic H-Ras but not K-Ras
We next determined the activation status of H- and K-Ras in n-6 and n-3 PUFA-fed mice using a Ras/Raf pull-down assay and subsequent immunoblotting with Ras isoform-specific antibodies. Figure 2 shows that n-3 PUFA feeding significantly suppresses (P<0.05) EGF-induced activation of H-Ras but not K-Ras in mouse colon. Total cellular expression of both isoforms was confirmed to be unaffected by diet (Fig. 1) . These results are in parallel with the distinct effect of n-3 PUFA on H- vs. K-Ras microlocalization, suggesting that n-3 PUFA differentially modulate cell signaling by selective displacement of caveolar proteins.

View larger version (24K): [in this window] [in a new window]   Figure 2. Ras activation in n-3 and n-6 PUFA-enriched colonocytes. A) After EGF stimulation, cell lysates were probed for GTP-bound Ras using a Ras/Raf pull-down assay, followed by immunoblotting with H- or K-Ras-specific antibodies. B) Results are expressed as fold change in EGF-stimulated Ras activation relative to unstimulated colonocytes (mean±SE, n=4). *Significant difference between diets (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

Although a growing body of evidence suggests that perturbations in caveolae lipid composition alter protein microlocalization and function, the biological relevance of using pharmacological treatments and in vitro systems remains unknown. In this study, we demonstrate for the first time in vivo modulation of caveolae lipid composition and protein localization and function in the colon using membrane lipid-modifying dietary n-3 PUFA.

n-3 PUFA feeding significantly altered colonic caveolae microenvironment compared with the n-6 PUFA control diet by changing fatty acyl composition and reducing cholesterol and caveolin-1. Enrichment of n-3 PUFA in caveolae microdomains is noteworthy since many acylated signaling proteins interact directly with this lipid environment by means of their saturated acyl moieties. The cholesterol lowering effect of n-3 PUFA was selective to caveolae, unlike pharmacological depletion of membrane cholesterol, since total cellular and plasma membrane cholesterol levels were unaltered. In light of the marked steric incompatibility between the rigid sterol moiety of cholesterol and conformationally flexible long-chain n-3 PUFA (22:6 n-3 and 20:5 n-3), n-3 PUFA-induced caveolar cholesterol reduction may be explained in part by poor solubility of cholesterol in phospholipids enriched with highly disordered long chain n-3 PUFA. The lack of changes in total cellular caveolin-1 content suggests that n-3 PUFA-induced caveolin-1 displacement may be mediated by alterations in caveolin-1 trafficking or recycling rather than by changes in protein expression.

The n-3 PUFA-induced modification of caveolae microenvironment coincided with selective displacement of caveolae resident acylated proteins, H-Ras (Fig. 1) and eNOS. H-Ras and eNOS are highly concentrated in caveolae by associating directly with caveolin-1, and caveolar localization regulates their activation. Displacement of H-Ras and eNOS after caveolar cholesterol and caveolin-1 reduction is consistent with previous studies. Displacement of acylated proteins from caveolae after n-3 PUFA feeding may be attributed to alterations in caveolae environment or to changes in protein acylation. We have shown that the inhibitory effect of n-3 PUFA on colonic H-Ras signaling does not involve alterations in H-Ras palmitoylation. Other studies have demonstrated that changes in protein palmitoylation do not necessarily result in protein displacement, suggesting that although an effect on protein acylation by n-3 PUFA is possible, it is unlikely to cause protein displacement from membrane microdomains. We conclude that alterations in the colonic caveolae environment are primarily responsible for acylated protein displacement from caveolae after n-3 PUFA treatment.

In contrast to H-Ras and eNOS, the distribution of non-caveolae proteins K-Ras and clathrin was unaffected by diet, indicating the selectivity of n-3 PUFA-induced alterations in caveolar protein microlocalization. We demonstrated that alterations in protein localization translate to selective modulation of protein function by showing that n-3 PUFA suppress EGF-stimulated activation of H-Ras but not K-Ras (Fig. 2) . More work is required to extend our findings on the specificity of n-3 PUFA effects on protein localization and function and to unravel the underlying mechanisms of n-3 PUFA-induced alterations in caveolae environment. Considering the differential compartmentalization of numerous signaling proteins within the plasma membrane, we speculate that our findings here may help develop a new paradigm to better understand the complexity of n-3 PUFA modulation of signaling networks.

In a major step toward developing a unifying mechanistic hypothesis addressing why n-3 PUFA suppress colon cancer compared with n-6 PUFA (the major dietary form of PUFA in the U.S. diet), we demonstrate for the first time that n-3 PUFA feeding can markedly alter lipid/protein composition of colonic caveolae microdomains, thereby selectively modulating localization and function of caveolar proteins. Our findings highlight a novel modality by which n-3 PUFA influence membrane micro-organization, thereby modulating biological responses.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram of the effects of dietary n-3 PUFA on caveolae resident protein localization in mouse colon. Incorporation of n-3 PUFA into caveolae membranes alters its biochemical composition by reducing cholesterol and caveolin-1. Acylated signaling proteins are displaced from caveolae, resulting in modified downstream signaling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1430fje;

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This Article Full Text (PDF) All Versions of this Article: 18/9/1040 03-1430fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MA, D. W. L. Articles by CHAPKIN, R. S. Search for Related Content PubMed PubMed Citation Articles by MA, D. W. L. Articles by CHAPKIN, R. S.