Free fatty acids exhibit diverse biological effects such as the regulation of immune responses in humans and animals. To investigate the biological effect of fatty acids in the model eukaryotic organism yeast, we examined the activity of various fatty acids in a yeast-based drug-screening system designed to detect the small-molecule compounds that inhibit Ca2+-signal-mediated cell-cycle regulation. Among the fatty acids examined, ricinoleic acid markedly alleviated the deleterious physiological effects induced by the compelled activation of Ca2+ signaling by external CaCl2, such as the polarized bud growth and the growth arrest in the G2 phase. In accordance with the physiological consequences induced by ricinoleic acid, it diminished the Ca2+-induced phosphorylation of Cdc28p at Tyr-19, concomitant with the decrease in the Ca2+-stimulated expression levels of Cln2p and Swe1p.
Dietary fatty acids exhibit diverse physiological effects in humans and animals. The polyunsaturated fatty acids are especially widely used as a complementary diet for the prevention or treatment of diseases such as cardiovascular diseases, rheumatoid arthritis, asthma and inflammatory diseases (Ferranto, 1994). The elevation of serum free unsaturated fatty acids in humans diminishes the Ca2+ responses and inhibits the T-lymphocyte signaling, suggesting that serum free fatty acids may exert an immunosuppressive effect (Chow, 1990; Richieri & Kleinfeld, 1990; Stulnig, 2000). Omega-3 unsaturated fatty acids affect the inflammatory responses (Caterina & Basta, 2001). Moreover, topical application of ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid) exerts analgesic and anti-inflammation effects on the induced acute and subchronic inflammation in an animal model (Vieira, 2000). An advantage of using polyunsaturated fatty acids as a complementary diet is their low side-effects. Although the mechanisms by which the fatty acids elicit these physiological responses are not understood, some of these effects may be attributable to the inhibition of cellular Ca2+ responses. Consistent with this notion, it was reported that bovine brain calcineurin is sensitive to polyunsaturated lipids in vitro (Abigail & Bruce, 2006).
Yeast-based drug screening that uses the powerful genetic techniques available in this organism offers a convenient and target-oriented methodology for discovering small-molecule bioactive compounds. Because the yeast genome contains a substantial number of orthologs of human drug targets, the bioactive compounds uncovered by the yeast screening systems are potential candidates for lead compounds of pharmacological interest. Based on these ideas, we previously developed a yeast-based screening procedure by which the small molecules that alleviate the Ca2+-signal-mediated growth inhibition of a Saccharomyces cerevisiae mutant (zds1Δ strain on a drug-supersensitive genetic background) can be detected with high specificity and efficiency. According to this procedure, the active compounds can be detected on solid medium containing a high concentration of CaCl2 by the ability to confer the Ca2+ resistance to a Ca2+-sensitive zds1Δ strain yeast whose growth is compromised by the compelled activation of Ca2+ signaling. This procedure was designated as ‘positive screening’ by the principle of drug detection (Mizunuma, 1998; Shitamukai, 2000; Miyakawa & Mizunuma, 2007).
For a better understanding of the molecular basis for the biological effects of free fatty acids, we investigated the effect of various fatty acids in the model eukaryotic organism yeast. Using the yeast-based positive-screening system, we examined various fatty acids for the activity to modulate the Ca2+-signal-mediated cell-cycle regulation. Of the fatty acids examined, ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid) exhibited a particularly prominent activity by the assay system. To confirm that ricinoleic acid inhibited the Ca2+-signal-mediated cell-cycle regulation, we further characterized the action mechanism of ricinoleic acid.
Materials and methods
Strains and chemicals
The strains used in this study were as follows: YAT1 (MATazds1∷TRP1), YNS17 (MATazds1∷TRP1 erg3∷HIS3 pdr1∷hisG URA3 hisG pdr3∷hisG) and YRC3 (MATaswe1∷HIS3∷SWE1-9xMyc∷URA3 CLN2-3xHA∷LEU zds1∷Genr erg3∷HIS3 pdr1∷hisG pdr3∷hisG TRP1) (Chanklan, 2008b). Fatty acids were purchased from Wako Pure Chemical Industries.
Assay procedure for positive screening
The screening was performed on yeast–peptone–dextrose (YPD) soft-agar plates containing CaCl2 using the halo assay according to the procedure described previously (Shitamukai, 2000; Miyakawa & Mizunuma, 2007; Chanklan, 2008b). Fatty acids were dissolved in methanol (1 mg mL−1) and a 5-μL aliquot of the solution was spotted on the surface of a soft-agar plate (about 5 mm thickness) containing the assay cells (YNS17 strain yeast) and 150 mM CaCl2. After 2 days of incubation at 30 °C, halo (growth zone) formation was observed around the samples. The effect of ricinoleic acid on cell growth was also determined in YPD liquid medium containing 150 mM CaCl2. Cell density was monitored by a Bio-photorecorder (Toyo Engineering Works, Tokyo). The cell morphology was observed under a light microscope.
A zds1Δ strain (YAT1), carrying a plasmid-borne construct (pKC190) containing a LacZ reporter gene driven by four tandem copies of the calcineurin-dependent response element, was incubated in YPD medium containing 100 mM CaCl2 with ricinoleic acid for 3 h, and β-galactosidase activity was measured according to the procedure described previously (Chanklan, 2008a).
Protein levels of Swe1p and Cln2p and Tyr19-phosphorylation levels of Cdc28p (Nasmyth & Reed, 1980) were determined by Western blot analysis. The procedures for the preparation of cell extracts, immunoprecipitation and immunodetection were as described previously (Chanklan, 2008b). For precipitation and the detection of the hemagglutinin-tagged protein, Myc-tagged protein, Cdc28p and phosphorylated Cdc28p (Tyr19), monoclonal antibodies 12CA5 against the hemagglutinin-epitope (Babco Construction Inc.), 9E10 against the Myc epitope (Babco Construction Inc.), anti-PSTAIRE antibody (Santa Cruz Biotechnology Inc.) and anti-phospho-cdc2 (Tyr15) antibody (Cell Signaling Technology), respectively, were used.
Fluorescence-activated cell sorting (FACS) analysis
Cultured cells were stained with propidium iodide to measure the cellular DNA content using FACScalibur (Becton Dickinson Co.) as described previously (Shitamukai, 2004).
Results and discussion
Screening of fatty acids that alleviate the deleterious physiological effects of external CaCl2 on zds1Δ strain yeast
To examine whether fatty acids affect Ca2+ signaling in yeast, we examined the effect of various saturated, unsaturated and hydroxylated fatty acids using the positive screening, a drug-screening procedure that detects the small molecules alleviating the Ca2+-signal-mediated growth inhibition of a zds1Δ strain yeast. The fatty acids examined were butyric acid (C4:0), hexanoic acid (C6:0), decanoic acid (C10:0), myristic acid (C14:0), stearic acid (octadecanoic acid, C18:0), oleic acid (C18:1), ricinoleic acid (C18:1; 12-hydroxy-9-cis-octadecenoic acid), linoleic acid (C18:2), conjugated isomer of linoleic acid (C18:2), α-linolenic acid (C18:3), γ-linolenic acid (C18:3), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6). For the screening assay, 5-μL aliquots of a 1 mg mL−1 solution of each fatty acid dissolved in methanol were spotted (about 3 mm in diameter) on solidified plates of YPD soft agar containing 150 mM CaCl2 and the drug-sensitive, Ca2+-sensitive assay strain (YNS17; zds1Δerg3Δpdr1Δpdr3Δ) (Shitamukai, 2000; Miyakawa & Mizunuma, 2007; Chanklan, 2008b), in which several genes involved in multidrug resistance (Nitiss & Wang, 1988; Balzi & Goffeau, 1995; Hemmi, 1995) and the resistance to Ca2+ (Mizunuma, 1998; Miyakawa & Mizunuma, 2007) had been deleted, was used. In the presence of 150 mM CaCl2 in the medium, the growth of assay cells was compromised due to compelled activation of cellular Ca2+ signaling. After 2 days of incubation at 30 °C, a halo of growth zone varying in size and intensity appeared around the spots where ricinoleic acid, eicosapentaenoic acid and oleic acid were applied, but none of the other fatty acids examined (data not shown). The activities of the three fatty acids that gave the positive effects in the screening assay were compared semi-quantitatively by a similar assay with a twofold serial dilution of the fatty acids (Fig. 1a–c). Ricinoleic acid exhibited the highest activity in this assay (Fig. 1a). When 0.312 μg (5 μL of 0.0625 mg mL−1 solution) of ricinoleic acid was applied, a halo of growth zone appeared around the spot. When 2.5 μg (5 μL of 0.5 mg mL−1 solution) or more of it was applied, a ring-shaped halo containing a clear (or less turbid) zone in the center of the halo appeared. These observations suggested that ricinoleic acid exerted a concentration-dependent biphasic effect on the growth of CaCl2-treated cells; namely, the compromised cell growth was rescued by ricinoleic acid at an appropriate concentration range, but cell growth was inhibited when its concentration was high, presumably due to its cytotoxic effect. The activities of eicosapentaenoic acid and oleic acid were much weaker than that of ricinoleic acid, showing a growth-promoting effect at 2.5 and 5 μg, respectively (Fig. 1b and c). In contrast to the potent effect of ricinoleic acid, ricinoleic acid methyl ester (5 μg) failed to exhibit activity by the assay (Fig. 1d). Because ricinoleic acid showed the highest activity among all the fatty acids examined, we focused on this fatty acid in further studies.
The effect of ricinoleic acid was also examined in a liquid culture in the presence of varying amounts of ricinoleic acid added to the culture of YNS17 strain in YPD medium containing 150 mM CaCl2. The cell density of shaking cultures was monitored using an automated cell-density photorecorder. The growth rate was significantly reduced by the presence of CaCl2, and the growth inhibition was partially alleviated by 0.025 μg mL−1 of ricinoleic acid (Fig. 2a). In YPD medium without added CaCl2, the same concentration of ricinoleic acid showed no significant positive or negative effect on cell growth.
We further characterized the physiological effect of ricinoleic acid on the cells cultivated in the presence of CaCl2 in a liquid culture. Morphologically, the Ca2+-induced polarized bud growth was alleviated by ricinoleic acid, both in the percentage and in the length of the elongated cells (Fig. 2b). The Ca2+-induced G2 cell-cycle arrest as determined by FACS analysis of cellular DNA content was also partially restored, with the increase in the 1C peak and the decrease in the 2C peak (Fig. 2c). These results demonstrated that each of the Ca2+-induced physiological alterations of zds1Δ cells was partially suppressed by ricinoleic acid.
The effect of ricinoleic acid on cellular Ca2+ levels
High external CaCl2 induces an elevation of cellular Ca2+ (Chanklan, 2008a). To examine whether ricinoleic acid affect the cellular Ca2+ level, we used the YAT1 strain containing a LacZ reporter construct in which the LacZ gene was placed under the control of four tandem copies of the calcineurin-dependent response element. The extent of the activation of the Ca2+-signaling pathway by external CaCl2 was monitored by the activity of β-galactosidase in the cells cultivated in the presence of CaCl2 and various concentrations of ricinoleic acid. In a control experiment, 1 mM of MgCl2 that inhibits the Ca2+-induced activation of the reporter gene was added (S. Koga, unpublished data). Ricinoleic acid had no significant effect on the increase in the cellular Ca2+ level at a concentration higher than that required for the suppression of Ca2+-induced physiological effects. The observation that the LacZ expression was unaffected by ricinoleic acid suggested that ricinoleic acid did not inhibit the activity of the Ca2+-signal mediator calcineurin per se, because calcineurin mediates the Ca2+-induced LacZ expression. These results suggested that ricinoleic acid inhibited the Ca2+-mediated cell-cycle regulation at a step following the elevation of the cytosolic Ca2+ concentration (Fig. 3).
Effect of ricinoleic acid on Ca2+-stimulated expression of Swe1p and Cln2p
The compelled activation of Ca2+-signaling pathways by external CaCl2 leads to the elevation of the Swe1p and Cln2p proteins and induces the characteristic physiological alterations, such as polarized bud growth and G2 cell-cycle arrest (Mizunuma, 1998, 2001, 2004). Swe1p, a homolog of Saccharomyces pombe wee1, is a negative regulator of the Cdc28p/Clb complex (Booher, 1993). Cln2p is a G1 cycline involved in the regulation of the cell cycle (Hadwiger, 1989). To observe whether ricinoleic acid suppressed the Ca2+-sensitive phenotypes through the inhibition of this mechanism, we examined the effect of ricinoleic acid on the Ca2+-stimulated expression levels of Swe1p and Cln2p that were detected by Western blot analysis using YRC3 strain harboring chromosomally integrated genes for Myc-tagged Swe1p and hemagglutinin-tagged Cln2p on a genetic background similar to that of the YNS17 strain (Chanklan, 2008a). We determined the Swe1p and Cln2p levels after shift of the culture medium from YPD to YPD containing CaCl2 and ricinoleic acid. As expected, the Ca2+-induced accumulation of both Swe1p and Cln2p was significantly diminished by ricinoleic acid (Fig. 4). In a parallel experiment, the Swe1p-dependent phosphorylation levels of Cdc28p were also determined using anti-phospho-cdc2 (Tyr15) antibody. In accordance with the inhibition of Swe1p accumulation, the Ca2+-induced Cdc28p phosphorylation was diminished (Fig. 4). These observations were consistent with the notion that ricinoleic acid downregulated both the Ca2+-induced, Swe1p- and Cln2p-mediated inhibition of the Cdc28p/Clb G2 cell-cycle engine and polarized bud growth, and led to the suppression of the Ca2+ phenotypes.
In our previous studies, it was shown that the level of Swe1p is upregulated in response to external CaCl2 in a manner dependent on calcineurin via three distinct pathways: (1) through the activation of SWE1 transcription by a yet to be identified calcineurin-dependent transcription factor (Mizunuma, 2001); (2) through the calcineurin-dependent delocalization and destabilization of Hsl1p, a negative regulatory kinase of Swe1p (Mizunuma, 2001); and (iii) through the downregulation of the proteasome system that is responsible for the degradation of Swe1p (Yokoyama, 2006). Through these multilateral mechanisms, the calcineurin-mediated pathways appear to ensure the activation of Swe1p, at the transcriptional, post-translational and protein degradation levels. The accumulation of Cln2p may be induced in a similar manner (Miyakawa & Mizunuma, 2007). Detailed mechanisms by which ricinoleic acid downregulated the Swe1p and Cln2p proteins still remain to be investigated.
Castor oil is a laxative agent that increases the intestinal motility (Stewart, 1975) and gastrointestinal mucosal permeability (Phillips, 1965; Stewart, 1975; Celme, 2001), and is also known as an anti-inflammatory agent (Celme, 2001; Rahman, 2006). Moreover, ricinoleic acid, the major fatty acid constituent of castor oil, exhibits unique physiological effects. Topical application of ricinoleic acid exerts analgesic and anti-inflammation effects on the induced acute and subchronic inflammation in an animal model (Vieira, 2000). Because ricinoleic acid differs structurally from oleic acid only by the presence of a 12-hydroxyl group, the occurrence of this residue and the conjugated double bond seems to be important for the biological effect of ricinoleic acid in yeast (Abigail & Bruce, 2006). For the understanding of the action mechanism of ricinoleic acid, it would be of interest to investigate the effect of ricinoleic acid in the mammalian cells on the activation of cell-cycle regulators such as wee1 and G1 cyclins.
We thank R. Chanklan, M. Kakito, K. Shiozaki and S. Koga for comments and technical assistance. This work was supported by the Royal Golden Jubilee PhD Program (PHD/0241/2548) by grants from the Thailand Research Fund to BWT and in part by the Faculty of Medicine, Srinakharinwirot University. S.A. is a recipient of the Royal Golden Jubilee Scholarship.