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Signal Transduction Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science and University of Adelaide, Adelaide, South Australia 5000, Australia
Address all correspondence and requests for reprints to: Pu Xia, Signal Transduction Laboratory, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia. E-mail: pu.xia{at}imvs.sa.gov.au.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The biological activity of E2 is widely considered to be primarily mediated through specific high-affinity estrogen receptors (ER) including 
- and ß-isoforms located within cell nuclei. In spite of the clarity with which the ER has been shown to act as transcription factors, it has been apparent that not all physiological effects of E2 are accomplished through a direct effect on gene transcription (1, 2). Indeed, evidence is accumulating that E2 has not only genotropic function but also nongenotropic effects through distinct cytoplasmic signaling pathways. For instance, E2 can induce very rapid increases in intracellular second messengers such as calcium (Ca2+) and cAMP (3, 4, 5). In cells expressing ER, the MAPK family members Erk1 and Erk2 were activated within 5–10 min in response to E2 treatment (6, 7). Furthermore, cell membrane-impermeable estrogen (E2-conjugated BSA) was capable of activating Erk1/2, which cannot merely be explained by activation of well-described nuclear ER (8). Experiments conducted with several different cell types led to the suggestion that membrane associated ER-like receptors and G proteins may be involved in E2 activities (9, 10, 11). In addition, E2 and ER complexes may also interact with other cytoplasmic signaling components, such as, Raf-1 kinase (12), phosphatidylinositol 3-kinase (13), protein kinase A (14), and protein kinase C (PKC) (15). Together, although the mechanisms for these actions of E2 remain elusive, the activation of these survival and mitogenic signals independent on ER transcription activity could account for, at least partially, the mitogenic effect of E2.
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, has recently received considerable attention as a novel signal lipid that regulates cell survival, proliferation and differentiation (reviewed in Refs. 16 and 17). The enzyme, sphingosine kinase (SphK) that catalyzes S1P formation, is activated by a variety of growth factors, cytokines and mitogenic factors (16, 17). Inhibition of this enzyme activity markedly inhibits cell growth or induces apoptosis (18). Addition of exogenous S1P or increases in intracellular level of S1P by overexpression of SphK regulated cell cycle via expediting the G1/S transition and increasing DNA synthesis, further suggesting a mitogenic effect of SphK (19, 20). More recently, we have reported that overexpression of SphK in NIH 3T3 cells had an increased enzymatic activity and accelerated cell growth in serum- and anchorage-independent manner (21). These transfected cells acquired the transformed phenotype and the ability to form tumors in nude mice, demonstrating an oncogenic potential of SphK. In addition, our previous work has shown that SphK activity is critically involved in the oncogen Ras-promoted oncogenesis (21), suggesting the existence of a novel signal transduction pathway trigged by SphK in mediating cell growth and tumorigenesis.
In this study, we explore a potential role of SphK in the mitogenic effect of E2 in MCF-7 human breast cancer cells. We have found that SphK activity was significantly stimulated in the cells treated with E2 in a dose-dependent manner. The increased SphK activity has been noticed to not only coincide with enhanced cell growth, but also serve as a necessary mediator to signal E2-dependent activation of MAPK and intracellular Ca2+ mobilization. Thus, the present report suggest that SphK pathway is an important cytoplasmic signaling to mediate E2 action in human breast cancer cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Detected bands demonstrated apparent molecular weight consistent with the predicted size of FLAG-tagged human SphK1. Overexpression of SphK in the cells resulted in an over 10-fold increase in the basal SphK activity, whereas the SphKG82D-transfected cells had a similar basal SphK activity to the control cells (Fig. 1B). To confirm the transgenes of wild-type SphK and SphKG82D functionally expressed in MCF-7 cells, SphK activity was measured after the cells were stimulated with phorbol 12-myristate 13-acetate (PMA), a known activator of SphK through PKC activation (23, 24). PMA stimulation resulted in an approximately 2-fold increase in SphK activity over the basal levels in MCF-7 cells transfected with SphK or empty-vector (Fig. 1B). No SphK activation was observed in the cells expressing SphKG82D in response to PMA stimulation (Fig. 1B), confirming that SphKG82D acts as a dominant-negative SphK in the transfected MCF-7 cells.
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, A and B). Even in serum-free medium for up to 5 d, the SphK-transfected cells survived and continued to grow (Fig. 2A), indicating a mitogenic effect resulted from SphK overexpression. Because E2 has been considered as an independent mitogenic factor for MCF-7 breast cancer cell growth, we next examined the effect of SphK on E2-dependent cell growth. Growth curves shows that treatment MCF-7 cells with E2 stimulated cell growth in the absence of serum (Fig. 2B). Remarkably, the E2-responsive cell growth was significantly enhanced in the wild-type SphK-transfected cells (Fig. 2B). In contrast, the SphKG82D-transfected cells not only lost the mitogenic activity of SphK, but also dramatically inhibited the cell growth in responses to serum (Fig. 2A) or E2 stimulation (Fig. 2B), suggesting a critical role of SphK in regulation of cell growth in MCF-7 breast cancer cells.
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, E2 stimulation was able to form solid cell foci within 10-d culture of MCF-7 cells. Overexpression of SphK resulted in more than 2-fold increases in the number of foci formed compared with the empty vector-transfected cells (Fig. 3A). The size of foci was also markedly increased in the SphK-transfected MCF-7 cells. Conversely, under the same culture conditions with E2 treatment, the cells transfected with SphKG82D were unable to form foci (Fig. 2A
) even after the cultures were prolonged to 14 d. Additionally, a similar result was obtained in the experiments with the colony formation in soft agar (Fig. 3B). These data suggest a critical involvement of SphK activity in the E2-dependent neoplastic growth of MCF-7 cells.
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, E2 stimulation of MCF-7 cells caused a rapid and transient increase in SphK activity, reaching a maximum of 240 ± 25% (P < 0.01) of basal within 15 min and returning to background level after 30 min treatment. The E2-induced SphK activity was dose dependent, reaching a maximum at 1 µM of E2 (Fig. 4B). To determine prolonged effects of E2 on SphK activity, we treated the MCF-7 cells for 1, 6, 18, 24, and 48 h with 10 nM E2. A significant increase in SphK activity was detected after 6 h of E2 treatment and remained for more than 24 h (Fig. 4C). Thus, two peaks of SphK activities were observed in the MCF-7 cells treated with E2. The first peak happened very quickly within 5–15 min of E2 stimulation, suggesting a nongenomic effect of E2. The second prolonged peak required at last 6 h of E2 exposure and could possibly be connected to the genomic effect of ER. In support of this hypothesis, pretreatment with actinomycin D, a transcriptional inhibitor, had no overt effect on the first peak of E2-induced SphK activity (Fig. 4D). Whereas, the delayed activation of SphK was abrogated by actinomycin D (Fig. 4C), suggesting an involvement of transcriptional activity in the delayed but prolonged effect of E2 on SphK. Furthermore, after pretreatment with E2 for 16 h, cells were pulsed in an additional dose of E2 (10 nM), which resulted in an additive increase in SphK activity with a same profile as the primary rapid response to E2 (Fig. 4D). Collectively, these data indicated that E2 has a dual effect on SphK activity dependent on E2 genotropic and nongenotropic action, respectively.
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(26). The lack of ER expression in MDA-MB-231 cell line was confirmed by immunoblotting analysis (Fig. 5A). In this ER-negative cell line, no increased activity of SphK was detected after treatment with E2 for either 15 min or 6 h (Fig. 5B), suggesting a requirement of ER, especially ER, for the E2-induced SphK activation. In addition, when the cellular levels of ER in ER-positive MCF-7 cells were down-regulated by pretreatment for 16 h with the pure estrogen antagonist ICI 182,780 (ICI) (Fig. 5A), the E2-induced SphK activity was blocked (Fig. 5B). By contrast, pretreatment of MCF-7 cells with ICI for a short term (30 min) had no influence on E2-induced SphK activation (Fig. 5C), which was consistent with previous findings that the rapid nongenomic effects of E2 were often preserved after ICI treatment (9, 27). Thus, these data along with the inability of actinomycin D on the SphK activation (Fig. 4D) suggest that the E2-induced rapid activation of SphK is mediated by ER but independent on their transcriptional activity. To further verify whether E2 signaling through putative membrane ER, we examined the effect of pertussis toxin (PTX), a Gi-protein inhibitor, on the E2-induced rapid activation of SphK. Remarkably, pretreatment of MCF-7 cells with PTX strongly abrogated E2-mediated rapid activation of SphK (Fig. 5C), implying a critical role for Gi protein-coupled membrane-associated ER (mER) complexes.
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) or assayed by Ca2+-imaging confocal microscopy in single cells (data not shown). The E2-induced rise of [Ca2+]i was significantly enhanced in the cells overexpressing SphK, whereas it was markedly decreased in the cells transfected with SphKG82D or pretreated with N,N-dimethylsphingosine (DMS), a specific competitive inhibitor of SphK (Fig. 6, A and B), suggesting a critical role of SphK in the E2-dependent Ca2+ mobilization. Furthermore, addition of exogenous S1P to MCF-7 cells resulted in an increase of [Ca2+]i (Fig. 6B), which is similar to what has been observed in other cell types (29, 30), supporting the role of SphK activity in Ca2+ signaling. In agreement with previous reports (9, 31), the E2-induced Ca2+ signaling was not prevented by ICI (Fig. 6C), indicating a nongenomic pathway operated in the action of E2.
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) or pretreatment with the inorganic Ca2+-channel blocker Ni2+ (data not shown), E2 was still able to increase [Ca2+]i albeit slightly less, suggesting that the increase of [Ca2+]i resulted chiefly from intracellular Ca2+ stores. As expected, pretreatment with 100 nM of tert-butylhydroquinone (BHQ), an endoplasmic reticulum Ca2+-ATPase blocker, strongly reduced E2-dependent increase in [Ca2+]i (Fig. 6D). Interestingly, 2-aminoethoxydiphenyl borate (2-APB), a compound known to block inositol 1,4,5-triphosphate (IP3) receptor (32), was unable to effectively abolish the effects of E2 on [Ca2+]i (Fig. 6E). However, in the presence of DMS plus 2-APB, the E2-promoted [Ca2+]i was almost completely abrogated (Fig. 6E), which supports previous finding that S1P can directly stimulate Ca2+ release from intracellular stores via a novel pathway that does not involve other mediators of Ca2+ release such as IP3 or cyclic ADP ribose (33).
SphK Activity Is Required for E2-Dependent Erk Activation
Erk activation has been well documented as an important cytoplasmic signaling for E2-mediated cell proliferation (6, 7, 8). Our previous work has revealed a role of SphK in activation of Ras and Erk induced by cytokines and mitogens (21, 22, 34). It was of interest to define whether SphK is involved in E2-induced Erk activation. Erk activity is tightly controlled by dual phosphorylation of residues Thr-183 and Tyr-185 of the proteins that is recognized on the immunoblot by using specific antibodies against the phosphorylated forms of Erk1/2. In agreement with previous reports (6, 7), treatment of MCF-7 cells with E2 resulted in a significant increase in Erk 1/2 activity, with maximal activity being observed within 15 min treatment and returning to basal levels by 60 min (Fig. 7). Remarkably, the E2-induced Erk1/2 phosphorylation was elevated in the wild-type SphK-transfected MCF-7 cells, whereas the activation of Erk was completely abrogated in the cells expressing dominant-negative SphK (Fig. 7). As a control in these experiments, E2-promoted phosphorylation of Erk1/2 was blocked by PD098095, a specific inhibitor of MEK, the upstream activator of Erk1/2 (Fig. 7). Additionally, the E2-induced Erk1/2 activation was significantly inhibited by pretreatment with ICI for 16 h, whereas it was insensitive to the short-term (30 min) treatment of ICI (Fig. 7B), which was consistent with the effect of ICI on E2-induced SphK activity (Fig. 5, A and B). Together, these data suggest that SphK activation is critically involved in the E2-mediated Erk1/2 activation in MCF-7 breast cancer cells.
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DISCUSSION |
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SphK is a unique and highly conserved lipid kinase that phosphorylates sphingosine resulting in formation of S1P. The importance of S1P as a bioactive lipid in regulation of cell growth was initially reported by Spiegel’s group who proposed that S1P is a second messenger to mediate platelet-derived growth factor’s mitogenic effect (19). Over the ensuing decade, there is accumulating evidence showing that SphK, via the production of S1P, is an important signal molecule in regulation of a wide range of cellular functions including Ca2+ mobilization, cell proliferation, differentiation as well as prevention of apoptosis (reviewed in Ref. 16). SphK activity in cells can be stimulated by certain agonists, including growth factors, cytokines and hormones, among which the mitogenic or survival factors are prominent (16, 17). We show here for the first time that E2 is a potent stimulator of SphK and that the E2-induced SphK activation serves as a mitogenic signal for the E2-dependent cell growth in MCF-7 cancer cells. Although the precise mechanism of SphK activation induced by E2 is unknown, it appears that this effect is mediated by putative plasma mER. The nature of mER is currently controversial because it has not yet been molecularly identified. It was suggested that mER is similar to cytosolic/nuclear ER/ß (35), or belongs to GPCR family (9), or form a large GPCR-ER complex (10, 11). Our data show that E2-induced SphK activity is profoundly inhibited by pretreatment with the G protein inhibitor PTX and insensitive to ER antagonist ICI, supporting the hypothesis of G protein-coupled mER that could account for the activation of SphK in rapid response to E2. In addition to the fast effect of E2 on activation of SphK, peaking within the first approximately 5–15 min, E2 also had a delayed effect to induce SphK activity that peaked at 6 h after treatment and prolonged more than 24 h. The data that the rapid response of SphK superimposed on the delayed effect of E2 (Fig. 4D), indicating that the biphasic activation of SphK was driven by different mechanisms. Even though the rapid response remained intact, the delayed effect of E2 was abrogated by a transcription inhibitor, suggesting a requirement of transcriptional activity for the delayed action. Whether this genomic action of E2 has a direct effect on SphK gene transcription or an indirect effect involved in the activation of SphK requires further investigation.
The activation of SphK has been demonstrated as a mitogenic signal to regulate cell proliferation. More recently our studies have shown an oncogenic potential of SphK that is able to transform rodent fibroblasts and form tumors in nude mice (21). Our previous work has also shown that SphK activity is implicated in Ras, but not Src, mediated oncogenesis, suggesting a specific signaling role of SphK in regulation of neoplastic cell growth. E2 exerts a mitogenic effect on MCF-7 cells without the addition of any other growth factors, which facilitates the study of the mitogenic signal of E2 in isolation. In the present study, we found that overexpression of wild-type SphK in MCF-7 cells resulted in a significant increase in SphK activity and cell growth rate in response to E2 stimulation. Additionally, the E2-dependent foci formation and colony growth in soft agar were significantly potentiated in the cells overexpressing SphK (Fig. 3). In contrast, enforced expression of a dominant-negative mutant SphKG82D in MCF-7 cells not only lost the SphK mitogenic potential of SphK, but also abrogated the neoplastic cell growth in responses to serum or E2 stimulation. Importantly, consistent with our previous reports (21, 22), whereas SphKG82D blocked the SphK activation induced by E2 or PMA, the basal SphK activity in unstimulated MCF-7 cells was unaffected by SphKG82D (Fig. 1). Thus, by blocking SphK activation the SphKG82D inhibited cell growth, demonstrating that SphK activation, but not its basal activity, is responsible for the mitogenic and oncogenic effect of SphK. Taken together, these data not only further confirmed our previous finding of the oncogenic potential of SphK in NIH 3T3 cells, but also strongly suggested that SphK activation is a key intracellular signal to mediate the E2-dependent and -independent neoplastic cell growth in breast cancer cells. These findings could allow development of a new strategy to target SphK for the treatment of breast cancer. While this paper was under preparation, Nava et al. (36) reported that enforced overexpression of murine SphK1 in MCF-7 cells enhanced E2-dependent cell growth and tumorogenesis in nude mice.
The biological action of SphK is based on catalysis of sphingosine to generate S1P that functions either as a ligand for the Edg family of GPCR or as a intracellular second messenger. The binding of S1P to Edg receptor family has been shown to initiate diverse cell responses, such as platelet activation, melanoma cell motility, neurite retraction, endothelial cell migration and tube formation (reviewed in Ref. 37). However, the involvement of these receptors in the regulation of cell growth and transformation remains to be defined. Indeed, S1P promoted mitogenic and antiapoptotic effects seem not to be correlated with the binding to cell surface receptors in several cell types (16, 17). In addition, our previous work (21) and others (20, 38) have shown that whilst enforced expression of SphK resulted in significant increases in cellular S1P levels there was no detectable S1P in the media as measured either by detection of radiolabeled S1P or by S1P bioactivity in the conditioned media. It is noted that the secretion of S1P, i.e. autocrine, is not necessary for the access of S1P to the receptors. Furthermore, whereas the G protein inhibitor PTX was able to abolish the receptor-mediated effects of S1P (e.g. cell migration), it did not suppress proliferation induced by overexpression of SphK (20). Even so, due to the complexity of GPCR, the limit of methodology in detection of S1P secretion, and a possible intracrine fashion of action on the receptors, a potential involvement of G protein-coupled S1P receptors in regulation of cell growth needs further examination.
Regardless of the dual action (intra- and intercellular) of S1P, several downstream targets of SphK signaling have been implicated in the regulation of cell growth and survival including activation of Ras/Raf/Erk, PI-3K/Akt, and nuclear factor-
B pathways and intracellular Ca2+ mobilization (16, 17). The ability of E2 to mobilize intracellular Ca2+ has been documented as a key cytoplasmic signaling independent of nuclear ER (3). Our data show that treatment of MCF-7 cells with E2 leads to both a rapid activation of SphK and a quick mobilization of intracellular Ca2+. Although the peak of SphK activation is not coincident with the increase of [Ca2+]i, it appears that SphK activation is required for E2-promoted Ca2+ mobilization. This view is supported by the following findings. First, S1P has a similar effect to E2 in rising of [Ca2+]i in MCF-7 cells. The effective concentration of S1P for the increase of [Ca2+]i is very low within nanomolar range, which suggests that when SphK is just activated by E2 it is ready to trigger the response of Ca2+. Second, overexpression of SphK in MCF-7 cells resulted in an increase in SphK activity which significantly potentiated the effect of E2 on the Ca2+ mobilization. Third, our data are consistent with previous reports that the E2-induced rapid rise in [Ca2+]i is due to both influx of extracellular Ca2+ and release from intracellular Ca2+ stores, with the latter appearing the preferential source of the E2-induced increase of [Ca2+]i. However, neither BHQ nor 2-APB effectively blocked the E2-induced increase in [Ca2+]i, which along with the previous studies showing that the phospholipase C inhibitor only partially prevented the E2-induced rise of [Ca2+]i (3), suggests additional mechanism(s) operating for the E2-promoted Ca2+ mobilization. Fourth, the inhibition of SphK by either SphKG82D expression or a specific chemical inhibitor (DMS) profoundly inhibited the E2-induced increase of [Ca2+]i, strongly implicating SphK activation in the action of E2. Indeed, multiple lines of evidences have indicated that S1P is an important signal molecule for Ca2+ regulation in animal cells, yeast, and plants (16). It was initially suggested that S1P mobilizes Ca2+ from internal sources in an IP3-independent manner (39). Many studies appear to support this concept and suggest a novel mechanism for the S1P-induced increases of [Ca2+]i (28, 29). Furthermore, the activation of SphK catalyzes sphingosine to S1P. Not only can S1P mobilize Ca2+, but also sphingosine has been shown to block the store-operated Ca2+ release-activated calcium current activated by agonists (40). Hence, upon SphK activation, it is speculated that E2 induced an increase in S1P level directly activating Ca2+ channels and also a simultaneous decrease of sphingosine leading to the deinhibition of Ca2+ release-activated calcium current and a composite rise in [Ca2+]i.
The activation of MAPK family members Erk1 and Erk2 has been well documented in the mitogenic action of E2 independent on transcription activity of ER (6, 7, 8). Recent studies have shown that the mER was capable of interacting with c-Src directly (7) or indirectly via the complexes of ER and progestin receptors (41, 42), leading to activation of Ras/Erk pathway. The findings that E2-dependent Erk1/2 activation was markedly increased in the SphK-transfected MCF-7 cells, whereas it was totally abrogated in the cells expressing SphKG82D (Fig. 7), place SphK in the up-stream of Erk activation induced by E2. Indeed, our previous work and others have shown that inhibition of SphK by its inhibitors or expression of a dominant-negative mutant SphK prevented the activation of Ras/Erk in response to a variety of stimuli including cytokines and growth factors (21, 22, 34, 44), suggesting an ability of SphK to converge diverse signals to MAPK activation. Additionally, c-Src tyrosine kinases have been suggested to function as intermediates between S1P/SphK and the Ras/Erk pathway to participate in the regulation of cell growth (43). Recently, Shu et al. (44) reported that VEGF activated SphK which subsequently mediates signaling from PKC to Ras/Erk activation via a mechanism that appears not to utilize Ras-guanine nucleotide exchange factors but rather modulates Ras GTPase-activating protein activity to favor Ras activation. Moreover, it has been demonstrated that the E2-induced release of Ca2+ from intracellular stores is a critical event to trigger MAPK activation in MCF-7 cells (5). Therefore, in view of the role of SphK in the E2-dependent Ca2+ mobilization as discussed above, the increased [Ca2+]i is a possible mediator to conjunct signals between SphK and MAPK activation in response to E2 stimulation.
Cumulatively, the data presented in this study suggest that the activation of SphK is an important cytoplasmic signal to mediate estrogen action, particularly its nongenomic effect, in human breast cancer cells. It thus provides a potential linkage of SphK and the metabolism of sphingolipids to the (patho)physiology of estrogens, which will allow creation of new strategies for the management of the relevant diseases, such as breast cancer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]ATP was purchased from Geneworks (Adelaide, South Australia). The antibodies against phosphorylated forms of Erk1/2 as well as total Erk1/2 were purchased from Promega (Madison, WI).
Cell Cultures and Transfection
Strains of the human adenocarcinoma MCF-7 cell line (ER+/ß+) [ATCC (Manassas, VA) no. HTB-22] and MDA-MB-453 (ER-/ß+) (ATCC no. HTB-131) were obtained from the ATCC. Cells were cultured on phenol red-free DMEM (CSL Biosciences, Parkville, Australia) containing 4.5 g/liter glucose, 2 mM L-glutamine, 10 mM nonessential amino acids, 1.6 mg/ml penicillin-streptomycin, 10 ng/ml insulin, and 1–10% fetal bovine serum (where mentioned). Human SphK1 (GenBank accession no. AF200328) and mutant SphKG82D cDNA was FLAG epitope-tagged and subcloned into pcDNA3 vector (Invitrogen, Melbourne, Australia) as described previously (22). Transient transfections were performed using the LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s protocols at a cell density of 106 cells/ml with 20 µg of DNA. Stable transfectants were selected in medium containing 0.2 g/liter G418 (Invitrogen).
Cell Growth Assay
To assess cell proliferation, we used the CellTiter 96 AQueous proliferation assay (Promega Corp.) that is based on metabolic conversion of a tetrazolium compound 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to a colored product by living cells as described in the manufacturer’s protocols. Cells (2000 cells/well) were seeded in four to six replicates into 96-well plates and incubated at 37 C in 5% CO2 with or without E2 (10 nM) in phenol-red free media for a desired time period. The medium was changed every 2–3 d. The absorbance intensity of the MTS product is directly proportional to the number of viable cells in culture when cell number is between 2,000 and 200,000; otherwise, the exponential dependence was determined. Total cell numbers were calculated based on calibration curves.
Focus Formation and Colony Formation Assays
MCF-7 cells were seeded into 24-well plastic tissue culture plates at a density of 1 x 105 cells/ml/well, and maintained in phenol-red free media containing 10% FCS until the cells reached confluent. The cells were then refed at 24 h before first treatment with or without 10 nM E2 and then every 2–3 d thereafter with 1% FCS medium. After 5, 10, and 14 d the cultures were photographed and number of foci were counted. For colony assay, cells (1 x 104) suspended in 2 ml of 0.36% agar (Becton Dickinson, Sparks, MD) with phenol-red free growth medium were added on a base layer of 0.72% agar containing culture medium as described previously (21). After 14 d of incubation, the colonies were stained with MTT and counted. All experiments were done at least three times with triplicates per experimental point. Average was assessed by counting the number of colonies under low magnification (x20) at four fields of each well.
Western Blotting
Cells were harvested and lysed by sonication in lysis buffer containing 50 mM Tris/HCl (pH 7.4), 10% glycerol, 0.05% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na3VO4, 10 mM NaF, 1 mM EDTA, and protease inhibitors (Complete; Roche Molecular Biochemicals, Mannheim, Germany). Protein concentrations of the cell lysates were determined with a Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of cell lysates containing 30–50 µg of proteins were resolved by 12% SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences, Melbourne, Australia). The membranes were then probed with appropriate antibodies according to standard method as described previously (24). The immunocomplexes were detected with an enhanced chemiluminescence PLUS kit (Amersham Biosciences) and by using Typhoon 9410 Variable Mode Imager (Amersham Biosciences).
SphK Activity Assay
Briefly, after desired treatments the cells were washed with ice-cold PBS, scraped from the culture dishes and homogenized in lysis buffer containing 20 mM Tris (pH 7.4), 20% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 10 µM MgCl2, 1 mM Na3VO4, 15 mM NaF, 10 µg/ml leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine. After centrifugation at 13,000 x g for 30 min, SphK activity was measured by incubating the cytosolic fraction with 5 µM D-erythro-sphingosine dissolved in 0.1% Triton X-100 and [32P]ATP (1 mM, 0.5 Ci/ml) for 30 min at 37 C as described previously (34). Reactions were terminated and S1P extracted by the addition of 800 µl of chloroform/methanol/HCl (100:200:1, vol/vol), followed by vigorous mixing, addition of 200 µl of chloroform and 200 µl of 2 M KCl, and phase separation by centrifugation. The labeled S1P in the organic phase was isolated by thin-layer chromatography on Silica Gel 60 with 1-butanol/ethanol/acetic acid/water (8:2:1:2, vol/vol) and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The enzyme activity was normalized by total protein concentration of each sample.
Measurement of [Ca2+]i
MCF-7 cells (1 x 108) were trypsinized, washed, and resuspended in 1 ml of phenol-red free media without serum and incubated with 2 µM Sulfinpyrazone for 15 min at room temperature. Then cells were incubated for 60 min at 37 C with 20 µM Fluo-3/AM combined with 20% Pluronic F-127 (1:1) in the dark, washed and removed for immediate analysis on Perkin-Elmer luminescence spectrophotometer (LS 50B) (Perkin-Elmer Corp., Norwalk, CT). The fluorescence of the cells was monitored at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. Each recording was calibrated by determining the maximal uptake of calcium (Fmax) in the presence of 0.1% Triton X-100 as well as the level of autofluorescence (Fmin), after quenching Fluo-3/AM fluorescence with 1 mM Mn2+. Intracellular calcium concentration, [Ca2+]i, was calculated as previously described (45) using the equation: [Ca2+]i = Kd (F -Fmin)/(Fmax -F), where Kd is the dissociation constant of fluo-3/AM set to 400 nM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; BHQ, tert-butylhydroquinone; DMS, N, N-dimethylsphingosine; E2, 17ß-estradiol; ER, estrogen receptor; GPCR, G protein-coupled receptor; ICI, ICI 182,780; IP3, inositol 1,4,5-triphosphate; mER, membrane-associated ER; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; S1P, sphingosine 1-phosphate; SphK, sphingosine kinase.
Received for publication April 4, 2003. Accepted for publication July 14, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 142:2336–2342(i). J Biol Chem 276:27071–27076
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 B. S. Paugh, L. Bryan, S. W. Paugh, K. M. Wilczynska, S. M. Alvarez, S. K. Singh, D. Kapitonov, H. Rokita, S. Wright, I. Griswold-Prenner, et al. Interleukin-1 Regulates the Expression of Sphingosine Kinase 1 in Glioblastoma Cells J. Biol. Chem., February 6, 2009; 284(6): 3408 - 3417. [Abstract] [Full Text] [PDF]
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 J. Li, H.-Y. Guan, L.-Y. Gong, L.-B. Song, N. Zhang, J. Wu, J. Yuan, Y.-J. Zheng, Z.-S. Huang, and M. Li Clinical Significance of Sphingosine Kinase-1 Expression in Human Astrocytomas Progression and Overall Patient Survival Clin. Cancer Res., November 1, 2008; 14(21): 6996 - 7003. [Abstract] [Full Text] [PDF]
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 D. Shida, X. Fang, T. Kordula, K. Takabe, S. Lepine, S. E. Alvarez, S. Milstien, and S. Spiegel Cross-talk between LPA1 and Epidermal Growth Factor Receptors Mediates Up-regulation of Sphingosine Kinase 1 to Promote Gastric Cancer Cell Motility and Invasion Cancer Res., August 15, 2008; 68(16): 6569 - 6577. [Abstract] [Full Text] [PDF]
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 D. A. Lebman and S. Spiegel Thematic Review Series: Sphingolipids. Cross-talk at the crossroads of sphingosine-1-phosphate, growth factors, and cytokine signaling J. Lipid Res., July 1, 2008; 49(7): 1388 - 1394. [Abstract] [Full Text] [PDF]
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 K. Takabe, S. W. Paugh, S. Milstien, and S. Spiegel "Inside-Out" Signaling of Sphingosine-1-Phosphate: Therapeutic Targets Pharmacol. Rev., June 1, 2008; 60(2): 181 - 195. [Abstract] [Full Text] [PDF]
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T. M. Leclercq, P. A. B. Moretti, M. A. Vadas, and S. M. Pitson Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity J. Biol. Chem., April 11, 2008; 283(15): 9606 - 9614. [Abstract] [Full Text] [PDF]
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 X. Li, M. Stankovic, C. S. Bonder, C. N. Hahn, M. Parsons, S. M. Pitson, P. Xia, R. L. Proia, M. A. Vadas, and J. R. Gamble Basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1 Blood, April 1, 2008; 111(7): 3489 - 3497. [Abstract] [Full Text] [PDF]
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 M. Kim, M. Kim, N. Kim, V. D. D'Agati, C. W. Emala Sr, and H. T. Lee Isoflurane mediates protection from renal ischemia-reperfusion injury via sphingosine kinase and sphingosine-1-phosphate-dependent pathways Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1827 - F1835. [Abstract] [Full Text] [PDF]
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 J. Min, A. Mesika, M. Sivaguru, P. P. Van Veldhoven, H. Alexander, A. H. Futerman, and S. Alexander (Dihydro)ceramide Synthase 1 Regulated Sensitivity to Cisplatin Is Associated with the Activation of p38 Mitogen-Activated Protein Kinase and Is Abrogated by Sphingosine Kinase 1 Mol. Cancer Res., August 1, 2007; 5(8): 801 - 812. [Abstract] [Full Text] [PDF]
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 D. Leiber, Y. Banno, and Z. Tanfin Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms Am J Physiol Cell Physiol, January 1, 2007; 292(1): C240 - C250. [Abstract] [Full Text] [PDF]
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 K. J. French, J. J. Upson, S. N. Keller, Y. Zhuang, J. K. Yun, and C. D. Smith Antitumor Activity of Sphingosine Kinase Inhibitors J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 596 - 603. [Abstract] [Full Text] [PDF]
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 O. Sukocheva, C. Wadham, A. Holmes, N. Albanese, E. Verrier, F. Feng, A. Bernal, C. K. Derian, A. Ullrich, M. A. Vadas, et al. Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1 J. Cell Biol., April 24, 2006; 173(2): 301 - 310. [Abstract] [Full Text] [PDF]
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F. Acconcia, C. J. Barnes, and R. Kumar Estrogen and Tamoxifen Induce Cytoskeletal Remodeling and Migration in Endometrial Cancer Cells Endocrinology, March 1, 2006; 147(3): 1203 - 1212. [Abstract] [Full Text] [PDF]
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 M. N. Chernova, D. H. Vandorpe, J. S. Clark, J. I. Williams, M. A. Zasloff, L. Jiang, and S. L. Alper Apparent receptor-mediated activation of Ca2+-dependent conductive Cl- transport by shark-derived polyaminosterols Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1644 - R1658. [Abstract] [Full Text] [PDF]
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 S. K. Goparaju, P. S. Jolly, K. R. Watterson, M. Bektas, S. Alvarez, S. Sarkar, L. Mel, I. Ishii, J. Chun, S. Milstien, et al. The S1P2 Receptor Negatively Regulates Platelet-Derived Growth Factor-Induced Motility and Proliferation Mol. Cell. Biol., May 15, 2005; 25(10): 4237 - 4249. [Abstract] [Full Text] [PDF]
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 S. M. Pitson, P. Xia, T. M. Leclercq, P. A.B. Moretti, J. R. Zebol, H. E. Lynn, B. W. Wattenberg, and M. A. Vadas Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling J. Exp. Med., January 3, 2005; 201(1): 49 - 54. [Abstract] [Full Text] [PDF]
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M. Yamanaka, D. Shegogue, H. Pei, S. Bu, A. Bielawska, J. Bielawski, B. Pettus, Y. A. Hannun, L. Obeid, and M. Trojanowska Sphingosine Kinase 1 (SPHK1) Is Induced by Transforming Growth Factor-{beta} and Mediates TIMP-1 Up-regulation J. Biol. Chem., December 24, 2004; 279(52): 53994 - 54001. [Abstract] [Full Text] [PDF]
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D. R. Herr, H. Fyrst, M. B. Creason, V. H. Phan, J. D. Saba, and G. L. Harris Characterization of the Drosophila Sphingosine Kinases and Requirement for Sk2 in Normal Reproductive Function J. Biol. Chem., March 26, 2004; 279(13): 12685 - 12694. [Abstract] [Full Text] [PDF]
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T. Sanchez, T. Estrada-Hernandez, J.-H. Paik, M.-T. Wu, K. Venkataraman, V. Brinkmann, K. Claffey, and T. Hla Phosphorylation and Action of the Immunomodulator FTY720 Inhibits Vascular Endothelial Cell Growth Factor-induced Vascular Permeability J. Biol. Chem., November 21, 2003; 278(47): 47281 - 47290. [Abstract] [Full Text] [PDF]
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