Ligand-induced nuclear translocation of S1P1 receptors mediates Cyr61 and CTGF transcription in endothelial cells
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Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P1), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P1 receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P1 receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P1 receptors were internalized from plasma membrane S1P1 after ligand treatment. Moreover, nuclear S1P1 is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P–S1P1 signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.
KeywordsS1P S1P1 Endothelial cells
- S1P1 (old nomenclature EDG-1, endothelial differentiating gene-1)
The high affinity GPCR for S1P
G-protein coupled receptor
Human umbilical vein endothelial cells
Cultured endothelial cells
Green fluorescent protein
Cysteine-rich angiogenic protein 61
Connective tissue growth factor
Nuclear localization sequences
Sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid mediator secreted by activated platelets (Yatomi et al. 1995), regulates a variety of cellular responses including proliferation, survival, cytoskeletal remodeling, adhesion, and chemotaxis (Wu et al. 1995, 1999, 2008; Spiegel et al. 1998; Lee et al. 1999, 2001, 2006; ). In endothelial cells (ECs), most of these responses are mediated by the S1P receptor subtype 1 (S1P1, old nomenclature EDG-1), a prototype of the S1P family of G-protein coupled receptors (GPCRs) (Lee et al. 1999, 2001, 2006; Paik et al. 2001). The S1P1 was cloned from cultured human umbilical vein endothelial cells (HUVECs) stimulated with the tumor promoter phorbol 12-myristate 13-acetate (PMA) (Hla and Maciag 1990). Subsequently, S1P1 was shown to activate mitogen-activated protein kinase (MAPK or ERK1/2) and phospholipase A2 (PLA2) and inhibit adenylate cyclase via Gi heterotrimeric G proteins (Lee et al. 1996). In addition, recent studies suggest that S1P/S1P1 signaling plays an important role in regulating blood vessel development and maturation (Lee et al. 1999; Liu et al. 2000). For example, in cultured ECs, S1P/S1P1 signaling regulates endothelial survival, migration, cytoskeletal reorganization, and adherens junction formation, all of which result in endothelial morphogenesis to form capillary-like structures in vitro (Lee et al. 1999, 2001, 2006). Furthermore, deletion of S1P1 results in embryonic lethality in transgenic mice due to a defect in the vasculature (Liu et al. 2000). These results together suggest that S1P/S1P1 signaling plays a critical role in neo-vessel formation and vasculature maintenance.
Ligand-induced internalization and intracellular trafficking of GPCRs is thought to be important in the regulation of signal transduction and biological functions mediated by GPCRs. The spatiotemporal relationship of S1P-induced intracellular trafficking of the S1P1 receptor was previously examined in HEK293 cells heterologously expressing green fluorescent protein (GFP) tagged S1P1 receptor (HEK293S1P 1 GFP) (Liu et al. 1999). In that study, it was shown that S1P1 receptors were internalized into endosomal compartments and subsequently recycled back to the plasma membrane after S1P stimulation (Liu et al. 1999). However, the relevance of this plasma membrane–endosome–plasma membrane trafficking route of S1P1 receptors has not yet been characterized in a truly physiological setting. Moreover, the functional significance of S1P1 internalization/trafficking in response to S1P stimulation is presently unknown.
There is increasing evidence suggesting nuclear localization of GPCRs (Marrache et al. 2005; Cook et al. 2006; Gobeil et al. 2002, 2003a, b; Lee et al. 2004; Pickard et al. 2006; Liao et al. 2007). However, the underlying mechanisms and functional significance of nuclear-localized GPCRs are poorly understood. In this report, we show that S1P1 is present in the nuclear compartment of ECs. Also, we demonstrate that S1P treatment results in nuclear translocation of S1P1 receptors. Furthermore, we demonstrate that the nuclear S1P–S1P1 signaling axis is able to activate transcriptional activities in endothelial cells. This study presents evidence for the first time showing the functional significance of nuclear-localized S1P1 in ECs. These data together suggest that nuclear S1P1 may play a novel role in mediating S1P-regulated endothelial functions.
Materials and methods
Sphingosine-1-phosphate (Biomol) was dissolved in fatty acid-free bovine serum albumin (BSA, Sigma, 0.4%). Mouse-derived S1P1 monoclonal antibody (E1-49) was generated by conventional hybridoma technology using bacteria-expressed S1P1 as an immunogen (Lee et al. 2006). E1-49 was shown to specifically react with the human S1P1 receptor by immunostaining, immunoblotting, and immunoprecipitation (Lee et al. 2006). Polyclonal rabbit anti-GFP antibody was purchased from Molecular Probes. Monoclonal mouse anti-calnexin was purchased from BD Transduction Laboratories. Mouse anti-human Na+/K+ ATPase (α6F) came from the Development Studies Hybridoma bank. Rabbit anti-human Lamin A/C was purchased from Cell Signaling. Rabbit anti-human Rab4 was purchased from Chemicon. Other reagents, unless otherwise specified, were from Sigma.
HUVECs were purchased from Cambrex Bioproducts. HUVECs and HEK293 stably expressing GFP-tagged S1P1 (S1P1GFP) or GFP polypeptides were cultured as previously described (Lee et al. 1999, 2001, 2006; Liu et al. 1999).
Cell fractionation and nuclei isolation
HUVECs were collected between passages 6–13 at ~80% confluency unless otherwise stated. Isolation of nuclei was achieved by using the hypotonic/Nonidet P-40 lysis method (Kaufmann et al. 1983; Gobeil et al. 2002; Lee et al. 2004; Liao et al. 2007). Briefly, cultures were rinsed twice with ice-cold PBS and collected with cell scrapers. Cells (5 × 106) were resuspended in 0.5 ml of hypotonic/Nonidet P-40 buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40) and incubated on ice for 5 min. After centrifuging at 500g for 5 min, nuclear pellets were washed twice with 0.5 ml of hypotonic/Nonidet P-40 buffer. The morphological integrity of isolated nuclei (> 90%) was assessed by light microscopy after trypan blue staining. The purity of subcellular fractions was routinely verified by immunoblotting with antibodies specific for markers of different subcellular organelles, e.g., CD61 and Na+/K+ ATPase for the plasma membrane, calnexin for the endoplasmic reticulum, Rab4 for the endosome, and lamin A/C for the nucleus.
Immunoprecipitation and immunoblotting
Cellular or nuclear extracts were prepared with TBST/OG buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 10 mM MgCl2, 1% Triton X-100, 60 mM n-octyl β-d-glucopyranoside) (Lee et al. 1999, 2001, 2006). Extracts were then incubated with anti-S1P1 at 4°C overnight. Subsequently, the immunocomplexes were precipitated by protein-A beads at 4°C for 1 h. After washing, precipitated polypeptides were released by boiling in double-strength Laemmli SDS-sampling buffer (4.6% (w/v) SDS, 10% (v/v) β-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCl, pH 6.8, 0.01% (w/v) bromphenol blue) at 95°C for 5 min and resolved with SDS-PAGE. The electrophoretically separated polypeptides were transferred to nitrocellulose membrane, blocked in 5% non-fat dry milk (LabScientific), and incubated with the indicated primary antibodies on a rotary shaker at 4°C overnight. After washing, the blots were incubated with HRP-conjugated second antibodies and detected with Enhanced Chemiluminescence Reagent (ECL) (Amersham).
Immunostaining and confocal microscope analysis
Cells were cultured in 35-mm glass bottom dishes (MatTek) and serum-starved in M199 medium for 2 h (for HUVECs) or in DMEM supplemented with 0.05% FBS for 24 h (for HEK293). Cells were then stimulated with S1P (200 nM) for various times, fixed with 4% paraformaldehyde, and permeabilized with 0.25% Triton X-100. S1P1 receptors were stained with the mouse E1-49 antibody (1:1,000 dilutions), followed by staining with Alexa488-conjugated goat anti-mouse antibody (1:1,000 dilutions). GFP-tagged S1P1 polypeptides (Lee et al. 1998; Liu et al. 1999) were directly visualized with an Axiovert 200 M epi-fluorescence microscope (Carl Zeiss). Confocal microscopic analysis was carried out as previously described (Lee et al. 1999; Liu et al. 1999; Wang et al. 2008).
Quantitative nuclear run-off transcription assay
Nuclear run-off transcriptional assays were performed essentially as described (Rolfe and Sewell 1997; Li and Chaikof 2002; Green and Bender 2000). Nuclei (2 × 106) were collected from HUVECs stimulated with or without S1P (1 μM, 1 h) and suspended in 200 μl of DEPC-treated water. The nuclear fraction (150 μg proteins) was mixed with an equal volume of 2 × reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.3 M KCl, 1 mM each of ATP, CTP, GTP, and UTP) containing RNase inhibitor (6 U/sample, Promega). Transcription was carried out at 30°C for 2 h in the presence or absence of S1P (1 μM) with gentle shaking. The transcription reaction was then terminated by the addition of DNase I (24 μg/sample) at 30°C for 10 min. Subsequently, 200 μl of SDS-containing buffer (5% SDS, 0.5 M Tris-HCl, pH 7.4, 0.125 M EDTA) was added and proteins were digested by the addition of proteinase K (200 μg/sample). RNAs were then isolated using STAT-60 reagent (Tel-Test Inc.) according to the manufacturer’s instructions. One microgram of RNAs was reverse-transcribed with the oligo-dT15 primer (Roche Applied Science) by using M-MLV reverse transcriptase (Promega) for first strand cDNA synthesis. PCR detection of gene expression was performed with 1 μl (or 20 ng) of reverse-transcribed cDNAs in a total volume of 20 μl of reaction mixture containing 200 nM of each primer, 0.2 mM of each dNTP and 1 unit of Taq Polymerase (Promega). Reactions were performed using the Gene Amp 9700 PCR System (Applied Biosystems). The PCR conditions were 30 cycles of denaturation at 95°C (30 s), annealing at 58°C (30 s), and extension at 72°C (30 s). Amplified PCR products were analyzed by electrophoresis on a 1.5% agarose gel. PCR primer pairs used were: human Cyr61, (sense) cctcggctggtcaaagttac, (antisense) aggctccattccaaaaacag; human CTGF: (sense) aaggtgtggctttaggagca, (antisense) tcttgatggctggagaatgc; human actin: (sense) aaactggaacggtgaaggtg, (antisense) tcaagttgggggacaaaaag.
S1P1 receptors are present in the nuclear compartment
S1P treatment results in nuclear translocation of S1P1 receptors
Subsequently, biotin-labeled cultures were washed and incubated at 37°C in the presence or absence of S1P for various times. Cell nuclei were prepared, solubilized, and incubated with avidin beads to isolate biotinylated polypeptides. As shown in Fig. 7b, there are no biotin-labeled S1P1 receptors observed in the isolated nuclei in the absence of S1P treatment (top panel, Fig. 7b). In sharp contrast, the biotinylated S1P1 receptors were clearly detected in the isolated nuclei at 10 min after S1P stimulation (top panel, Fig. 7b). In a parallel control immunoblot, nuclear S1P1 polypeptides were shown to be quantitatively increased after S1P stimulation (second panel, Fig. 7b). Together, these data suggest that the nuclear S1P1 was translocated from plasma membrane after ligand stimulation.
Nuclear S1P–S1P1 signaling stimulates Cyr61 and CTGF transcription in ECs
Intracellular trafficking plays an important role in GPCRs signaling and function. Using HEK293 cells expressing S1P1GFP polypeptides, we showed that S1P treatment results in the internalization of S1P1 receptors into the intracellular endosomal compartment (Liu et al. 1999). Also, it was recently demonstrated that S1P1 is relocated to caveolae for regulation of cortical actin formation (Singleton et al. 2005) and to proteasomes, where the receptor was ubiquitinylated and degraded (Oo et al. 2007). These results together suggest that S1P1 may be redistributed to multiple intracellular organelles to execute distinct signaling pathways. Thus, we utilized time-lapse microscope analysis to examine the S1P-stimulated intracellular trafficking of S1P1 receptors in a spatiotemporal manner (Fig. 6; Supplemental data 1). Surprisingly, we constantly observed that S1P1 receptors were rapidly internalized into intracellular compartments upon ligand stimulation, traveled to nuclear regions, and then recycled back to plasma membrane (Fig. 6; Supplemental data 1). This observation suggests that cellular nucleus may be a novel intracellular destination of ligand-induced S1P1 trafficking.
Subsequently, we rigorously examined this speculation by utilizing various biochemical and microscopic techniques. Cellular fractionation studies showed that S1P1 receptors were present in the nuclei of ECs and HEK293 cells stably expressing S1P1GFP polypeptides (Fig. 1). The isolated nuclear fraction was lack of contaminated polypeptides from other cellular compartments (e.g., CD61, Na/K ATPase for the plasma membrane, Rab4 for the endosome, and calnexin for endoplasmic reticulum; Fig. 1a, c). Moreover, lamin A/C polypeptides (a specific nuclear marker) were only detected in the isolated nuclear fraction (Fig. 1a, c). These data suggest that the presence of S1P1 receptors in the nuclei is not an artifact of isolation. Furthermore, anti-S1P1 immunogold labeling followed by high resolution electron microscopic analysis showed that S1P1 receptors are specifically present in both plasma membrane and nuclear envelop of ECs (Fig. 2). In addition, S1P1 polypeptides were clearly present in the nuclei when the isolated nuclei were examined by confocal microscope analysis (Fig. 3). Collectively, these data suggest that nuclear presence of S1P1 receptors is a truly physiological phenomenon.
There are increasing lines of evidence showing the presence of GPCRs in the nuclear compartment (Marrache et al. 2005; Cook et al. 2006; Gobeil et al. 2002, 2003a, b; Lee et al. 2004; Pickard et al. 2006; Liao et al. 2007). The topological aspects of nuclear GPCRs are completely unknown at present, because GPCRs are highly hydrophobic due to their seven trans-membrane segments. Moreover, since the canonical nuclear localization sequences (NLS) have not been identified in GPCRs, the mechanisms underlying nuclear translocation of GPCRs have not been defined. Immunogold labeling showed that S1P1 receptors are mainly located at the nuclear envelop region (arrows, Figs. 2, 5). Thus, it is possible that the structure of nuclear S1P1 might be stabilized in the nuclei by integration into nuclear membrane, which is similar to the topological characteristics of plasma membrane S1P1.
Specific nuclear translocation is generally thought to occur via NLS, which contains either a stretch of basic amino acids or bipartite clusters of basic amino acids (Silver 1991; Dingwall and Laskey 1991). A close examination of the primary sequence of S1P1 polypeptides does not reveal the presence of such a canonical NLS. Interestingly, 11 of 34 amino acids (32.4%) in the third intracellular loop (i3 domain) of the S1P1 receptors are either Arg or Lys (RIYSLVRTRSRRLTFRKNISKGSRSSEKSLALLK). It suggests that the i3 domain of S1P1 receptor might function as a non-canonical NLS; and thus, mediate nuclear translocation of S1P1 receptors. However, this notion is highly speculative and awaits empirical determination by site-directed mutagenesis of i3 domain of S1P1 receptors in the future.
It should be noted that the size of the S1P1 receptor in the nucleus is the same as that in the plasma membrane as detected by immunoblotting (Fig. 1a), which is consistent with a full-length receptor (Fig. 1a). Also, the nuclear S1P1 receptor could be detected by either anti-GFP for the C-terminal GFP tag (Figs. 1b, c, 7) or an antibody for the N-terminal Flag tag (data not shown). This data indicates that nuclear S1P1 has not been proteolytically processed and is full length during the process of nuclear translocation. However, it is presently unknown whether subtle modifications (e.g., phosphorylation) of S1P1 receptors are required for nuclear translocation.
Confocal microscopic analysis and live cell imaging showed that S1P treatment stimulates the translocation of plasma membrane S1P1 receptors to nuclear compartment (Figs. 4, 6; supplemental data 1). The ligand-induced plasma membrane–nucleus trafficking route was further confirmed by the membrane biotinylation technique (Fig. 7). We used the cell impermeable Sulfo-NHS-SS-Biotin reagent to exclusively label the extracellular domains of plasma membrane polypeptides (Fig. 7a). After ligand stimulation, the biotin-labeled S1P1 receptors appeared in the isolated nuclei in a time-dependent manner (top panel, Fig. 7b). Thus, these data suggest that the nuclear S1P1 was translocated from plasma membrane after ligand stimulation.
In summary, we show that S1P1 is present in the nuclear compartment of normal growing ECs. Also, we provide evidence supporting that S1P stimulation induces the plasma membrane–nucleus trafficking of S1P1 receptors in ECs. Importantly, we show that the nuclear S1P–S1P1 signaling axis in ECs directly induces expression of Cyr61 and CTGF, two growth factors which are functionally implied in angiogenic responses (Leu et al. 2002; Gao and Brigstock 2004). These data together strongly suggest that nuclear localization of the S1P1 receptor is a truly physiological phenomenon.
We thank Dr. Arthur R. Hand (Central Electron Microscope Facility/University of Connecticut Health Center) for the EM-immunogold labeling. This work is supported by NIH grants R01HL071071 and AHA grant-in-aid 0755245B (M.L).
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Supplemental data 1 Live cell imaging of ligand induced intracellular trafficking of S1P1 receptors. HEK293S1P1GFP cells were transfected with NLS tagged YFP polypeptides (blue) and serum-starved for 24 hrs. After S1P (200 nM) addition, the intracellular trafficking of S1P1GFP polypeptides (green) was recorded by live cell imaging technique as described (Jala et al., 2005). The time (min) after S1P addition is shown. This movie is a representative of seven independent experiments (AVI 4511 kb)
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