HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/2/341 03-0302fjev1    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 BAUDHUIN, L. M. Articles by XU, Y. Search for Related Content PubMed PubMed Citation Articles by BAUDHUIN, L. M. Articles by XU, Y.

S1P₃-mediated Akt activation and cross-talk with platelet-derived growth factor receptor (PDGFR)¹

LINNEA M. BAUDHUIN^*,,2,3, YING JIANG^*,3, ALEXANDER ZASLAVSKY^*,, ISAO ISHII, JEROLD CHUN and YAN XU^*,,||,4

^* Department of Cancer Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA;
Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA;
Department of Molecular Genetics, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan;
Department of Molecular Biology, Scripps Research Institute, La Jolla, California, USA; and
^|| Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, USA

⁴ Correspondence: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: xuy{at}cf.org

SPECIFIC AIMS

We examined Akt signaling pathways induced by sphingosine-1-phosphate (S1P) in mouse embryonic fibroblasts (MEFs) and other cells. We observed a novel cross-talk between S1P and the PDGF receptor (PDGFR) mediated by S1P₃ that is, signaling pathway, receptor subtype, and cell type specific.

PRINCIPAL FINDINGS

1. S1P₃ was required for PDGFR-dependent Akt S473 phosphorylation in MEFs
We recently reported that both S1P and PDGF induce a MEK-dependent Akt activation only in S1P₃-expressing cells. We postulated that S1P₃ is required for this MEK (and ERK)-dependent Akt phosphorylation and proposed that a potential cross-talk exists between S1P₃ and PDGFR. We used S1P₃ null MEFs, which lack endogenous expression of S1P₄ and S1P₅ but have normal expression of S1P₁ and S1P₂. In wild-type MEFs, which express S1P_1-3, S1P (1 µM) and PDGF (10 ng/mL) induced phosphorylation of ERK and Akt (S473) (Fig. 1 A). AG1296, a specific inhibitor of PDGFR, inhibited the phosphorylation of Akt and ERK induced by PDGF-BB (Fig. 1A ). Phosphorylation of Akt, but not ERK, induced by S1P was also sensitive to AG1296 (50 µM), suggesting that S1P requires PDGFR activity to stimulate Akt S473 phosphorylation in MEFs whereas ERK phosphorylation by S1P is independent of PDGFR. On the other hand, we found that S1P was unable to induce Akt phosphorylation in S1P₃ null MEFs whereas ERK activation by S1P was unaffected (Fig. 1B ). Reestablishment of S1P₃ expression (by infection of S1P₃ in a retroviral vector) restored the ability of S1P to induce Akt activation, which again was sensitive to AG1296 (Fig. 1C ), strongly suggesting that S1P₃ is required for S473 Akt phosphorylation in MEFs; this activity is dependent on the activation of PGDFR. We confirmed this notion in S1P_2/3 double null MEFs, where S1P was unable to induce Akt phosphorylation but ERK activation by S1P was unaffected (data not shown). S1P-induced Akt signaling could be restored by infecting S1P₃ alone in S1P_2/3 double null MEFs (Fig. 1D ), supporting the necessity of S1P₃ for Akt signaling and suggesting that S1P₂ is unnecessary for this activity in MEFs. In S1P₃ null and S1P_2/3 double null MEFs, vector-only infected cells did not change cell responsiveness to either S1P or PDGF (data not shown). Figure 1E shows mean densitometric traces of several experiments.

View larger version (38K): [in this window] [in a new window]   Figure 1. S1P3 and PDGFR activity are required for Akt phosphorylation induced by S1P. A) MEFs were stimulated by S1P (1 µM) and PDGF-BB (10 ng/mL) for 10 and 5 min, respectively. Western blot analyses were performed using antibodies against phosphorylated Akt (p-S473-Akt), phosphorylated ERK (p-ERK), or total Akt and total ERK. AG1296 (30 min pretreatment) was used to block the PDGFR activity. B) S1P3 null MEFs were stimulated by S1P and PDGF-BB with or without AG1296 (50 µM) pretreatment. C) pMSCV-S1P3 was infected into S1P3 null MEFs. Stimulation by S1P and PDGF-BB and Western blot analyses were performed as described above. D) Same as panel C except that pMSCV-S1P3 was infected into S1P2/3 double null MEFs. Expression of S1P3 was confirmed by detecting the Flag-tag using an M2 antibody. E) A graph of the mean densitometric traces of several experiments. ***P < 0.001; Student’s t test.

2. S1P is dependent on PDGFR for Akt and ERK activation in HEY ovarian cancer cells
We examined the PDGFR dependence of S1P-induced activation of Akt and ERK in HEY cells, which endogenously express S1P₃. AG1296 (10 µM) pretreatment of HEY cells resulted in complete inhibition of Akt activation (measured by Akt enzymatic activity) induced by S1P and PDGF (Fig. 2 A). To confirm that PDGFR activity was required for this Akt activation, we examined the effects of AG1296, 2A1E2 (a neutralizing antibody to PDGFR-ß), and 2H7C5 (a neutralizing antibody to PDGFR-) on S473 and T308 phosphorylation of Akt. A combination of 2A1E2 and 2H7C5 (both at 0.3 nM) completely blocked PDGF-BB-induced Akt activation/phosphorylation; we used this concentration in subsequent studies. The dependence on PDGFR activity for Akt activation was S1P specific, as lysophosphatidic acid, a similar lipid signaling molecule, was not dependent on PDGFR for Akt phosphorylation at S473 or T308 (Fig. 2B ). We also found that S473 phosphorylation of Akt induced by endothelin-1 and thrombin in HEY cells was not affected by pretreatment with AG1296 (data not shown). The dependence on a tyrosine kinase receptor by S1P was specific to PDGFR, since AG1478 and AG1024 (specific inhibitors of EGFR and IGFR, respectively), had no effect on S1P-induced Akt activation (Fig. 2C ). In contrast, we demonstrated that PDGFR was required for ERK phosphorylation induced by S1P in HEY cells, as determined by Western blot analysis (Fig. 2D ). This is consistent with our observation that MEK and ERK are upstream of Akt in S1P-induced Akt activation in HEY cells. These results suggest that upstream signaling mechanisms leading to Akt activation may be different in different cell types.

View larger version (27K): [in this window] [in a new window]   Figure 2. S1P activates Akt in a PDGFR-dependent manner. HEY cells were pretreated with AG1296 (10 µM), AG1478 (200 nM), AG1024 (10 µM) for 30 min or 2A1E2 and 2H7C5 (0.3 nM) for 15 min, then treated with S1P (1 µM) for 20 min (Akt activation/phosphorylation) or 5 min (ERK phosphorylation), PDGF (10 ng/mL), EGF (10 ng/mL), or IGF-I (20 ng/mL) for 5 min. A) Nonradioactive immunoprecipitation Akt kinase assay. B–D) Western blot analyses using antibodies against phosphorylated Akt (p-S473-Akt, p-T308-Akt), phosphorylated ERK (p-ERK), or total Akt. Western blot analyses are representative of at least 3 independent experiments.

3. S1P induces tyrosine phosphorylation of PDGFR
To directly assess the involvement of PDGFR and whether S1P could actually stimulate tyrosine phosphorylation of PDGFR, HEY cells were treated with S1P or PDGF and tyrosine phosphorylation of PDGFR-ß was assessed. PDGFR was strongly phosphorylated by PDGF (see Fig. 3 in the full text article). S1P also induced phosphorylation of PDGFR, although its kinetics was slower. Phosphorylation of PDGFR by S1P occurred as early as 1 min and maximized at 20 min. Treatment of cells with PTX attenuated S1P- but not PDGF-induced PDGFR tyrosine phosphorylation, suggesting that the former occurred in a G_i-dependent manner and the latter did not require G_i. S1P- and PDGF-induced tyrosine phosphorylation of PDGFR were both insensitive to LY294002, a specific inhibitor of phosphatidylinositol-3 kinase (PI3-K), indicating that PI3-K activation is not required for PDGFR phosphorylation.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of different types of interactions between S1P receptors and PDGFR. Left panel: "sequential" model proposed by Spiegel’s group. SPH, sphingosine; SPHK, sphingosine kinase. Middle panel: the "integrative" model proposed by Pyne’s group. Right panel summarizes results in the paper. Different S1P receptor subtypes are involved in different models.

4. PDGFR-dependent Akt activation by S1P is cell line specific
S1P phosphorylated Akt in a PDGFR-dependent manner in other endogenous S1P₃-expressing cell lines (A2780, T474D, and an immortalized human microvascular endothelial cell line, HMEC-1), but in a PDGFR-independent manner in cell lines (PC-3 and GI-101A) expressing no or very low levels of S1P₃. These results suggest that PDGFR-dependent phosphorylation of Akt by S1P is mediated by S1P₃ specifically. Other S1P receptors (such as S1P₁ and S1P₂) may mediate PDGFR-independent Akt activation by S1P in a cell type-dependent manner.

CONCLUSIONS AND SIGNIFICANCE

S1P stimulates or inhibits numerous cellular functions and signaling pathways, leading to different cellular functions. Akt plays an important role in cell survival and tumorigenesis. We have introduced important insights into cell type-, receptor subtype-, and signaling pathway-dependent cellular effects of S1P. We have revealed a new type of cross-talk between S1P₃ and PDGFR. Interactions between the signaling pathways of S1P and PDGF have been shown in two different models, the sequential model by Spiegel’s group and the integrative model by Pyne’s group (Fig. 3 ). The sequential model suggests that production of S1P induced by PDGF mediates the interaction and that PDGF is unidirectionally dependent on S1P₁ to induce cell motility. The integrative model indicates that a physical interaction between S1P₁ and PDGFR, but not S1P production, plays the central role and that signaling efficiencies of both S1P and PDGF are increased (bidirectional) (Fig. 3) . In studies from Pyne’s group, PDGF did not induce S1P production in either HEK293 or smooth muscle cells. Our work here adds important, interesting information to models of interactions of S1P-PDGF signaling. We show that 1) another S1P receptor subtype (S1P₃, rather than S1P₁) is involved in the interaction between S1P and PDGF signaling; 2) a "reciprocal" requirement exists, i.e., S1P-induced Akt phosphorylation via S1P₃ requires PDGFR; and 3) S1P can activate PDGFR by induction of tyrosine phosphorylation of PDGFR (Fig. 3) . Transactivation of tyrosine kinase receptors by GPCRs has drawn considerable attention in recent years. The published data and our novel observations may not necessarily be mutually exclusive and the interactions between S1P and PDGF are likely to be signaling pathway, S1P receptor subtype, and cell type specific. These interactions between S1P and PDGFR may play important physiological and pathological regulatory roles.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0302fje

² Current address: Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA.

³ These authors contributed equally to this work.

This article has been cited by other articles:

				S. Balthasar, N. Bergelin, C. Lof, M. Vainio, S. Andersson, and K. Tornquist Interactions between sphingosine-1-phosphate and vascular endothelial growth factor signalling in ML-1 follicular thyroid carcinoma cells Endocr. Relat. Cancer, June 1, 2008; 15(2): 521 - 534. [Abstract] [Full Text] [PDF]

				C. K. Means, S. Miyamoto, J. Chun, and J. H. Brown S1P1 Receptor Localization Confers Selectivity for Gi-mediated cAMP and Contractile Responses J. Biol. Chem., May 2, 2008; 283(18): 11954 - 11963. [Abstract] [Full Text] [PDF]

				Z. Zhao, Y. Xiao, P. Elson, H. Tan, S. J. Plummer, M. Berk, P. P. Aung, I. C. Lavery, J. P. Achkar, L. Li, et al. Plasma Lysophosphatidylcholine Levels: Potential Biomarkers for Colorectal Cancer J. Clin. Oncol., July 1, 2007; 25(19): 2696 - 2701. [Abstract] [Full Text] [PDF]

				L. K. Landeen, N. Aroonsakool, J. H. Haga, B. S. Hu, and W. R. Giles Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2698 - H2711. [Abstract] [Full Text] [PDF]

				C. D. Keller, P. Rivera Gil, M. Tolle, M. van der Giet, J. Chun, H. H. Radeke, M. Schafer-Korting, and B. Kleuser Immunomodulator FTY720 Induces Myofibroblast Differentiation via the Lysophospholipid Receptor S1P3 and Smad3 Signaling Am. J. Pathol., January 1, 2007; 170(1): 281 - 292. [Abstract] [Full Text] [PDF]

				J. S. Kang, Y. D. Yoon, M. H. Han, S.-B. Han, K. Lee, K. H. Lee, S.-K. Park, and H. M. Kim Glabridin Suppresses Intercellular Adhesion Molecule-1 Expression in Tumor Necrosis Factor-{alpha}-Stimulated Human Umbilical Vein Endothelial Cells by Blocking Sphingosine Kinase Pathway: Implications of Akt, Extracellular Signal-Regulated Kinase, and Nuclear Factor-{kappa}B/Rel Signaling Pathways Mol. Pharmacol., March 1, 2006; 69(3): 941 - 949. [Abstract] [Full Text] [PDF]

				R. N. Kumar, J. H. Ha, R. Radhakrishnan, and D. N. Dhanasekaran Transactivation of Platelet-Derived Growth Factor Receptor {alpha} by the GTPase-Deficient Activated Mutant of G{alpha}12 Mol. Cell. Biol., January 1, 2006; 26(1): 50 - 62. [Abstract] [Full Text] [PDF]

				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]

				M. E. Winters, A. I. Mehta, E. F. Petricoin III, E. C. Kohn, and L. A. Liotta Supra-additive Growth Inhibition by a Celecoxib Analogue and Carboxyamido-triazole Is Primarily Mediated through Apoptosis Cancer Res., May 1, 2005; 65(9): 3853 - 3860. [Abstract] [Full Text] [PDF]

				J.-M. Peng, S.-M. Liang, and C.-M. Liang VP1 of Foot-and-Mouth Disease Virus Induces Apoptosis via the Akt Signaling Pathway J. Biol. Chem., December 10, 2004; 279(50): 52168 - 52174. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/2/341 03-0302fjev1    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 BAUDHUIN, L. M. Articles by XU, Y. Search for Related Content PubMed PubMed Citation Articles by BAUDHUIN, L. M. Articles by XU, Y.