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: 19/7/819 04-2988fjev1    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 Kim, K.-s. Articles by Xu, Y. Search for Related Content PubMed PubMed Citation Articles by Kim, K.-s. Articles by Xu, Y.

GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine

Kwan-sik Kim^*,1,2, Juan Ren^*,1, Ying Jiang^*,1, Quteba Ebrahem, Russell Tipps^*, Kelly Cristina^*, Yi-jin Xiao^*, Jing Qiao, Kevin L. Taylor, Hazel Lum, Bela Anand-Apte and Yan Xu^*,||,3

^* Department of Cancer Biology, The Lerner Research Institute and
Department of Ophthalmic Research, Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA;
Department of Pharmacology, Rush University Medical Center, Chicago, Illinois, USA;
Center for Drug Development and Discovery, Taussig Cancer Center, The Cleveland Clinic Foundation, Cleveland, Ohio, USA; and
^|| Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, USA

³ Correspondence: Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland OH 44195, USA. E-mail: xuy{at}ccf.org

SPECIFIC AIMS

We tested the in vivo angiogenic activity of sphingosylphosphorylcholine (SPC) and determined the role of GPR4 in endothelial cells (EC). We found that 1) GPR4 is the major receptor mediating the angiogenic effects of SPC in EC; 2) GPR4 plays a pivotal role in cell survival, growth, migration, and tube formation through both SPC-dependent and -independent pathways; and 3) biological effects resulting from SPC/GPR4 interactions involve the activation of phosphatidylinositol-3 kinase (PI3K), Akt, and vascular endothelial growth factor receptor 2 (VEGFR2).

PRINCIPAL FINDINGS

1. SPC stimulates angiogenesis in vivo
We used chick embryo chorioallantoic membrane (CAM) assays to determine the effect of SPC exposure on angiogenesis in vivo. SPC and VEGF, but not lysophosphatidic acid (LPA) or the H₂O control, induced increased branching and tortuosity of the blood vessels.

2. Modulation of GPR4 function in EC
The receptor(s) mediating SPC effects in EC has not been previously identified. Quantitative PCR was used to examine the expression of OGR1-related GPCRs in different types of EC including primary HUVEC, dermal microvascular EC, pulmonary artery EC, human aortic EC, and the immortalized human microvascular endothelialcell line 1 (HMEC-1). GPR4 is the only G-protein-coupled receptor (GPCR) of the OGR1 related GPCRs expressed at significant levels in these EC, suggesting it may be the primary receptor involved in SPC effects in EC.

The role of GPR4 in HUVEC and HMEC-1 was determined using a small interfering RNA (siRNA) to inhibit expression of endogenous GPR4. RNAi targeting LPA₃ was used as a negative control. Using an antibody generated against a C-terminal peptide of human GPR4, we found the level of GPR4 was significantly reduced in GPR4-RNAi cell lines compared with all control cell lines. We generated a mutant GPR4 (mtGPR4) by changing 8 nucleotides in the region of GPR4 targeted by our siRNA molecule without changing the encoded amino acids. As predicted, after the introduction of mtGPR4 into GPR4-RNAi cell lines, GPR4 expression was restored.

3. GPR4 plays a critical role in SPC-induced tube formation in HUVEC
To determine whether GPR4 is involved in angiogenesis, we measured EC tube formation as an in vitro indicator of angiogenic potential. As tube formation was induced similarly in parental, vector, and LPA₃-RNAi-HUVEC by S1P (0.1–1 µM), VEGF (10 ng/mL), and SPC (1–5 µM), only results from LPA₃-RNAi-HUVEC are shown (Fig. 1 A, a–d). VEGF and S1P induced tube formation in GPR4-RNAi-HUVEC (Fig. 1A, f, g ). In contrast, down-regulation of GPR4 via RNAi completely blocked SPC-induced tube formation (Fig. 1A, h ). Reconstitution of wild-type GPR4 by expression of mtGPR4 fully restored SPC-induced tube formation, indicating that GPR4 is indeed the receptor mediating the tube formation activity of SPC in HUVEC (Fig. 1A, i-l ). LPA (3–10 µM) or lysophosphatidylcholine (LPC; 1–10 µM) did not induce, or only weakly induced, tube formation (Fig. 1A, m, n and data not shown).

View larger version (82K): [in this window] [in a new window]   Figure 1. Involvement of GPR4 in tube formation in HUVEC. A) Tube formation in HUVEC-derived cell lines using the in vitro angiogenesis assay kit (fibrin) from Chemicon. Cells cultured in starvation medium with no further additions treated with S1P (1 µM), VEGF (10 ng/mL) or SPC (3 µM), LPA (3 µM) or LPC (3 µM), respectively. B) Quantitative summary of results from three independent experiments depicted in (A). ***P <0.001 (Student’s t test).

4. GPR4 is critically important for the tube formation in microvascular EC
To confirm that the effect of GPR4 on SPC-induced tube formation was not limited to HUVEC, we performed similar assays on HMEC-1. In contrast to HUVEC, HMEC-1 cells only form tubes on fibrin matrix in the presence of 5% FBS. We found that FBS-induced tube formation was completely inhibited in GPR4-RNAi-HMEC but not in LPA₃-RNAi-HMEC. FBS-induced tube formation was fully restored in GPR4-RNAi-mtGPR4-HMEC, confirming it is a GPR4-dependent process.

We analyzed tube formation by HMEC-1 using Chemicon’s ECMatrix gel. On this substrate, HMEC-1 formed tubes even in the absence of FBS. Inclusion of 5% FBS, VEGF (10–20 ng/mL) or SPC (1–10 µM) did not further enhance tube formation. The vector and LPA₃-RNAi-HMEC cell lines displayed tube formation abilities on ECMatrix not significantly different from those of parental HMEC-1. However, in GPR4-RNAi-HMEC, the ability of cells to form tubes was lost. Introduction of mtGPR4 into GPR4-RNAi-HMEC fully restored the tube formation capacity of the cells, confirming that the observed effects were GPR4 specific.

5. GPR4 plays a pivotal role in survival and proliferation of microvascular EC
We found that siRNA-mediated down-regulation of GPR4 in HUVEC did not significantly affect proliferation. In contrast, the cellular growth rate of GPR4-RNAi-HMEC was dramatically reduced compared with control cell lines. Doubling times for parental, vector, LPA₃-RNAi-, and GPR4-RNAi-HMEC were 0.97, 1.35, 1.1, and 6.6 days, respectively. Expression of mtGPR4 fully restored the cellular growth rate to a doubling time of 0.94 days. These results further validate our approach for testing GPR4-specific functions and indicate that GPR4 plays a pivotal role in HMEC-1 survival and proliferation. GPR4-RNAi-HMEC did not respond to SPC. Re-expression of GPR4 restored SPC responsiveness beyond that of the control RNAi cell lines due to overexpression of mtGPR4.

6. GPR4 mediates SPC-induced migration of EC
SPC induced significant chemotactic cell migration in parental and control cells. SPC-induced cell migration was completely blocked in GPR4-RNAi-HMEC. Re-expression of GPR4 restored SPC responsiveness above and beyond that of the parental and control RNAi cell lines Similar results were obtained in HUVEC. SPC/GPR4-mediated cell migration was dependent on PI3K, ERK, Akt, and VEGFR2.

7. SPC effects on EC require PI3K and VEGF receptor 2 (VEGFR2) activities and may involve transactivation of VEGFR2
SPC induced activation of ERK and Akt in HMEC-1-derived cell lines (Fig. 2 A, lanes 1, 2). GPR4 was required for activation of Akt (solid arrows) but not ERK (dashed arrows, Fig. 2A ). Using specific signaling molecule inhibitors, we found that activation of ERK required G_i and MEK, but not PI3K (Fig. 2A , lanes 3–8). In contrast, SPC-induced Akt activation was dependent on PI3K activity but independent of MEK. The Rho kinase inhibitor (Y27632) inhibited neither ERK nor Akt activation by SPC. SPC-induced activation of Akt was suppressed by two specific inhibitors of VEGFR2 (VEGFR-TK-i and oxindole1; Fig. 2A ). SPC-induced phosphorylation of ERK was affected, albeit to a lesser extent, by the VEGFR2 inhibitors. Cross-talk between SPC and VEGFR2 pathways was demonstrated in that SPC (5 µM) induced tyrosine phosphorylation of VEGFR2 in HUVEC, albeit to a lesser extent than VEGF itself (Fig. 2B ). SPC-induced tube formation in HUVEC was completely inhibited by PI3K (LY294002) and VEGFR2 (VEGFR-TK-i) inhibitors and partially inhibited by MEK (PD98059) and Akt (Akt-i) inhibitors.

View larger version (57K): [in this window] [in a new window]   Figure 2. PI3K, VEGFR2, ERK, and Akt signaling activities are involved in SPC/GPR4 effects on EC. A) Western blot analyses of SPC-induced activation of ERK and Akt in LPA3-RNAi-HMEC (top panel), GPR4-RNAi-HMEC (middle), and GPR4-RNAi-mtGPR4-HMEC (lower). Cells were treated with SPC (3 µM, lane 2) or without SPC (Control, lane 1) for 10 min. Cells were pretreated with Y27632 (10 µM), LY294002 (10 µM), PD98059 (10 µM), oxindole-1 (10 µM), or VEGFR-TK-i (10 µM) for 30 min followed by SPC (3 µM) treatment. For PTX (100 ng/mL), cells were pretreated for 16 h. The optimal concentration of each inhibitor was chosen based on previous experiments (data not shown). SPC-induced phosphorylation of Akt and ERK is noted by the solid and dashed arrows, respectively. B) SPC induced transphosphorylation of VEGFR2. VEGF and SPC induced tyrosine phosphorylation of VEGFR2. Coimmunoprecipitation and Western blot analyses were conducted. HUVEC cells were starved from serum for 24 h, then treated with VEGF (20 ng/mL for 5 min) or SPC (5 µM for 5 or 10 min, respectively).

CONCLUSIONS AND SIGNIFICANCE

This work describes the first cellular function of GPR4. All other work published relating to GPR4 has not directly addressed its endogenous cellular functions. In contrast to the overexpression systems used in all previous studies, we used "loss of function" (RNAi) and "gain of function" (reconstitution with mtGPR4) approaches, thus providing a better controlled system to study the endogenous cellular and physiological role of GPR4. Second, a recent paper published suggests that OGR1 and GPR4 are proton-sensing receptors. This becomes a major issue in the field of bioactive lipid signaling. Our data clearly show that GPR4 is required for SPC’s action in EC. This provides pivotal and novel information on the cellular function and the signaling properties of GPR4. Third, showing that SPC has in vivo angiogenesis activity is not a simple extension of the in vitro work published earlier. SPC effects have been suggested due to its detergent nature and/or its conversion to S1P. We have provided evidence to argue against this. Linking its activity to its receptor is important to further understand the mechanisms of action. Finally, we provide the first line of evidence that SPC transactivates VEGFR2, one of the most important growth factor receptors in endothelial function.

The apparent discrepancies between our findings and work related to proton sensing may be interpreted in different ways. First, overexpression of GPR4 may introduce constitutive activity, which may sense pH changes. Second, GPR4, OGR1, and other related GPCRs may have dual roles, which are cell type and signaling pathway specific. We found that HMEC-1 and HUVEC, which express endogenous GPR4, did not respond to pH changes to regulate cAMP. Our results and the proton sensing work may not be mutually exclusive.

Angiogenesis, the growth of new capillary blood vessels, is critical for many biological processes, including embryonic development, reproduction, and tumor development. We show here for the first time that GPR4 plays a pivotal role in endothelial functions. In vivo studies under physiological and pathological conditions are the next key step to define the role and potential clinical usage of the SPC/GPR4 axis.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic of the role and signaling pathways of SPC and GPR4 in EC. SPC activates its receptor, GPR4, to induce a Gi-dependent transactivation (phosphorylation) of VEGFR2. VEGFR2 is required for SPC-induced Akt activation. SPC may induce ERK activation via a GPR4-independent pathway. ERK and Akt are involved in SPC-stimulated angiogenic effects in EC.

FOOTNOTES

¹ These authors contributed equally in this work

² Current address: Department of Obstetrics and Gynecology, Chonbuk National University Medical School, Chonju, Chonbuk, Korea

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2988fje;

This article has been cited by other articles:

				C. Peter, M. Waibel, C. G. Radu, L. V. Yang, O. N. Witte, K. Schulze-Osthoff, S. Wesselborg, and K. Lauber Migration to Apoptotic "Find-me" Signals Is Mediated via the Phagocyte Receptor G2A J. Biol. Chem., February 29, 2008; 283(9): 5296 - 5305. [Abstract] [Full Text] [PDF]

				L. S. Singh, M. Berk, R. Oates, Z. Zhao, H. Tan, Y. Jiang, A. Zhou, K. Kirmani, R. Steinmetz, D. Lindner, et al. Ovarian Cancer G Protein Coupled Receptor 1, a New Metastasis Suppressor Gene in Prostate Cancer J Natl Cancer Inst, September 5, 2007; 99(17): 1313 - 1327. [Abstract] [Full Text] [PDF]

				H. Kinoshita, N. Matsuda, Y. Kimoto, S. Tohyama, K. Hama, K. Nakahata, and Y. Hatano Sevoflurane, but Not Propofol, Prevents Rho Kinase-Dependent Contraction Induced by Sphingosylphosphorylcholine in the Porcine Coronary Artery Anesth. Analg., August 1, 2007; 105(2): 325 - 329. [Abstract] [Full Text] [PDF]

				L. V. Yang, C. G. Radu, M. Roy, S. Lee, J. McLaughlin, M. A. Teitell, M. L. Iruela-Arispe, and O. N. Witte Vascular Abnormalities in Mice Deficient for the G Protein-Coupled Receptor GPR4 That Functions as a pH Sensor Mol. Cell. Biol., February 15, 2007; 27(4): 1334 - 1347. [Abstract] [Full Text] [PDF]

				E. S. Jeon, H. J. Moon, M. J. Lee, H. Y. Song, Y. M. Kim, Y. C. Bae, J. S. Jung, and J. H. Kim Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-{beta}-dependent mechanism J. Cell Sci., December 1, 2006; 119(23): 4994 - 5005. [Abstract] [Full Text] [PDF]

				J. Qiao, F. Huang, R. P. Naikawadi, K. S. Kim, T. Said, and H. Lum Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4 Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L91 - L101. [Abstract] [Full Text] [PDF]

				F. Huang, P. V. Subbaiah, O. Holian, J. Zhang, A. Johnson, N. Gertzberg, and H. Lum Lysophosphatidylcholine increases endothelial permeability: role of PKC{alpha} and RhoA cross talk Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L176 - L185. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/7/819 04-2988fjev1    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 Kim, K.-s. Articles by Xu, Y. Search for Related Content PubMed PubMed Citation Articles by Kim, K.-s. Articles by Xu, Y.