Skip to main content

Advertisement

SpringerLink
Log in

The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin-Polyplex Transfection of tGAS1

  • Original Research
  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Glioblastoma is the most malignant primary brain tumor and is very resistant to treatment; hence, it has a poor prognosis. Neurotensin receptor type 1 (NTSR1) plays a key role in cancer malignancy and has potential therapeutic applications. However, the presence and function of neurotensin (NTS) receptors in glioblastoma is not clearly established. RT-PCR assays showed that healthy (non-tumor) astroglial cells and C6 glioma cells express NTSR2 and its isoform (vNTSR2) rather than NTSR1. In glioma cells, NTS promotes the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2), an effect that was completely abolished by blocking the internalization of the NTS/NTSR complex. We demonstrated pharmacologically that the internalization is dependent on the activation of NTSR2 receptors and it was prevented by levocabastine, a NTSR2 receptor antagonist. The internalization of NTSR2 and vNTSR2 was further demonstrated by its ability to mediate gene transfer (transfection) via the NTS-polyplex system. Expression of reporter transgenes and of the pro-apoptotic soluble form of growth arrest specific 1 (tGAS1) was observed in glioma cells. A significant reduction on the viability of C6 cells was determined when tGAS1 was transfected into glioma cells. Conversely, astroglial cells could neither internalize NTS nor activate ERK 1/2 and could not be transfected by the NTS-polyplex. These results demonstrate that the internalization process of NTSR2 receptors is a key regulator necessary to trigger the activation of the ERK 1/2. Our data support a new internalization pathway in glioma C6 cells that involve NTSR2/vNTSR2, which can be used to selectively transfer therapeutic genes using the NTS-polyplex system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ahn S, Shenoy SK, Wei H, Lefkowitz RJ (2004) Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J Biol Chem 279:35518–35525

    Article  CAS  PubMed  Google Scholar 

  • Alifano M et al (2010) Neurotensin receptor 1 determines the outcome of non-small cell lung cancer. Clin Cancer Res 16:4401–4410

    Article  CAS  PubMed  Google Scholar 

  • Alvarez-Maya I, Navarro-Quiroga I, Meraz-Rios MA, Aceves J, Martinez-Fong D (2001) In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor. Mol Med 7:186–192

    CAS  PubMed Central  PubMed  Google Scholar 

  • Arango-Rodriguez ML et al (2006) Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection. Biochim Biophys Acta 1760:1009–1020

    Article  CAS  PubMed  Google Scholar 

  • Benitez JA, Arregui L, Vergara P, Segovia J (2007) Targeted-simultaneous expression of Gas1 and p53 using a bicistronic adenoviral vector in gliomas. Cancer Gene Ther 14:836–846

    Article  CAS  PubMed  Google Scholar 

  • Breunig M, Lungwitz U, Liebl R, Goepferich A (2007) Breaking up the correlation between efficacy and toxicity for nonviral gene delivery. Proc Natl Acad Sci USA 104:14454–14459

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Camby I et al (1996) Neurotensin-mediated effects on astrocytic tumor cell proliferation. Neuropeptides 30:133–139

    Article  CAS  PubMed  Google Scholar 

  • Carraway R, Leeman SE (1973) The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 248:6854–6861

    CAS  PubMed  Google Scholar 

  • Castillo-Rodriguez RA, Arango-Rodriguez ML, Escobedo L, Hernandez-Baltazar D, Gompel A, Forgez P, Martinez-Fong D (2014) Suicide HSVtk gene delivery by neurotensin-polyplex nanoparticles via the bloodstream and GCV treatment specifically inhibit the growth of human MDA-MB-231 triple negative breast cancer tumors xenografted in athymic mice. PLoS One 9:e97151

    Article  PubMed Central  PubMed  Google Scholar 

  • Cortez N, Trejo F, Vergara P, Segovia J (2000) Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental parkinsonism. J Neurosci Res 59:39–46

    Article  CAS  PubMed  Google Scholar 

  • DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK (2007) Beta-arrestins and cell signaling. Annu Rev Physiol 69:483–510

    Article  CAS  PubMed  Google Scholar 

  • Diaz-Coranguez M et al (2013) Transmigration of neural stem cells across the blood brain barrier induced by glioma cells. PLoS One 8:e60655

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dominguez-Monzon G, Benitez JA, Vergara P, Lorenzana R, Segovia J (2009) Gas1 inhibits cell proliferation and induces apoptosis of human primary gliomas in the absence of Shh. Int J Dev Neurosci 27:305–313 The official journal of the International Society for Developmental Neuroscience

    Article  CAS  PubMed  Google Scholar 

  • Dupouy S et al (2009) The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression. PLoS One 4:e4223

    Article  PubMed Central  PubMed  Google Scholar 

  • Dupouy S, Mourra N, Doan VK, Gompel A, Alifano M, Forgez P (2011) The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers. Biochimie 93:1369–1378

    Article  CAS  PubMed  Google Scholar 

  • Evdokiou A, Cowled PA (1998) Tumor-suppressive activity of the growth arrest-specific gene GAS1 in human tumor cell lines. Int J Cancer 75:568–577

    Article  CAS  PubMed  Google Scholar 

  • Evers BM, Ishizuka J, Chung DH, Townsend CM Jr, Thompson JC (1992) Neurotensin expression and release in human colon cancers. Ann Surg 216:423–430 Discussion 430–421

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gendron L, Perron A, Payet MD, Gallo-Payet N, Sarret P, Beaudet A (2004) Low-affinity neurotensin receptor (NTS2) signaling: internalization-dependent activation of extracellular signal-regulated kinases 1/2. Mol Pharmacol 66:1421–1430

    Article  CAS  PubMed  Google Scholar 

  • Gobeil S, Zhu X, Doillon CJ, Green MR (2008) A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene. Genes Dev 22:2932–2940

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gonzalez-Barrios JA et al (2006) Neurotensin polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats. Mol Ther 14:857–865

    Article  CAS  PubMed  Google Scholar 

  • Heuser JE, Anderson RG (1989) Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389–400

    Article  CAS  PubMed  Google Scholar 

  • Jeong M et al (2009) Possible novel therapy for malignant gliomas with secretable trimeric TRAIL. PLoS One 4:e4545

    Article  PubMed Central  PubMed  Google Scholar 

  • Jimenez A, Lopez-Ornelas A, Estudillo E, Gonzalez-Mariscal L, Gonzalez RO, Segovia J (2014) A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model. Exp Cell Res 327:307–317

    Article  CAS  PubMed  Google Scholar 

  • Kesari S (2011) Understanding glioblastoma tumor biology: the potential to improve current diagnosis and treatments. Semin Oncol 38(Suppl 4):S2–S10

    Article  PubMed  Google Scholar 

  • Kitabgi P, Rostene W, Dussaillant M, Schotte A, Laduron PM, Vincent JP (1987) Two populations of neurotensin binding sites in murine brain: discrimination by the antihistamine levocabastine reveals markedly different radioautographic distribution. Eur J Pharmacol 140:285–293

    Article  CAS  PubMed  Google Scholar 

  • Lepee-Lorgeoux I, Betancur C, Rostene W, Pelaprat D (1999) Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization. Brain Res Dev Brain Res 113:115–131

    Article  CAS  PubMed  Google Scholar 

  • Lopez-Ornelas A, Mejia-Castillo T, Vergara P, Segovia J (2011) Lentiviral transfer of an inducible transgene expressing a soluble form of Gas1 causes glioma cell arrest, apoptosis and inhibits tumor growth. Cancer Gene Ther 18:87–99

    Article  CAS  PubMed  Google Scholar 

  • Lopez-Ornelas A, Vergara P, Segovia J (2014) Neural stem cells producing an inducible and soluble form of Gas1 target and inhibit intracranial glioma growth. Cytotherapy 16:1011–1023

    Article  CAS  PubMed  Google Scholar 

  • Lv H, Zhang S, Wang B, Cui S, Yan J (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 114:100–109

    Article  CAS  PubMed  Google Scholar 

  • Martin S, Vincent JP, Mazella J (2003) Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia. J Neurosci 23:1198–1205

    CAS  PubMed  Google Scholar 

  • Martinez-Fong D, Navarro-Quiroga I (2000) Synthesis of a non-viral vector for gene transfer via the high-affinity neurotensin receptor. Brain Res Brain Res Protoc 6:13–24

    Article  CAS  PubMed  Google Scholar 

  • Mazella J et al (1998) The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor. J Biol Chem 273:26273–26276

    Article  CAS  PubMed  Google Scholar 

  • Miggin SM, Kinsella BT (1998) Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms. Biochim Biophys Acta 1425:543–559

    Article  CAS  PubMed  Google Scholar 

  • Miller WE, Lefkowitz RJ (2001) Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking. Curr Opin Cell Biol 13:139–145

    Article  CAS  PubMed  Google Scholar 

  • Moussa O, Ashton AW, Fraig M, Garrett-Mayer E, Ghoneim MA, Halushka PV, Watson DK (2008) Novel role of thromboxane receptors beta isoform in bladder cancer pathogenesis. Cancer Res 68:4097–4104

    Article  CAS  PubMed  Google Scholar 

  • Navarro V, Martin S, Mazella J (2006) Internalization-dependent regulation of HT29 cell proliferation by neurotensin. Peptides 27:2502–2507

    Article  CAS  PubMed  Google Scholar 

  • Navarro-Quiroga I, Antonio Gonzalez-Barrios J, Barron-Moreno F, Gonzalez-Bernal V, Martinez-Arguelles DB, Martinez-Fong D (2002) Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal. Brain Res Mol Brain Res 105:86–97

    Article  CAS  PubMed  Google Scholar 

  • Nouel D, Faure MP, St Pierre JA, Alonso R, Quirion R, Beaudet A (1997) Differential binding profile and internalization process of neurotensin via neuronal and glial receptors. J Neuroscience 17:1795–1803

    CAS  Google Scholar 

  • Nouel D, Sarret P, Vincent JP, Mazella J, Beaudet A (1999) Pharmacological, molecular and functional characterization of glial neurotensin receptors. Neuroscience 94:1189–1197

    Article  CAS  PubMed  Google Scholar 

  • Parent JL, Labrecque P, Orsini MJ, Benovic JL (1999) Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization. J Biol Chem 274:8941–8948

    Article  CAS  PubMed  Google Scholar 

  • Perron A, Sarret P, Gendron L, Stroh T, Beaudet A (2005) Identification and functional characterization of a 5-transmembrane domain variant isoform of the NTS2 neurotensin receptor in rat central nervous system. J Biol Chem 280:10219–10227

    Article  CAS  PubMed  Google Scholar 

  • Raychowdhury MK, Yukawa M, Collins LJ, McGrail SH, Kent KC, Ware JA (1994) Alternative splicing produces a divergent cytoplasmic tail in the human endothelial thromboxane A2 receptor. J Biol Chem 269:19256–19261

    CAS  PubMed  Google Scholar 

  • Reubi JC, Waser B, Schaer JC, Laissue JA (1999) Neurotensin receptors in human neoplasms: high incidence in Ewing’s sarcomas. Int J Cancer 82:213–218

    Article  CAS  PubMed  Google Scholar 

  • Roskoski R Jr (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res 66:105–143

    Article  CAS  PubMed  Google Scholar 

  • Rubio-Zapata HA, Rembao-Bojorquez JD, Arango-Rodriguez ML, Dupouy S, Forgez P, Martinez-Fong D (2009) NT-polyplex: a new tool for therapeutic gene delivery to neuroblastoma tumors. Cancer Gene Ther 16:573–584

    Article  CAS  PubMed  Google Scholar 

  • Saada S et al (2012) Differential expression of neurotensin and specific receptors, NTSR1 and NTSR2, in normal and malignant human B lymphocytes. J Immunol 189:5293–5303

    Article  CAS  PubMed  Google Scholar 

  • Sarret P, Gendron L, Kilian P, Nguyen HM, Gallo-Payet N, Payet MD, Beaudet A (2002) Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem 277:36233–36243

    Article  CAS  PubMed  Google Scholar 

  • Schade B et al (2013) β-Catenin signaling is a critical event in ErbB2-mediated mammary tumor progression. Cancer Res 73:4474–4487

    Article  CAS  PubMed  Google Scholar 

  • Schotte A, Leysen JE, Laduron PM (1986) Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain. Naunyn Schmiedebergs Arch Pharmacol 333:400–405

    Article  CAS  PubMed  Google Scholar 

  • Schotte A, Rostene W, Laduron PM (1988) Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system. J Neurochem 50:1026–1031

    Article  CAS  PubMed  Google Scholar 

  • Servotte S et al (2006) The in vitro influences of neurotensin on the motility characteristics of human U373 glioblastoma cells. Neuropathol Appl Neurobiol 32:575–584

    Article  CAS  PubMed  Google Scholar 

  • Shy BR, Wu CI, Khramtsova GF, Zhang JY, Olopade OI, Goss KH, Merrill BJ (2013) Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/beta-catenin signaling. Cell Rep 4:1–9

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sobolesky PM, Moussa O (2013) The role of beta-arrestins in cancer. Prog Mol Biol Transl Sci 118:395–411

    Article  CAS  PubMed  Google Scholar 

  • Somai S, Gompel A, Rostene W, Forgez P (2002) Neurotensin counteracts apoptosis in breast cancer cells. Biochem Biophys Res Commun 295:482–488

    Article  CAS  PubMed  Google Scholar 

  • Souaze F et al (2006a) Expression of neurotensin and NT1 receptor in human breast cancer: a potential role in tumor progression. Cancer Res 66:6243–6249

    Article  CAS  PubMed  Google Scholar 

  • Souaze F et al (2006b) Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas. Carcinogenesis 27:708–716

    Article  CAS  PubMed  Google Scholar 

  • St-Gelais F, Jomphe C, LE Trudeau (2006) The role of neurotensin in central nervous system pathophysiology: what is the evidence? J Psychiatry Neurosci 31:229–245

    PubMed Central  PubMed  Google Scholar 

  • Swift SL, Burns JE, Maitland NJ (2010) Altered expression of neurotensin receptors is associated with the differentiation state of prostate cancer. Cancer Res 70:347–356

    Article  CAS  PubMed  Google Scholar 

  • Vandenbulcke F, Nouel D, Vincent JP, Mazella J, Beaudet A (2000) Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor. J Cell Sci 113(Pt 17):2963–2975

    CAS  PubMed  Google Scholar 

  • Vincent JP, Mazella J, Kitabgi P (1999) Neurotensin and neurotensin receptors. Trends Pharmacol Sci 20:302–309

    Article  CAS  PubMed  Google Scholar 

  • Walker N, Lepee-Lorgeoux I, Fournier J, Betancur C, Rostene W, Ferrara P, Caput D (1998) Tissue distribution and cellular localization of the levocabastine-sensitive neurotensin receptor mRNA in adult rat brain. Brain Res Mol Brain Res 57:193–200

    Article  CAS  PubMed  Google Scholar 

  • Wu Z, Martinez-Fong D, Tredaniel J, Forgez P (2012) Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy. Front Endocrinol 3:184

    Google Scholar 

  • Younes M et al (2014) Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib. Oncotarget 5:8252–8269

    PubMed Central  PubMed  Google Scholar 

  • Zamorano A, Lamas M, Vergara P, Naranjo JR, Segovia J (2003) Transcriptionally mediated gene targeting of gas1 to glioma cells elicits growth arrest and apoptosis. J Neurosci Res 71:256–263

    Article  CAS  PubMed  Google Scholar 

  • Zamorano A, Mellstrom B, Vergara P, Naranjo JR, Segovia J (2004) Glial-specific retrovirally mediated gas1 gene expression induces glioma cell apoptosis and inhibits tumor growth in vivo. Neurobiol Dis 15:483–491

    Article  CAS  PubMed  Google Scholar 

  • Zarco N, Gonzalez-Ramirez R, Gonzalez RO, Segovia J (2012) GAS1 induces cell death through an intrinsic apoptotic pathway. Apoptosis 17:627–635

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Instituto de Ciencia y Tecnología del Gobierno del Distrito Federal (ICyTDF) Grant #ICYTDF/228/2010, ARN-CONACYT Grant #142947 (DMF), and Conacyt Grant #127357 (JS). AEAS was a recipient of CONACYT fellowship #244983.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN # 2508, 07360, Mexico, DF, Mexico

    Alberto E. Ayala-Sarmiento, Daniel Martinez-Fong & José Segovia

  2. Programa de Nanociencias y Nanotecnología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. IPN # 2508, 07360, Mexico, DF, Mexico

    Daniel Martinez-Fong

Authors
  1. Alberto E. Ayala-Sarmiento
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Martinez-Fong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José Segovia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José Segovia.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ayala-Sarmiento, A.E., Martinez-Fong, D. & Segovia, J. The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin-Polyplex Transfection of tGAS1. Cell Mol Neurobiol 35, 785–795 (2015). https://doi.org/10.1007/s10571-015-0172-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10571-015-0172-z

Keywords

Search

Navigation