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Identification of TGFβ-induced proteins in non-endocrine mouse pituitary cell line TtT/GF by SILAC-assisted quantitative mass spectrometry

  • Takehiro TsukadaEmail author
  • Yukinobu Isowa
  • Keiji Kito
  • Saishu Yoshida
  • Seina Toneri
  • Kotaro Horiguchi
  • Ken Fujiwara
  • Takashi Yashiro
  • Takako Kato
  • Yukio KatoEmail author
Regular Article
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Abstract

TtT/GF is a mouse cell line derived from a thyrotropic pituitary tumor and has been used as a model of folliculostellate cells. Our previous microarray data indicate that TtT/GF possesses some properties of endothelial cells, pericytes and stem/progenitor cells, along with folliculostellate cells, suggesting its plasticity. We also found that transforming growth factor beta (TGFβ) alters cell motility, increases pericyte marker transcripts and attenuates endothelial cell and stem/progenitor cell markers in TtT/GF cells. The present study explores the wide-range effect of TGFβ on TtT/GF cells at the protein level and characterizes TGFβ-induced proteins and their partnerships using stable isotope labeling of amino acids in cell culture (SILAC)-assisted quantitative mass spectrometry. Comparison between quantified proteins from TGFβ-treated cells and those from SB431542 (a selective TGFβ receptor I inhibitor)-treated cells revealed 51 upregulated and 112 downregulated proteins (|log2| > 0.6). Gene ontology and STRING analyses revealed that these are related to the actin cytoskeleton, cell adhesion, extracellular matrix and DNA replication. Consistently, TGFβ-treated cells showed a distinct actin filament pattern and reduced proliferation compared to vehicle-treated cells; SB431542 blocked the effect of TGFβ. Upregulation of many pericyte markers (CSPG4, NES, ACTA, TAGLN, COL1A1, THBS1, TIMP3 and FLNA) supports our previous hypothesis that TGFβ reinforces pericyte properties. We also found downregulation of CTSB, EZR and LGALS3, which are induced in several pituitary adenomas. These data provide valuable information about pericyte differentiation as well as the pathological processes in pituitary adenomas.

Keywords

Isotopic tracer Pituitary gland Proteomics Transforming growth factor beta (TGFβ) Pituitary adenoma 
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Notes

Acknowledgments

We would like to thank Tom Kouki (Jichi Medical University) for his support in transmission electron microscopy and Editage (www.editage.jp) for English language editing.

Funding information

This work was partially supported by the Japan Society for the Promotion of Science KAKENHI Grants (Numbers 16K18818 to SY, 26460281 to KF, 16K08475 to KH, 26292166 to YK and 15K07771 to TK), the MEXT-supported Program for the Strategic Research Foundation at Private Universities (2014–2018), the Meiji University International Institute for BioResource Research (MUIIR) and start-up funds to TT from the Faculty of Science Department at Toho University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

441_2018_2989_MOESM1_ESM.docx (210 kb)
Electronic Supplementary Material Fig. 1 Heavy medium containing 13C6-labeled lysine and arginine have no effect on TGFβ-induced Smad2 nuclear translocation. After a 3-day culture in light (top) or heavy medium (bottom), TtT/GF cells were treated with vehicle, TGFβ (10 ng/mL), or TGFβ (10 ng/mL) and selective TGFβ receptor inhibitor (SB431542, 10 μM) for 30 min. Treated cells were stained for Smad2 (green) and DAPI (blue) (for staining protocol see Tsukada et al. 2018). Diffuse cytoplasmic staining for Smad2 was observed with vehicle treatment (a, a′); however, intense nuclear staining was observed with 10 ng/mL TGFβ (b, b′). The TGFβ-induced Smad2 nuclear translocation was completely blocked by 10 μM SB431542 (c, c′). No significant difference was observed between light and heavy medium in terms of the efficiency of TGFβ and TGFβ receptor inhibitor. Bar = 100 μm (DOCX 209 kb)
441_2018_2989_MOESM2_ESM.docx (188 kb)
Electronic Supplementary Material Fig. 2 TGFβ attenuates transcript levels of stem cell marker genes and promotes pericyte markers. Total RNA was extracted after a 3-day treatment with TGFβ/SB431542 in light medium using the RNeasy Mini Kit and RNase-free DNase Set according to the manufacturer’s instructions (Qiagen, Hilden, Germany). cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan) with oligo-(dT)20 primer (Life Technologies). Quantitative PCR (AriaMx, Agilent Technologies) was performed using SYBR Green Real-time PCR Master Mix Plus (Toyobo, Osaka, Japan) and specific primer sets at 0.6 μM for each target gene (Electronic Supplementary Material, Table S6). Each sample was measured in duplicate and results are based on five independent experiments; data were analyzed by the comparative CT method (ddCt method) to estimate gene copy number relative to that of the TATA box-binding protein (Tbp), used as an internal standard. Genes included stem cell markers (Sca-1, Cd34), pericyte markers (Nes, Cspg4, Col1a1) and a folliculostellate cell marker (S100b). TGFβ increased pericyte marker gene expression and decreased stem cell marker gene expression. *p < 0.05 (Tukey’s test) (DOCX 188 kb)
441_2018_2989_MOESM3_ESM.docx (75 kb)
Electronic Supplementary Material Table S1 (DOCX 75 kb)
441_2018_2989_MOESM4_ESM.docx (78 kb)
Electronic Supplementary Material Table S2 (DOCX 78 kb)
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Electronic Supplementary Material Table S3 (DOCX 104 kb)
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Electronic Supplementary Material Table S4 (DOCX 140 kb)
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Electronic Supplementary Material Table S5 (DOCX 142 kb)
441_2018_2989_MOESM8_ESM.doc (106 kb)
Electronic Supplementary Material Table S6 (DOC 106 kb)

References

  1. Andoniadou CL, Matsushima D, Mousavy Gharavy SN, Signore M, Mackintosh AI, Schaeffer M, Gaston-Massuet C, Mollard P, Jacques TS, Le Tissier P, Dattani MT, Pevny LH, Martinez-Barbera JP (2013) Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell 13:433–445CrossRefGoogle Scholar
  2. Azuma M, Tofrizal A, Maliza R, Batchuluun K, Ramadhani D, Syaidah R, Tsukada T, Fujiwara K, Kikuchi M, Horiguchi K, Yashiro T (2015) Maintenance of the extracellular matrix in rat anterior pituitary gland: identification of cells expressing tissue inhibitors of metalloproteinases. Acta Histochem Cytochem 48:185–192CrossRefGoogle Scholar
  3. Berger SJ, Lee S-W, Anderson GA, Pasa-Tolić L, Tolić N, Shen Y, Zhao R, Smith RD (2002) High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS. Anal Chem 74:4994–5000CrossRefGoogle Scholar
  4. Beyer TA, Narimatsu M, Weiss A, David L, Wrana JL (2013) The TGFβ superfamily in stem cell biology and early mammalian embryonic development. Biochim Biophys Acta 1830:2268–2279CrossRefGoogle Scholar
  5. Chapman L, Nishimura A, Buckingham JC, Morris JF, Christian HC (2002) Externalization of annexin I from a folliculo-stellate-like cell line. Endocrinology 143:4330–4338CrossRefGoogle Scholar
  6. Chen J, Gremeaux L, Fu Q, Liekens D, Van Laere S, Vankelecom H (2009) Pituitary progenitor cells tracked down by side population dissection. Stem Cells (Dayton, Ohio) 27:1182–1195CrossRefGoogle Scholar
  7. Chen Y, Chuan HL, Yu SY, Li CZ, Wu ZB, Li GL, Zhang YZ (2017) A novel invasive-related biomarker in three subtypes of nonfunctioning pituitary adenomas. World Neurosurgery 100:514–521CrossRefGoogle Scholar
  8. Denef C (2008) Paracrinicity: the story of 30 years of cellular pituitary crosstalk. J Neuroendocrinol 20:1–70CrossRefGoogle Scholar
  9. Deutsch EW, Csordas A, Sun Z, Jarnuczak A, Perez-Riverol Y, Ternent T, Campbell DS, Bernal-Llinares M, Okuda S, Kawano S, Moritz RL, Carver JJ, Wang M, Ishihama Y, Bandeira N, Hermjakob H, Vizcaíno JA (2017) The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. Nucleic Acids Res 45:D1100–D1106CrossRefGoogle Scholar
  10. Devnath S, Inoue K (2008) An insight to pituitary folliculo-stellate cells. J Neuroendocrinol 20:687–691CrossRefGoogle Scholar
  11. Domogatskaya A, Rodin S, Boutaud A, Tryggvason K (2008) Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells 26:2800–2809CrossRefGoogle Scholar
  12. Fauquier T, Rizzoti K, Dattani M, Lovell-Badge R, Robinson IC (2008) SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland. Proc Natl Acad Sci U S A 105:2907–2912CrossRefGoogle Scholar
  13. Fujiwara K, Jindatip D, Kikuchi M, Yashiro T (2010) In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats. Cell Tissue Res 342:491–495CrossRefGoogle Scholar
  14. Gonzalez DM, Medici D (2014) Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 7:re8CrossRefGoogle Scholar
  15. Gressner OA, Weiskirchen R, Gressner AM (2007) Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options. Comp Hepatol 6:7CrossRefGoogle Scholar
  16. Hezel AF, Deshpande V, Zimmerman SM, Contino G, Alagesan B, O’Dell MR, Rivera LB, Harper J, Lonning S, Brekken RA, Bardeesy N (2012) TGF-β and αvβ6 integrin act in a common pathway to suppress pancreatic cancer progression. Cancer Res 72:4840–4845CrossRefGoogle Scholar
  17. Horiguchi K, Kouki T, Fujiwara K, Kikuchi M, Yashiro Y (2011) The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland. J Endocrinol 208:225–232Google Scholar
  18. Horiguchi K, Nakakura T, Yoshida S, Tsukada T, Kanno N, Hasegawa R, Takigami S, Ohsako S, Kato T, Kato Y (2016) Identification of THY1 as a novel thyrotrope marker and THY1 antibody-mediated thyrotrope isolation in the rat anterior pituitary gland. Biochem Biophys Res Commun 480:273–279CrossRefGoogle Scholar
  19. Huang CX, Zhao JN, Zou WH, Li JJ, Wang PC, Liu CH, Wang YB (2014) Reduction of galectin-3 expression reduces pituitary tumor cell progression. Genet Mol Res 13:6892–6898CrossRefGoogle Scholar
  20. Hughes S, Chan-Ling T (2004) Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo. Invest Opthalmol Vis Sci 45:2795–2806CrossRefGoogle Scholar
  21. Inman GJ, Nicolás FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62:65–74CrossRefGoogle Scholar
  22. Inoue K, Matsumoto H, Koyama C, Shibata K, Nakazato Y, Ito A (1992) Establishment of a folliculo-stellate-like cell line from a murine thyrotropic pituitary tumor. Endocrinology 131:3110–3116CrossRefGoogle Scholar
  23. Inoue K, Mogi C, Ogawa S, Tomida M, Miyai S (2002) Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells? Arch Physiol Biochem 110:50–53CrossRefGoogle Scholar
  24. Järvinen PM, Laiho M (2012) LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation. Cell Signal 24:819–825CrossRefGoogle Scholar
  25. Kito K, Ito H, Nohara T, Ohnishi M, Ishibashi Y, Takeda D (2016) Yeast interspecies comparative proteomics reveals divergence in expression profiles and provides insights into proteome resource allocation and evolutionary roles of gene duplication. Mol Cell Proteomics 15:218–235CrossRefGoogle Scholar
  26. Krylyshkina O, Chen J, Mebis L, Denef C, Vankelecom H (2005) Nestin-immunoreactive cells in rat pituitary are neither hormonal nor typical folliculo-stellate cells. Endocrinology 146:2376–2387CrossRefGoogle Scholar
  27. Lin H, Zhang Y, Wang H, Xu D, Meng X, Shao Y, Lin C, Ye Y, Qian H, Wang S (2012) Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells. Cancer Gene Ther 19:845–851CrossRefGoogle Scholar
  28. Lu J, Shenoy AK (2017) Epithelial-to-pericyte transition in cancer. Cancers 9:1–13CrossRefGoogle Scholar
  29. Meng X-M, Nikolic-Paterson DJ, Lan HY (2016) TGF-β: the master regulator of fibrosis. Nat Rev Nephrol 12:325–338CrossRefGoogle Scholar
  30. Mitsuishi H, Kato T, Chen M, Cai L-Y, Yako H, Higuchi M, Yoshida S, Kanno N, Ueharu H, Kato Y (2013) Characterization of a pituitary-tumor-derived cell line, TtT/GF, that expresses Hoechst efflux ABC transporter subfamily G2 and stem cell antigen 1. Cell Tissue Res 354:563–572CrossRefGoogle Scholar
  31. Mollard P, Hodson DJ, Lafont C, Rizzoti K, Drouin J (2012) A tridimensional view of pituitary development and function. Trends Endocrinol Metab 23:261–269CrossRefGoogle Scholar
  32. Okada M, Kusunoki S, Ishibashi Y, Kito K (2017) Proteomics analysis for asymmetric inheritance of preexisting proteins between mother and daughter cells in budding yeast. Genes Cells 22:591–601CrossRefGoogle Scholar
  33. Ong S-E, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386CrossRefGoogle Scholar
  34. Ozawa H, Miyachi M, Ochiai I, Tsuchiya S, Morris JF, Kawata M (2002) Annexin-1 (lipocortin-1)-immunoreactivity in the folliculo-stellate cells of rat anterior pituitary: the effect of adrenalectomy and corticosterone treatment on its subcellular distribution. J Neuroendocrinol 14:621–628CrossRefGoogle Scholar
  35. Renner U, Gloddek J, Arzt E, Inoue K, Stalla GK (1997) Interleukin-6 is an autocrine growth factor for folliculostellate-like TtT/GF mouse pituitary tumor cells. Exp Clin Endocrinol Diabetes 105:345–352CrossRefGoogle Scholar
  36. Righi A, Morandi L, Leonardi E, Farnedi A, Marucci G, Sisto A, Frank G, Faustini-Fustini M, Zoli M, Mazzatenta D, Agati R, Foschini MP (2013) Galectin-3 expression in pituitary adenomas as a marker of aggressive behavior. Hum Pathol 44:2400–2409CrossRefGoogle Scholar
  37. Rizzoti K, Akiyama H, Lovell-Badge R (2013) Mobilized adult pituitary stem cells contribute to endocrine regeneration in response to physiological demand. Cell Stem Cell 13:419–432CrossRefGoogle Scholar
  38. Saunders WB, Bohnsack BL, Faske JB, Anthis NJ, Bayless KJ, Hirschi KK, Davis GE (2006) Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3. J Cell Biol 175:179–191CrossRefGoogle Scholar
  39. Schor AM, Canfield AE, Sutton AB, Arciniegas E, Allen TD (1995) Pericyte differentiation. Clin Orthop Relat Res 313:81–91Google Scholar
  40. Shojaee N, Patton WF, Chung-Welch N, Su Q, Hechtman HB, Shepro D (1998) Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes. Electrophoresis 19:323–332CrossRefGoogle Scholar
  41. Stilling GA, Bayliss JM, Jin L, Zhang H, Lloyd RV (2005) Chromogranin A transcription and gene expression in Folliculostellate (TtT/GF) cells inhibit cell growth. Endocr Pathol 16:173–186CrossRefGoogle Scholar
  42. Ström A, Olin A, Aspberg A, Hultgårdh-Nilsson A (2006) Fibulin-2 is present in murine vascular lesions and is important for smooth muscle cell migration. Cardiovasc Res 69:755–763CrossRefGoogle Scholar
  43. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452CrossRefGoogle Scholar
  44. Tanase C, Albulescu R, Codrici E, Calenic B, Popescu ID, Mihai S, Necula L, Cruceru ML, Hinescu ME (2014) Decreased expression of APAF-1 and increased expression of cathepsin B in invasive pituitary adenoma. Onco Targets Ther 8:81–90CrossRefGoogle Scholar
  45. ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869CrossRefGoogle Scholar
  46. Tierney T, Christian HC, Morris JF, Solito E, Buckingham JC (2003) Evidence from studies on co-cultures of TtT/GF and AtT20 cells that Annexin 1 acts as a paracrine or juxtacrine mediator of the early inhibitory effects of glucocorticoids on ACTH release. J Neuroendocrinol 15:1134–1143CrossRefGoogle Scholar
  47. Tofrizal A, Fujiwara K, Yashiro T, Yamada S (2016) Alterations of collagen-producing cells in human pituitary adenomas. Med Mol Morphol 49:224–232CrossRefGoogle Scholar
  48. Tsukada T, Yoshida S, Kito K, Fujiwara K, Yako H, Horiguchi K, Isowa Y, Yashiro T, Kato T, Kato Y (2018) TGFβ signaling reinforces pericyte properties of the non-endocrine mouse pituitary cell line TtT/GF. Cell Tissue Res 371:339–350CrossRefGoogle Scholar
  49. Ueharu H, Yoshida S, Kikkawa T, Kanno N, Higuchi M, Kato T, Osumi N, Kato Y (2017) Gene tracing analysis reveals the contribution of neural crest-derived cells in pituitary development. J Anat 230:373–380CrossRefGoogle Scholar
  50. Vankelecom H (2007) Non-hormonal cell types in the pituitary candidating for stem cell. Semin Cell Dev Biol 18:559–570CrossRefGoogle Scholar
  51. Vitale ML, Barry A (2015) Biphasic effect of basic fibroblast growth factor on anterior pituitary Folliculostellate TtT/GF cell coupling, and Connexin 43 expression and phosphorylation. J Neuroendocrinol 27:787–801CrossRefGoogle Scholar
  52. Vizcaíno JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Ríos D, Dianes JA, Sun Z, Farrah T, Bandeira N, Binz PA, Xenarios I, Eisenacher M, Mayer G, Gatto L, Campos A, Chalkley RJ, Kraus HJ, Albar JP, Martinez-Bartolomé S, Apweiler R, Omenn GS, Martens L, Jones AR, Hermjakob H (2014) ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol 32:223–226CrossRefGoogle Scholar
  53. Weiss A, Attisano L (2013) The TGFbeta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol 2:47–63CrossRefGoogle Scholar
  54. Yoshida S, Higuchi M, Ueharu H, Nishimura N, Tsuda M, Yako H, Chen M, Mitsuishi H, Sano Y, Kato T, Kato Y (2014) Characterization of murine pituitary-derived cell lines Tpit/F1, Tpit/E and TtT/GF. J Reprod Dev 60:295–303CrossRefGoogle Scholar
  55. Yoshida S, Kato T, Kato Y (2016) Regulatory system for stem/progenitor cell niches in the adult rodent pituitary. Int J Mol Sci 17:E75CrossRefGoogle Scholar
  56. Yoshida S, Kato T, Kanno N, Nishimura N, Nishihara H, Horiguchi K, Kato Y (2017) Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland. Cell Tissue Res 370:99–112CrossRefGoogle Scholar
  57. Zhu H, Pan S, Gu S, Bradbury EM, Chen X (2002) Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Commun Mass Spectrom 16:2115–2123CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Takehiro Tsukada
    • 1
    Email author
  • Yukinobu Isowa
    • 2
  • Keiji Kito
    • 3
    • 4
  • Saishu Yoshida
    • 2
    • 3
  • Seina Toneri
    • 1
  • Kotaro Horiguchi
    • 5
  • Ken Fujiwara
    • 6
  • Takashi Yashiro
    • 6
  • Takako Kato
    • 2
    • 3
  • Yukio Kato
    • 3
    • 4
    Email author
  1. 1.Department of Biomolecular Science, Faculty of ScienceToho UniversityFunabashiJapan
  2. 2.Organization for the Strategic Coordination of Research and Intellectual PropertiesMeiji UniversityKawasakiJapan
  3. 3.Institute for EndocrinologyMeiji UniversityKawasakiJapan
  4. 4.Department of Life Sciences, School of AgricultureMeiji UniversityKawasakiJapan
  5. 5.Laboratory of Anatomy and Cell Biology, Department of Health SciencesKyorin UniversityMitakaJapan
  6. 6.Division of Histology and Cell Biology, Department of AnatomyJichi Medical University School of MedicineShimotsukeJapan

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