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Cell and Tissue Research

, Volume 376, Issue 2, pp 281–293 | Cite as

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
  • 202 Downloads

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 

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)
441_2018_2989_MOESM5_ESM.docx (105 kb)
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)

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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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