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TGFβ signaling reinforces pericyte properties of the non-endocrine mouse pituitary cell line TtT/GF

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Abstract

The non-endocrine TtT/GF mouse pituitary cell line was derived from radiothyroidectomy-induced pituitary adenoma. In addition to morphological characteristics, because the cells are S100β-positive, they have been accepted as a model of folliculostellate cells. However, our recent microarray analysis indicated that, in contrast to folliculostellate cells, TtT/GF cells might not be terminally differentiated, as they share some properties with stem/progenitor cells, vascular endothelial cells and pericytes. The present study investigates whether transforming growth factor beta (TGFβ) can elicit further differentiation of these cells. The results showed that canonical (Tgfbr1 and Tgfbr2) and non-canonical TGFβ receptors (Tgfbr3) as well as all TGFβ ligands (Tgfb1–3) were present in TtT/GF cells, based on reverse transcription PCR. SMAD2, an intercellular signaling molecule of the TGFβ pathway, was localized in the nucleus upon TGFβ signaling. Furthermore, TGFβ induced cell colony formation, which was completely blocked by a TGFβ receptor I inhibitor (SB431542). Real-time PCR analysis indicated that TGFβ downregulated stem cell markers (Sox2 and Cd34) and upregulated pericyte markers (Nestin and Ng2). Double immunohistochemistry using mouse pituitary tissue confirmed the presence of NESTIN/NG2 double-positive cells in perivascular areas where pericytes are localized. Our results suggest that TtT/GF cells are responsive to TGFβ signaling, which is associated with cell colony formation and pericyte differentiation. As pericytes have been shown to regulate angiogenesis, tumorigenesis and stem/progenitor cells in other tissues, TtT/GF cells could be a useful model to study the role of pituitary pericytes in physiological and pathological processes.

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Acknowledgement

We would like to thank Editage (www.editage.jp) for English language editing.

Funding

This work was partially supported by Japan Society for the Promotion of Science KAKENHI Grants (Numbers 16 K18818 to SY, 26,460,281 to KF, 16 K08475 to KH, 26,292,166 to YK and 15 K07771 to TK), a MEXT-supported Program for the Strategic Research Foundation at Private Universities, 2014–2018, by the Meiji University International Institute for BioResource Research (MUIIR) and start-up funds to TT from the Faculty of Science Department at Toho University.

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Authors and Affiliations

  1. Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan

    Takehiro Tsukada

  2. Institute for Endocrinology, Meiji University, Kawasaki, Kanagawa, 214-8571, Japan

    Saishu Yoshida, Keiji Kito, Takako Kato & Yukio Kato

  3. Organization for the Strategic Coordination of Research and Intellectual Properties, Meiji University, Kawasaki, Kanagawa, 214-8571, Japan

    Saishu Yoshida, Yukinobu Isowa & Takako Kato

  4. Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571, Japan

    Keiji Kito & Yukio Kato

  5. Division of Histology and Cell Biology, Department of Anatomy, Jichi Medical University School of Medicine, Shimotsuke, Tochigi, 329-0498, Japan

    Ken Fujiwara & Takashi Yashiro

  6. Diabetic Neuropathy Project, Department of Sensory and Motor Systems, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo, 156-8506, Japan

    Hideji Yako

  7. Laboratory of Anatomy and Cell Biology, Department of Health Sciences, Kyorin University, Mitaka, Tokyo, 181-8612, Japan

    Kotaro Horiguchi

  8. Department of Life Science, School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-Ku, Kanagawa, 214-8571, Japan

    Yukio Kato

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  1. Takehiro Tsukada
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  2. Saishu Yoshida
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  3. Keiji Kito
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  4. Ken Fujiwara
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  5. Hideji Yako
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  7. Yukinobu Isowa
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  8. Takashi Yashiro
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  9. Takako Kato
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  10. Yukio Kato
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Correspondence to Takehiro Tsukada or Yukio Kato.

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Supplementary Fig. 1

The effect of TGFβ1 and TGFβ3 on Smad2 nuclear translocation was equivalent to that of TGFβ2. TtT/GF cells were cultured for 3 days and then treated with TGFβ1–3, and a TGFβ receptor inhibitor (SB431542) for 30 min at the indicated concentrations. Treated cells were stained for SMAD2 (a–g). Right panels show merged images (a’–g’; SMAD2: green, DAPI: blue). Similar to TGFβ2 (c, c’), TGFβ1 and TGFβ3 induced Smad2 nuclear translocation (b, b′ and d, d′, respectively). TGFβ-induced SMAD2 nuclear translocation was completely blocked by 10 μM SB431542 (e–g and e’–g’). Bar = 100 μm (JPEG 829 kb)

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Supplementary Fig. 2

TGFβ2 promotes VEGF expression in TtT/GF cells. TtT/GF cells were treated with TGFβ2 (10 ng/mL) and/or a selective TGFβ receptor I inhibitor (SB431542; 10 μM) for 3 days, and Vegf mRNA expression was determined by quantitative real-time PCR (n = 4, mean ± SEM). mRNA copy numbers were normalized to those of TATA-binding protein (Tbp) mRNA concentrations. TGFβ2 significantly increased Vegf expression, which was completely blocked by co-administration of SB431542. ***, ****p < 0.001, 0.0001, respectively (Tukey’s test) (JPEG 70 kb)

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Tsukada, T., Yoshida, S., Kito, K. et al. TGFβ signaling reinforces pericyte properties of the non-endocrine mouse pituitary cell line TtT/GF. Cell Tissue Res 371, 339–350 (2018). https://doi.org/10.1007/s00441-017-2758-x

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