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Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix

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Abstract

Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODC-SMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in α-actin and myosin, maker proteins of differentiation, growth of ODC-SMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of α-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of α-actin and myosin, resulting in differentiation through inhibition of focal adhesions.

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Abbreviations

DMEM:

Dulbecco’s modified Eagle’s medium

DTT:

Dithiothreitol

FAK:

Focal adhesion kinase

FBS:

Fetal bovine serum

ODC:

Ornithine decarboxylase

ODC-SMC:

ODC transfected SMC

PBS:

Phosphate-buffered saline

SMC:

Smooth muscle cells

Skp-2:

S-phase kinase-associated protein-2

pTracer-AZΔT205:

pTracer-CMV containing AZ1 without frameshift

pTracer-ODC:

pTracer-CMV encoding the ODC gene

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Acknowledgments

We thank Dr. O. A. Jänne for the kind supply of antibody against AZ1, and Dr. S. Matsufuji and Dr. Y. Murakami for the kind supply of antibody against ODC. We also thank Dr. A. J. Michael for his help in preparing the manuscript. This work was supported in part by Special Funds for Education and Research (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan

    Itsuko Ishii, Hiromi Kaneko, Masashi Uchida, Yukari Suzuki, Kyohei Higashi, Satoko Yagi & Kazuei Igarashi

  2. Division of Pharmacy, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8677, Japan

    Takaaki Suzuki, Noritaka Ariyoshi & Mitsukazu Kitada

Authors
  1. Itsuko Ishii
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  2. Takaaki Suzuki
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  3. Hiromi Kaneko
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  4. Masashi Uchida
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  5. Yukari Suzuki
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  6. Kyohei Higashi
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  7. Satoko Yagi
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  8. Noritaka Ariyoshi
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  9. Kazuei Igarashi
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  10. Mitsukazu Kitada
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Correspondence to Itsuko Ishii.

Additional information

I. Ishii, T. Suzuki and H. Kaneko contributed equally to this work.

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Ishii, I., Suzuki, T., Kaneko, H. et al. Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix. Amino Acids 42, 565–575 (2012). https://doi.org/10.1007/s00726-011-1034-8

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  • DOI: https://doi.org/10.1007/s00726-011-1034-8

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