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Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation

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Abstract

Calpains are a family of calcium-dependent proteases. Two isoforms, calpain 1 and 2, have been implicated in angiogenesis and endothelial cell adhesion and migration. Calpains regulate the function of eNOS; however, the relation of calpains and eNOS to lymphangiogenesis is still unclear. In the present study, we evaluated the role of calpain and eNOS in the formation of cords by lymphatic endothelial cells on Matrigel. Human lymphatic microvascular dermal-derived endothelial cells were transfected with siRNA against calpain 1 or 2. Calpain 2 knockdown, but not calpain 1 knockdown, significantly reduced cord formation, adhesion, and migration on Matrigel. These decreases correlated with a reduction in eNOS, and phosphorylated eNOS and Hsp90 levels, as assayed by immunoprecipitation and western blotting. In contrast, the knockdown of calpain 1, but not calpain 2, increased cell adhesion, enhanced migration, and stabilized late-stage cord formation by increasing cord length compared to the control. These differences correlated with an increase in the level of phosphorylated eNOS. The results indicated that the functions of calpains and eNOS are important for cord formation by lymphatic endothelial cells. For the first time, we have found different functions of calpain 1 and 2. Calpain 1 is involved in the degradation of eNOS and Hsp90 and the phosphorylation of eNOS, while calpain 2 regulates eNOS phosphorylation during cord formation by lymphatic endothelial cells on Matrigel.

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Acknowledgments

This study was supported by the president’s discretion expenses of the University of Toyama, a Grant-in-Aid for Young Scientists (B) (No. 15790089), Grants-in-Aid for Cancer Research (No. 16022224 and 16023225), and a grant for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sport, Science and Technology, Japan.

Conflict of interest

The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Department of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan

    Orawin Prangsaengtong, Jun Yeon Park, Michiko Jo, Naotoshi Shibahara & Keiichi Koizumi

  2. Department of Surgery (I), Faculty of Medicine, University of Toyama, Toyama, 930-0194, Japan

    Kazutaka Senda & Yoshinori Doki

  3. Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan

    Orawin Prangsaengtong, Hiroaki Sakurai, Ikuo Saiki & Keiichi Koizumi

  4. Department of Biopharmacy, Faculty of Pharmacy, Srinakharinwirot University, Nakhonnayok, 26120, Thailand

    Orawin Prangsaengtong

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  1. Orawin Prangsaengtong
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  2. Kazutaka Senda
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  3. Yoshinori Doki
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Correspondence to Keiichi Koizumi.

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Prangsaengtong, O., Senda, K., Doki, Y. et al. Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation. Human Cell 25, 36–44 (2012). https://doi.org/10.1007/s13577-012-0042-7

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  • DOI: https://doi.org/10.1007/s13577-012-0042-7

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