Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation.
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 lymphangiogenesisis 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.
Department of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan.
Orawin
O
Prangsaengtong
Kazutaka
K
Senda
Yoshinori
Y
Doki
Jun Yeon
JY
Park
Michiko
M
Jo
Hiroaki
H
Sakurai
Naotoshi
N
Shibahara
Ikuo
I
Saiki
Keiichi
K
Koizumi
D000328:Adult; D002154:Calpain / Q000494:pharmacology / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen; D004338:Drug Combinations; D042783:Endothelial Cells / Q000166:cytology*; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007797:Laminin; D042583:Lymphangiogenesis / Q000187:drug effects* / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011509:Proteoglycans
D004338:Drug Combinations; D018841:HSP90 Heat-Shock Proteins; D007527:Isoenzymes; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D052250:Nitric Oxide Synthase Type III; D002154:Calpain; C569870:CAPN2 protein, human