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Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

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Abstract

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G1 phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21Waf1/Cip1 and p27Kip1 and phosphorylation of p27Kip1 but rk-2 treatment inhibited translocation of p21Waf1/Cip1 and p27Kip1 from nucleus to cytoplasm and phosphorylation of p27Kip1. Also, rk-2 induced up-regulation of p53 and nuclear p21Waf1/Cip1 and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L-cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21Waf1/Cip1 expression induced by rk-2.

These data suggest that rk-2 induces the BCE cell cycle arrest at G0–G1 phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.

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Abbreviations

BCE:

bovine capillary endothelial

bFGF:

basic fibroblast growth factor

CDK:

cyclin-dependent kinase

CKIs:

cyclin-dependent kinase inhibitors

CS:

calf serum

DCFH-DA:

2′,7′-dichlorofluorescein diacetate

DMEM:

Dulbecco’s modified Eagle’s medium

FACS:

fluorescence-activated cell sorter

NAC:

N-Acetyl-L-cysteine

PBS:

phosphate-buffered saline

Rb:

retinoblastoma tumor suppressor gene product

rk-2:

recombinant human prothrombin kringle-2

ROS:

reactive oxygen species

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgements

This work was supported by Grant No. R01-2005-000-10179-0 from the Basic Research Program of the Korea Science & Engineering Foundation and the Brain Korea 21 Project in 2005.

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  1. Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea

    Tae Hyong Kim, Seunghyun Oh & Soung Soo Kim

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  1. Tae Hyong Kim
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  2. Seunghyun Oh
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  3. Soung Soo Kim
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Correspondence to Soung Soo Kim.

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Kim, T.H., Oh, S. & Kim, S.S. Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors. Angiogenesis 8, 307–314 (2006). https://doi.org/10.1007/s10456-005-9020-y

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  • DOI: https://doi.org/10.1007/s10456-005-9020-y

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