In Vitro Effects of Vaspin on Porcine Granulosa Cell Proliferation, Cell Cycle Progression, and Apoptosis by Activation of GRP78 Receptor and Several Kinase Signaling Pathways Including MAP3/1, AKT, and STAT3

被引:36
作者
Kurowska, Patrycja [1 ]
Mlyczynska, Ewa [1 ]
Dawid, Monika [1 ]
Opydo-Chanek, Malgorzata [2 ]
Dupont, Joelle [3 ]
Rak, Agnieszka [1 ]
机构
[1] Jagiellonian Univ Krakow, Dept Physiol & Toxicol Reprod, Inst Zool & Biomed Res, PL-30387 Krakow, Poland
[2] Jagiellonian Univ Krakow, Dept Expt Hematol, Inst Zool & Biomed Res, PL-30387 Krakow, Poland
[3] INRA, UMR85, Unite Physiol Reprod & Comportements, F-37380 Nouzilly, France
关键词
vaspin; proliferation; cell cycle; apoptosis; granulosa cells; ovary; pig; VASCULAR ENDOTHELIAL-CELLS; SERINE-PROTEASE INHIBITOR; IMMUNOHISTOCHEMICAL LOCALIZATION; OVARIAN-FOLLICLES; ER STRESS; IGF-I; EXPRESSION; LEPTIN; STEROIDOGENESIS; PROGESTERONE;
D O I
10.3390/ijms20225816
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Vaspin, a visceral adipose tissue-derived serine protease inhibitor, is expressed in the porcine ovary; it induces the activation of various kinases and steroidogenesis. The aim of this study was to examine the effect of vaspin on granulosa (Gc) proliferation, cell cycle regulation, and apoptosis. Porcine Gc was incubated with vaspin (0.01-10 ng/mL) for 24 to 72 h, proliferation was measured using alamarBlue assay, cell cycle progression was assessed using flow cytometry, and cyclin (D, E, and A) protein expression was measured using immunoblotting. Apoptosis was assessed by measuring caspase activity using Caspase-glo 3/7 assay. Furthermore, histone-associated DNA fragments levels were measured using a cell-death detection ELISA; BAX (bcl-2-like protein 4), BCL2 (B-cell lymphoma 2), caspases (-3, -8, and -9), p53 mRNA, and protein expression were assessed using real time PCR and immunoblotting. We found that vaspin significantly enhanced Gc proliferation and cell cycle progression into the S and G2/M phases and decreased apoptosis. We observed that siRNA silencing of the glucose-regulated protein (GRP78) receptor and pharmacological inhibitors of mitogen-activated kinase (MAP3/1/ERK1/2), Janus kinase (STAT3) and protein kinase B (AKT) blocked the ability of vaspin cell proliferation and enhanced caspase-3/7 activities. These results suggest that vaspin via mitogenic effect on porcine Gc acts as a new regulator of ovarian growth, development, or folliculogenesis.
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页数:18
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