Inhibitory effect of rapamycin on connective tissue growth factor-stimulated cell proliferation and fibronectin secretion in myofibroblasts
10.3760/cma.j.issn.1001-7097.2009.09.005
- VernacularTitle:雷帕霉素抑制结缔组织生长因子诱导的成肌纤维细胞增殖及细胞外基质蛋白的分泌
- Author:
Xuxia GAO
;
Haichang HUANG
;
Xiaomei LI
- Publication Type:Journal Article
- Keywords:
Cell proliferation;
Fibronectins;
Sirolimus;
Myofibroblasts;
Connective tissue growth factor;
ERK1/2 signaling pathway
- From:
Chinese Journal of Nephrology
2009;25(9):678-682
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the inhibitory effect and associated mechanism of rapamycin on proliferation and extracellular matrix (ECM) secretion in myofibroblasts stimulated by connective tissue growth factor (CTGF). Methods Primary cultivated myofibroblasts were divided into 6 groups: control, CTGF (100 μg/L), rapamycin 20 μg/L+CTGF 100 μg/L, rapamycin 40 μg/L +CTGF 100 μg/L, rapamycin 20 μg/L, and rapamycin 40 μg/L alone. 5'-bromodeoxyuridine (BrdU) incorporation assay was used to detect the myofibroblast proliferation.Western blot was used to analysis the secretory FN protein in the supernatant medium of cultured myofibroblasts and the ERK1/2 phosphorylation in myofibroblasts. Results CTGF (100 μg/L)incubation significantly increased the number of Brdu positive myofibroblasts(P<0.01) and the level of FN protein secretory (P<0.05) in cell supernatant medium compared with control group,respectively. The number of Brdu positive myofibroblasts markedly decreased by 62% and 70% (P <0.05) in rapamycin 20 μg/L+CTGF 100 μg/L and rapamycin 40 μg/L+CTGF 100 μg/L groups, respectively. The FN protein levels in supernatant were decreased by 15% and 44% compared with CTGF 100 μg/L group, respectively; but the difference of FN protein levels was significant only in rapamycin 40 μg/L group (P<0.05). CTGF could activate ERK1/2 at 10 minutes; but as myofibroblasts were pretreated with rapamycin 40 μg/L for 30 min, it abolished CTGF-induced ERK1/2 phosphoralation. PD98059, the specific inhibitor of ERK1/2, could block the effect of CTGF-induced proliferation (7%±5% vs 85%±7%, P<0.01) and FN secretion (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusions Rapamycin partially suppresses the proliferation and ECM secretion of myofibroblasts induced by CTGF. Its effect may be through inhibiting CTGF-induced activation of ERKI/2 signaling pathway.
