Shuai Zhang1, Jiang Hong Wang1, Li Jie Tian2, Bao Li Wang2, Juan Zhang1. 1. Dept. of Prosthetics, Stomatological Hospital of Tianjin Medical University, Tianjin 300070, China. 2. Zhu Xianyi Memorial Hospital of Tianjin Medical University & Endocrinology Institute, Tianjin 300134, China.
Abstract
OBJECTIVES: To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process. METHODS: Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR. RESULTS: E2 could significantly promote the proliferation of chondrocytes cultured in vitro, and maximum promotion was achieved at a concentration of 10-8 mol·L-1. RAPA could significantly inhibit cell proliferation. E2 at aconcentration of 10-8 mol·L-1 could greatly improve the gene expression levels of ERα and COLⅡ (P<0.01) with the protein levels of ERα and p-mTOR (P<0.05), and decrease the gene expression levels of Beclin-1 and ATG-5 (P<0.05) with the protein levels of Beclin-1 and LC3-Ⅱ (P<0.05). RAPA could also enhance the relative protein expression of Beclin-1 and LC3-Ⅱ (P<0.01), and reduce the expression of p-mTOR (P<0.01). Treatment with the ERα antagonist significantly reduced the expression of p-mTOR in cells (P<0.01). CONCLUSIONS: At a concentration of 10-8 mol·L-1, E2 could effectively activate the phosphorylation of mTOR through the ERα-p-mTOR pathway, inhibit cell autophagy, and promote the proliferation of condylar chondrocytes.
OBJECTIVES: To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process. METHODS: Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR. RESULTS: E2 could significantly promote the proliferation of chondrocytes cultured in vitro, and maximum promotion was achieved at a concentration of 10-8 mol·L-1. RAPA could significantly inhibit cell proliferation. E2 at aconcentration of 10-8 mol·L-1 could greatly improve the gene expression levels of ERα and COLⅡ (P<0.01) with the protein levels of ERα and p-mTOR (P<0.05), and decrease the gene expression levels of Beclin-1 and ATG-5 (P<0.05) with the protein levels of Beclin-1 and LC3-Ⅱ (P<0.05). RAPA could also enhance the relative protein expression of Beclin-1 and LC3-Ⅱ (P<0.01), and reduce the expression of p-mTOR (P<0.01). Treatment with the ERα antagonist significantly reduced the expression of p-mTOR in cells (P<0.01). CONCLUSIONS: At a concentration of 10-8 mol·L-1, E2 could effectively activate the phosphorylation of mTOR through the ERα-p-mTOR pathway, inhibit cell autophagy, and promote the proliferation of condylar chondrocytes.
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Authors: Giuseppe Musumeci; Paola Castrogiovanni; Francesca Maria Trovato; Annelie Martina Weinberg; Mohammad K Al-Wasiyah; Mohammed H Alqahtani; Ali Mobasheri Journal: Int J Mol Sci Date: 2015-08-31 Impact factor: 5.923