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TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells
Chu Chang Chua1, Balvin H L Chua, Zhongyi Chen
1Osteoporosis Center, James H Quillen College of Medicine, East Tennessee State University, and Veterans Affairs Medical Center, Box 70432, Johnson City, TN 37614, USA. chua.chu@med.va.gov
Abstract:
Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor beta1 (TGF-beta1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-beta1 on TNF-alpha-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-alpha, 10 ng/ml of TGF-beta1, or combination, for 6 h. TNF-alpha exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-alpha upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-alpha treatment. In addition, caspase-3-like activity was significantly activated following TNF-alpha treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-alpha. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-alpha induced apoptosis. In contrast, the addition of TGF-beta1 attenuated all of the aforementioned effects of TNF-alpha. Our results demonstrate that TGF-beta1 can decrease TNF-alpha-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-alpha-induced caspase gene expression.
Insights
Transforming growth factor beta1 (TGF-beta1) reduces tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in osteoblasts. TGF-beta1 inhibits TNF-alpha
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, induces apoptosis in osteoblasts.
- Transforming growth factor beta1 (TGF-beta1) is a growth factor known to stimulate bone formation.
Purpose of the Study:
- To investigate the role of TGF-beta1 in modulating TNF-alpha-induced apoptosis in murine osteoblastic MC3T3-E1 cells.
Main Methods:
- Cells were treated with TNF-alpha, TGF-beta1, or a combination.
- Gene expression was analyzed using Ribonuclease protection assays (RPA).
- Protein levels and activity were assessed via Western blot and enzyme assays. Apoptosis was confirmed using Hoechst staining, TUNEL assay, and ELISA.
Main Results:
- TNF-alpha upregulated mRNA levels of caspases-1, -7, -11, -12, and FAS.
- TNF-alpha treatment led to increased processing and activation of caspases and elevated levels of cleaved poly(ADP-ribose) polymerase and FAS protein.
- TNF-alpha induced apoptosis, as evidenced by multiple assays.
- TGF-beta1 significantly attenuated all TNF-alpha-induced apoptotic effects.
Conclusions:
- TGF-beta1 decreases TNF-alpha-induced apoptosis in murine osteoblasts.
- This protective effect is mediated, at least partly, by the attenuation of TNF-alpha-induced caspase gene expression.