TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells.
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.
Chua
Chu Chang
CC
Osteoporosis 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
Chua
Balvin H L
BH
Chen
Zhongyi
Z
Landy
Cathy
C
Hamdy
Ronald C
RC
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D006690:Bisbenzimidazole; D020169:Caspases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D020287:In Situ Nick-End Labeling; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D005456:Fluorescent Dyes; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases; D006690:Bisbenzimidazole