Apoptotic Effect Of TNF-alpha Research Articles

TNF-alpha induces apoptosis of lactotropes from female rats.

TNF-alpha is involved in the regulation of normal tissue homeostasis affecting cell proliferation, differentiation, and death. We previously reported that TNF-alpha reduces anterior pituitary cell proliferation and PRL release in an estrogen-dependent manner. In the present project we studied the induction of apoptosis by TNF-alpha in anterior pituitary cells from female rats. TNF-alpha (50 ng/ml) decreased the viability of anterior pituitary cells. Incubation with TNF-alpha for 24 h increased the percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive cells. TNF-alpha increased the percentage of somatotropes and lactotropes with apoptotic nuclear morphology without affecting the proportion of apoptotic corticotropes or gonadotropes. TNF-alpha increased the percentage of apoptotic lactotropes in cultured cells from rats killed in proestrus and estrus, but not in diestrus. This effect was significantly higher in cells from rats in proestrus than in estrus. In anterior pituitary cells from ovariectomized rats, TNF-alpha significantly increased the percentage of apoptotic lactotropes only when the cells were incubated in the presence of 17beta-estradiol. These results indicate that TNF-alpha induces apoptosis in somatotropes and lactotropes from female rats. The apoptotic effect of TNF-alpha on lactotropes is dependent on estrogens and could be involved in the regulation of anterior pituitary cell renewal during the estrous cycle.

Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes.

It has been demonstrated that tumor necrosis factor-alpha (TNF alpha) induces apoptosis in cardiac myocytes. However, its mechanism of action is still not well understood. In the present study, we hypothesized that TNF alpha induces myocardial apoptosis by induction of inducible nitric oxide synthase (iNOS). Neonatal cardiac myocytes were isolated from iNOS (-/-) mutant and C57BL6 wild type mice. Cells were cultured for 3 days before treatment with an NO donor or TNF alpha. Following treatment with S-nitroso-N-acetyl-penicillamine (SNAP) or TNF-alpha, cells were tested for apoptosis by terminal deoxynucleotidyl transfer-mediated end labeling (TUNEL) staining and cell death detection ELISA. NO production was measured by nitrite concentration in the culture medium. Cardiomyocyte expression of iNOS and TNF type 1 receptor (TNFR1) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). SNAP (0.01-100 microM) induced apoptosis of cardiac myocytes in a concentration-dependent manner in the wild type mice (n = 5, P < 0.01). TNFR1 mRNA was expressed in neonatal cardiomyocytes from both wild type and iNOS (-/-) mutant mice. TNF alpha induced a concentration-dependent increase in iNOS mRNA expression and nitrite production as well as significant apoptosis of cardiomyocytes in the wild type mice (n = 4, P < 0.01). However, without iNOS expression, the apoptotic effects of TNF-alpha were significantly attenuated in cardiomyocytes from iNOS (-/-) mutant mice (n = 4, P < 0.05). TNF alpha induces apoptosis via iNOS expression and NO production in neonatal mouse cardiomyocytes.

Involvement of Protein Kinase C-β and Ceramide in Tumor Necrosis Factor-α-induced but Not Fas-induced Apoptosis of Human Myeloid Leukemia Cells

The role of protein kinase C-beta (PKC-beta) in apoptosis induced by tumor necrosis factor (TNF)-alpha and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-beta. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-alpha-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-beta reestablished their susceptibility to TNF-alpha-induced apoptosis. The apoptotic effect of TNF-alpha in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-beta deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-alpha-induced apoptosis involves PKC-beta and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.

Nerve growth factor inhibits apoptosis induced by tumor necrosis factor in PC12 cells.

Tumor necrosis factor-alpha (TNFalpha) may play a role in at least some of the neuronal death that occurs following brain insults or in neurodegenerative diseases. It is therefore important to characterize the mechanism underlying apoptosis induced by TNFalpha in neuronal cells and to identify factors capable of protecting neurons from this death. In the present study, we characterized the apoptotic effect of TNFalpha in PC12 cells, a model system commonly used for studying neuronal apoptosis, and examined the role of Bcl-2 and caspases in this process. We show that TNFalpha induces apoptosis in both naive and primed PC12 cells. The TNFalpha-induced apoptosis was inhibited by nerve growth factor (NGF) but not by insulin. These findings suggest that the apoptotic effect of TNFalpha can be inhibited by trophic factors and that the survival-promoting effect of NGF is mediated by a specific pathway not shared by all tyrosine kinase receptors. The effect of Bcl-2 on TNFalpha-induced apoptosis was examined in PC12 cells overexpressing Bcl-2. These cells were resistant to TNFalpha-induced apoptosis, suggesting that the apoptotic effect of TNFalpha in PC12 cells is mediated via a pathway controlled by Bcl-2. Examination of the role of caspase-3 like activity in TNFalpha-induced apoptosis showed that caspase-3-like proteases are activated, and their substrate, poly (ADP-ribose) polymerase, is cleaved following TNFalpha treatment. In addition, the broad-spectrum inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), was found to inhibit the TNFalpha-induced apoptosis of PC12 cells. These results suggest that caspases are activated following TNFalpha treatment and are needed for TNFalpha-induced apoptosis in PC12 cells.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.