TNF-alpha promotes caspase activation and apoptosis in human fetal membranes.
OBJECTIVE
Increased amniotic fluid tumor necrosis factor (TNF) is a marker of infection when associated with preterm labor and preterm premature rupture of the amniochorionic membranes (PROM). We have noted increased apoptosis in membranes derived from women with PROM. This study examines the role of TNF in promoting fetal membrane apoptosis.


METHODS
Amniochorion (n = 8), collected at the time of elective repeat cesarean section prior to labor from normal term gestation, were placed in an organ explant system. After 48 h in culture, the membranes were stimulated with recombinant TNF-alpha (20 ng/mL) for 24 h. Tissue frozen after stimulation was subjected to RT-PCR to study the expression of TNF-induced caspase genes. ELISA assayed the levels of proapoptotic p53 in tissues and cell death related nuclear matrix protein (NMP) in tissue culture supernatants. The activity of caspases in tissue homogenates was measured using substrates specific for caspase 2, 3, 6, 8, and 9. Results were analyzed by using the Wilcoxon nonparametric test for paired samples. A p < 0.05 was considered significant.


RESULTS
RT-PCR showed induction of caspases 2, 8, and 9 (caspase cascade initiators) in human fetal membranes after TNF stimulation. Caspases 3 and 6 (effector caspases) expression was constitutive in both TNF stimulated- and control membranes. Caspases, 2, 3, 8, and 9 activity was significantly higher in TNF-stimulated tissues compared with control, whereas, no significant change in caspase 6 activity was noticed. TNF-stimulated tissues released increased levels of NMP (24.03 U/mL) compared with control (13.5U/mL) (p = 0.03). TNF also increased p53 levels in the tissues (0.05 ng/mL) compared with control cultures (0.03 ng/mL; p = 0.02).


CONCLUSIONS
TNF increases proapoptotic p53 levels and caspase activities in fetal membranes. Increased NMP reflects cell death.
Menon
Ramkumar
R
The Perinatal Research Center, Women's Health Research and Education Foundation, The Women's Hospital, Nashville, Tennessee, USA.
Lombardi
Salvatore J
SJ
Fortunato
Stephen J
SJ
D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism*; D046508:Culture Techniques; D004789:Enzyme Activation; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D005321:Extraembryonic Membranes / Q000502:physiology*; D006801:Humans; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D034521:Nuclear Matrix-Associated Proteins; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D020169:Caspases