Journal of Assisted Reproduction and Genetics

, Volume 19, Issue 4, pp 201–204 | Cite as

TNF-α Promotes Caspase Activation and Apoptosis in Human Fetal Membranes

  • Ramkumar Menon
  • Salvatore J. Lombardi
  • Stephen J. Fortunato
Article

Abstract

Purpose: 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-α (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.

amniochorion caspases preterm labor p53 PROM TNF 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

REFERENCES

  1. 1.
    Gibbs RS, Blanco JD: Premature rupture of the membranes. Obstet Gynecol 1982;60:671–679Google Scholar
  2. 2.
    Savitz DA, Blackmore CA, Thorp JM: Epidemiologic characteristics of preterm delivery: Etiologic heterogeneity. Am J Obstet Gynecol 1991;164:467–471Google Scholar
  3. 3.
    Gomez R, Ghezzi F, Romero R, Munoz H, Tolosa JE, Rojas I: Premature labor and intra-amniotic infection. Clinical aspects and role of the cytokines in diagnosis and pathophysiology. Clin Perinataol 1995;22:281–342Google Scholar
  4. 4.
    Fortunato SJ, Menon R: Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes. Am J Obstet Gynecol 2001;184:1399–1405Google Scholar
  5. 5.
    Fortunato SJ, Menon R, Lombardi SJ: Programmed cell death (apoptosis): A possible pathway to metalloproteinase activation and fetal membrane degradation in PROM. Am J Obstet Gynecol 2000;182:1468–1476Google Scholar
  6. 6.
    Cryns VL, Yuan J: The cutting edge: Caspases in apoptosis and dissease. In When Cell Die, RA Lockshin, Z Zakeri, JL Tilly (eds), New York, Wiley-Liss, 1998, p 177Google Scholar
  7. 7.
    Fortunato SJ, Menon R, Lombardi SJ: Support for an infectioninduced apoptotic pathway in human fetal membranes. Am J Obstet Gynecol 2001;184:1392–1397Google Scholar
  8. 8.
    Berezney R, Coffey DS: Identification of a nuclear protein matrix. Biochem Biophys Res Commun 1974;60:1410–1417Google Scholar
  9. 9.
    Vogelstein B, Pardoll DM, Coffey DS: Supercoiled loops and eucaryotic DNA replicaton. Cell 1980;22:79–85Google Scholar
  10. 10.
    Miller TE, Beausang LA, Winchell LF, Lidgard GP: Detection of nuclear matrix proteins in serum from cancer patients. Cancer Res 1992;52:422–427Google Scholar
  11. 11.
    Miller T, Beausang LA, Meneghini M, Lidgard G: Death induced changes to the nuclear matrix: The use of anti-nuclear matrix antibodies to study agents of apoptosis. Biotechniques 1993;15:1042–1047Google Scholar
  12. 12.
    Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wallach D: A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem 1995;270:7795–7798Google Scholar
  13. 13.
    Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, Dixit VM: FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 1996 14;85:817–827Google Scholar
  14. 14.
    Feng Gao C, Ren S, Zhang L, Nakajima T, Ichinose S, Hara T, Koike K, Tsuchida N: Caspase-dependent cytosolic release of cytochrome c and membrane translocation of bax in p53-induced apoptosis. Exp Cell Res 2001;265:145–151Google Scholar
  15. 15.
    Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479–489Google Scholar
  16. 16.
    O'Brien MA, Moravec RA, Riss TL: Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells. Biotechniques 2001;30:886–891Google Scholar

Copyright information

© Plenum Publishing Corporation 2002

Authors and Affiliations

  • Ramkumar Menon
    • 1
  • Salvatore J. Lombardi
    • 1
  • Stephen J. Fortunato
    • 1
  1. 1.The Perinatal Research CenterWomen's Health Research and Education Foundation, The Women's HospitalNashville

Personalised recommendations