Differential expression and regulation of inducible nitric oxide synthase (iNOS) mRNA in human trophoblasts in vitro.
OBJECTIVE
To determine the expression of inducible nitric oxide synthase (iNOS) in human trophoblast and to examine the possible regulation of iNOS gene by cytokines.


METHODS
Total RNA was isolated from: 1) homogenized placental tissue; from 2) isolated and purified cytotrophoblast cells; and 3) cytotrophoblast and syncytiotrophoblast cells treated with cytokines in vitro. RNA was reverse transcribed and amplified by polymerase chain reaction, using specific primers for iNOS. Trophoblast cells were treated in vitro by interferon-gamma (IFN-gamma) in a dose of 10 ng/mL, Interleukin 1beta (IL-1beta) (4 ng/mL) and leukemia inhibitory factor (LIF) (1 ng/mL). Trophoblasts were also subjected to immunocytochemistry using iNOS-specific antibody to detect iNOS protein expression in these cells.


RESULTS
The expression of iNOS mRNA was found both in placental tissue and isolated cytotrophoblast cells. In culture, the highly differentiated syncytiotrophoblast expressed more mRNA than cytotrophoblast cells. IFN-gamma and LIF, but not IL-1beta, induced iNOS mRNA expression in trophoblast cells in vitro. The effects of these cytokines on iNOS mRNA were only observed in syncytiotrophoblast cells, but not in cytotrophoblast cells. Immunocytochemical staining confirmed the trophoblast cells as a major source of the iNOS synthase production.


CONCLUSIONS
1) Human trophoblast cells are able to express the iNOS mRNA, hence suggesting a role for NO in placental growth and function. 2) LIF and IFN-gamma, but not IL-1beta, induce the iNOS mRNA expression in syncytiotrophoblast cells in vitro, suggesting possible similar regulatory mechanisms in vivo. 3) This study, for the first time, demonstrates the stimulating effect of LIF on iNOS gene expression in human tissue.
Hambartsoumian
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Fertility Center of New England/Faulkner Institute for Reproductive Medicine, Reading, MA 01867, USA. hambartsoumian@hotmail.com
Srivastava
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RK
Seibel
M M
MM
D002478:Cells, Cultured; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis*; D014327:Trophoblasts / Q000201:enzymology*
D006131:Growth Inhibitors; D007375:Interleukin-1; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D012333:RNA, Messenger; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II