Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20alpha-hydroxysteroid dehydrogenase.
Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.
Telleria
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Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota 57069, USA.
Goyeneche
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AA
Stocco
C O
CO
Gibori
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D043607:20-alpha-Hydroxysteroid Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016923:Cell Death; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D016328:NF-kappa B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D011247:Pregnancy; D011388:Prolactin / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020218:Response Elements; D051996:Transcription Factor RelA
D002457:Cell Extracts; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D011388:Prolactin; D043607:20-alpha-Hydroxysteroid Dehydrogenase