Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons.
Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from (3)H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [(3)H]-pregnenolone metabolism, yielding [(3)H]-progesterone and [(3)H]-5alpha-dihydroprogesterone ([(3)H]-5alpha-DHP), was time-dependent and inhibited by trilostane, a 3beta-hydroxysteroid-dehydrogenase (3beta-HSD) inhibitor. Conversely, (3)H-progesterone metabolism, which yielded [(3)H]-5alpha-DHP > [(3)H]-3beta,5alpha-THP > [(3)H]-3alpha,5alpha-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3beta-HSD type I mRNA expression and 3beta-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5alpha-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.
Pistritto
Giuseppa
G
Department of Neuroscience, University of Rome Tor Vergata-Medical School, Via Montpellier, 1, 00133-Rome, Italy.
Papacleovoulou
Georgia
G
Ragone
Gianluca
G
Di Cesare
Silvia
S
Papaleo
Veruska
V
Mason
J Ian
JI
Barbaccia
Maria Luisa
ML
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019898:Autocrine Communication / Q000502:physiology*; D001921:Brain / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D005968:Glutamate Decarboxylase / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011863:Radioimmunoassay; D013234:Stem Cells / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D011374:Progesterone; D014212:Tretinoin; D011284:Pregnenolone; D015096:3-Hydroxysteroid Dehydrogenases; C477269:3 beta-hydroxy-delta 5-steroid dehydrogenase, rat; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1