In vitro study of placental trophoblast calcium uptake using JEG-3 human choriocarcinoma cells.

During human fetal development, placental syncytiotrophoblasts actively transport calcium from the maternal to the fetal circulation. Two functional components, a cytosolic Ca2(+)-binding protein (CaBP) and a Ca2(+)-ATPase have been identified in the syncytiotrophoblasts of the chorionic villi. We report here the calcium uptake properties of a human choriocarcinoma cell line, JEG-3, which was used as an in vitro model cell system for the syncytiotrophoblasts. In culture, JEG-3 proliferated as large syncytial aggregates expressing typical syncytiotrophoblast markers. 45Ca uptake by JEG-3 was a substrate- and temperature-dependent, membrane-mediated active process that exhibited linear kinetics for up to 7 min. Both the CaBP and the Ca2(+)-ATPase were expressed by JEG-3, on the basis of biochemical, histochemical, immunochemical and or mRNA assays. Immunohistochemistry and in situ hybridization revealed that JEG-3 cells were heterogeneous with respect to the expression of the CaBP. The Ca2(+)-ATPase activity of JEG-3 was similar to the placental enzyme in terms of sensitivity to specific inhibitors, and was detected histochemically along the cell membrane. Fura-2 Ca2+ imaging revealed that calcium uptake by JEG-3 was not accompanied by a concomitant increase in cytosolic [Ca2+], suggesting a specific Ca2+ sequestration mechanism. The involvement of calciotropic hormonal regulation was evaluated by studying the response of JEG-3 to 1,25-dihydroxy vitamin D3. Calcium uptake was significantly stimulated in a dose-dependent manner by a 24-h treatment of the cells with 1,25-dihydroxy vitamin D3 (optimal dose approximately 0.5 nM); the CaBP level doubled whereas steady-state CaBP mRNA did not, suggesting that CaBP expression was regulated by 1,25-dihydroxy vitamin D3. These observations strongly suggest that the JEG-3 human choriocarcinoma cells should serve as a convenient in vitro model system for studying the cellular mechanism and regulation of transplacental calcium transport.

Hemin enhances differentiation and maturation of cultured regenerated skeletal myotubes.

Satellite cells, isolated from marcaine-damaged rat skeletal muscle, differentiate in culture to form contracting, cross-striated myotubes. Addition of 20 microM hemin (ferriprotoporphyrin IX chloride) to the culture medium resulted in increases in the number, size, and alignment of myotubes; in the number of myotubes that exhibited cross-striations; and in the strength and frequency of myotube contractions. Hemin increased satellite cell fusion by 27%, but decreased cell proliferative rate by 30%. Hemin increased the specific activity of creatine kinase (CK), a sensitive indicator of muscle differentiation, by 157%. Separation of CK isoenzymes by agarose gel electrophoresis showed that hemin increased only the muscle-specific CK isoenzymes (MM-CK and MB-CK). Thus, hemin seems to duplicate some of the effects of innervation on cultured myotubes by increasing contraction frequency and strength, appearance of cross-striations, and muscle-specific isoenzymes. In contrast, 3-amino-1,2,4-triazole, an inhibitor of heme biosynthesis, decreased the number of cross-striated myotubes, the strength and frequency of myotube contractions, and CK activity. These inhibitory effects were reversed by hemin. Collectively, these results demonstrate a physiologically significant role for heme in myotube maturation.

Mouse placental 57-kDa calcium-binding protein: I. Cloning of cDNA and characterization of developmental expression.

A cDNA clone to the mouse placental 57-kDa calcium-binding protein (MCaBP) [29] was isolated by immunoscreening a mouse placenta cDNA library constructed in the expression phage vector, lambda gt 11. The MCaBP cDNA was 0.7 kb in size, with restriction sites for StuI and Bg/II, and its identity to the MCaBP was confirmed by mRNA hybrid selection. RNA blot hybridization revealed a predominant, 3.9-kb transcript of the MCaBP in day-18 mouse placenta. The expression of the MCaBP during development was analyzed with respect to the levels of protein activity, translatable MCaBP mRNA, and total MCaBP transcript.