IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): evidence for MAP kinase-dependent and MAP kinase-independent mechanisms.
OBJECTIVE
IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model.


METHODS
The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo(b30) choriocarcinoma cells using the non-metabolizing amino acid analog, [(3)H]-alpha-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation.


RESULTS
Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30-40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 miccroM), an inhibitor of MAP kinase activation, inhibited 20-30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms.


CONCLUSIONS
These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo(b30) cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.
Fang
J
J
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
Mao
D
D
Smith
C H
CH
Fant
M E
ME
D026924:Amino Acid Transport System A / Q000235:genetics; D021542:Amino Acids, Neutral / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000963:Antimetabolites / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D014594:Uterine Neoplasms / Q000235:genetics / Q000378:metabolism*; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D026924:Amino Acid Transport System A; D021542:Amino Acids, Neutral; D000621:Aminoisobutyric Acids; D000963:Antimetabolites; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C498797:SLC38A2 protein, human; D015091:beta-Alanine; C012924:2,2-dimethyl-beta-alanine; C100049:2-aminoisobutyric acid; D007334:Insulin-Like Growth Factor I