Insulin and Prolactin Synergistically Stimulate β-Casein Messenger Ribonucleic Acid Translation by Cytoplasmic Polyadenylation

0888-8809/04/$15.00/0 Printed in U.S.A. Molecular Endocrinology 18(7):1670–1686 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2003-0483 Insulin and Prolactin Synergistically Stimulate ␤Casein Messenger Ribonucleic Acid Translation by Cytoplasmic Polyadenylation KYOUNG MOO CHOI, ITAMAR BARASH, AND ROBERT E. RHOADS Department of Biochemistry and Molecular Biology (K.M.C., R.E.R.), Louisiana State University Health Sciences Center, Shreveport, Louisiana, 71130-3932; and Institute of Animal Science (I.B.), The Volcani Center, 50250 Bet-Degan, Israel stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of ␤-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3ⴕ-untranslated region of ␤-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity. (Molecular Endocrinology 18: 1670–1686, 2004) T Transcription of the ␤-casein gene is highly dependent on prolactin and less so on glucocorticoids, whereas the reverse is true for the WAP gene (reviewed in Ref. 2). Hydrocortisone further increases prolactin-stimulated ␤-casein mRNA accumulation but has no effect in cells grown with insulin alone. The principal mechanism by which prolactin activates milk protein gene transcription is via the Jak2-Stat5 pathway. Milk protein synthesis is also regulated by changes in mRNA stability. Addition of prolactin to rat mammary gland explants grown in the presence of insulin plus hydrocortisone increases the stability of casein mRNA 17- to 25-fold (4). For cultured mouse mammary epithelial cells growing on collagen gels, addition of all three lactogenic hormones increases the rate of ␤-casein gene transcription by 2.5-fold but increases the accumulation of ␤-casein mRNA by 73fold. Conversely, insulin has a major effect on transcription of the ␤-casein gene but little effect on ␤casein mRNA stability (5). The increase in casein mRNA stability may result from increased polyadenylation. Kuraishi et al. (6) found that the poly(A) tract of mouse ␤- and ␥-casein mRNAs is shorter after weaning but longer when pups are again allowed to nurse. The length of the poly(A) tract is correlated with casein mRNA half-life both in vitro (6) and in vivo (7). Furthermore, there is an increase in the mRNA for poly(A) polymerase (PAP) during pregnancy and lactation, in parallel with the increase in poly(A) length of casein HE MAMMARY GLAND develops in distinct stages during the embryonic period, puberty, pregnancy, and lactation and regresses during weaning (1). The lactation stage is characterized by the expression of milk proteins, the major ones being ␣-, ␤-, and ␥-caseins, ␣-lactalbumin, ␤-lactoglobulin, and whey acidic protein (WAP). Both development and lactation are regulated by a variety of hormones. Insulin, glucocorticoids, and prolactin play major roles in the accumulation of milk protein mRNAs (2). The functions of individual hormones in the onset and maintenance of lactation have been extensively studied at several levels, but one of the most intriguing aspects is the synergy exhibited by combinations of hormones (e.g. Ref. 3). At present, the mechanisms responsible for this synergy are only partially understood. Abbreviations: 3-BSD, 3␤-Hydroxysteroid dehydrogenase; CPE, cytoplasmic polyadenylation element; CPEB, CPE binding protein; CPSF, polyadenylation specificity factor; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK-3, glycogen synthase kinase-3; mTOR, mammalian target of rapamycin; nt, nucleotide; PAC, puromycin acetyltransferase; PABP, poly(A)-binding protein; PAP, poly(A) polymerase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; RNase H, ribonuclease H; UTR, untranslated region; WAP, whey acidic protein. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 1670 Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in ␤-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and ␤-casein synthesis but not the synergistic Choi et al. • Translational Control of Casein Synthesis 1671 RESULTS Total Protein Synthesis Is Synergistically Stimulated by Insulin Plus Prolactin Cultured cell lines have been established that partially replicate the hormone-dependent changes in gene expression of intact mammary glands. The COMMA-1D cell line was developed from primary mammary epithelial cells of midpregnant mice (12). The clonal cell line CID 9 was derived from COMMA-1D cells by enriching for casein-producing cells (13). Culture of CID 9 cells in the presence of lactogenic hormones on Matrigel, a laminin-rich basement membrane preparation, causes them to stop proliferating, differentiate into alveoli-like structures (mammospheres), and secrete milk proteins (14). We investigated protein synthesis during differentiation of CID 9 cells on Matrigel in the presence of either insulin alone or insulin plus prolactin by pulselabeling for 30 min with [35S]Met each day. Western blotting indicated that ␣- and ␤-caseins began to accumulate by d 1 to d 2 in the presence of insulin plus prolactin but not insulin alone, whereas immunoprecipitation of proteins pulse labeled with [35S]Met indicated an increase in the instantaneous rate of ␣- and ␤-casein synthesis from d 3 to d 5 (data not shown). The increase in ␤-casein synthesis was correlated with an increased accumulation of ␤-casein mRNA. We chose d 5 cells cultured in the presence of insulin and prolactin for further experiments because milk proteins are clearly expressed. To test whether lactogenic hormones affected milk protein synthesis at the translational level, it was necessary to measure their effect over a period of time too short for changes in mRNA levels. (The absence of mRNA changes was confirmed in experim (...truncated)