THE MAMMARY GLAND develops in distinct

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1 /04/$15.00/0 Molecular Endocrinology 18(7): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /me 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, ; and Institute of Animal Science (I.B.), The Volcani Center, Bet-Degan, Israel 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 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 3 -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: , 2004) THE 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 ( the foremost professional society serving the endocrine community. 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 73- fold. 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 1670

2 Choi et al. Translational Control of Casein Synthesis Mol Endocrinol, July 2004, 18(7): mrna, suggesting that the poly(a) tract length is determined by PAP gene expression. Various types of evidence have suggested that milk gene expression may also be regulated at the translational level. Travers et al. (8) found that involution of the rat mammary gland could be induced by either litter removal or deprivation of GH and prolactin. Litter removal for 48 h caused a decrease in casein mrna levels and an increased association of casein mrna with the monosome fraction, whereas deprivation of hormones caused no change in the association of casein mrna with the polysome fraction. Baruch et al. (9) expressed in transgenic mice a chimeric gene derived from the promoter of ovine -lactoglobulin and the structural gene of human serum albumin. When mammary explants from virgin mice were cultured in the absence of hormones, human serum albumin mrna continued to accumulate, but synthesis of the corresponding protein required insulin and prolactin. This phenomenon was pursued in mouse mammary epithelial cells (CID 9) cultured in the presence of insulin, where it was observed that addition of prolactin gradually increased the rate of protein synthesis over 24 h (10). This study also implicated a role for phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I) in prolactin-stimulated translation. PHAS-I binds to and sequesters initiation factor eif4e, the cap-binding protein, but phosphorylation of PHAS-I in response to a variety of hormones and growth factors causes release of eif4e and stimulation of cap-dependent translation (11). Addition of prolactin to CID 9 cells increased phosphorylation of PHAS-I and its release from eif4e (10). These previous studies in whole animals, mammary gland explants, and cultured cells suggest, but do not prove, an effect of lactogenic hormones on the process of translation itself. Due to the long time courses over which these experiments have been conducted (days to weeks), it is not possible to determine the extent to which changes in milk protein synthesis are due to mrna levels, elaboration of endoplasmic reticulum, or other long-term cellular alterations rather than a direct translational effect. We therefore undertook a study of cultured mammary epithelial cells to test for changes in the protein synthesis rate immediately after hormone addition (0 30 min), verifying that there were no changes in mrna levels. The results indicated that insulin alone stimulates the rate of total protein synthesis whereas prolactin alone has no effect, but insulin plus prolactin stimulate protein synthesis more than the sum of either hormone alone. These effects are even more striking for -casein synthesis. The mrna-specific translational enhancement occurs through lengthening of the poly(a) tract via the cytoplasmic polyadenylation element binding protein (CPEB), not through a change in PHAS-I phosphorylation. 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 [ 35 S]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 [ 35 S]Met indicated an increase in the instantaneous rate of - and -casein synthesis from d3tod5(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 experiments presented below.) We deprived the cells of insulin and prolactin for various lengths of time and then measured protein synthesis for the first 30 min after readdition of hormones (Fig. 1). After 1, 3, or 6hofhormone deprivation, there was no significant difference in the rate of protein synthesis between cells cultured for 30 min with no hormone (open bars), with prolactin alone (cross-hatched bars), with insulin alone (shaded bars), or with both hormones (striped bars). After 9 h of hormone deprivation, protein synthesis was stimulated by readdition of insulin, reaching a plateau after 14 h of hormone removal (1.66-fold over no hormone readdition). There was no increase in protein synthesis caused by readdition of prolactin alone. However, the combination of insulin plus prolactin stimulated protein synthesis more than the sum of insulin alone and prolactin alone (1.59-fold stimulation at 14 h compared with insulin alone, 2.64-fold compared with no hormone). It was unexpected that the rate of hormone-stimulated protein synthesis would be higher after 14 h of hormone deprivation compared with no hormone deprivation (Fig. 1, striped bars at 14 h vs. 1 h). This may

3 1672 Mol Endocrinol, July 2004, 18(7): Choi et al. Translational Control of Casein Synthesis Fig. 1. Development of Protein Synthesis Dependence on Lactogenic Hormones CID 9 cells were allowed to differentiate in the presence of 5 g/ml insulin and 0.05 g/ml prolactin for 5 d after transfer to Matrigel-coated dishes. Hormones were removed for the indicated times, and then various combinations of hormones were added back simultaneously with 25 Ci/ml [ 35 S]Met. The rate of protein synthesis was measured by incorporation of [ 35 S]Met into protein for 30 min. Open bars, No hormone; cross-hatched bars, 0.5 g/ml prolactin alone; solid bars, 5 g/ml insulin alone; striped bars,5 g/ml insulin plus 0.5 g/ml prolactin. A repeated measures two-way ANOVA indicated significant differences (P 0.01) for all comparisons at 9, 12, 14, and 16 h except prolactin vs. no hormone. reflect hypersensitization of signal transduction components when hormones are removed. Attenuation of hormonal responses upon long-term exposure to either insulin (e.g. Ref. 15) or prolactin (e.g. Ref. 16) has been documented. When cells were subsequently maintained in the presence of insulin plus prolactin for 30 h, the protein synthesis rate returned to that observed with no deprivation of hormones (data not shown), supporting the explanation that protein synthesis is attenuated after long-term exposure to hormones. It is conceivable that the increase in protein labeling could merely reflect a different rate of [ 35 S]Met uptake in the presence of hormones, rather than a true increase in the translational rate. To test this, we measured the trichloroacetic acid-soluble radioactivity in CID 9 cells over a 30-min time course after addition of [ 35 S]Met to the medium, comparing the four hormone treatments used in Fig. 1. The results indicated that [ 35 S]Met uptake was the same for all treatments 8.2% (data not shown). The dose response of total protein synthesis to prolactin, with and without insulin, is shown in Fig. 2A. Addition of prolactin alone failed to stimulate protein synthesis over a range of g/ml (open bars). Addition of insulin alone increased protein synthesis 1.8-fold compared with no hormone (striped vs. open bars at far left). However, addition of a constant amount of insulin and increasing amounts of prolactin stimulated protein synthesis 3.0-fold up to 0.5 g/ml prolactin, after which it decreased (striped bars). This latter effect is similar to the biphasic action of prolactin in other systems, e.g. the human chorionic gonadotropin-stimulated secretion of progesterone by Leydig cells (17). Insulin and Prolactin Synergistically Stimulate Milk Protein Synthesis The spectrum of proteins synthesized in the presence of either insulin alone or insulin plus prolactin was investigated by labeling with [ 35 S]Met for 30 min after a 14-h deprivation and re-addition of hormones (Fig. 3A). Most bands increased in intensity after insulin treatment. When both hormones were added, some bands remained the same compared with insulin alone and some increased further, e.g. at approximately 46 kda and approximately 30 kda. To determine the effects of lactogenic hormones on the synthesis of individual proteins, we used antibodies to milk proteins ( -casein, -casein, and WAP) and a typical non-milk protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Fig. 3B). Synthesis of the three milk proteins was stimulated by insulin alone and was further stimulated by insulin plus prolactin. Synthesis of GAPDH, on the other hand, was stimulated by insulin alone, but no further stimulation occurred when prolactin was included. The dose-response of -casein synthesis to prolactin in the presence of a constant amount of insulin is shown in Fig. 2B. Prolactin alone failed to stimulate -casein synthesis (open bars). Insulin alone increased -casein synthesis 9.5-fold compared with no hormone (striped vs. open bars at far left). Addition of a constant amount of insulin and increasing amounts of prolactin stimulated -casein synthesis 8.7-fold over insulin alone (striped bars at 0.5 g/ml vs. 0 g/ml) and 83-fold over prolactin alone (striped vs. open bars at 0.5 g/ml). As with total protein synthesis (Fig. 2A), the stimulation was less at 0.8 g/ml than at 0.5 g/ml prolactin (Fig. 2B).

4 Choi et al. Translational Control of Casein Synthesis Mol Endocrinol, July 2004, 18(7): Fig. 2. Dependence of Protein Synthesis on Prolactin Concentration Cells were allowed to differentiate as in Fig. 1 and deprived of hormones for 14 h. Hormones were added back simultaneously with 50 Ci/ml [ 35 S]Met. Radioactivity incorporated into total proteins (A) or -casein (B) was measured 30 min later. Open bars, Readdition of prolactin alone at the indicated concentrations; striped bars, readdition of prolactin plus 5 g/ml insulin. A two-sample t test assuming equal or unequal variances demonstrated that there were highly significant differences (P 0.01) between insulin alone and insulin plus prolactin at all concentrations. To verify that these hormonal effects were due to changes in translational efficiency, it was necessary to show that mrna levels did not change. We employed real-time PCR to quantitate levels of casein mrna relative to rrna (Table 1). The concentration of -casein mrna increased fold when prolactin was included at 0.5 g/ml during differentiation. When a lower concentration of prolactin was used (0.05 g/ml), mrna induction was correspondingly lower ( fold). During the 14 h of hormone deprivation, -casein mrna decayed to a relative value of Most significantly for the question of translational control, adding back either insulin alone, prolactin alone, or insulin plus prolactin did not increase the level of Fig. 3. Changes in Total and Milk Protein Synthesis Caused by Readdition of Lactogenic Hormones CID 9 cells were allowed to differentiate and then deprived of hormones as in Fig. 2. Either 5 g/ml insulin, 0.5 g/ml prolactin, or both were added back simultaneously with [ 35 S]Met followed by incubation for 30 min. A, Protein (50 g) was subjected to SDS-PAGE on 12% gels followed by autoradiography. B, Protein (250 g) was subjected to immunoprecipitation with a mixture of antibodies against - and -caseins, WAP, and GAPDH. The immunoprecipitate was dissolved and subjected to SDS-PAGE on 12% gels, and proteins were visualized by autoradiography (lower section). Radioactive bands were scanned and quantitated using ImageQuant 5.0 software (Molecular Dynamics, Inc., Sunnyvale, CA) (upper section). -casein mrna during the 30 min over which protein synthesis was measured. Comparable results were obtained when GAPDH mrna rather than rrna was used as a control RNA (data not shown). Thus, the increase in the rate of -casein synthesis observed upon hormone readdition in Figs. 2 and 3 is due to changes in the rate of mrna translation, not mrna levels. -Casein, But Not GAPDH, mrna Is Shifted to Higher Polysomes by Insulin Plus Prolactin We wished to determine whether the increase in protein synthesis was due to an effect on initiation, elongation, or termination. Polysomal distribution of individual mrnas is an independent method of measuring changes in protein synthesis rate that is complementary to metabolic labeling, because it does not depend on amino acid pool size. An increase in the rate of initiation relative to elongation or termination results in a shift of the mrna from smaller to larger polysomes, whereas the opposite occurs if the rate of elongation or termination is increased relative to initiation (18). For cells undergoing each hormonal treatment, we examined the polysomal distribution of the mrnas for casein, as an example of a protein that is synergisti-

5 1674 Mol Endocrinol, July 2004, 18(7): Choi et al. Translational Control of Casein Synthesis cally increased by insulin plus prolactin, and GAPDH, as an example of one that is not. To determine polysome size accurately, we employed isokinetic sucrose gradient ultracentrifugation (19, 20). The distance traveled in an isokinetic sucrose gradient is directly proportional to the polyribosome number, making it possible to calculate polysome size even when individual peaks are not resolved. The overall polysome distribution, as measured by OD 254, indicated that the fraction of ribosomes in polysomes increased in cells incubated for 30 min with insulin (Fig. 4, B vs. A) and increased even more with insulin plus prolactin (Fig. 4, C vs. B). The polysomes were disaggregated when EDTA was included in the gradients, indicating that these profiles do not represent nonpolysomal ribonucleoprotein complexes (data Table 1. Measurement of -Casein mrna in CID 9 Cells by Real-Time PCR Differentiated on a Removal of Hormones Readdition of Hormones Relative Concentration of -Casein mrna b Insulin c None None 1.0 Insulin plus 0.5 g/ml prolactin None None 3,200, ,000 d Insulin plus 0.05 g/ml prolactin None None 1,500, ,000 Insulin plus 0.05 g/ml prolactin 14 h None 260,000 20,000 Insulin plus 0.05 g/ml prolactin 14 h 0.5 g/ml prolactin 250,000 10,000 Insulin plus 0.05 g/ml prolactin 14 h Insulin 260,000 10,000 Insulin plus 0.05 g/ml prolactin 14 h Insulin plus 0.5 g/ml prolactin 250,000 10,000 a Cytodifferentiation on Matrigel was carried out over 5dinthepresence of the indicated hormones. b Relative -casein mrna levels were determined by real-time PCR. The level of -casein mrna in CID 9 cells allowed to differentiate in the absence of prolactin is arbitrarily set to 1.0. A one-way ANOVA and a Bonferroni post hoc multiple comparison test of the fourth through seventh rows indicated no significant differences. c Insulin was used at 5 g/ml in all cases. d Mean SD (n 3). Fig. 4. Changes in Polysomal Distribution of -Casein and GAPDH mrnas after Stimulation with Lactogenic Hormones CID 9 cells were allowed to differentiate and deprived of hormones, and the indicated hormones were restored as in Fig. 3. Cells were lysed 30 min later and the lysates were subjected to ultracentrifugation on isokinetic sucrose gradients. Polysome sedimentation (from left to right) was monitored by absorbance at 254 nm (A C). The distribution of -casein (D F) and GAPDH (G I) mrnas was determined by real-time PCR. Polysome gradients were run three times for each hormonal condition (total of nine gradients), and the mrna content of each polysomal fraction was averaged for the triplicates. The means and standard deviations of mrna content, expressed as a percentage of the total mrna present in all fractions, are shown. A two-way ANOVA indicated that the polysomal distribution of -casein mrna was significantly different (P 0.01) between each hormonal treatment.

6 Choi et al. Translational Control of Casein Synthesis Mol Endocrinol, July 2004, 18(7): not shown). -Casein mrna was biphasically distributed when no hormones were added back to cells, with peaks in fractions 2 and 5 (Fig. 4D). By analysis of distances sedimented, we calculated (19, 20) that fraction 2 corresponds to the untranslated messenger ribonucleoprotein complex (mrnp) pool, and fraction 5 corresponds to low polysomes (5.1 ribosomes per mrna). Insulin shifted most of the -casein mrna out of mrnps and into polysomes (Fig. 4E), and the average polysome size increased to 6.5 ribosomes per mrna. Insulin plus prolactin caused an even more pronounced loss of -casein mrna from mrnps and a further shift to heavy polysomes (Fig. 4F), with a peak at 9.7 ribosomes per mrna. Translation of GAPDH mrna was also initiated more efficiently in cells treated with insulin compared with no hormone (Fig. 4, H vs. G), the peak shifting from 8.2 to 11.2 ribosomes per mrna. However, the polysome distribution did not change when prolactin was included (Fig. 4, I vs. H). Overall, these data indicate that the combination of insulin plus prolactin increases the initiation rate of -casein mrna relative to the elongation/termination rate but has no effect on GAPDH mrna. Signaling Pathways Leading to the Synergistic Stimulation of Protein Synthesis by Insulin Plus Prolactin We used specific inhibitors to investigate signal transduction pathways leading to the synergistic stimulation of milk protein synthesis (Fig. 5). Rapamycin inhibits the mammalian target of rapamycin (mtor), an insulin-stimulated kinase that promotes initiation of highly cap-dependent mrnas and 5 -TOP mrnas (21). PD98059 inhibits MAPK (22), which is required for insulin-stimulated protein synthesis (23, 24). LY is an inhibitor of phosphatidylinositol 3- kinase [PI3K (25)], which is upstream of the mtor regulation of PHAS-I and p70 S6K as well as the protein kinase B (PKB) regulation of glycogen synthase kinase-3 (GSK-3). LY also inhibits mtor directly (26, 27). The preparation of cells was the same as in Figs However, in this experiment, the inhibitors were added 30 min before the simultaneous addition of hormones and [ 35 S]Met. At 25 ng/ml, rapamycin reduced insulin-stimulated synthesis of total protein (Fig. 5A, solid triangles) as well as -casein (Fig. 5E) to the level seen with no hormone (open circles). This concentration is similar to that observed to inhibit insulinstimulated protein synthesis in other systems (24). At higher concentrations of rapamycin, translation in the absence of hormones or in the presence of prolactin alone (solid circles) was inhibited further, presumably because mtor has some basal activity in the absence of insulin. Most significantly, translation in the presence of insulin plus prolactin (open triangles) remained higher than with insulin alone at all rapamycin concentrations. This indicates that the pathway responsible for synergistic translational stimulation of total protein and -casein is not sensitive to rapamycin. Fig. 5. Effects of Inhibitors on Hormone-Stimulated Synthesis of Total Proteins and -Casein CID 9 cells were allowed to differentiate and then deprived of hormones as in Fig. 2. The indicated concentrations of rapamycin, PD98059, LY294002, or cordycepin were added 30 min before the addition of 15 Ci/ml [ 35 S]Met, in the absence of hormones (open circles), with 0.5 g/ml prolactin alone (solid circles), with 5 g/ml insulin alone (solid triangles), or with 5 g/ml insulin plus 0.5 g/ml prolactin (open triangles). (When open and solid circles overlap, only the former are shown.) Incorporation of radioactivity into total protein (A D) or -casein (E H) was measured after 30 min.

7 1676 Mol Endocrinol, July 2004, 18(7): Choi et al. Translational Control of Casein Synthesis Insulin-stimulated synthesis of total proteins and -casein was also eliminated by 50 M PD98059 (Fig. 5, panels B and F, solid triangles vs. open circles), which is similar to the concentration needed to inhibit MAPK in other systems (28), but the synergistic hormonal stimulation was maintained even up to 100 M (open triangles vs. solid triangles). This indicates that the MAPK branch of the insulin pathway to translational stimulation is active in these cells but not responsible for the synergistic hormonal stimulation. LY at 25 M produced the same effect on insulin-stimulated synthesis of total proteins and casein as did rapamycin and PD98059 (Fig. 5, C and G). This LY concentration is similar to that needed to inhibit PI3K in other systems (26), but the synergistic hormonal stimulation was maintained even up to 50 M. This eliminates pathways downstream of PI3K as being responsible for this phenomenon. The opposite behavior was seen with the chain terminator cordycepin (3 -deoxyadenosine), an inhibitor of polyadenylation (29, 30). There was no significant effect of cordycepin on synthesis of total proteins (Fig. 5D) or -casein (Fig. 5H) in the absence of hormone readdition (open circles), in the presence of insulin alone (solid triangles), or in the presence of prolactin alone (solid circles). However, cordycepin progressively inhibited synthesis of total proteins as well as -casein in the presence of insulin plus prolactin (open triangles). By 100 g/ml, the rate was the same as with insulin alone. This concentration is similar to that observed to block cytoplasmic polyadenylation in other systems (31). These results suggest that the increase in translational efficiency of -casein mrna is due to cytoplasmic polyadenylation. Insulin Plus Prolactin Induces Cytoplasmic Poly(A) Elongation of -Casein, But Not GAPDH, mrna Fig. 6. Cytoplasmic Polyadenylation of -Casein mrna in Response to Lactogenic Hormones CID 9 cells were allowed to differentiate and deprived of hormones, and the indicated hormones were restored as in Fig. 3. Cells were lysed 30 min later. A, -casein mrna was detected by Northern blotting either as the full-length mrna (lanes 1 4) or after digestion with RNase H in the presence of a complementary oligodeoxynucleotide that approximately bisects the mrna (lanes 5 15). In lanes 9 11, cordycepin was added to the cell culture medium at the indicated concentrations (in micrograms/ml) 30 min before hormone addition. In lanes 12 15, the RNase H reaction also contained oligo(dt) to remove poly(a). The positions and lengths of marker RNAs are shown. B, GAPDH was similarly detected by northern blotting with or without cleavage using a different complementary oligonucleotide. C, The 3 -terminal fragment of -casein mrna was detected by northern blotting after digestion with RNase H as described in panel A. The indicated drugs, at the highest concentrations shown in Fig. 5, were added to the medium 30 min before hormone addition. To test directly for a hormone-dependent change in the poly(a) length of -casein mrna, we isolated total RNA from cells prepared as in Figs. 2 4 and examined mrna length by northern blotting (Fig. 6A). The mobility of full-length -casein mrna was the same in cells treated for 30 min with either no hormone, prolactin alone, or insulin alone, but it decreased slightly for cells treated with insulin plus prolactin (lanes 1 4). Because small differences in length are difficult to visualize with intact mrnas, we examined only the 3 -portion of -casein mrna by first cleaving specifically with ribonuclease H (RNase H) (32, 33) in the presence of an oligonucleotide complementary to nucleotide (nt) (lanes 5 11). The decrease in mobility of the 3 terminal RNA fragment for cells treated with insulin plus prolactin could be seen more clearly (lane 8 vs. lanes 5 7). The mobility shift was diminished by pretreatment of the cells with cordycepin in a dosedependent manner (lanes 9 11). By 100 g/ml of cordycepin, a concentration that eliminated synergistic stimulation of translation (Fig. 5, D and H), the mobility was the same as that from cells treated with no hormone (Fig. 6A, lane 10 vs. lane 5). To confirm that the observed mobility change of -casein mrna was due to a lengthening of the poly(a) tract, we also digested the mrna with RNase H in the presence of the sequence-specific oligonucleotide plus oligo(dt) (Fig. 6A, lanes 12 15). This results in site-specific cleavage of -casein mrna as well as removal of the poly(a) tract. The 3 -terminal mrna fragments from cells treated under all four hormonal conditions had the same mobility. Thus, the reduced mobility observed in lane 8 is due to poly(a) length and not other structural changes (e.g. RNA editing, methylation, alternative splicing).

8 Choi et al. Translational Control of Casein Synthesis Mol Endocrinol, July 2004, 18(7): As a control, the poly(a) length of GAPDH mrna was measured (Fig. 6B). No change in full-length GAPDH mrna was detected with any of the hormonal treatments (lanes 1 4). Similarly, when only the 3 terminal fragment was visualized, there was no change in length (lanes 5 8). Treatment with cordycepin did not change the length of the GAPDH mrna fragment (lanes 9 11). Including oligo(dt) in the RNase H digestion resulted in a slight increase in mobility, reflecting the loss of a short poly(a) tract. Thus, the hormoneinduced increase in poly(a) length observed with a typical milk protein mrna, -casein, does not occur with a typical nonmilk protein mrna, GAPDH. The effects of various signal transduction inhibitors on hormone-stimulated polyadenylation were tested. Rapamycin and LY294002, which inhibit PHAS-I phosphorylation in CID 9 cells (10), were tested for an effect on the hormone-induced poly(a) lengthening of -casein mrna (Fig. 6C). In addition, PD98059, which inhibits insulin-stimulated protein synthesis but not PHAS-I phosphorylation, was tested. None of these inhibitors affected poly(a) length under the four hormonal conditions tested. In particular, the poly(a) lengthening produced only in the presence of insulin and prolactin was not prevented (lanes 4, 8, 12, and 16). The magnitude of poly(a) lengthening can be estimated from electrophoretic mobilities. The -casein mrna fragment with no poly(a) (Fig. 6A, lanes 12 15) migrated at approximately 665 nt, similar to the 657 nt calculated from sequence information (34). The mrna fragment from cells treated with no hormone, insulin alone, or prolactin alone (lanes 5, 6, and 7, respectively) was about 690 nt, whereas that from cells treated with insulin plus prolactin (lane 8) was approximately 869 nt. The kinetics of poly(a) lengthening of -casein mrna after the addition of insulin and prolactin were investigated. As shown in Fig. 7, the poly(a) tract progressively increased from about 20 to approximately 200 nt over 30 min. The 3 -Untranslated Region (UTR) of -Casein mrna Is Sufficient for Poly(A) Elongation and Translational Enhancement in the Presence of Insulin and Prolactin Studies in other systems have identified cytoplasmic poly(a) elongation elements (CPEs) in the 3 -UTRs of mrnas that undergo regulated cytoplasmic polyadenylation (reviewed in Refs. 35 and 36). We therefore tested whether the 3 -UTR of -casein mrna could confer hormone-stimulated polyadenylation to a reporter mrna. A vector containing the coding region for puromycin acetyltransferase (PAC) followed by the 3 -UTR of -casein mrna, termed ppur-3, was used to transfect proliferating CID 9 cells. The cells were seeded 1 d later on Matrigel for differentiation. On d 3, the cells were deprived of hormones for 14 h, after which various combinations of hormones were restored for 30 min. Fig. 7. Time Course of Poly(A) Lengthening for -Casein mrna after Exposure to Hormones CID 9 cells were allowed to differentiate with insulin plus prolactin and then deprived of hormones overnight, after which insulin plus prolactin was restored as in Fig. 3. Cells were lysed at the indicated times after hormone addition. The 3 -terminal fragment of -casein mrna was detected by northern blotting after digestion with RNase H as described in Materials and Methods. To detect polyadenylation changes in the exogenous PAC mrna, RNase H was again employed, but this time with an oligonucleotide complementary to nt , immediately upstream of the -casein 3 - UTR. Northern blotting with a -casein mrna probe detected the cleaved 3 -UTR (Fig. 8A). A reduction in mobility occurred in the presence of both insulin and prolactin (lane 4) but not with the other hormonal treatments (lanes 1 3). The mobility shift was prevented by pretreatment of the cells with 200 g/ml cordycepin (lane 5). Controls with oligo(dt) in combination with the PAC-specific oligonucleotide indicated that the change in mobility was due to polyadenylation (lanes 6 9). PAC activity was measured in CID 9 cell extracts (Fig. 8B). In cells transfected with ppur-3, insulin alone produced a small but significant increase in PAC activity compared with no hormone (1.25-fold; shaded vs. open bars), whereas prolactin alone produced no change (cross-hatched vs. open bars). However, insulin plus prolactin caused a 2-fold increase in PAC activity (striped vs. open bars). In cells transfected with the empty vector ppur, insulin alone increased PAC activity, but there was no further increase with insulin plus prolactin. -Casein mrna contains two sequences that resemble previously described mouse CPEs, at nt and , as well as the AAUAAA hexanucleotide sequence at nt (37). We wished to determine whether mutation of one or both of these putative CPE sequences resulted in loss of hormone-stimulated translation. The sequence of ppur-3 was modified to generate an RNA in which the -casein 3 -UTR at nt

9 1678 Mol Endocrinol, July 2004, 18(7): Choi et al. Translational Control of Casein Synthesis Insulin Plus Prolactin Increases PHAS-I Phosphorylation, but This Is Not Responsible for the Synergistic Stimulation of Milk Protein Synthesis When CID 9 cells are differentiated in the presence of insulin alone for 5 d and then prolactin is added to the medium, phosphorylation of PHAS-I increases (10). This was offered as a possible mechanism for regulation of milk protein synthesis by lactogenic hormones. We therefore investigated whether PHAS-I phosphorylation might be responsible for the synergistic stimulation of translation. Insulin alone caused an increase in the more highly phosphorylated - and -forms of PHAS-I, and insulin plus prolactin converted most of the PHAS-I into the -form (data not shown). However, cordycepin did not affect the higher phosphorylation induced by insulin plus prolactin. Furthermore, agents that prevent PHAS-I phosphorylation did not block synergistic hormone-stimulated translation (Fig. 5) or poly(a) elongation (Fig. 6C). Thus, insulin plus prolactin causes two events: phosphorylation of PHAS-I and mrna-specific poly(a) lengthening, but neither is a prerequisite for the other. Fig. 8. Polyadenylation and Translation of a Reporter mrna Containing the -Casein 3 -UTR Proliferating CID 9 cells were transfected with either ppur, which encodes puromycin acetyltransferase (PAC), with ppur-3, in which the PAC coding sequence is followed by the mouse -casein 3 -UTR, with ppur-3 M1, in which a potential CPE at nt of the -casein 3 -UTR is mutated, or with ppur-3 M2, in which a potential CPE at nt is mutated. Cells were allowed to differentiate for 3 d and deprived of hormones, and the indicated hormones were added back for 30 min as in Fig. 3. A, The -casein 3 -UTR in cells transfected with ppur-3 was detected by northern blotting after digestion with RNase H in the presence of an oligodeoxynucleotide complementary to sequences in PAC mrna immediately upstream of the -casein 3 -UTR (lanes 1 4). For cells analyzed in lane 5, cordycepin (200 g/ml) was added to the medium for 30 min before hormone addition. In lanes 6 9, the RNase H reaction also contained oligo(dt) to remove poly(a). B, Extracts from the cells transfected with the indicated plasmids were assayed for PAC activity. was changed from UUUUAU to UUUGGU (ppur- 3 M1). Similarly, the sequence was changed from UUUUUUUU to UUUGGUUU at nt , resulting in ppur-3 M2. In cells transfected with ppur-3 M1, the increase in PAC activity for insulin plus prolactin was the same as for insulin alone (Fig. 8), indicating that the CPE at nt is responsible. Transfection with ppur-3 M2, on the other hand, gave the same result as transfection with ppur-3, indicating that the putative CPE at nt is not functional. Insulin Plus Prolactin Causes Phosphorylation of CPEB but Does Not Change PAP Activity In Xenopus oocytes, mrna-specific polyadenylation is triggered by the phosphorylation of CPEB (38). We tested for a similar mechanism in CID 9 cells treated with insulin plus prolactin. Cells were prepared as described in Figs. 2 4 and then tested for CPEB phosphorylation after a 30-min exposure to each of the hormone combinations (Fig. 9A). Neither insulin alone nor prolactin alone caused a detectable change in the mobility of CPEB (lanes 2 and 3). However, the combination of the two hormones resulted in a portion of the CPEB shifting into the phosphorylated form (lane 4). A previous study in whole animals documented a correlation between poly(a) length of mouse - and -casein mrnas with the levels of PAP mrna and proposed that the poly(a) length is determined by expression of PAP (7). We therefore tested whether the hormone-induced increase in -casein mrna polyadenylation in CID 9 cells was due to a change in the activity of PAP. Cells were deprived of hormones overnight, and then various combinations of hormones were restored for 30 min. Cytoplasmic extracts were then assayed for PAP activity (Fig. 9B). The endlabeled poly(a) primer was elongated during a 10-min incubation with CID 9 cell extract (lanes 2 5) and elongated even more over 20 min (lane 6). Yeast poly(a) polymerase served as a positive control (lane 7). However, there was no difference in poly(a) polymerase activity among the four hormonal treatments (lanes 2 5).

10 Choi et al. Translational Control of Casein Synthesis Mol Endocrinol, July 2004, 18(7): Fig. 9. Phosphorylation of CPEB and Activity of PAP in Response to Lactogenic Hormones CID 9 cells were allowed to differentiate and deprived of hormones, and the indicated hormones were restored as in Fig. 2. A, Cell lysates were immunoprecipitated with antibodies against CPEB and subjected to SDS-PAGE on 8% gels followed by Western blotting to detect CPEB phosphorylation. B, Elongation of 32 P-labeled poly(a) was detected after incubation with either no extract (lane 1) or with cytoplasmic extracts of CID 9 cells for 10 min (lanes 2 5) or 20 min (lane 6). In lane 7, yeast PAP was substituted for CID 9 extract. The reaction products were subjected to TBE-PAGE on a 6% gel containing 7 M urea followed by autoradiography. DISCUSSION The mechanisms and signaling pathways by which insulin stimulates protein synthesis have been extensively studied in fibroblasts, myeloid precursors, and Chinese hamster ovary cells (reviewed in Refs. 11 and 39). Insulin regulates the initiation phase of protein synthesis by increasing the activity of eif2b, the guanine nucleotide-exchange factor that is necessary for recycling eif2. Activation of eif2b occurs via GSK-3 through both PKB- and protein kinase C- -mediated routes, both of which are downstream of phosphatidylinositol-dependent kinase-1 and PI3K (39, 40). Insulin also decreases eif2 phosphorylation in some cells (e.g. Ref. 41), which prevents its sequestration in an inactive complex with eif2b. Another mechanism for insulin-stimulated initiation is via recruitment of mrna to the 43S initiation complex. This involves phosphorylation of p70 S6K and PHAS-I, both of which are downstream of mtor, PKB, phosphatidylinositol-dependent kinase-1, and PI3K. Insulin-stimulated initiation of protein synthesis also requires MAPK (23, 24), possibly via Src homology domain protein tyrosine phosphatase 2 (SH-PTP2) (42) or p90 S6K, the latter of which in turn regulates GSK-3 (43). Rapamycin blocks the insulin-stimulated pathway to PHAS-I phosphorylation but not the MAPK pathway (44, 45). The elongation phase of protein synthesis is also stimulated by insulin via two mechanisms: phosphorylation of eef1 and inhibition of phosphorylation of eef2 (46). The present study shows that protein synthesis in CID 9 cells is stimulated by insulin, and the signaling pathways involve mtor, PI3K, and MAPK, based on the ability of rapamycin, LY294002, and PD98059, respectively, to inhibit insulin-stimulated translation (Fig. 5). Thus, many of the same pathways identified in other cells operate in CID 9 cells. To our knowledge, the effect of prolactin on the translational machinery, on the other hand, has been the subject of only one prior investigation (10). When CID 9 cells were differentiated in the presence of insulin alone for 5 d, and then prolactin was added to the medium, the most highly phosphorylated -form of PHAS-I was enriched compared with the less phosphorylated - and -forms. Accumulation of the -form was abolished by mtor and PI3K inhibitors but not MAPK inhibitors. Furthermore, addition of prolactin increased the rate of protein synthesis approximately 3-fold over a period of 24 h. This correlation suggested that selective translation of milk protein mrnas may proceed via PHAS-I phosphorylation and the concomitant release of eif4e. The present study indicates that even though prolactin stimulates the phosphorylation of PHAS-I in the presence of insulin, this is not responsible for the synergistic stimulation of protein synthesis. Inhibitors of mtor and PI3K, which prevent PHAS-I phosphorylation in CID 9 cells (10), also inhibit the stimulation of protein synthesis by insulin alone or insulin plus prolactin, but they do not eliminate the differential translational stimulation (Fig. 5) or poly(a) elongation (Fig. 6C) conferred by prolactin. On the other hand, cordycepin has no effect on PHAS-I phosphorylation in response to insulin or insulin plus prolactin (data not shown) but completely inhibits

11 1680 Mol Endocrinol, July 2004, 18(7): Choi et al. Translational Control of Casein Synthesis the synergistic stimulation of total protein and -casein synthesis (Fig. 5, D and H). Thus, the phosphorylation of PHAS-I is likely to contribute to insulinstimulated translation (47, 48) but not synergistic hormone-stimulated translation. Rather than PHAS-I, the increase in rate of -casein mrna initiation is due to poly(a) lengthening because 1) cordycepin prevents the synergistic stimulation of translation but not the stimulation caused by insulin alone (Fig. 5, D and H); 2) the poly(a) length of casein, but not GAPDH, mrna increases with prolactin plus insulin but not with either hormone alone (Fig. 6); and 3) the dose response of cordycepin is the same for inhibition of synergistic translation and cytoplasmic polyadenylation of -casein mrna (Figs. 5 and 6). Poly(A) lengthening increases the rate of translational initiation due to the binding of poly(a)-binding protein (PABP) to a specific site near the N terminus of eif4g (49, 50). Poly(A) stabilizes the PABP-eIF4GeIF4E complex, in effect causing the circularization of mrnas by association of these proteins with the 5 cap and 3 -poly(a) tract, which in turn leads to mrna stabilization and enhanced translational reinitiation (51, 52). The translational stimulation conferred by the cap acts synergistically rather than additively with that conferred by the poly(a) tract (53, 54). In our system, we envision that the combination of insulin and prolactin causes two events that increase the rate of initiation: the release of eif4e by phosphorylation of PHAS-I and the lengthening of the poly(a) of selected mrnas. The shift of -casein mrna to higher polysomes after insulin addition (Fig. 4) confirms that the stimulation is primarily through initiation rather than elongation. The observation that polysome size is further increased for -casein, but not GAPDH, mrna in the presence of both hormones is consistent with the stimulatory effect of poly(a) lengthening on initiation rate (53, 54). Meiotic maturation of stage VI Xenopus oocytes provides the best studied precedent for hormoneactivated mrna-specific increases in poly(a) length leading to a stimulation of translational initiation (reviewed in Ref. 35). During vertebrate oogenesis, gene expression is governed primarily by translational control (55, 56). The developing oocyte accumulates maternal components, including mrnas, proteins, and ribosomes, that will be required during the rapid development that follows fertilization (57). The stored maternal mrnas encode cell cycle-regulatory proteins such as c-mos, Cdk2, and cyclins A, B1, and B2, which play a role in early embryonic development (38, 58, 59). These translationally dormant mrnas reside in stable ribonucleoprotein complexes and contain short 3 -poly(a) tracts (60, 61). Upon progesterone stimulation, some of the stored maternal mrnas are recruited to the protein synthetic machinery whereas many of the housekeeping mrnas cease to be translated (62, 63). Progesterone initiates a series of events that result in the cytoplasmic addition of A residues to these mrnas (64). The activated aurora kinase Eg2 phosphorylates CPEB at Ser-174 (65, 66). Phosphorylated CPEB recruits the cleavage and polyadenylation specificity factor (CPSF) as well as PAP and PABP, which are necessary for polyadenylation (67). Cytoplasmic polyadenylation requires two cisacting elements in the 3 -UTR: the CPE, which is bound by CPEB, and the hexanucleotide AAUAAA (35). Translation is repressed by binding of eif4e to maskin, which interacts with nonphosphorylated CPEB (68, 67). Phosphorylation of CPEB also leads to dissociation of maskin from eif4e, a step that is prevented by cordycepin (69). This is followed by the binding of PABP to the newly elongated poly(a) tract, the interaction of PABP with eif4g, and the replacement of the inhibitory maskin-eif4e complex with the active eif4e-eif4g complex that promotes capdependent translation. Similar mechanisms for regulated polyadenylation appear to operate in the embryonic development of other organisms. In the surf clam Spisula solidissima, three abundant mrnas encoding cyclin A, cyclin B, and ribonucleotide reductase are held in an inactive or masked state in the oocyte (70). The masked mrnas are loaded onto polysomes after fertilization at a time when translationally regulated mrnas undergo poly(a) extension (71, 72). These translationally up-regulated mrnas contain several copies of CPE-like U 4 6 AA/U motifs in their 3 -UTRs (73). Deletion of the CPE of the ribonucleotide reductase mrna prevents the binding of p82, the functional homolog of Xenopus CPEB (74). In Drosophila embryos, regulated polyadenylation is essential for correct embryonic patterning. The mrnas for bicoid, Toll, and torso, which play important roles in body patterning, undergo poly(a) elongation and translational activation (75). Toll mrna has a 192-nt element in the 3 -UTR that is necessary, but not sufficient, for polyadenylation (76). Cytoplasmic polyadenylation also takes place in maturing mouse oocytes. Several dormant maternal mrnas possess short poly(a) tracts, but they are elongated to nt after the induction of oocyte maturation, after which translation ensues (77). Mouse cyclin B1 mrna contains functional CPEs that direct polyadenylation-stimulated translation in maturing oocytes. Mouse oocytes have factors homologous to those involved in Xenopus oocyte maturation, including CPEB, CPSF, maskin, and IAK1/ Eg2 (78). The activities of both the aurora kinase and protein phosphatase PP1 are tightly regulated during prophase I progression. Mouse CPEB is dephosphorylated by PP1 at embryonic stage E18.5, thereby rendering it, and CPE-mediated mrna translation, inactive until oocyte maturation (79). Active aurora kinase phosphorylates CPEB on Thr-171 (78). Although cytoplasmic polyadenylation is a hallmark of early animal development, evidence for this mechanism for modulation of gene expression in late development or in adult tissues is much less