Regulation of Expression of the Chicken Ovalbumin Gene: Interactions between Steroid Hormones and Second Messenger Systems
Emmanouil
Skoufos
and
Michel
M. Sanders
Department of Biochemistry University of Minnesota Minneapolis, Minnesota 55455
The chicken ovalbumin gene is subject to multihormonal regulation. Maximal expression of it requires not only the synergistic effects of estrogen and corticosterone, but also the permissive effects of insulin. In addition to effects on transcription, the stability of its message is greatly enhanced by estrogen. Furthermore, two signal transduction pathways involving protein kinases have been implicated in the regulation of the ovalbumin gene. To better define the role of second messengers on expression of the ovalbumin gene, the effects of the protein kinase-C (PKC) and the CAMP-dependent protein kinase (PKA) pathways on the endogenous levels of ovalbumin mRNA and the transcription of an ovalbumin fusion gene were investigated. Primary cultures of oviduct cells were treated with phorbol 12myristilate 13-acetate (an activator of PKC) or with forskolin and 3-isobutyl-1-methylxanthine (an activator of PKA) alone, activators plus estrogen and corticosterone, or activators plus both steroids and insulin. The results indicate that phorbol 12-myristilate 13-acetate causes a dramatic destabilization of ovalbumin message, resulting in a reduction in ovalbumin mRNA levels. In contrast, the activators of the PKA system can substitute for insulin and, thereby, increase expression of the ovalbumin gene synergistically with the steroids. The effect of the activators of the PKA system is at the level of transcription. Thus, in chicken oviduct cell cultures, the PKA and PKC signal transduction pathways act in opposing ways to modulate the steroid-induced expression of the ovalbumin gene. (Molecular Endocrinology 6: 1412-1417,1992)
and differentiation by steroid hormones (for a review, see Ref.1). Estrogen promotes differentiation of the tubular gland cells and induces expression of the genes coding for the major egg-white proteins ovalbumin, lysozyme, ovomucoid, and transferrin (1). After primary exposure to estrogen, secondary exposure to three other classes of steroids androgens, glucocorticoids, and progestins results in induction of the ovalbumin gene (Ref. 1 and references therein). In primary chicken oviduct cultures all four classes of steroids induce the gene; however, synergistic effects occur when they are administered together (2). Cell and tissue culture experiments showed that in addition to estrogen, corticosterone and insulin are required for maximal expression of the ovalbumin gene (l-3). In addition, estrogen causes a lo-fold increase in the stability of ovalbumin mRNA, increasing its half-life from 2-3 h in untreated chicks to 24 h in estrogen-treated chicks (4, 5). The transcription of the ovalbumin gene is controlled by a complex array of 5’-flanking elements, including binding sites for the COUP transcription factor as well as the CCAAT and TATA box transcription factors (6). A steroid-dependent regulatory element (SDRE) is necessary to confer responsiveness to steroids, and a negative regulatory element down-regulates the basal levels of expression of the gene (2, 7). Although the SDRE is required for induction by both steroids, it does not contain any canonical steroid receptor-binding sites, nor it does appear to bind purified receptors (7). In addition, protein synthesis is required for induction of the gene-by steroids (8). This suggests that activation of the ovalbumin gene by the hormones is an indirect effect that involves prior induction of a steroid-responsive protein which, in turn, binds to the SDRE and induces the ovalbumin gene. Protein phosphorylation may also be involved in the induction of the ovalbumin gene. Two pathways that result in phosphorylation of proteins, the protein kinaseC (PKC) and the CAMP-dependent protein kinase (PKA) pathways, have been implicated in modulating the transcription of the ovalbumin gene. Evans and McKnight
INTRODUCTION For almost 3 decades the chicken oviduct has served as model for studying the regulation of gene expression O&38-8809/92/1412-14,7$03.00/0 Molecular Endocrmology CopyrIght 0 1992 by The Endocrine
Society
1412
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Regulation
of Ovalbumin
Gene
by Second
(3) demonstrated, using an oviduct explant tissue culture system, that forskolin, an activator of adenylate cyclase, can substitute for insulin in inducing the ovalbumin gene. A recent study by Gaub et al. (9) reported that phorbol 12-myristilate 13-acetate (PMA) can increase the expression of a reporter gene controlled by 58 basepairs of the basal ovalbumin promoter when it is transfected into chicken embryonic fibroblast cells overexpressing the estrogen receptor. To determine whether one or both second messenger pathways modulate the expression of the endogenous ovalbumin gene by steroids in viva, the effects of PMA, forskolin, and 3-isobutyl-1 -methylxanthine (IBMX), an inhibitor of CAMP phosphodiesterase, on the levels of endogenous ovalbumin mRNA and the transcription of an ovalbumin fusion gene in a chicken oviduct cell culture system were examined. The results indicate that activation of the PKC system destabilizes ovalbumin mRNA, while activation of the PKA system results in an increase in the steroid-induced transcription of the ovalbumin gene.
RESULTS Interactions
AND DISCUSSION between
Steroids
1413
Messengers
and PMA
To determine whether agents such as PMA that activate the PKC pathway regulate the endogenous ovalbumin gene, primary oviduct cells were cultured with PMA (Fig. 1). Treatment with insulin alone did not significantly elevate the levels of ovalbumin mRNA. The addition of estrogen and corticosterone caused a IOto 12-fold induction of ovalbumin mRNA compared to insulin alone. As previously described (2), addition of insulin further potentiated the effects of the steroids by a factor of 2. Although PMA by itself enhanced the amount of ovalbumin mRNA over that observed with insulin alone, addition of 500 nM PMA to the steroids in the medium abolished the steroid-dependent increase in ovalbumin mRNA. Addition of insulin or removal of PMA from the medium (data not shown) did not reverse this inhibition. This demonstrates that the PKC pathway inhibits the steroid-mediated induction of the ovalbumin gene and drastically represses the level of its mRNA. To ensure that the reduction in the levels of the endogenous ovalbumin mRNA by PMA was not due to toxicity, the effects of varying the concentration and the duration of exposure of cells to PMA (Fig. 2) were examined. The dose-response curve for PMA is bellshaped, with maximal inhibition occurring at 500 nM. Concentrations as high as 1 PM and as low as 50 nM were effective. High concentrations of PMA can desensitize the PKC pathway, accounting for the reduced inhibition by 1 PM PMA (10, 11). Minimal differences in the effects of PMA between a 24- or a 48-h incubation were observed. Furthermore, PMA did not kill the cells, as the amounts of total nucleic acid recovered per dish were unaffected by PMA (data not shown). These results indicate that inhibition of the steroid-dependent
1000
500
0
I
S
S+I
P
S+P
S+P+I
Treatment Fig. 1. PMA, an Activator of the PKC Pathway, Inhibits the Induction of Ovalbumin mRNA by Steroids Oviduct cells were cultured in the presence of 50 rig/ml insulin (I), lo-’ M estrogen and 10e6 M corticosterone (S), insulin and steroids (S+I), 500 nM PMA (P), steroids and PMA (S+P), or steroids, insulin, and PMA (S+I+P) for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per fig total nucleic acid (tNA) extracted. Each bar represents the mean f SEM of the results from two experiments in duplicate. Additional experiments confirmed these results.
induction of the ovalbumin gene by PMA is not an artifact due to toxic effects of PMA. To ascertain whether the PKC pathway was affecting the actions of estrogen or corticosterone, oviduct ceils were cultured with PMA plus estrogen or PMA plus corticosterone (Fig. 3). As with both steroids combined (Fig. l), PMA negated the induction of the ovalbumin gene by either estrogen or corticosterone. Thus, it seems that activation of the PKC pathway can independently inhibit the increase in ovalbumin mRNA levels caused by both estrogen and corticosterone. To determine whether activation of the PKC pathway decreases the levels of ovalbumin mRNA by repressing the transcription of the gene or by destabilizing its mRNA, the effects of PMA on the expression of a chimeric plasmid were examined (Fig. 4). Primary oviduct cells were transiently transected with the pOvCAT,900 plasmid that contains the sequences between -900 and +9 from the ovalbumin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The transfected cells were cultured with the steroids alone, steroids plus insulin, or steroids plus PMA for 48 h. The addition of insulin resulted in a 2fold increase in the expression of the reporter gene, as previously reported (2). Surprisingly, the addition of PMA to the steroid-containing medium also resulted in
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MOL 1414
ENDO.
1992
Vol6
No. 9
4
e?-
80
3
.-6 z .c +
8
60
A .= .->
: z d
2
z u
40
2 0
1
20 0
200
400
600
PMA
800
1000
(nh4)
2. The Effects of Concentration and Length of Exposure of PMA on the Levels of Ovalbumin mRNA Oviduct cells were cultured in the presence of estrogen and corticosterone with the indicated concentrations of PMA for 24 (0) or 48 (0) h. Samples were assayed for ovalbumin mRNA, as described in Materials and Methods. The results are expressed as percent inhibition (+ SEM for two experiments in duplicate for each ooint) of the induction of the ovalbumin mRNA by the steroids. Fig.
0 S+P
Treatment Fig. 4. PMA Causes an Increase in the Expression of a Reporter Gene Controlled by the Ovalbumin Promoter Primary oviduct cells were transiently transfected with the pOvCAT-,900 plasmid, which contains the region of the ovalbumin gene between -900 and +9 fused to the CAT gene. The transfected cells were cultured in the presence of estrogen and corticosterone (S), insulin and steroids (S+I), or steroids and 500 nM PMA (S+P) for 48 h. Total protein was extracted and assayed for CAT activity. The bars represent the mean f SEM of two experiments, performed in triplicate.
3OOd
LJ n
-P
0
+P
200 -
loo-
04 C
E
Hormones Fig. 3. PMA Independently Interacts with Both Steroids to Repress the Endogenous Levels of Ovalbumin mRNA Oviduct cells were cultured in the presence of estrogen (E) or corticosterone (C) with 500 nM PMA (Cl) or without PMA (m) for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per pg total nucleic acid (tNA) extracted. Each bar represents the mean + SEM of the results from two experiments, performed in triplicate.
an increase in CAT activity comparable to that achieved with insulin and steroids. The same result was observed when primary oviduct cells were transfected with a plasmid that contained 8 kilobases of the 5’-sequence of the ovalbumin gene linked to the CAT reporter gene (data not shown). This indicates that activation of the PKC pathway may cause a modest 2-fold increase in the expression of the ovalbumin gene. Thus, since the net result of activation of the PKC pathway is a dramatic repression of the steady state levels of ovalbumin mRNA, presumably the major effect of PMA is posttranscriptional. To determine whether PMA might be destabilizing the ovalbumin mRNA, a time-course experiment was performed (Fig. 5). Exposure of cells to PMA for a period as short as 2 h resulted in a 35% decrease in the levels of the mRNA, which is consistent with destabilization of the message. The half-life of ovalbumin mRNA after the addition of PMA was estimated to be 2.75 h (? 0.25 h). Since the half-life of ovalbumin mRNA decreases by lo-fold (from 24 to -2.4 h) upon withdrawal of estrogen (4, 5), these results suggest that PMA is negating the capacity of estrogen to stabilize ovalbumin mRNA. Thus, the drastic reduction in the levels of endogenous ovalbumin mRNA caused by activation of the PKC pathway is due to rapid destabili-
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Regulation of Ovalbumin Gene by Second Messengers
Ttme In culture
(hrs)
Fig. 5. PMA Rapidly Represses the Amounts of Ovalbumin mRNA Oviduct cells were cultured in the presence of insulin, estrogen, and corticosterone (W) for 24 h. At that time, the medium was discarded, and new medium containing only insulin and steroids (S+I; 0) or insulin, steroids, and 500 nM PMA (S+I+P; 0) was added. The cells were cultured for the additional times indicated. Samples were assayed for ovalbumin mRNA, as described in Materials and Methods. Each point represents the mean + SEM from a typical experiment, performed in triplicate.
zation of the ovalbumin mRNA, rather than diminished transcription. Gaub et a/. (9) reported that activation of PKC by 200 nM PMA causes induction of transcription from the basal (-58 to 1) ovalbumin promoter in chicken embryonic fibroblasts overexpressing the estrogen receptor. Our transfection results also indicate that PMA can increase the transcription of an ovalbumin fusion gene (Fig. 4). However, the major effect of activation of the PKC pathway is a rapid repression of the levels of endogenous ovalbumin mRNA due to message destabilization. This effect masks any direct increase in transcription of the gene in normal oviduct cells. In addition to chicken ovalbumin mRNA, activation of the PKC pathway has recently been implicated in alteration of the stability of several mRNAs. Phorbol esters increase the stability of interleukin-2 mRNA in human lymphocytes (12) and colony-stimulating factor mRNA in human fibroblasts (13). However, activation of the PKC pathway by phorbol esters results in destabilization of the actin, troponin, and myosin mRNAs in chicken myoblasts (14) as well as estrogen receptor mRNA in MCF-7 cells (15). Thus, a number of genes are regulated, both positively and negatively, at the level of mRNA stability by the PKC pathway. This introduces a third level of regulation by this very diverse signal transduction pathway in addition to regulation through direct substrate phosphorylation and control of transcription. Interactions
among
Steroids,
Insulin,
and CAMP
To determine whether activators of the PKA pathway affect the levels of ovalbumin mRNA, primary oviduct cells were treated with forskolin or IBMX. As shown in
1415
Fig. 6, treatment of cultured oviduct cells with forskolin or IBMX alone for 48 h did not cause a marked change in the basal levels of ovalbumin mRNA. However, the addition of either forskolin or IBMX to a steroid-containing medium potentiated the induction of the ovalbumin gene by steroids in a manner similar to that of insulin. The addition of forskolin or IBMX to culture medium containing both insulin and the steroids did not further enhance the induction of ovalbumin mRNA produced by the cooperation of steroids and insulin. These results show that activators of PKA synergize with the steroids and substitute for insulin in isolated cells, which is in agreement with observations in oviduct explant cultures (3). In addition, the effects of the activators and insulin are not additive, suggesting that insulin and CAMP use convergent pathways to modulate the expression of the ovalbumin gene. To investigate the independent interactions of the PKA pathway with each of the steroids, oviduct cells were cultured with forskolin and estrogen or with forskolin and corticosterone (Fig. 7). The results suggest that activation of the CAMP pathway synergizes independently with each of the steroids to increase the levels of ovalbumin mRNA. This ability of the PKA pathway is very similar to that of insulin, which also independently synergizes with each of the steroids to increase the levels of ovalbumin mRNA (2). To determine whether the increase in the levels of ovalbumin mRNA by activation of the PKA pathway is a result of enhanced transcription, the effects of forskolin on the expression of pOvCAT-.900 were examined (Fig. 8). Treatment with forskolin causes an increase in
200
Treatment Fig. 6. Agents That Activate the PKA Pathway Synergize with the Steroids to Potentiate the Production of Ovalbumin mRNA Oviduct cells were cultured for 48 h in the presence of insulin (I), estrogen and corticosterone (S), insulin and steroids (S+I), 33 PMforskolin (F), steroids and forskolin (S+F), steroids, insulin, and forskolin (S+I+F), 125 PMIBMX (X), steroids and IBMX (S+X), or steroids, insulin, and IBMX (S+I+X). The bars represent mean relative amounts of ovalbumin mRNA obtained from three different experiments with duplicate samples + SEM, using the amounts of ovalbumin mRNA induced by steroids and insulin as 100%.
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MOL 1416
END0.1992
Vol6
No. 9
1000
T
I-n
-I=
600
S
S+I
S+F
Treatment Hormones Fig. 7. Forskolin Interacts with Both Steroids Independently to Induce the Ovalbumin Gene Oviduct cells were cultured with estrogen (E) or corticosterone (C) with (8) or without (m) the addition of 33 FM forskolin for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per pg total nucleic acid (tNA) extracted. Each bar represents the mean + SEM of the results from two experiments, performed in triplicate.
the expression of this chimeric plasmid, resulting in an increase in CAT activity. This suggests that the increase in the endogenous steady state levels of ovalbumin
mRNA caused by activation of the PKA pathway is solely due to modulation of transcription, because CAT activity and ovalbumin mRNA levels are increased to the same extent. This modulation of the expression of the ovalbumin gene by interactions between the insulin or PKA pathways and steroid-mediated events may occur at the level of transcription factors. The activities of several transcription factors can be modulated by phosphorylation (16-l 8). Similarly, insulin or the PKA pathway may phosphorylate a transcription factor and, thus, increase its DNA binding or transactivation activities. This transcription factor may be 1) the one induced by the steroid hormones, which binds to the SDRE, 2) one that interacts with the steroid-responsive protein, or 3) a factor completely independent of the steroid-responsive protein. Studies are under way to distinguish among these possibilities. Considerable evidence suggests that steroid hormones increase transcription of the ovalbumin gene through the binding of proteins to the SDRE (2, 7). Insulin enhances that activity through a phosphorylation
Fig. 8. Activation of the CAMP Pathway Causes an Increase in the Expression of a Reporter Gene Controlled by the Ovalbumin Promoter Primary oviduct cells were transiently transfected with the pOvCAT-,900 plasmid, which contains the region of the ovalbumin gene between -900 and +9 fused to the CAT gene. The transfected cells were cultured with estrogen and corticosterone (S), insulin and steroids (S+I), or steroids and 33 ELM forskolin (S+F) for 48 h. Total protein was extracted and assayed for CAT activity. The bars represent the mean + SEM of two experiments, performed in triplicate.
event that may or may not involve these proteins. Because activators of the PKA pathway can mimic the effects of insulin, they are presumably affecting the same events enhanced by insulin. In contrast, although PMA can increase transcription to the extent achieved by insulin and the activators of the PKA system, the principal action of the PKC system is to destabilize the ovalbumin mRNA. Because the stability of the ovalbumin mRNA decreases to the half-life seen in the absence of steroids, the most likely scenario is that the PKC pathway negates the effects of steroids on ovalbumin mRNA stability. Analysis of the effects of PMA on mRNA stability may thus provide a tool for determining how estrogen enhances that stability.
MATERIALS AND METHODS Animals
and Cell Culture
Immature White Leghorn chicks were implanted with diethylstilbesterol pellets, which were then withdrawn, and restimulated as previously described (19). Pellets were withdrawn 48 h before the tissue was harvested. The magnum portion of the oviduct was removed, minced with scissors, and incubated for 1 h in dissociation medium (Ham’s F-12 nutrient medium; Gibco-BRL, Gaithersburg, MD) supplemented with 1.7 mg/ml collagenase, 20 pg/ml trypsin, 25 pg/ml DNase, and 0.192 U/
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Regulation
of Ovalbumin
Gene
by Second
Messengers
1417
ml protease). The supernatant was removed, and single cells were harvested by centrifugation, washed twice in F-12 medium, and resuspended in culture medium [F-l 2 mediumDulbecco’s Modified Eagle’s medium (Gibco-BRL; 1 :l , vol/vol) supplemented with 0.1% (wt/vol) BSA, 5 U/ml penicillin (GibcoBRL), 5 fig/ml streptomysin (Gibco-BRL), and 200 pg/ml fungizone (Gibco-BRL)]. The cells were plated in culture medium supplemented with 50-1000 nM PMA as indicated, 33 PM forskolin, or 125 FM IBMX in addition to 1 O-’ M 17P-estradiol, 1O-6 M corticosterone, or 50 ng,/ml insulin. All hormones and reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. RNA
Isolation
and
cRNA
Hybridization
Ovalbumin cDNA sequences from 82-1347 (20) were subcloned in both orientations into pTZ18R and transcribed with T7 RNA oolvmerase. The cRNA was labeled with f?SlUTP and became ihe probe, while the mRNA was used to construct a standard curve (10-5000 ng). Total cellular nucleic acid was isolated, as previously described (19) and hybridized for at least 16 h with the radiolabeled cRNA probe at 80 C in 0.6 M NaCI, 20 mM Tris-HCI (pH 7.5) 5 mM EDTA, 2.5% (vol/vol) ethanol, 75 pg/ml salmon testis DNA, and 0.1% (wt/vol) sodium dodecyl sulfate (21). The samples were digested with a ribonuclease (RNase) solution [25 rig/ml RNase-A, 250 U/ml RNase-T,, 0.3 M NaCI, 10 mM Tris-HCI (pH 7.5) 2 mM EDTA, and 75 pg/ml salmon testis DNA), precipitated with 10% (wt/ vol) trichloroacetic acid, and quantified by liquid scintillation counting (7). Transfection
of Oviduct
Cells
and
CAT
Assays
Tubular oviduct cells were isolated and cultured as described above. They were transfected with pOVCAT-,900 (7) using CaP04 coprecipitation, as described previously (2). All cells transfected with a single DNA were pooled before aliquoting them into medium to rule out variations in transfection efficiency. CAT assays were used to measure the expression of the transfected plasmids and were performed as previously described (2).
Acknowledgments We want to thank Sasa Dragas-Graonic, and Natalie Hayes for their technical
Christopher assistance.
Carlson,
Received December 20, 1991. Revision received May 25, 1992. Accepted June 30,1992. Address requests for reprints to: Dr. Michel Sanders, Department of Biochemistry, 4-225 Millard Hall, University of Minnesota, Minneapolis, Minnesota 55455. This work was supported by American Cancer Society Grant BE-68 (to M.M.S.).
REFERENCES 1. Sanders MM, McKnight GS 1986 Egg white genes: hormonal regulation in homologous and heterologous cells. In: Malacinski GM (ed) Molecular Genetics of Mammalian Cells. Macmillan Press, New York, pp 183-216 2. Sanders MM. McKniaht GS 1988 Positive and neaative regulatory elements control the steroid-responsive”ovalbumin promoter. Biochemistry 27:6550-6557
3. Evans ME, McKnight GS 1984 Regulation of the ovalbumin gene: effects of insulin, adenosine 3’, 5’-monophosphate, and estrogen. Endocrinology 115:368-377 4. Palmiter RD 1973 Rate of ovalbumin messenger ribonucleic acid synthesis in the oviduct of estrogen-primed chicks. J Biol Chem 248:8260-8270 5. Palmiter RD, Carey NH 1974 Rapid inactivation of ovalbumin messenqer ribonucleic acid after acute withdrawal of estrogen. Proc Natl Acad Sci USA 71:2357-2361 6. Saaami I. Tsai S. Wana H. Tsai M-J. 0’ Mallev BW 1986 Identification of- two f&tom required for transcription of the ovalbumin gene. Mol Cell Biol 6:4259-4267 7. Schweers LA, Frank DE, Weigel NL, Sanders MM 1990 The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element. J Biol Chem 265:7590-7595 8. McKnight GS 1978 The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in oviduct explant cultures. Cell 14:403-413 9. Gaub M-P, Bellard M, Scheuer I, Chambon P, SassoneCorsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves the FosJun complex. Cell 63:1267-l 276 10. Hoefler J, Deutch P, Lin J, Habener J 1990 Distinctive adenosine 3’,5’ monophosphate and phorbol ester responsive signal transduction pathways converge at the level of transcriptional activation by the interaction of DNA binding proteins. Mol Endocrinol 3:868-880 11. Moore C, Brentano S, Miller W 1990 Human P45Oscc gene transcription is induced by cyclic AMP and repressed by 12-Ctetradecanoylphorbol-13-acetate and A231 87 through independent cis elements. Mol Cell Biol lo:601 36023 12. Ohmura T, Onoue K 1990 Stability of IL-2 mRNA in Tlymphocytes is controlled by a protein kinase C-regulated mechanism. Int lmmunol2:1073-1079 13. Akashi M, Saito M, Koeffler HP 1989 Lymphotoxin stimulation and regulation of colony-stimulating factors in fibroblasts. Blood 74:2383-2390 14. Zhu Y-Y, Schwartz RJ, Crow MT 1991 Phorbol esters selectively deregulate contractile protein gene expression in terminately differentiated myotubes through trancriptional repression and message destabilization. J Cell Biol 1151745-754 15. Saceda M, Knabbe C, Dickson RB, Lippman ME, Bronzert D, Lindsey RK, Gottardis MM, Martin MB 1991 Posttranslational destabilization of estrogen receptor mRNA in MCF-7 cells by 12-0-tetradecanoylphorbol-13-acetate. J Biol Chem 266:17809-l 7814 16. Merino A, Buckbinder L, Mermelstein FH, Reinberg D 1989 Phosphorylation of cellular proteins regulates their binding to the CAMP response element. J Biol Chem 264:21266-21276 17. Luscher B, Christenson E, Litchfield DW, Krebs EG, Eisenman RN 1990 Myb DNA binding is inhibited by phosphorylation at a site deleted during oncogenic activation. Nature 334:517-522 18. Jackson SP, MacDonald JJ, Lees-Miller S, Tijian R 1990 GC box binding induces phosphorylation of Spl by a DNA-dependent protein kinase. Cell 63:155-l 65 19. Sanders MM, McKnight GS 1985 Chicken egg white genes: multihormonal regulation in a primary cell culture system. Endocrinology 116:398-405 20. Humphries P, Cachet M, Krust A, Gerlinger P, Kourilsky P, Chambon P 1977 Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene. Nucleic Acids Res 4:2389-2406 21. ldzerda RL, Huebers H, Finch CA, McKnight GS 1986 Rat transferrin gene expression: tisssue-specific regulation by iron deficiency. Proc Natl Acad Sci USA 83:3723-3727
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Emmanouil
Skoufos
and
Michel
M. Sanders
Department of Biochemistry University of Minnesota Minneapolis, Minnesota 55455
The chicken ovalbumin gene is subject to multihormonal regulation. Maximal expression of it requires not only the synergistic effects of estrogen and corticosterone, but also the permissive effects of insulin. In addition to effects on transcription, the stability of its message is greatly enhanced by estrogen. Furthermore, two signal transduction pathways involving protein kinases have been implicated in the regulation of the ovalbumin gene. To better define the role of second messengers on expression of the ovalbumin gene, the effects of the protein kinase-C (PKC) and the CAMP-dependent protein kinase (PKA) pathways on the endogenous levels of ovalbumin mRNA and the transcription of an ovalbumin fusion gene were investigated. Primary cultures of oviduct cells were treated with phorbol 12myristilate 13-acetate (an activator of PKC) or with forskolin and 3-isobutyl-1-methylxanthine (an activator of PKA) alone, activators plus estrogen and corticosterone, or activators plus both steroids and insulin. The results indicate that phorbol 12-myristilate 13-acetate causes a dramatic destabilization of ovalbumin message, resulting in a reduction in ovalbumin mRNA levels. In contrast, the activators of the PKA system can substitute for insulin and, thereby, increase expression of the ovalbumin gene synergistically with the steroids. The effect of the activators of the PKA system is at the level of transcription. Thus, in chicken oviduct cell cultures, the PKA and PKC signal transduction pathways act in opposing ways to modulate the steroid-induced expression of the ovalbumin gene. (Molecular Endocrinology 6: 1412-1417,1992)
and differentiation by steroid hormones (for a review, see Ref.1). Estrogen promotes differentiation of the tubular gland cells and induces expression of the genes coding for the major egg-white proteins ovalbumin, lysozyme, ovomucoid, and transferrin (1). After primary exposure to estrogen, secondary exposure to three other classes of steroids androgens, glucocorticoids, and progestins results in induction of the ovalbumin gene (Ref. 1 and references therein). In primary chicken oviduct cultures all four classes of steroids induce the gene; however, synergistic effects occur when they are administered together (2). Cell and tissue culture experiments showed that in addition to estrogen, corticosterone and insulin are required for maximal expression of the ovalbumin gene (l-3). In addition, estrogen causes a lo-fold increase in the stability of ovalbumin mRNA, increasing its half-life from 2-3 h in untreated chicks to 24 h in estrogen-treated chicks (4, 5). The transcription of the ovalbumin gene is controlled by a complex array of 5’-flanking elements, including binding sites for the COUP transcription factor as well as the CCAAT and TATA box transcription factors (6). A steroid-dependent regulatory element (SDRE) is necessary to confer responsiveness to steroids, and a negative regulatory element down-regulates the basal levels of expression of the gene (2, 7). Although the SDRE is required for induction by both steroids, it does not contain any canonical steroid receptor-binding sites, nor it does appear to bind purified receptors (7). In addition, protein synthesis is required for induction of the gene-by steroids (8). This suggests that activation of the ovalbumin gene by the hormones is an indirect effect that involves prior induction of a steroid-responsive protein which, in turn, binds to the SDRE and induces the ovalbumin gene. Protein phosphorylation may also be involved in the induction of the ovalbumin gene. Two pathways that result in phosphorylation of proteins, the protein kinaseC (PKC) and the CAMP-dependent protein kinase (PKA) pathways, have been implicated in modulating the transcription of the ovalbumin gene. Evans and McKnight
INTRODUCTION For almost 3 decades the chicken oviduct has served as model for studying the regulation of gene expression O&38-8809/92/1412-14,7$03.00/0 Molecular Endocrmology CopyrIght 0 1992 by The Endocrine
Society
1412
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 18:41 For personal use only. No other uses without permission. . All rights reserved.
Regulation
of Ovalbumin
Gene
by Second
(3) demonstrated, using an oviduct explant tissue culture system, that forskolin, an activator of adenylate cyclase, can substitute for insulin in inducing the ovalbumin gene. A recent study by Gaub et al. (9) reported that phorbol 12-myristilate 13-acetate (PMA) can increase the expression of a reporter gene controlled by 58 basepairs of the basal ovalbumin promoter when it is transfected into chicken embryonic fibroblast cells overexpressing the estrogen receptor. To determine whether one or both second messenger pathways modulate the expression of the endogenous ovalbumin gene by steroids in viva, the effects of PMA, forskolin, and 3-isobutyl-1 -methylxanthine (IBMX), an inhibitor of CAMP phosphodiesterase, on the levels of endogenous ovalbumin mRNA and the transcription of an ovalbumin fusion gene in a chicken oviduct cell culture system were examined. The results indicate that activation of the PKC system destabilizes ovalbumin mRNA, while activation of the PKA system results in an increase in the steroid-induced transcription of the ovalbumin gene.
RESULTS Interactions
AND DISCUSSION between
Steroids
1413
Messengers
and PMA
To determine whether agents such as PMA that activate the PKC pathway regulate the endogenous ovalbumin gene, primary oviduct cells were cultured with PMA (Fig. 1). Treatment with insulin alone did not significantly elevate the levels of ovalbumin mRNA. The addition of estrogen and corticosterone caused a IOto 12-fold induction of ovalbumin mRNA compared to insulin alone. As previously described (2), addition of insulin further potentiated the effects of the steroids by a factor of 2. Although PMA by itself enhanced the amount of ovalbumin mRNA over that observed with insulin alone, addition of 500 nM PMA to the steroids in the medium abolished the steroid-dependent increase in ovalbumin mRNA. Addition of insulin or removal of PMA from the medium (data not shown) did not reverse this inhibition. This demonstrates that the PKC pathway inhibits the steroid-mediated induction of the ovalbumin gene and drastically represses the level of its mRNA. To ensure that the reduction in the levels of the endogenous ovalbumin mRNA by PMA was not due to toxicity, the effects of varying the concentration and the duration of exposure of cells to PMA (Fig. 2) were examined. The dose-response curve for PMA is bellshaped, with maximal inhibition occurring at 500 nM. Concentrations as high as 1 PM and as low as 50 nM were effective. High concentrations of PMA can desensitize the PKC pathway, accounting for the reduced inhibition by 1 PM PMA (10, 11). Minimal differences in the effects of PMA between a 24- or a 48-h incubation were observed. Furthermore, PMA did not kill the cells, as the amounts of total nucleic acid recovered per dish were unaffected by PMA (data not shown). These results indicate that inhibition of the steroid-dependent
1000
500
0
I
S
S+I
P
S+P
S+P+I
Treatment Fig. 1. PMA, an Activator of the PKC Pathway, Inhibits the Induction of Ovalbumin mRNA by Steroids Oviduct cells were cultured in the presence of 50 rig/ml insulin (I), lo-’ M estrogen and 10e6 M corticosterone (S), insulin and steroids (S+I), 500 nM PMA (P), steroids and PMA (S+P), or steroids, insulin, and PMA (S+I+P) for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per fig total nucleic acid (tNA) extracted. Each bar represents the mean f SEM of the results from two experiments in duplicate. Additional experiments confirmed these results.
induction of the ovalbumin gene by PMA is not an artifact due to toxic effects of PMA. To ascertain whether the PKC pathway was affecting the actions of estrogen or corticosterone, oviduct ceils were cultured with PMA plus estrogen or PMA plus corticosterone (Fig. 3). As with both steroids combined (Fig. l), PMA negated the induction of the ovalbumin gene by either estrogen or corticosterone. Thus, it seems that activation of the PKC pathway can independently inhibit the increase in ovalbumin mRNA levels caused by both estrogen and corticosterone. To determine whether activation of the PKC pathway decreases the levels of ovalbumin mRNA by repressing the transcription of the gene or by destabilizing its mRNA, the effects of PMA on the expression of a chimeric plasmid were examined (Fig. 4). Primary oviduct cells were transiently transected with the pOvCAT,900 plasmid that contains the sequences between -900 and +9 from the ovalbumin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The transfected cells were cultured with the steroids alone, steroids plus insulin, or steroids plus PMA for 48 h. The addition of insulin resulted in a 2fold increase in the expression of the reporter gene, as previously reported (2). Surprisingly, the addition of PMA to the steroid-containing medium also resulted in
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MOL 1414
ENDO.
1992
Vol6
No. 9
4
e?-
80
3
.-6 z .c +
8
60
A .= .->
: z d
2
z u
40
2 0
1
20 0
200
400
600
PMA
800
1000
(nh4)
2. The Effects of Concentration and Length of Exposure of PMA on the Levels of Ovalbumin mRNA Oviduct cells were cultured in the presence of estrogen and corticosterone with the indicated concentrations of PMA for 24 (0) or 48 (0) h. Samples were assayed for ovalbumin mRNA, as described in Materials and Methods. The results are expressed as percent inhibition (+ SEM for two experiments in duplicate for each ooint) of the induction of the ovalbumin mRNA by the steroids. Fig.
0 S+P
Treatment Fig. 4. PMA Causes an Increase in the Expression of a Reporter Gene Controlled by the Ovalbumin Promoter Primary oviduct cells were transiently transfected with the pOvCAT-,900 plasmid, which contains the region of the ovalbumin gene between -900 and +9 fused to the CAT gene. The transfected cells were cultured in the presence of estrogen and corticosterone (S), insulin and steroids (S+I), or steroids and 500 nM PMA (S+P) for 48 h. Total protein was extracted and assayed for CAT activity. The bars represent the mean f SEM of two experiments, performed in triplicate.
3OOd
LJ n
-P
0
+P
200 -
loo-
04 C
E
Hormones Fig. 3. PMA Independently Interacts with Both Steroids to Repress the Endogenous Levels of Ovalbumin mRNA Oviduct cells were cultured in the presence of estrogen (E) or corticosterone (C) with 500 nM PMA (Cl) or without PMA (m) for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per pg total nucleic acid (tNA) extracted. Each bar represents the mean + SEM of the results from two experiments, performed in triplicate.
an increase in CAT activity comparable to that achieved with insulin and steroids. The same result was observed when primary oviduct cells were transfected with a plasmid that contained 8 kilobases of the 5’-sequence of the ovalbumin gene linked to the CAT reporter gene (data not shown). This indicates that activation of the PKC pathway may cause a modest 2-fold increase in the expression of the ovalbumin gene. Thus, since the net result of activation of the PKC pathway is a dramatic repression of the steady state levels of ovalbumin mRNA, presumably the major effect of PMA is posttranscriptional. To determine whether PMA might be destabilizing the ovalbumin mRNA, a time-course experiment was performed (Fig. 5). Exposure of cells to PMA for a period as short as 2 h resulted in a 35% decrease in the levels of the mRNA, which is consistent with destabilization of the message. The half-life of ovalbumin mRNA after the addition of PMA was estimated to be 2.75 h (? 0.25 h). Since the half-life of ovalbumin mRNA decreases by lo-fold (from 24 to -2.4 h) upon withdrawal of estrogen (4, 5), these results suggest that PMA is negating the capacity of estrogen to stabilize ovalbumin mRNA. Thus, the drastic reduction in the levels of endogenous ovalbumin mRNA caused by activation of the PKC pathway is due to rapid destabili-
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Regulation of Ovalbumin Gene by Second Messengers
Ttme In culture
(hrs)
Fig. 5. PMA Rapidly Represses the Amounts of Ovalbumin mRNA Oviduct cells were cultured in the presence of insulin, estrogen, and corticosterone (W) for 24 h. At that time, the medium was discarded, and new medium containing only insulin and steroids (S+I; 0) or insulin, steroids, and 500 nM PMA (S+I+P; 0) was added. The cells were cultured for the additional times indicated. Samples were assayed for ovalbumin mRNA, as described in Materials and Methods. Each point represents the mean + SEM from a typical experiment, performed in triplicate.
zation of the ovalbumin mRNA, rather than diminished transcription. Gaub et a/. (9) reported that activation of PKC by 200 nM PMA causes induction of transcription from the basal (-58 to 1) ovalbumin promoter in chicken embryonic fibroblasts overexpressing the estrogen receptor. Our transfection results also indicate that PMA can increase the transcription of an ovalbumin fusion gene (Fig. 4). However, the major effect of activation of the PKC pathway is a rapid repression of the levels of endogenous ovalbumin mRNA due to message destabilization. This effect masks any direct increase in transcription of the gene in normal oviduct cells. In addition to chicken ovalbumin mRNA, activation of the PKC pathway has recently been implicated in alteration of the stability of several mRNAs. Phorbol esters increase the stability of interleukin-2 mRNA in human lymphocytes (12) and colony-stimulating factor mRNA in human fibroblasts (13). However, activation of the PKC pathway by phorbol esters results in destabilization of the actin, troponin, and myosin mRNAs in chicken myoblasts (14) as well as estrogen receptor mRNA in MCF-7 cells (15). Thus, a number of genes are regulated, both positively and negatively, at the level of mRNA stability by the PKC pathway. This introduces a third level of regulation by this very diverse signal transduction pathway in addition to regulation through direct substrate phosphorylation and control of transcription. Interactions
among
Steroids,
Insulin,
and CAMP
To determine whether activators of the PKA pathway affect the levels of ovalbumin mRNA, primary oviduct cells were treated with forskolin or IBMX. As shown in
1415
Fig. 6, treatment of cultured oviduct cells with forskolin or IBMX alone for 48 h did not cause a marked change in the basal levels of ovalbumin mRNA. However, the addition of either forskolin or IBMX to a steroid-containing medium potentiated the induction of the ovalbumin gene by steroids in a manner similar to that of insulin. The addition of forskolin or IBMX to culture medium containing both insulin and the steroids did not further enhance the induction of ovalbumin mRNA produced by the cooperation of steroids and insulin. These results show that activators of PKA synergize with the steroids and substitute for insulin in isolated cells, which is in agreement with observations in oviduct explant cultures (3). In addition, the effects of the activators and insulin are not additive, suggesting that insulin and CAMP use convergent pathways to modulate the expression of the ovalbumin gene. To investigate the independent interactions of the PKA pathway with each of the steroids, oviduct cells were cultured with forskolin and estrogen or with forskolin and corticosterone (Fig. 7). The results suggest that activation of the CAMP pathway synergizes independently with each of the steroids to increase the levels of ovalbumin mRNA. This ability of the PKA pathway is very similar to that of insulin, which also independently synergizes with each of the steroids to increase the levels of ovalbumin mRNA (2). To determine whether the increase in the levels of ovalbumin mRNA by activation of the PKA pathway is a result of enhanced transcription, the effects of forskolin on the expression of pOvCAT-.900 were examined (Fig. 8). Treatment with forskolin causes an increase in
200
Treatment Fig. 6. Agents That Activate the PKA Pathway Synergize with the Steroids to Potentiate the Production of Ovalbumin mRNA Oviduct cells were cultured for 48 h in the presence of insulin (I), estrogen and corticosterone (S), insulin and steroids (S+I), 33 PMforskolin (F), steroids and forskolin (S+F), steroids, insulin, and forskolin (S+I+F), 125 PMIBMX (X), steroids and IBMX (S+X), or steroids, insulin, and IBMX (S+I+X). The bars represent mean relative amounts of ovalbumin mRNA obtained from three different experiments with duplicate samples + SEM, using the amounts of ovalbumin mRNA induced by steroids and insulin as 100%.
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MOL 1416
END0.1992
Vol6
No. 9
1000
T
I-n
-I=
600
S
S+I
S+F
Treatment Hormones Fig. 7. Forskolin Interacts with Both Steroids Independently to Induce the Ovalbumin Gene Oviduct cells were cultured with estrogen (E) or corticosterone (C) with (8) or without (m) the addition of 33 FM forskolin for 48 h. RNA was extracted and hybridized to a specific ovalbumin probe, as described in Materials and Methods. The amount of endogenous ovalbumin mRNA induced by each treatment is expressed as picograms of RNA bound to the specific ovalbumin probe per pg total nucleic acid (tNA) extracted. Each bar represents the mean + SEM of the results from two experiments, performed in triplicate.
the expression of this chimeric plasmid, resulting in an increase in CAT activity. This suggests that the increase in the endogenous steady state levels of ovalbumin
mRNA caused by activation of the PKA pathway is solely due to modulation of transcription, because CAT activity and ovalbumin mRNA levels are increased to the same extent. This modulation of the expression of the ovalbumin gene by interactions between the insulin or PKA pathways and steroid-mediated events may occur at the level of transcription factors. The activities of several transcription factors can be modulated by phosphorylation (16-l 8). Similarly, insulin or the PKA pathway may phosphorylate a transcription factor and, thus, increase its DNA binding or transactivation activities. This transcription factor may be 1) the one induced by the steroid hormones, which binds to the SDRE, 2) one that interacts with the steroid-responsive protein, or 3) a factor completely independent of the steroid-responsive protein. Studies are under way to distinguish among these possibilities. Considerable evidence suggests that steroid hormones increase transcription of the ovalbumin gene through the binding of proteins to the SDRE (2, 7). Insulin enhances that activity through a phosphorylation
Fig. 8. Activation of the CAMP Pathway Causes an Increase in the Expression of a Reporter Gene Controlled by the Ovalbumin Promoter Primary oviduct cells were transiently transfected with the pOvCAT-,900 plasmid, which contains the region of the ovalbumin gene between -900 and +9 fused to the CAT gene. The transfected cells were cultured with estrogen and corticosterone (S), insulin and steroids (S+I), or steroids and 33 ELM forskolin (S+F) for 48 h. Total protein was extracted and assayed for CAT activity. The bars represent the mean + SEM of two experiments, performed in triplicate.
event that may or may not involve these proteins. Because activators of the PKA pathway can mimic the effects of insulin, they are presumably affecting the same events enhanced by insulin. In contrast, although PMA can increase transcription to the extent achieved by insulin and the activators of the PKA system, the principal action of the PKC system is to destabilize the ovalbumin mRNA. Because the stability of the ovalbumin mRNA decreases to the half-life seen in the absence of steroids, the most likely scenario is that the PKC pathway negates the effects of steroids on ovalbumin mRNA stability. Analysis of the effects of PMA on mRNA stability may thus provide a tool for determining how estrogen enhances that stability.
MATERIALS AND METHODS Animals
and Cell Culture
Immature White Leghorn chicks were implanted with diethylstilbesterol pellets, which were then withdrawn, and restimulated as previously described (19). Pellets were withdrawn 48 h before the tissue was harvested. The magnum portion of the oviduct was removed, minced with scissors, and incubated for 1 h in dissociation medium (Ham’s F-12 nutrient medium; Gibco-BRL, Gaithersburg, MD) supplemented with 1.7 mg/ml collagenase, 20 pg/ml trypsin, 25 pg/ml DNase, and 0.192 U/
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Regulation
of Ovalbumin
Gene
by Second
Messengers
1417
ml protease). The supernatant was removed, and single cells were harvested by centrifugation, washed twice in F-12 medium, and resuspended in culture medium [F-l 2 mediumDulbecco’s Modified Eagle’s medium (Gibco-BRL; 1 :l , vol/vol) supplemented with 0.1% (wt/vol) BSA, 5 U/ml penicillin (GibcoBRL), 5 fig/ml streptomysin (Gibco-BRL), and 200 pg/ml fungizone (Gibco-BRL)]. The cells were plated in culture medium supplemented with 50-1000 nM PMA as indicated, 33 PM forskolin, or 125 FM IBMX in addition to 1 O-’ M 17P-estradiol, 1O-6 M corticosterone, or 50 ng,/ml insulin. All hormones and reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. RNA
Isolation
and
cRNA
Hybridization
Ovalbumin cDNA sequences from 82-1347 (20) were subcloned in both orientations into pTZ18R and transcribed with T7 RNA oolvmerase. The cRNA was labeled with f?SlUTP and became ihe probe, while the mRNA was used to construct a standard curve (10-5000 ng). Total cellular nucleic acid was isolated, as previously described (19) and hybridized for at least 16 h with the radiolabeled cRNA probe at 80 C in 0.6 M NaCI, 20 mM Tris-HCI (pH 7.5) 5 mM EDTA, 2.5% (vol/vol) ethanol, 75 pg/ml salmon testis DNA, and 0.1% (wt/vol) sodium dodecyl sulfate (21). The samples were digested with a ribonuclease (RNase) solution [25 rig/ml RNase-A, 250 U/ml RNase-T,, 0.3 M NaCI, 10 mM Tris-HCI (pH 7.5) 2 mM EDTA, and 75 pg/ml salmon testis DNA), precipitated with 10% (wt/ vol) trichloroacetic acid, and quantified by liquid scintillation counting (7). Transfection
of Oviduct
Cells
and
CAT
Assays
Tubular oviduct cells were isolated and cultured as described above. They were transfected with pOVCAT-,900 (7) using CaP04 coprecipitation, as described previously (2). All cells transfected with a single DNA were pooled before aliquoting them into medium to rule out variations in transfection efficiency. CAT assays were used to measure the expression of the transfected plasmids and were performed as previously described (2).
Acknowledgments We want to thank Sasa Dragas-Graonic, and Natalie Hayes for their technical
Christopher assistance.
Carlson,
Received December 20, 1991. Revision received May 25, 1992. Accepted June 30,1992. Address requests for reprints to: Dr. Michel Sanders, Department of Biochemistry, 4-225 Millard Hall, University of Minnesota, Minneapolis, Minnesota 55455. This work was supported by American Cancer Society Grant BE-68 (to M.M.S.).
REFERENCES 1. Sanders MM, McKnight GS 1986 Egg white genes: hormonal regulation in homologous and heterologous cells. In: Malacinski GM (ed) Molecular Genetics of Mammalian Cells. Macmillan Press, New York, pp 183-216 2. Sanders MM. McKniaht GS 1988 Positive and neaative regulatory elements control the steroid-responsive”ovalbumin promoter. Biochemistry 27:6550-6557
3. Evans ME, McKnight GS 1984 Regulation of the ovalbumin gene: effects of insulin, adenosine 3’, 5’-monophosphate, and estrogen. Endocrinology 115:368-377 4. Palmiter RD 1973 Rate of ovalbumin messenger ribonucleic acid synthesis in the oviduct of estrogen-primed chicks. J Biol Chem 248:8260-8270 5. Palmiter RD, Carey NH 1974 Rapid inactivation of ovalbumin messenqer ribonucleic acid after acute withdrawal of estrogen. Proc Natl Acad Sci USA 71:2357-2361 6. Saaami I. Tsai S. Wana H. Tsai M-J. 0’ Mallev BW 1986 Identification of- two f&tom required for transcription of the ovalbumin gene. Mol Cell Biol 6:4259-4267 7. Schweers LA, Frank DE, Weigel NL, Sanders MM 1990 The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element. J Biol Chem 265:7590-7595 8. McKnight GS 1978 The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in oviduct explant cultures. Cell 14:403-413 9. Gaub M-P, Bellard M, Scheuer I, Chambon P, SassoneCorsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves the FosJun complex. Cell 63:1267-l 276 10. Hoefler J, Deutch P, Lin J, Habener J 1990 Distinctive adenosine 3’,5’ monophosphate and phorbol ester responsive signal transduction pathways converge at the level of transcriptional activation by the interaction of DNA binding proteins. Mol Endocrinol 3:868-880 11. Moore C, Brentano S, Miller W 1990 Human P45Oscc gene transcription is induced by cyclic AMP and repressed by 12-Ctetradecanoylphorbol-13-acetate and A231 87 through independent cis elements. Mol Cell Biol lo:601 36023 12. Ohmura T, Onoue K 1990 Stability of IL-2 mRNA in Tlymphocytes is controlled by a protein kinase C-regulated mechanism. Int lmmunol2:1073-1079 13. Akashi M, Saito M, Koeffler HP 1989 Lymphotoxin stimulation and regulation of colony-stimulating factors in fibroblasts. Blood 74:2383-2390 14. Zhu Y-Y, Schwartz RJ, Crow MT 1991 Phorbol esters selectively deregulate contractile protein gene expression in terminately differentiated myotubes through trancriptional repression and message destabilization. J Cell Biol 1151745-754 15. Saceda M, Knabbe C, Dickson RB, Lippman ME, Bronzert D, Lindsey RK, Gottardis MM, Martin MB 1991 Posttranslational destabilization of estrogen receptor mRNA in MCF-7 cells by 12-0-tetradecanoylphorbol-13-acetate. J Biol Chem 266:17809-l 7814 16. Merino A, Buckbinder L, Mermelstein FH, Reinberg D 1989 Phosphorylation of cellular proteins regulates their binding to the CAMP response element. J Biol Chem 264:21266-21276 17. Luscher B, Christenson E, Litchfield DW, Krebs EG, Eisenman RN 1990 Myb DNA binding is inhibited by phosphorylation at a site deleted during oncogenic activation. Nature 334:517-522 18. Jackson SP, MacDonald JJ, Lees-Miller S, Tijian R 1990 GC box binding induces phosphorylation of Spl by a DNA-dependent protein kinase. Cell 63:155-l 65 19. Sanders MM, McKnight GS 1985 Chicken egg white genes: multihormonal regulation in a primary cell culture system. Endocrinology 116:398-405 20. Humphries P, Cachet M, Krust A, Gerlinger P, Kourilsky P, Chambon P 1977 Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene. Nucleic Acids Res 4:2389-2406 21. ldzerda RL, Huebers H, Finch CA, McKnight GS 1986 Rat transferrin gene expression: tisssue-specific regulation by iron deficiency. Proc Natl Acad Sci USA 83:3723-3727
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