(Received for publication, August 18, 1995; and in revised form, January 29, 1996)
From the
Transcription of the 252-base pair-long mouse -casein gene
promoter is induced by the synergistic action of insulin, prolactin,
and glucocorticoid in a primary mammary epithelial cell culture. The
promoter contains a region termed block C having a highly conserved
sequence and position among many casein genes. Mutation of block C
reduced the response of the promoter to lactogenic hormones 84%.
Nuclear extracts from lactating mouse mammary glands contained both a
double-stranded and a single-stranded DNA binding protein complex (DS1
and SS), which specifically bind to the sequences AAATTAGCATGT and
CCACAA of block C, respectively. The DS1 and the SS protein complexes
were approximately 400 and 280 kDa, respectively. Each complex
contained a DNA-binding component(s) having a molecular mass of
approximately 120 kDa for DS1 and 80 and 65 kDa for SS. Deoxycholate,
which interferes with the protein-protein interactions, inhibited the
binding activities of DS1 and SS. The maximal increase in the binding
activity of DS1 and SS in the mammary gland occurred during pregnancy
and during lactation, respectively. In organ culture, the DS1 activity
is increased by epidermal growth factor or prolactin in combination
with insulin, whereas the SS activity is enhanced by insulin,
prolactin, and glucocorticoid. These results suggest that multiprotein
complexes binding to the double- and single-stranded DNA of block C
mediate hormonal induction of
-casein gene transcription.
Caseins are the major milk proteins produced by the lactating
mammary gland. The transcription of casein genes is regulated by the
interplay of various hormones such as insulin, prolactin, and
glucocorticoid. This system serves as a good model to study the
molecular mechanisms whereby hormones regulate mammalian gene
transcription. Prolactin and glucocorticoid are required for
enhancement of both transcription of casein genes and stability of the
transcripts(1) , whereas insulin is essential for transcription
but not for stabilization of casein mRNA(2) . Transfection of
reporter plasmids containing 5`-flanking region of the -casein
gene to either primary mouse mammary epithelial cells (3) or a
rat mammary epithelial cell line, HC11(4) , also demonstrated
that all three hormones are required for full induction of the casein
gene transcription and that the proximal region of the promoter,
-258/+7 of mouse gene (5) or -355/-1 of
rat gene(4) , is sufficient for hormonal induction.
At least seven promoter regions of mouse, rat, bovine, and rabbit casein genes have been cloned(6, 7, 8, 9, 10, 11) . Alignment of their DNA sequences has revealed three highly conserved sequences, designated as blocks A, B, and C, in each promoter region ( (6) and Fig. 1A). Blocks A and B share a similar sequence, whereas the sequence of block C is quite different from those of block A and B. The presence of nuclear proteins binding to these three regions was detected in the lactating mouse mammary glands (5) as well as a rat mammary epithelial cell line, HC11(12) . These proteins are likely candidates for transcription factors involved in casein gene expression.
Figure 1:
Structure of the proximal promoter
region of mouse -casein gene (A) and alignment of
sequence between block C and TATA box of various casein promoters (B). The positions of blocks A, B (MGF binding site) and C and
TATA box are shown by base pair distance from the transcriptional
initiation site, denoted as +1. Sequences of block C and TATA box
are shown by boldface letters.
Recently a
protein that binds to block B was cloned and sequenced(13) .
This protein has been named mammary gland-specific factor (MGF ()or STAT5) and was shown to be a member of the
cytokine-regulated transcription factor gene family(13) .
Several lines of evidence indicate that prolactin stimulates the
phosphorylation of MGF, which then participates in the transcriptional
activation of the
-casein gene by binding to block B and probably
block A(4, 14) . These findings relating to MGF
suggest that the highly conserved regions of the casein gene promoter
serve as a major site for transcriptional regulation whereupon
hormonally induced transcription factors act.
Another well conserved
sequence, block C, is present approximately 20 base pairs upstream of a
TATA box and approximately 40 base pairs downstream of block B. Its
position also is well conserved among casein gene promoters (Fig. 1B). At present, however, the role of block C in
regulating casein gene transcription has not been elucidated. Moreover,
the nuclear protein(s) that bind to this region have not been
characterized. Recently several groups reported that single-stranded
DNA binding proteins play a role in transcriptional
regulation(15, 16, 17, 18, 19, 20, 21) .
For example, a sequence-specific, single-stranded DNA binding factor
(STR; (16) and (22) ) that binds to the distal region
of rat -casein gene promoter has been shown to have a suppressive
role. These studies led us to investigate both double- and
single-stranded DNA binding proteins that bind to block C.
In this
study we examined the functional role of block C in the hormonal
induction of mouse -casein gene transcription. Transfection
experiments using a primary mammary epithelial cell (PMME) culture
system indicated that block C is crucial for the
-casein gene
transcription induced by lactogenic hormones. Our studies also revealed
the presence of nuclear protein complexes in the lactating mammary
gland that bind to both double-stranded and single-stranded DNA within
block C. The appearance of these binding complexes in the mammary gland
is hormonally regulated and undergoes developmental changes during the
periods of pregnancy and lactation.
Gel shift assays were performed using either
double-stranded or single-stranded oligonucleotides corresponding to
the -69/-38 region of the mouse -casein gene promoter.
The coding strand was end-labeled with
[
-
P]ATP (Amersham Corp.; 6000 Ci/mmol)
using T4 polynucleotide kinase (Stratagene). To prepare the
double-stranded probe, the labeled oligonucleotide was hybridized to
the noncoding strand. Both oligonucleotides were purified by
electrophoresis in a 10% polyacrylamide gel to remove unincorporated
nucleotides and nonhybridized oligonucleotides. Nuclear extracts were
incubated with a
P-labeled probe (3
10
cpm) in 25 µl of binding buffer (12 mM Hepes, pH
7.9, 12% glycerol, 60 mM NaCl, 1.5 mM MgCl
, and 2.5 mM DTT) with 3 µg of bovine
serum albumin (Promega), 1.5 µg of poly(dI-dC) (Sigma), and with or
without competitor oligonucleotides bearing wild-type or mutated
sequences (see Table 1). Following a 30-min incubation at 25
°C, the reaction mixtures were subjected to electrophoresis in a 6%
polyacrylamide (29:1, acrylamide/bisacrylamide) gel in TBE buffer (pH
8.3, 90 mM Tris base, 90 mM boric acid, and 0.5
mM EDTA). The gels were dried, and the bands were detected by
autoradiography. For quantitation of band activities, gels
corresponding to retarded bands were cut out, and their radioactivities
were measured by a liquid scintillation counter (Beckman LS3801).
Figure 2:
Effect of various mutations within block C
on transcriptional induction of mouse -casein gene by lactogenic
hormones. A, effect of various hormone combinations on
induction of CAT activity of pD30 wild-type construct (bearing
-245/+7 region of
-casein promoter). Mouse mammary
epithelial cells were prepared from mice in midpregnant stage
(12-14 days of gestation) and cultured for 5 days in the presence
of the indicated hormones. Concentration of insulin (I),
hydrocortisone (H), and prolactin (P) used were 5
µg/ml, 1 µg/ml, and 5 µg/ml, respectively. B, the
basal (black bar) and induced (shaded bar) CAT
activities of various constructs. The basal and induced activities were
determined in culture with insulin and with
insulin/hydrocortisone/prolactin, respectively. The activities were
calculated relative to the induced activity of pD30wt construct
(=1) in each transfection experiment. Means of relative values
± S.E. from three to six independent transfection experiments
are presented.
The DS probe formed at least three bands when incubated with mammary nuclear extracts (Fig. 3A). The upper major band (DS1) showed clear sequence specificity because the band was competed completely by wild type or M7 and partially by M3, M8, or M10 but not by the others (Fig. 3A, Table 1). The intensity of the middle minor band (DS2) was rather faint in this experiment. However, other experiments using a partially purified fraction of nuclear proteins, in which DS2 binding activity was enriched, indicated that DS2 had the same sequence specificity as DS1 (data not shown). The lowest band was considered to be nonspecific because it did not show clear sequence specificity in competition experiments.
Figure 3:
Competition gel shift assay of the binding
complex formation on the block C region. Three micrograms of nuclear
extract from mammary glands obtained from mice in the 3rd through the
7th days of lactation was incubated with an end-labeled double-stranded (A) or a single upper strand (B) DNA corresponding to
the -69/-38 region of mouse -casein promoter in the
absence or the presence of 50-fold molar excess of double-stranded (A) or 20-fold molar excess of single-stranded (B)
unlabeled competitors. The sequence of competitors (M1 through M10) is
shown in Table 1. Three, five, or two micrograms of nuclear
extracts from lactating mice was allowed to bind to DS, SS(+), or
SS(-) probe, respectively (C). Thirty-fold molar excess
of unlabeled DS (lanes 2 and 6), SS(+) (lanes 3 and 5) or SS(-) (lane 10)
wild-type competitor was added to the binding mixture before the
addition of labeled probes. WT, wild-type; NO, no
competitors. Specific complexes, DS1, DS2, and SS, are shown by arrows.
The SS(+) probe formed two binding complexes (Fig. 3B). The lower band was judged to be nonspecific because wild type and all mutated competitors were equally effective. The upper band (SS) was specific because wild type, M1, M2, M9, and M10 all competed efficiently, M5, M6, M7, and M8 competed less efficiently, and M4 showed no competition. M3, which showed substantial competition in DS1 binding, was a poor competitor in SS binding. Fig. 3C showed that SS(+) did not compete in DS1 complex formation (lane 3), suggesting that DS1 did not bind to SS(+). On the other hand, the SS complex formation was inhibited by the wild type of DS competitor (lane 7), suggesting that SS did bind to DS. These results implied that DS1 required double-stranded DNA for binding, whereas SS could bind to both single- and double-stranded DNA in block C. These findings suggested that these two binding factors are different entities.
The SS(-) probe formed a broad band, which was considered to be nonspecific because of no competition by a 30-fold molar excess of SS(-) competitor (Fig. 3C, lanes 7 and 10). In the presence of SS(+) competitor, appearance of the DS complex was found (Fig. 3C, lane 9). This was likely due to formation of DS probe as a result of annealing of SS(+) competitor to SS(-) probe during the binding reaction.
To confirm the formation of binding complexes and to localize the direct binding sites in the block C region, DEPC interference assays were performed. DEPC modifies the N-7 position of free purines in single- and double-stranded DNA, which results in interfering protein-DNA interaction(27) . DNA fragments bound to proteins were isolated, cleaved at the position of modification by the treatment of piperidine, and subjected to gel electrophoresis. Nuclear extracts from lactating mammary glands were incubated with either DS or SS(+) oligonucleotides chemically modified at A and G residues by DEPC. When a DS probe labeled at either the coding or noncoding strand was used, interference of A and G residues occurred at positions corresponding to those mutated in M4, M5, and M6 competitors (Fig. 4, A and B, closed circle). When a labeled SS(+) probe was used, residues at positions corresponding to those mutated in M4 were interfered with (Fig. 4C, closed circle). We found hypersensitive sites at adenine residues positioned at -55 and -57 on the double-stranded probe (Fig. 4B), and also at -49 on the single-stranded probe (Fig. 4C). These hypersensitive sites could result from binding of proteins to the adjacent nucleotides. The results of interference assays indicated that the binding sequences of DS1 and SS involved 5`-AAATTAGCATGT-3` and 5`-CCACAA-3`, respectively.
Figure 4:
DEPC interference assay to determine
direct contact sites of DS1 or SS binding to the block C region.
Fifteen micrograms of nuclear extract prepared from lactating mouse
mammary gland was incubated with double-stranded DNA corresponding to
the -66/-29 region (A), double-stranded (B) or coding strand DNA (C) corresponding to the
-75/-38 region of mouse -casein promoter as a probe.
Free and bound DNA probes were recovered and subjected to 18%
polyacrylamide gel electrophoresis. Results from noncoding strand (A) and coding strand (B) using double-stranded DNA,
and coding strand using single-stranded DNA (C) are shown with
A + marker. Results of DEPC interference assays are summarized (D). Obstructed residues (closed circle) of
double-stranded (upper) and single-stranded (lower)
DNA are shown.
Figure 5:
Developmental change of DS1 and SS binding
activity in mammary glands. Nuclear extracts were prepared from mouse
mammary glands of various reproductive stages. The term of gestation
was determined retrospectively by stature and characteristics of fetus,
and the term of lactation was determined by the day after parturition.
Postlactating mice were sacrificed 3 weeks after removal of their pups.
Nuclear extracts of virgin and postlactating mice were prepared from a
mixture of mammary glands of 10 and 3 mice, respectively. Nuclear
extracts of pregnant or lactating mice were prepared independently from
three different mice of each stage. Three micrograms of each nuclear
extract was allowed to bind to either double-stranded (A) or
coding strand (B) DNA probe corresponding to the
-69/-38 region of the mouse -casein promoter. Specific
complexes, DS1 and SS, are shown by arrows. The same nuclear
extract used in Fig. 3was used in lane 8 as a control. C, the level of binding activity in each band was evaluated by
determining its radioactivity. These results were presented relative to
the mean of the peak activity, 17th to 19th day of pregnancy for DS1
and 7th to 10th day of lactation for SS. The relative values of mean
± S.E. of each time point were obtained from three independent
experiments.
Figure 6:
Hormonal regulation of DS1 and SS binding
activities in cultured mammary glands. Mammary gland explants were
prepared from mice in midpregnant stage (12th to 14th day of
gestation), and cultured in the medium containing the indicated
combination of hormones up to 3 days. Three micrograms of nuclear
extract from cultured tissues was allowed to bind to either
double-stranded (A) or coding strand (B) DNA probe
corresponding to the -69/-38 region of mouse -casein
promoter. Concentrations of hormones used were as follows: 5 µg/ml
of insulin (I), 1 µg/ml of hydrocortisone (H), 5
µg/ml of prolactin (P), and 50 ng/ml of EGF (E).
Specific complexes are shown by arrows. The same nuclear
extract used in Fig. 3was used in lanes 7 and 13 (A) and lanes 6 and 12 (B) as
a control.
The level of SS binding activity was increased by the combination of insulin, prolactin, and hydrocortisone, whereas the combination of insulin and prolactin was effective in maintaining the initial level during culture. The activity decreased in the presence of insulin alone or the combination of insulin and hydrocortisone or insulin and EGF (Fig. 6B).
In addition, we attempted to examine the effect of hormones on the binding activities of DS1 and SS in mouse mammary epithelial cells in primary culture. However, it was difficult to obtain nuclear extracts from cultured cells in adequate quantity and quality because those cells cultured on the Matrigel needed to be extensively treated with trypsin and EDTA to detach them from the substratum. This treatment produced a number of problems in isolating nuclear binding proteins from cultured cells.
Figure 7:
Molecular weight determination of the DS
and the SS binding protein(s) by gel filtration column chromatography.
The mixture of marker proteins, including thyroglobulin (669 kDa),
ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67
kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease
A (13.7 kDa), was applied onto a Superdex 200 column. The peak position
of elution for each protein was determined by monitoring 280-nm
absorbance and plotted against its molecular weight (closed
circle) (A). The peak fraction having DS1, DS2, or SS
binding activity is indicated by arrows. An aliquot (5 mg of
proteins) of the 45% ammonium sulfate-precipitated fraction of nuclear
extract prepared from lactating mouse mammary glands was applied onto
the same column. One-milliliter fractions of eluent were collected, and
5 µl of each fraction was used for a gel shift assay. DS and SS
binding activities were determined using double-stranded (B)
or coding strand (C) DNA probe corresponding to the
-69/-38 region of mouse -casein promoter,
respectively. Specific complexes are shown by arrows. AP and
FT refer to the original sample applied and the flow-through fraction,
respectively.
In order to detect protein molecules binding directly to DNA probes, cross-linking experiments were performed. Using nuclear extracts, we found a single protein binding to the DS probe in the DS1 band (Fig. 8A) and two proteins binding to the SS(+) probe (Fig. 8C). We also performed similar experiments using partially purified extracts in which DS2 binding activity was enriched. Again, a single protein binding to the DS probe was found (Fig. 8B). The molecular mass of the DNA binding protein was approximately 120 kDa for DS1 and 80 kDa for DS2. The molecular masses of two DNA binding proteins for the SS complex were estimated to be 80 and 65 kDa. These values were much smaller than those of the corresponding complexes determined by gel filtration experiments.
Figure 8:
Characterization of DS or SS complex by
two-dimensional/UV cross-linking experiments and the effect of
detergent treatment. Thirty micrograms of nuclear extracts from
lactating mouse mammary glands was allowed to bind to either
double-stranded (A) or coding strand (C)
5-bromo-2`-deoxyuridine-substituted DNA probe corresponding to the
-69/-38 region of the mouse -casein promoter. Thirty
micrograms of partially purified extract (mixture of fractions
25-28 obtained from gel filtration column chromatography (see Fig. 7)) was allowed to bind to a double-stranded (B)
5-bromo-2`-deoxyuridine-substituted DNA probe. The reaction mixture was
separated by 6% polyacrylamide gel electrophoresis (one-dimensional)
and then UV-irradiated in situ by placing the gel directly
under a UV transilluminator (345 nm). The lane containing samples was
then excised, equilibrated in a buffer containing SDS, and resolved by
8% SDS-polyacrylamide gel electrophoresis (two-dimensional). Proteins
cross-linked to
P-labeled probe were detected by
autoradiography (indicated by arrows). The positions of
molecular mass markers (kDa) are shown on the left. The effect
of detergents, deoxycholate (DOC) and Nonidet P-40 (NP40), were determined by incubating 3 µg of nuclear
extract prepared from lactating mouse mammary glands with either
double-stranded (D) or coding strand (E) DNA probe
corresponding to the -69/-38 region of the mouse
-casein promoter in the presence of indicated concentrations of
detergents. Specific complexes are shown by arrows.
Deoxycholate is a mild ionic detergent which, when used at low concentrations, is known to disrupt protein-protein interactions but not DNA-protein interactions(30, 31) . As shown in Fig. 8D, the addition of deoxycholate at very low concentrations to the binding reaction inhibited the formation of DS or SS bands and produced no bands migrating faster (Fig. 8, D and E). This suggested that disruption of DNA-binding complexes by deoxycholate produced no DNA binding component of smaller size. The effect of Nonidet P-40, a nonionic detergent, was also analyzed. Nonidet P-40 (0.3%) had no effect on the DS complex formation by itself but prevented the disruptive effect of deoxycholate in the complex formation. This might be due to the effect of Nonidet P-40 to sequester deoxycholate from solution(31) . As for the SS complex, Nonidet P-40 like deoxycholate, inhibited the formation of the complex. The results presented above suggested that all three DNA-binding complexes, DS1, DS2, and SS, are composed of multiple proteins.
Previously we reported the presence of three highly conserved
regions, designated as blocks A, B, and C, in the promoters of many
casein genes(6) . The functional role of block C has not been
elucidated, whereas block B and probably block A have been suggested to
serve as an acting site of a prolactin-dependent transcription factor,
MGF(12) . We demonstrate here that the mouse -casein gene
promoter (-245/+7) containing all three blocks responded
fully to lactogenic hormones, insulin, prolactin, and hydrocortisone
when transfected into mouse PMME. Mutational disruption of block C
impaired the response of the promoter to the lactogenic hormones as
much as 84%, although the mutated promoter still retained its hormonal
response (2.7-fold). These observations indicated that block C, like
the other two conserved regions, is important for enhancement of the
transcriptional activation by lactogenic hormones.
The results of gel shift assays using DNA probes corresponding to the block C sequence revealed the presence of two double-stranded DNA binding activities (DS1 and DS2) and one single-stranded (coding strand) DNA binding activity (SS) in the lactating mammary gland. The results obtained by competition experiments in gel shift assay and by DEPC interference assay indicated that the binding sequences of these complexes involved 5`-AAATTAGCATGT-3` for DS1 and 5`-CCACAA-3` for SS. Nine out of 12 nucleotides of the DS1 binding sequence and seven out of eight nucleotides of the SS binding sequence are conserved among seven casein gene promoters (see Table 1). As for DS2 complex, DEPC interference assay gave inconclusive results because of low levels of the complex formation. However, the results obtained by competition experiments suggested that DS2 complex involves the same binding sequence as DS1. At present, the relationship between DS1 and DS2 remains to be elucidated.
The formation of the double-stranded
binding complex DS1 and the single-stranded binding complex SS involved
a contiguous, partially overlapping element, located at
-54/-43 and -59/-53, respectively, on the mouse
-casein gene promoter. Competition gel shift assays revealed that
the sequence mutated in M3 was important for the binding of SS but less
so for DS1 binding and that the sequence mutated in M5 was important
for the binding of DS1 but not for SS binding. The data in Fig. 3C suggested that SS could bind to both single-
and double-stranded DNA in block C, whereas DS1 could bind only to the
double-stranded DNA. Transfection experiments showed that disruption of
SS binding site (pD30.3) resulted in a greater decrease in hormonal
induction of CAT activity than that of DS1 binding site (pD30.5),
suggesting that the binding of SS was important for the induction.
Moreover, disruption of both sites (pD30.35) caused an even greater
decrease in the CAT activity, suggesting the possible interplay between
DS1 and SS binding factors. On the other hand, these mutations did not
affect the basal CAT activities. These results suggested that the DS1
and the SS complexes are formed on a contiguous element within block C
and that they work cooperatively as positive regulators of hormonal
induction of mouse
-casein gene transcription. It is possible that
the initial binding of DS1 to block C results in conformational changes
such as partial dissociation of the double-stranded DNA and
subsequently allow SS to bind to block C. The difference in strand
requirements between the two binding factors may provide the mean
whereby the two factors interact with block C in turn to induce casein
gene transcription. Replacement of DS1 binding by SS may occur at or
near their binding site(s), which have the overlapping recognition
sequence.
Sequence-specific, single-stranded DNA binding proteins
have been reported to play an important role in the expression of
various genes (15, 16, 17, 18, 19, 20, 21) .
Most of them bind solely to DNA. Recently novel single-stranded binding
proteins were shown to bind to the contiguous sequence of a TTF-1
binding site in thyrotoropin receptor gene promoter. TTF-1 is a
double-stranded binding transcriptional factor. The authors speculated
that single-stranded binding proteins positively regulate gene
transcription in cooperation with TTF-1(32) . Another
sequence-specific, single-stranded DNA binding factor (STR; Refs. 16
and 22) was reported to play a role in -casein gene transcription.
This factor was found to bind to the distal region of the
-casein
gene promoter and to exert negative regulation. On the other hand, we
found that the SS complex binds to the proximal region of the promoter
and exerts positive regulation. Although the molecular mechanisms of
action of these regulators have not yet been clarified, these findings
suggest the importance of single-stranded DNA binding proteins for
-casein gene regulation.
UV cross-linking experiments indicated that a 120-kDa protein bound directly to the double-stranded DNA of block C. On the other hand, the molecular mass of the DS1 protein(s) complex was estimated to be 400 kDa by gel filtration column chromatography. As for the SS complex, 80- and 65-kDa proteins were found to bind directly to single-stranded DNA, whereas the molecular mass of the entire binding complex was approximately 280 kDa. Thus, the values of molecular mass of the entire binding complex and DNA-binding component(s) are quite different. Although the reason for this difference is not known at the present time, one possibility is that the DS1 and SS complex formation involves multiple proteins, which include DNA binding and non-DNA binding proteins. The inhibitory effect of deoxycholate on the binding activities of both complexes suggested that these complexes were composed of multiple components. Moreover, deoxycholate treatment produced no DNA-binding complexes having smaller molecular weights, suggesting that full assembly is necessary for their DNA binding activities. Many transcription factors including Fos/Jun, ATF/CREB, MyoD, and E12/E47-like proteins are known to be multimeric complexes formed by protein-protein interactions(33, 34, 35) .
Our studies showed that the appearance of the DS1 and SS binding complexes in the mammary gland undergoes developmental changes under the influence of hormones. The DS1 binding activity increased to a maximal level during pregnancy and was maintained at somewhat reduced levels during lactation. In contrast, the SS binding activity increased maximally during lactation. It is noteworthy that the levels of the two binding activities in the gland increase sequentially during the periods of pregnancy and lactation when casein gene expression increases progressively. On the other hand, their levels are low in virgin and postlactating periods when casein gene is not expressed. Thus, the appearance of the two binding activities correlate well with the change in casein gene expression.
It has been well established that the combination of EGF and insulin stimulates the formation of daughter cells in the mammary gland that express casein gene in the presence of insulin, prolactin, and hydrocortisone in vitro(29) . Organ culture experiments using mouse mammary gland explants showed that the DS1 binding activity was increased by the combination of insulin and EGF, and to a somewhat lesser extent by the combination of insulin and prolactin. The SS binding activity was enhanced by the synergistic actions of insulin, prolactin, and hydrocortisone. These results indicated that the binding activities of the two complexes are differentially regulated by the different combinations of hormones during mammary gland development.
Prolactin, hydrocortisone, and EGF
are also implicated in the mammary gland development during the periods
of pregnancy and lactation. For example, both the plasma concentration
of EGF and the number of EGF receptors in the gland increase during
pregnancy(36, 37) . During the lactating period, the
number of EGF receptors in mammary tissue decreases, but both the
plasma concentration of prolactin and the number of prolactin receptors
in the gland increase(38) . These changes in the levels of EGF
and prolactin and their receptors could, at least in part, account for
changes in the DS1 and the SS binding activity in the mammary gland
during the periods of pregnancy and lactation. The findings presented
in this study suggest that the hormonal induction of -casein gene
expression involves stimulation of binding activities of DS1 and SS,
which, in turn, enhance the gene transcription via block C. These
binding proteins are likely candidates for transcription factors that
participate in the tissue- and stage-specific casein gene expression.
Purification of SS and DS1 complexes would allow us to examine their
predicted transcriptional activities using the in vitro transcription system. Moreover, cloning of these factors would
make it possible to determine the relative roles of each by expressing
them in hormone-responsive cell lines that are deficient in the SS/DS1
binding activities.