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Endocrinology Vol. 144, No. 6 2683-2694
Copyright © 2003 by The Endocrine Society

Developmental and Hormonal Signals Dramatically Alter the Localization and Abundance of Insulin Receptor Substrate Proteins in the Mammary Gland

A. V. Lee, P. Zhang, M. Ivanova, S. Bonnette, S. Oesterreich, J. M. Rosen, S. Grimm, R. C. Hovey1, B. K. Vonderhaar, C. R. Kahn, D. Torres, J. George, S. Mohsin, D. C. Allred and D. L. Hadsell

The Breast Center, Departments of Medicine, Molecular and Cellular Biology, Pediatrics, and Pathology, Baylor College of Medicine, Houston, Texas 77030; U.S. Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research, Center (S.B., D.L.H.), and Molecular and Cellular Endocrinology Section, Center for Cancer Research, National Cancer Institute (R.C.H., B.K.V.), National Institutes of Health, Bethesda, Maryland 20892; and Research Division, Joslin Diabetes Center, Department of Medicine, Harvard Medical School (C.R.K.), Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Adrian V. Lee, Ph.D., The Breast Center, Room N1110, Baylor College of Medicine, One Baylor Plaza, MS 600, Houston, Texas 77030. E-mail: avlee{at}breastcenter.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POSTNATAL development of the mammary gland in virgin female mice is characterized by the progressive infiltration of the mammary fat pad by terminal endbuds (TEB), highly proliferative structures that are responsible for completing the mammary ductal network (1). The development and differentiation of these and other epithelial structures within the mammary gland are critically dependent upon both ovarian hormones [estrogen (E) and progesterone (P)] and pituitary hormones (GH and PRL). These hormones work in concert with locally acting growth factors to coordinate development of the gland (2).

Powerful techniques, such as gene-targeted deletion of hormones receptors and reconstitution and transplantation of mammary glands, have determined the relative importance of stromal vs. epithelial receptors in mammary gland development. Estrogen receptor {alpha} (ER{alpha})-null mice (ERKO) fail to undergo ductal elongation (3, 4), and stromal and epithelial ER{alpha} are implicated as the major mediator of ductal morphogenesis (5, 6). GH receptor (GHR)-null mice have retarded ductal development that is mediated by stromal GHR (7), probably acting indirectly via IGF-I (8). Consistent with this, IGF-I-null mice have severely retarded mammary ductal development and branching similar to ERKO mice (9, 10). IGF-I receptor (IGF-IR)-null mice die perinatally (11), but grafting of IGF-IR-null embryonic mammary anlage into cleared fat pads demonstrated reduced growth (12), associated with reduced TEB size, number, and epithelial cell proliferation. Progesterone receptor (PR)-null mice have normal ductal development, but fail to undergo pregnancy-induced lobuloalveolar development (13). This action of PR is thought to be mediated by epithelial PR (7) working in a juxtacrine manner, possibly via IGF-II (14, 15). PRL receptor (PRLR)-null mice have decreased branching of pregnant mammary glands and fail to lactate (16).

Insulin receptor substrates (IRS) are a family of intracellular proteins that integrate and coordinate hormone, cytokine, and growth factor signaling. To date four IRS proteins (IRS-1 to IRS-4) have been identified (17, 18, 19, 20). All contain conserved pleckstrin homology and phosphotyrosine-binding domains at the amino termini. The carboxyl termini are less conserved and contain multiple tyrosine phosphorylation sites that act as binding sites for Src homology domain 2-containing proteins (21). The IRS proteins were first identified as substrates and presumed signaling intermediates of the insulin receptor, and mice that are null for either IRS-1 (22) or IRS-2 (23) display a variety of defects, including insulin resistance and growth retardation. However, it is now clear that IRS proteins can be activated and phosphorylated by a number of other signaling pathways, including those that are critical for mammary gland development, e.g. GH and PRL (24, 25).

IRS protein levels are regulated by both gene transcription and protein degradation. We and others have shown that E increases IRS-1 mRNA and protein levels in breast cancer cells in vitro and in vivo (26, 27, 28). Similarly, IRS-2 mRNA levels are induced by PR (29). After our studies showing that high IRS-1 expression indicates a poor prognosis for ER-positive breast cancer patients (26), we examined the hormonal and developmental regulation of IRS expression in normal mouse mammary gland. We show here that 1) IRS proteins show distinct patterns of expression in virgin mammary glands; 2) IRS levels change dramatically during mammary gland development; 3) sex steroid hormones increase IRS levels; and 4) IRS-1 levels are altered 4-fold during the estrous cycle. These studies lay a foundation for further work investigating the signaling capacity and role of IRS-1 and -2 in mammary gland development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
FVB mice used in these studies were from Charles River Laboratories, Inc. (Wilmington, MA). Animal studies were conducted using procedures outlined in the NIH Guide to the Care and Use of Experimental Animals as approved by the Baylor College of Medicine animal care and use committee. For developmental studies mammary glands were harvested from FVB female mice at 6 wk of age; 8, 16, or 18 d of pregnancy; 10 d of lactation; 10 d of lactation followed by 3 d of forced involution; and 21 d of lactation, followed by 3 d of forced involution. Studies of IRS colocalization with proliferating cells were performed on mammary glands from mice treated as previously described (14, 30). The IRS-1-null mice used in these studies were on the B6/129 background and were genotyped by PCR as previously described (22).

Whole-mount analysis
Virgin mammary development was studied at 6 wk of age in hematoxylin-stained whole mounts (12). Quantitative morphometry was conducted on TIF images of whole mounts prepared from wild-type (n = 6) and IRS-1-null (n = 4). The end points measured included fat pad area, area of the fat pad occupied by epithelium, number of branch points, and number of TEB. Body weights were determined for each animal at the time of sampling so that results could be adjusted for differences in body weight.

Hormone stimulation
To determine of loss of IRS-1 causes diminished epithelial cell proliferation in response to treatment with ovarian hormones, wild-type (n = 5) and IRS-1-null (n = 3) females were treated at 13 wk of age with a single sc injection of sesame oil containing E (1 µg) and P (1 mg). After 48 h these hormone-treated mice were labeled for 2 h with bromodeoxyuridine (BrdU; 100 mg/kg), and then mammary glands were harvested and processed for immunohistochemistry (IHC).

To establish a requirement for ovarian hormones and to determine whether exogenous administration of these hormones mimics the pregnancy-dependent changes observed in IRS expression, 6-wk-old nulliparous female FVB mice (n = 20) were either ovariectomized (ovex; n = 10) or left intact (n = 10). Mice then had a beeswax pellet implanted sc that contained E (20 µg) and P (20 mg; n = 5) or vehicle (beeswax pellet alone; n = 5). After 8 d mice were labeled for 2 h with BrdU (100 mg/kg), and mammary glands were harvested for IHC and immunoblotting.

Estrous staging
Six-week-old nulliparous female FVB mice (n = 20) were classified according to their stage in the estrous cycle by vaginal lavage according to previously published morphological criteria (31). Mice were injected with BrdU (100 mg/kg) 2 h before they were killed. Mice were then anesthetized, and 1 ml blood was withdrawn directly from the heart. Blood was left overnight at 4 C, and serum was separated after centrifugation at 3000 x g for 15 min. We recovered between 175–250 µl serum from each mouse. Serum E and P levels were then determined using RIA kits (Diagnostic Systems Laboratories, Inc., Webster, TX; DSL-39100 for E and DSL-3400 for P) according to the manufacturer’s instructions. The sensitivities of these assays were 0.1 ng/ml and 0.6 pg/ml for P and E, respectively. Mammary glands were either frozen in liquid nitrogen for future DNA or RNA analysis or fixed overnight (together with ovary and uterus) in 4% paraformaldehyde before IHC. The vaginal lavage was corroborated by additional parameters, including histology and proliferation of the uterus and ovary.

RNA analysis
Total RNA was isolated from approximately 100 mg tissue using Ultraspec (Biotecx Laboratories, Inc., Houston, TX) as described by the manufacturer. Levels of IRS-1 and IRS-2 mRNAs were determined by ribonuclease protection assays (RPA). The probes for murine IRS-1 and -2 have been described previously (32). The pTRI-cyclophilin-mouse antisense control template (Ambion, Inc., Austin, TX) was used to measure cyclophilin mRNA as a loading control. Antisense cRNAs were produced using T3 RNA polymerase, yielding probes of 502, 384, and 138 nucleotides for IRS-1, IRS-2, and cyclophilin, respectively. RPAs were conducted using the RPA II kit (Ambion, Inc.). Densitometry was performed with a phosphorimager.

IHC
Both IRS-1 and -2 antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) used in this study are very specific by immunoblotting, giving single bands at 175 and 185 kDa, respectively, and we have previously shown that immunohistochemistry of lactating mammary glands reveals a specific cytoplasmic staining that was absent in control immunoglobulin G incubations (32). In addition, the IRS-1 antibody gives no signal on IRS-1-null mammary tissue (data not shown). All incubations were preformed at room temperature, and all washing was performed with 0.15 M NaCl, 0.01 M Tris-HCl (pH 7.4), and 0.05% Tween 20 unless otherwise stated. Slides were cut at 3–4 µm, baked overnight at 58 C, and deparaffinized using a Shandon-Lipshaw Varistain (program 2). Heat-induced antigen retrieval was performed in 0.1 M Tris-HCl (pH 9.0) for 5 min. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide solution for 5 min. Endogenous biotin was blocked using the avidin/biotin blocking kit according to the manufacturer’s instructions (Vector Laboratories, Inc., Burlingame, CA). Slides were then incubated with IRS-1 antibody (1:400 dilution in Tris-buffered saline and 1% BSA) or IRS-2 antibody (1:200 dilution in Tris-buffered saline and 1% BSA) for 1 h, biotinylated secondary antibody (1:200) for 30 min, and then horseradish peroxidase-labeled avidin (1:200) for 30 min. As a negative control, slides were incubated with purified rabbit immunoglobulin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Detection was achieved by incubation with diaminobenzidine (DAKO Corp., Carpenteria, CA) for 15 min, followed by enhancement with 0.2% osmium tetroxide for 30 sec. Slides were counterstained with 0.05% methylene green for 30 sec, dehydrated, and mounted using cytoseal. IRS-1 and -2 were also detected by indirect immunofluorescence (IF) on paraffin sections as previously described (14, 32). BrdU was detected by direct IF on either paraffin or frozen sections as previously described (14). Visualization and analysis of IF samples was conducted as previously described (15).

Immunoblotting
Mammary tissue lysates were prepared for immunoblot analysis as previously described (26, 32). Total protein (50 µg) were electrophoresed by 8% SDS-PAGE, transferred to nitrocellulose, and then probed with antibodies specific to IRS-1 (1:1000; Upstate Biotechnology, Inc.); IRS-2 (1:1000; Upstate Biotechnology, Inc.) using previously described procedures (26, 32). The blots were also probed with an antibody to ß-actin (Sigma-Aldrich Corp., St. Louis, MO) and cytokeratin 18 (Progen, Heidelberg, Germany) to control for protein loading. After enhanced chemiluminescence images were captured using a CCD video camera (Fluorimager 8000, {alpha} Innotech, San Leandro, CA), and pixel intensity values were obtained using this machine.

Statistical analysis
For comparisons among IRS-1 genotypes or hormonal treatment groups we performed a two-tailed t test. To determine changes in IRS-1 levels throughout the estrous cycle we performed a one-way ANOVA. Mean separation was accomplished with Tukey’s highest significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct patterns of IRS-1 and -2 expression in the mammary glands of immature nulliparous mice
Mammary gland development is regulated by both hormones and growth factors. Because IRS proteins are important mediators of growth factor and cytokine signaling and are also hormonally regulated in breast cancer cells, we used IHC to examine the expression of IRS-1 and -2 in the mammary glands of immature nulliparous mice. Rigorous optimization and quality control were first performed for utilization of these antibodies by IHC (see Materials and Methods). In the mammary gland IRS-1 showed intense nonuniform staining of mammary epithelial cells (MECs; Fig. 1AGo). No staining was observed in mammary glands from IRS-1-null mice, confirming the specificity of this signal (data not shown). Every duct examined in a nulliparous mouse at this age showed a unique pattern of staining, generally with IRS-1-positive cells residing next to IRS-1-negative cells. IRS-1-positive cells appeared thinner than IRS-1-negative cells, which were more round and cuboidal. Although the IRS-1 staining appeared as if the whole cell was immunoreactive, dilution of the primary antibody showed that the majority of the signal was in the cytoplasm and that the whole cell staining probably reflected the intense staining of the cytoplasm covering the nucleus (consistent with the cytoplasmic staining of IRS-1 in Figs. 2AGo and 4BGo). However, it is possible that there may be some nuclear IRS-1, as shown recently by many groups (33, 34, 35, 36, 37). IRS-1 did not appear to be highly expressed in the myoepithelial cells surrounding the ductal epithelial cells. Analysis of TEBs (Fig. 1BGo) revealed nonuniform IRS-1 staining within the body of the TEB. IRS-1 was not found in the cap cell layer (which is ER negative) that encapsulates the TEB.



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  		Figure 1. IRS-1 and IRS-2 exhibit distinct patterns of expression in mammary glands from nulliparous immature mice. IHC of IRS-1 (top panels) and IRS-2 (bottom panels) from 6-wk-old virgin FVB mice. Images are from serial sections from ducts (A and C) and TEBs (B and D). The arrow indicates the cap cell layer. Positive staining is brown, and the counterstain is green. The scale bar indicates 50 µm.

 


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  		Figure 2. IRS expression correlates poorly with proliferation. Double IF staining was performed for BrdU (green) and IRS-1 (C, G, and I) or IRS-2 (D, H, and J; red). A–H, TEBs within the glands of a 6-wk-old virgin mouse. I and J, Ductal epithelium from a 13-wk-old mouse treated with E plus P for 2 d. Dapi staining is shown in A, B, E, and F to illustrate the structural integrity of the TEBs shown in C, D, G, and H. The scale bar in A represents 50 µm in A–D. The scale bar in E represents 50 µm for E–H. The scale bar in I represents 50 µm for I and J.

 


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  		Figure 4. IRS expression is increased during pregnancy and lactation, but lost during involution. IHC of IRS-1 (top panels) and IRS-2 (bottom panels) from mice at 16 d of pregnancy (A and D), 10 d of lactation (B and E), and 3 d of involution (C and F). Positive staining is brown, and the counterstain is green. The scale bar represents 50 µm.

 
In contrast to IRS-1, IRS-2 showed more homogeneous staining of MECs (Fig. 1CGo). IRS-2 was less intensely stained than IRS-1 and was specifically present only in the cytoplasm. Staining for IRS-2 in the TEB was similar to that in the mature duct, being more homogeneous throughout all cells. In contrast to IRS-1, IRS-2 was present in the cap cell layer.

Interestingly the nonuniform pattern of IRS-1 staining is very similar to that previously reported for steroid hormone receptors, and none of these proteins (IRS-1, ER, PR, and PRLR) is found in the cap cell layer (30, 38, 39, 40). Due to the unique nature of the pattern of IRS-1 staining, we also examined IRS-1 expression in nulliparous mice at different ages, which represent the major stages of virgin mammary gland development. At the earliest age examined (3.5 wk), when a rudimentary ductal mammary structure is present, IRS-1 appeared in a punctuate pattern in both TEBs and ducts (data not shown). This pattern was found at 4.5 and 6.5 wk and also at 10.5 wk (data not shown). Thus, the pattern of IRS-1 expression does not change during virgin mammary gland development despite the critical importance of steroid hormones in this process.

To examine whether IRS expression was associated with cells that were actively replicating DNA (S-phase), we identified IRS-positive cells by IF, and then detected cells in S-phase (an indirect measure of proliferation) by labeling with BrdU for 2 h (Fig. 2Go). Visualization of mammary tissue sections that were labeled for both IRS-1 and BrdU revealed that IRS-1 was found primarily in the most inner body cells of the TEB adjacent to the lumen, and most (but not all) of the IRS-1-positive cells were not in S-phase (Fig. 2Go, C and G). Interestingly, this area of the TEB has been found to have the highest levels of apoptosis (41). IRS-2 was also mainly present in the luminal surface of the TEB, but was detected in cap cells, where it colocalized with proliferating cells as shown in Fig. 2Go, D and H. In mature ducts, there was very little proliferation, and again, these proliferating cells were either IRS positive or negative (data not shown). Treatment of mice with E and P is known to induce dramatic proliferation within the mammary gland. We therefore colocalized proliferating cells and IRS-1 (Fig. 2IGo) and IRS-2 (Fig. 2JGo) in mature mice treated for 48 h with E and P. Analysis of IF revealed that the proliferating cells could be either IRS positive or negative. Similar results were found with IRS-1 using [3H]thymidine incorporation in tissue from mice treated with E and P for 5 d (data not shown). These data show that IRS-1 positivity is not exclusively associated with cells in S-phase and suggests that IRS-1 heterogeneity may be related to factors other than cell cycle progression.

To examine whether IRS-1 is important for mammary ductal outgrowth, we performed whole mount analysis on mammary glands from 6-wk-old virgin IRS-1 null mice (Fig. 3Go). Analysis of the whole mounts demonstrated that the overall size of the mammary fat pad was smaller (P < 0.001) in IRS-1-null mice than in their wild-type littermates (Fig. 3Go, A and B). Epithelial area, when expressed in absolute terms, tended to be smaller in IRS-1-null mice but was much more variable than fat pad area (P > 0.05). The percentage of the fat pad occupied by epithelium was virtually identical between IRS-1-null and wild-type mice (44 ± 10% and 44 ± 6%, respectively). Because the body weight of IRS-1 null mice was less (P < 0.005) than that of their wild-type siblings (12.0 ± 0.3 and 17.0 ± 0.8 g, respectively), the mammary gland measurements were adjusted for body weight (Fig. 3CGo). When adjusted for body weight, both fat pad and epithelial areas were nearly identical in IRS-1-null and wild-type mice. In addition, mammary glands of IRS-1-null and wild-type mice had similar (P > 0.05) numbers of branch points (106 ± 27 and 118 ± 31, respectively) and TEBs (9 ± 2 and 10 ± 2, respectively), suggesting that mammary gland growth is reduced in the IRS-1-null mice in proportion to overall body size, but that ductal patterning is normal. We also studied the ability of the mammary epithelium from IRS-1-null mice to proliferate in response to exogenous E plus P. The percentage of BrdU-labeled epithelial cells at 48 h after a single injection of E plus P was similar in mammary glands of IRS-1-null and wild-type mice (26.88 ± 10.46% and 29.53 ± 8.04%, respectively). Further analysis of the role of IRS-1 in pregnancy and lactation in the mammary gland has been hampered by reduced fertility in IRS-1-null mice but will be addressed in more detail in future studies with transplanted IRS-null mammary tissue.



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  		Figure 3. Normal virgin mammary ductal development in IRS-1-null mice. Whole-mount analysis of mammary glands from 6-wk-old virgin mice. A, Representative whole mount of wild-type (+/+) and IRS-1-null (-/-) mammary glands. The scale bar represents 1 cm. B, Morphometric analysis showing the total fat pad and epithelial areas within the whole mounted glands prepared from +/+ and -/- mice. C, Fat pad and epithelial areas expressed as a ratio to body weight. Bars represent the mean ± SEM (n = 6 and 4 for wild-type and IRS-1-null mice, respectively).

 
IRS distribution and abundance changes during mammary gland development
We next examined IRS expression during the latter three stages of mammary development: pregnancy, lactation, and involution (Fig. 4Go). Mammary glands from pregnant mice (d 8) showed intense IRS-1 staining that was present in nearly all MECs (Fig. 4AGo), with a more uniform pattern of staining relative to glands from immature nulliparous females (Fig. 1AGo). The uniform pattern during pregnancy was seen in both ducts and alveoli. IRS-2 levels also increased during pregnancy, and staining was present in all MECs (Fig. 4DGo), similar to that observed in ducts from nulliparous females (Fig. 1CGo). Midlactating (d 10) mammary glands showed a further dramatic increase in staining for both IRS-1 and -2 (Fig. 4Go, B and E), where IRS-1 was clearly present in all cells. In contrast, mammary glands from mice undergoing forced involution (after 10 d of lactation pups were removed from the dam, and the dam was killed 3 d later) showed negligible IRS-1 or -2 staining (Fig. 4Go, C and F). We have previously reported that this dramatic reduction in IRS-1 expression during forced involution coincides with an inability of overexpressed des(1, 2, 3)-human IGF-I to protect these glands from apoptosis (32).

IRS-1 and IRS-2 protein levels alter more than 200-fold during mammary gland development
To quantitate the developmental changes in IRS expression determined by IHC, we performed immunoblotting on lysates from mammary glands taken from mice at the same developmental time points (Fig. 5Go). IRS-1 and IRS-2 levels were weakly detectable in 6-wk nulliparous mammary glands. There was a 5- to 10-fold increase in IRS-1 and -2 levels during pregnancy, followed by a further 20-fold increase during lactation. After lactation, there was a rapid and dramatic loss of IRS-1 and -2 protein levels until IRS-1 and -2 became undetectable. Figure 5BGo shows the fold changes after correction for loading by normalization to ß-actin. To confirm that the changes in IRS levels were not simply due to changes in epithelial cell number, we also immunoblotted the samples for cytokeratin 18 (Fig. 5CGo). Similar to ß-actin, cytokeratin-18 levels did not change dramatically, and if IRS levels are corrected for this protein, then the induction at lactation remains the same, but the loss at involution becomes even more pronounced. The changes in IRS levels determined by immunoblotting mirrored the changes recorded by IHC (Figs. 1Go and 4Go), again confirming that this is not simply due to changes in cell populations within the gland.



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  		Figure 5. Immunoblot analysis of IRS expression during mammary gland development. A, Mammary glands from mice at seven different developmental stages were lysed and immunoblotted for IRS-1 (top panel), IRS-2 (middle panel), and ß-actin (bottom panel) as a loading control. B, Graph showing the pixel density values obtained from A. IRS-1 and -2 were corrected for ß-actin. Each bar represents the mean ± SEM for each of the groups shown in A. C, Immunoblot of cytokeratin 18 (K18). We show here a representative lysate from one animal per developmental time point.

 
We next examined how IRS mRNA levels were altered during the same developmental time points. For measurement of IRS mRNA levels, we pooled mammary glands (n = 3) from the developmental stages used in the previous experiment and performed ribonculease protection analysis (Fig. 6Go). Despite the dramatic 200-fold changes in IRS protein levels (Fig. 5Go), we found only minor changes in IRS mRNA levels during mammary development. The maximum change in IRS mRNA levels between any developmental stages was no more than 2-fold. We have previously shown that IRS protein levels fall dramatically during involution and that this is accompanied by insignificant alterations in mRNA levels (32). The results from our previous study were not expressed as a ratio to cyclophilin, which is known to decrease with lactation and increase with involution. Thus, the small decrease noted here probably reflects a denominator effect due to changes in the housekeeping gene that was used. Taken as a whole, these data indicate that dramatic changes in IRS protein levels during all stages of mammary development occur without equivalent changes in mRNA levels.



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  		Figure 6. IRS mRNA levels do not alter dramatically during mammary gland development. A, A ribonuclease protection assay was conducted on total RNA prepared from mammary tissue of mice at different stages of development. Each lane contains 40 µg pooled RNA representing two or three mice. Yeast tRNA served as a negative control, and undigested probe is shown. B, Graph showing quantitation of the data in A using a phosphorimager. IRS-1 and -2 were corrected for cyclophilin (cyclo).

 
IRS-1 levels are increased by E and P treatment
In breast cancer cell lines, IRS-1 and IRS-2 are regulated by E (26) and P (29). To determine whether the changes in IRS expression during pregnancy were induced by ovarian steroids, we treated ovex or intact mice with E plus P for 8 d to mimic early pregnancy. Due to the pronounced and unique pattern of IRS-1 expression and the changes in its expression associated with pregnancy, we focused on IRS-1. IRS-1 levels by immunoblotting varied dramatically between the mammary glands of intact nulliparous 6-wk-old mice (Fig. 7Go, vehicle). Treatment with E plus P resulted in a 2.5-fold increase in IRS-1 levels, although this did not reach significance (due to the wide variation of IRS-1 levels between the mice). Ovariectomy (ovex) reduced IRS-1 levels compared with those in intact mice (Fig. 7Go, vehicle vs. ovex); furthermore, IRS-1 levels now exhibited little variation between mice. Treatment of ovex mice with E plus P resulted in a 3-fold increase in IRS-1 levels (P < 0.05) similar to the effect seen in intact mice. The changes in IRS-1 levels seen by immunoblotting were also observed by IHC (Fig. 8Go). As shown previously in Fig. 1Go, IRS-1 exhibited a nonuniform pattern of staining in intact mice, with approximately 50% of cells being positive (Fig. 8AGo). In contrast, treatment with E plus P resulted in a more uniform pattern of expression, with 100% of MECs expressing IRS-1 (Fig. 8BGo). The uniformity of IRS-1 expression was seen in both alveoli and ducts. Interestingly, ovex did not alter the pattern or number of IRS-1-positive cells by IHC, although there was a general decrease in immunostaining (Fig. 8CGo). After the 8-d treatment with E plus P, there were very few TEBs remaining; however, in those present, IRS-1 changed from the nonuniform pattern of expression (Fig. 8Go, E and G) to a uniform pattern, with all body cells staining positive (Fig. 8Go, F and H). IRS-1 was still not found in the cap cells even in the presence of hormones, as these cells are ER and PR negative and thus unable to respond to hormone stimulation. To determine whether the increase in IRS-1 levels was due to E, P, or their combination, we examined IRS-1 levels in mice that were ovex and treated with E or P alone for 5 d. P had no effect on IRS-1 levels, but IRS-1 levels were induced by E treatment alone (data not shown). This was also confirmed by immunoblotting, which revealed a significant increase after 4 d of E treatment (data not shown). Analysis of IRS mRNA levels in mammary tissue from E- plus P-treated mice revealed that ovex did not alter IRS-1 mRNA levels, and ovex followed by hormone stimulation did not increase IRS-1 mRNA levels (data not shown). However, a small increase in IRS-1 mRNA levels (~0.5-fold; P < 0.05) was noted in intact mice treated with E plus P. These observations support the idea that regulation of IRS expression occurs largely at the protein level, possibly via increased protein synthesis or decreased protein degradation.



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  		Figure 7. The abundance of IRS-1 increases upon E plus P treatment of mice. Mammary glands from mice (n = 5/group) that were treated with vehicle, treated with E plus P, ovex, or ovex and treated with E plus P for 8 d were crushed, and lysates were prepared. Mammary gland lysate (50 µg/lane) was separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for IRS-1 (top panel) or ß-actin (bottom panel) as a loading control. B, Graph representing pixel density values from A. IRS-1 was corrected for ß-actin. Bars represent the mean ± SEM for five mice. *, P < 0.05.

 


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  		Figure 8. Treatment of mice with E plus P changes IRS-1 localization from nonuniform to uniform in both ducts and the body cells of TEBs. IHC for IRS-1 was performed on mice after the treatment described in Fig. 7Go. Glands are from mice after ovex (A, C, E, and G) or ovex and E plus P treatment for 8 d (B, D, F, and H). Positive staining is brown, and the counterstain is green. Images are shown of ducts (A–D) and TEBs (E–H). The scale bar represents 50 µm.

 
IRS-1 levels alter throughout the estrous cycle
The large variation in IRS-1 levels in intact nulliparous mammary glands (Fig. 7Go, vehicle) and the decrease in expression and variation after ovex (Fig. 7Go, ovex) led us to examine the regulation of IRS-1 expression throughout the estrous cycle. The stage of the estrous cycle was determined by vaginal lavage and correlated with E and P serum levels (Fig. 9AGo), uterine proliferation (Fig. 9BGo), and IRS-1 levels in the mammary gland (Fig. 9CGo). Serum E levels were highest during estrus, in contrast to P levels, which were highest during diestrus. The trends we observed are similar to those previously found during the mouse estrous cycle, although the estradiol levels we measureed were slightly lower (2–8 pg/ml) than those in other reports (42). This may be due to the difference in the strain of mouse used (most other studies used outbred strains) or to the insensitivity of the RIA kit. Proliferation in the uterus was highly dependent upon the stage of the estrous cycle and showed a cyclicity similar to that previously reported by others (43). Total levels of IRS-1 by immunoblotting changed more than 4-fold during the estrous cycle. The changes in IRS-1 levels throughout the estrous cycle were highly significant (P < 0.01). Analysis of IRS-1 by IHC revealed no dramatic changes in the pattern or number of positive cells (data not shown). This suggests that this change in total IRS-1 levels may not occur through an increase in the number of positive cells, but via an increase on a per cell basis. Overall, these data show regulation of IRS-1 expression throughout the estrous cycle and are consistent with hormonal regulation during the cycle, which is lost after ovex (Fig. 7Go).



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  		Figure 9. IRS-1 abundance is highest in the mammary gland during metestrus. The stage of the estrous cycle was determined in 6-wk-old female mice and correlated with serum E and P levels (A), uterine proliferation (B), and IRS-1 expression (C). Serum E and P were measured using RIA. Each point represents the mean ± SEM. Uterine proliferation was measured by immunostaining mammary glands for BrdU and counting a minimum of 300 glandular, luminal, and stromal cells. Data are represented as the percentage of positive BrdU cells, and each point represents the mean ± SEM. IRS-1 was measured by immunoblotting as described in Fig. 6Go and was corrected for ß-actin. Bars represent the mean ± SEM. Bars with different superscripts differ significantly (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe here the first report on the distribution and hormonal regulation of IRS-1 and -2 expression in the mouse mammary gland. IRS-1 showed a nonuniform pattern of expression in virgin ductal epithelium and was not found in cap cells. In contrast, IRS-2 was distributed evenly along ductal epithelium and was present in cap cells. IRS-1 abundance was increased by ovarian steroids (albeit possibly in an indirect manner), and levels changed throughout the estrous cycle. IRS protein abundance increased dramatically during pregnancy and lactation, and the change in these levels occurred mainly at the protein, rather than the mRNA, level. We also present data suggesting that IRS-1 plays a role in determining mammary gland size, just as it does in determining body size, but that IRS-1 is not required for normal mammary ductal patterning in virgin mice.

The pattern of IRS-1 expression in the mammary ducts of immature virgin mice is similar to that reported for certain growth factors, e.g. IGF-II (44), and hormone receptors such as ER (38) and PR (39). However, our data suggest that the IRS-1 pattern is not governed by these factors, as their patterning does not follow the same temporal regulation as IRS-1. Given the dramatic increase in IRS expression during pregnancy and lactation, the best candidate markers that may colocalize with IRS-1 are the milk protein genes. The expression of at least two of the milk protein genes, ß-casein and whey acidic protein, actually mimics most closely that observed for IRS-1 in that they are nonuniformly expressed in mammary ducts from virgin mice and become uniformly expressed during pregnancy and lactation (45). These observations suggest that IRS-1 expression within the mammary gland may be more linked to epithelial cell differentiation than to proliferation and ductal development. This is consistent with normal virgin mammary gland development in IRS-1-null mice (discussed below).

Despite the fact that ER or PR do not seem to control the pattern of IRS-1 expression in ductal epithelium in virgin mice, our data indicate that ovarian and pituitary hormones can up-regulate IRS expression. In this study we found that ovex reduced IRS-1 levels, whereas E plus P increased levels. In a separate set of studies we found that PRL or E alone, but not P, was able to induce levels of IRS-1 and -2 (46). Considering that E increases circulating PRL levels via the pituitary (47), it is possible that E induction of IRSs may be indirect via PRL. The regulation of PRLR through the estrous cycle (48, 49) may also be mediating the changes in IRS-1 levels. The observation that IRS-1 and -2 expression was highest during lactation is also consistent with the idea that PRL plays a role in the regulation of these proteins. The developmental regulation of IRS-1 and -2 expression is similar to that of the milk protein genes (50, 51) and mimics clustered gene expression profiles such as milk, protein biosynthesis, and protein transport (52). The expression of the milk protein genes is known to require PRL (53). However, the fact that changes in IRS expression occurs predominantly at the protein level suggests that if PRL is regulating IRS expression, the mechanism differs from that specific to other milk protein genes.

The discordance between changes in IRS proteins and mRNA abundance is a recurrent theme throughout these and other studies and is in stark contrast to many reports of transcriptional regulation of IRSs by steroid hormones in breast cancer cells (26, 27, 28). Despite dramatic changes in IRS protein levels, we never detected more than a 2-fold change in IRS levels during hormonal or developmental changes in the mammary gland. Consistent with this finding, a comprehensive analysis of gene expression of the mammary gland during different developmental stages found little or no variation in IRS-1 expression (IRS-1 Affymetrix microarray data available at www.abramsoninstitute.org/chodoshdata.html). It is possible, therefore, that there are changes in IRS mRNA translation or changes in IRS protein stability. Although there have been no previous studies on translation rates of IRS-1 mRNA (and the IRS-1 gene does not have a long 5'-untranslated region), there is evidence for posttranslational degradation of IRS-1 protein via the proteasome (54, 55).

Regarding the function of IRS-1 in the mammary gland, our data suggest that the loss of IRS-1 results in reduced mammary fat pad size, which is consistent with previous studies showing reduced overall body size in both IRS-1 null mice and Drosophila mutants (56, 57). In the Drosophila CHICO mutant, the reduced body size is due to both decreased cell size as well as decreased cell proliferation (56). The size reductions observed in the fat pads of the IRS-1-null mice were of the same magnitude as the decrease in body weight and were therefore not surprising. However, because IRS-1 has been demonstrated to be a central mediator of IGF-dependent proliferation in cultured breast cancer cells lines (58), we were surprised by the presence of normal mammary ductal development in IRS-1-null mice.

A possible explanation for the normal mammary ductal development in IRS-1-null mice is that IRS-2 may have compensated for the loss of IRS-1 expression. In fact, IRS-2 was first identified by its activation and phosphorylation in IRS-1-null mice (57). In contrast, IRS-2 may be the major signaling intermediate for IGF-IR and IGF-I in the TEB, as IRS-2 was found in the cap cells [which show reduced proliferation in IGF-IR-null mice (12)] and showed a higher degree of colocalization with BrdU labeling than did IRS-1. Although other studies have suggested that IRS-2 is not a major regulator of proliferation (59), and IRS-2-null mice do not exhibit growth retardation (23), the mammary gland may be an exception where it is IRS-2 that is important for proliferation.

Another possible reason for the normal ductal development in mammary glands of IRS-1-null mice is that IRS-1 may be more important for other stages of mammary gland development, such as pregnancy and lactation. Although our attempts to test these hypotheses have been hampered by fertility problems in the IRS-1-null mice, we did observe that BrdU labeling was similar in IRS-1 wild-type and null mammary epithelium 48 h after the administration of exogenous E plus P. This hormonal treatment is known to stimulate mammary epithelial cell proliferation similar to that which occurs during pregnancy (60). As such, these data suggest that IRS-1 may not be required for normal pregnancy-dependent mammary gland development. However, the most dramatic changes in IRS expression occurred during lactation and involution and coupled with our previously published observation that exogenous PRL can also induce IRS-1 expression support the hypothesis that IRS-1 may be important for lactogenesis. This hypothesis also has some support in the clinical observations that polycystic ovary syndrome in women is associated with polymorphisms in IRS-1 and -2 (61) and with an inability to undergo normal lactogenesis (62). Studies of lactation are not feasible in transplant experiments and will be best tested using tissue-specific, gene-targeting models for IRS-1 and -2 as they become available.

In summary, our data indicate that IRSs exhibit unique patterns of expression in ductal epithelium, and levels increase greatly during pregnancy and lactation. Our studies to date suggest that whereas loss of IRS-1 reduces mammary fat pad size (similar to other organs and body weight), IRS-1 expression is not critical for normal ductal development (branching and TEB number) or for pregnancy-induced proliferation. Further studies of the role of IRSs in lactation and cell differentiation are warranted and underway.


   Acknowledgments
 
We thank Ms. Nicole Lawrence for her technical assistance with morphometry and BrdU analysis of the IRS-1 null mice.


   Footnotes
 
This work was supported in part by federal funds from the NIH [DK-52197 (to D.L.H.) and CA-94118 (to A.V.L.)].

1 Current address: Lactation and Mammary Gland Biology Group, Department of Animal Science, University of Vermont, Burlington, Vermont 05405. Back

Abbreviations: BrdU, Bromodeoxyuridine; E, estrogen; ER, estrogen receptor; ERKO, estrogen receptor {alpha} knockout; GHR, GH receptor; IB, immunoblotting; IF, immunofluorescence; IGF-IR, IGF-I receptor; IHC, immunohistochemistry; MEC, mammary epithelial cell; ovex, ovariectomized, ovariectomy; P, progesterone; PR, progesterone receptor; PRLR, PRL receptor; RPA, ribonuclease protection assay; TEB, terminal end bud.

Received October 23, 2002.

Accepted for publication February 26, 2003.


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