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Department of Neural and Behavioral Sciences, Penn State College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Teresa L. Wood, Ph.D., Department of Neural and Behavioral Sciences H109, Penn State College of Medicine, P.O. Box 850, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: twood{at}psu.edu.
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Introduction |
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Although it is unclear whether circulating or locally produced IGFs are most critical for mammary gland development, several lines of evidence suggest that local expression of IGF-I and IGF-II is highly regulated. Treatment of mammary tissue with GH induces stromal expression of IGF-I mRNA in a dose-dependent manner (18, 19, 20). Recent studies from several laboratories strongly support the hypothesis that IGF-II is uniquely regulated by prolactin and is a critical mediator of prolactin-induced alveolar differentiation (3, 21, 22). Previous data from our own laboratory demonstrated that IGF-I, IGF-II, and the IGF-IR mRNAs are expressed in distinct patterns during postnatal periods of mammary development in mice, further supporting their differential regulation and function (8, 9).
Members of the high-affinity IGF binding protein (IGFBP) family also are present in mammary and breast tissue of mammals (23, 24, 25, 26, 27, 28), although data on their specific expression and function in mammary development is limited. The IGFBPs have a major role in modulating the bioavailability of the IGFs (for reviews see Refs.28, 29, 30, 31). In circulation or in the extracellular environment, IGFBPs prolong the half-life of the IGFs. In addition, free IGFs in solution preferentially bind to the IGFBPs and thus are inhibited from binding to their receptors. In contrast, association of the IGF-IGFBP complex with the cell surface or extracellular matrix results in decreased affinity of the IGFBPs for the IGFs. This resulting loss in IGF affinity allows release of the ligands for potential interaction with the IGF receptors.
There are limited data on the roles of the IGFBPs in normal development of the mammary gland with the exception of IGFBP-5. Several studies support proapoptotic functions for IGFBP-5 during the process of involution (24, 25, 26, 28, 32, 33), and a recent study on mammary tissue from mice carrying a heterozygous deletion of the prolactin receptor suggests that IGFBP-5 expression is normally repressed by prolactin during lactation (34). Prolactin regulation of IGFBP-5 is consistent with other recent data demonstrating that loss of the CAATT-enhancer binding protein-ß transcription factor leads to misexpression of the prolactin receptor, progesterone receptor, and IGFBP-5 in mammary epithelial cells (21). However, other than the limited data on IGFBP-5 expression, little is known about expression of the IGFBPs during the postnatal growth stages of ductal elongation and alveolar differentiation. The goal of the current study was to determine the developmental patterns of IGFBP expression during normal development of the mammary gland. Here we report the patterns of IGFBP mRNA expression and the levels of IGFBP expression during postnatal stages of mammary gland development in the mouse. We provide data demonstrating that members of the IGFBP family have distinct patterns and levels of expression that correlate with specific stages in the progression of mammary epithelial growth and maturation, supporting a role for these molecules in growth and differentiation of mammary tissue.
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Materials and Methods |
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-35S]UTP and the enhanced chemiluminescense detection system were from NEN Life Science Products (Boston, MA). Protease inhibitor cocktail was obtained from Sigma Chemical Co. (St. Louis, MO). Unless otherwise specified, standard laboratory reagents were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co.
Animals and tissue isolation
All animal experimentation protocols were approved by Penn State Hershey Medical Center Institutional Animal Care and Use Committees and follow the National Institutes of Health guidelines. C57BL6/J female virgin mice were killed at 4, 6, 8, 10, or 12 wk of age. For the pregnancy ages, adult C57BL6/J female virgin mice were placed in matings and checked daily for vaginal plugs. The morning of appearance of the vaginal plug was considered d 0.5. Pregnant dams were killed on d 8, 13, and 18 of pregnancy. Additional pregnant mice were allowed to deliver their pups for analyses of glands during lactation and involution. For lactation and involution studies, litters were culled or pups were cross-fostered on d 1 to normalize to eight pups per dam. Lactating glands were obtained from dams taken at 2 and 10 d of lactation. For the involution study, pups were removed from the dams after 10 d. The dams were then killed and glands removed at 2, 4, and 6 d after pup removal. In all studies, the number four abdominal glands were removed and flash frozen in liquid nitrogen for protein isolation or for cryostat sectioning for in situ hybridization studies. At least three glands from individual mice were analyzed for mRNA expression by in situ hybridization and for protein expression at each of the developmental time points. Frozen cryostat sections (10–15 µm) were mounted onto Superfrost Plus slides (Fisher) and stored at –80 C until use for in situ hybridization.
Whole-mount staining
The number four inguinal mammary glands were removed and placed in tissue cassettes between Whatman filter paper and sponges and fixed overnight in 3:1 100% ethanol/glacial acetic acid. The glands were washed three times in acetone for 1 h and 30 min in 100% ethanol and 95% ethanol and subsequently stained overnight in hematoxylin [0.13 g FeCl3, 13.5 ml dH20, 1.74 ml stock hematoxylin (10% hematoxylin in 95% ethanol) 200 ml 95% ethanol, brought to pH 1.25 with concentrated HCl). After staining, the cassettes were rinsed with tap water, and the glands were then dehydrated with acidic 50% ethanol two to three times for 1 h each. The 30-min washes were then performed with 70 and 95% ethanol, followed by 100% ethanol. Glands were placed in xylene for 20 min, removed from cassettes, coverslipped, and mounted with Cytoseal XYL (Stephens Scientific Division of Richard-Allan Scientific, Kalamazoo, MI).
Protein isolation and Western immunoblotting
Proteins were isolated by pulverizing the frozen glands in liquid nitrogen and homogenizing in protein extraction buffer (150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA, pH 8.0; 1 mM EGTA; 1% Triton X-100; 0.5% Nonidet P40; 100 mM NaF; 10 mM Na pyrophosphate; 10 mM Na orthovanadate; 2 mM phenylmethylsulfonyl fluoride; and 1:100 dilution of protease inhibitor cocktail). Samples were placed on ice for 30 min and then centrifuged at 600 rpm for 20 min at 4 C, followed by ultracentrifugation at 40,000 rpm for 45 min at 4 C. Protein concentrations were determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA).
Equal amounts of protein (50 µg/lane) were run on precast 12% Bis-Tris gels using the NuPage gel system (Invitrogen, Carlsbad, CA) along with rainbow molecular weight markers (Amersham Life Sciences, Piscataway, NJ). Serum samples (0.05 µl) from 4, 6, 8, and 10 wk of age were used to determine circulating levels of the IGFBPs. The proteins or serum were transferred electrophoretically from the gels to nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH). The membranes were dried and visualized with Ponceau S staining (Sigma Chemicals) to ensure an equal amount of protein loading. Membranes were blocked with 5% nonfat dry milk in 1x Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and incubated overnight at 4 C with primary antibodies diluted in TBST with 5% nonfat dry milk (IGFBP-2, 1:250; IGFBP-1, 1:250; IGFBP-4, 1:250; IGFBP-5, 1:250; ß-actin, 1:1000). After overnight incubation, the blots were washed with TBST (three times for 5 min each) and incubated with goat antirabbit-HRP (1:5000) or, for ß-actin, goat antimouse-HRP (1:500) in TBST with 5% nonfat dry milk for 1 h at room temperature. The blots were then washed with TBST (three times for 5 min each) and TBS (5 min) and visualized using the NEN Life Science Products Renaissance Chemiluminescence kit.
IGFBP levels were quantified on the immunoblots by determining the relative optical density after normalization to ß-actin levels. A standard protein sample was run on each gel and was used for normalization of ß-actin across samples on different gels. Relative optical densities were obtained using NIH Image, and the results are expressed as mean ± SE of a minimum of three different samples at each age.
In situ hybridization
[35S]-labeled RNA transcripts for IGFBPs 1–6 were synthesized from linearized plasmids containing partial cDNA inserts to rat [IGFBP-1 (35), -2 (36), -3 (37), -4 (38), -6 (39)] or mouse [IGFBP-5 (40)] sequences. Linearized DNAs were incubated with Sp6, T7, or T3 RNA polymerase in the presence of CTP, GTP, ATP, and [35S]UTP, according to standard RNA transcription protocols (Promega Corp., Madison, WI). The resulting RNA transcripts were purified on Sephadex G-50 (Roche Molecular Biochemicals, Indianapolis, IN) and used without hydrolysis.
In situ hybridizations were performed as previously described (8). After hybridizations, slides were coated with Kodak NTB-2 liquid autoradiograph emulsion diluted 1:1 with dH2O (Eastman Kodak, Rochester, NY), and sections were exposed at 4 C in a desiccated lightproof box. After either 6 or 9 wk of exposure, sections were developed (Kodak D19 for 3.5 min at 15 C), rinsed for 1 min in distilled water, and fixed (in Kodak fixer for 10 min at 15 C). After rinsing in running tap water for 30 min, sections were counterstained with contrast red, dehydrated through ethanols and xylenes, and mounted with Permount mounting media (Fisher).
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Results |
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). At 4 wk of age, growth of the ductal tree in the mammary gland was apparent as a rudimentary structure that had not yet reached the lymph node (Fig. 1A). By 8 wk of age, the leading edge of the ductal tree had extended beyond the lymph node, and the developing structures had expanded laterally (Fig. 1B). The growing ductal structures reached the limits of the fat pad between 10 and 12 wk of age in this strain of mice (Fig. 1C).
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). By the time lactation began after parturition, the fat pad was entirely filled with the milk-producing structures, and the loss of adipose tissue was apparent (Fig. 1E). After pup removal after 10 d of lactation, the mammary gland underwent forced involution that resulted in a loss of the lobuloalveolar ductal structures (Fig. 1F).
IGFBP expression during postnatal mammary development
Protein expression levels for IGFBPs 1–5 were investigated in the developing mammary gland using Western immunoblot analysis (Figs. 2 and 3). Because IGFBP-6 expression was low in mammary tissue during postnatal development, it was not included in the protein analysis. IGFBPs 1–5 were detectable in mammary tissue throughout pubertal growth stages from 4 wk through 12 wk of age (Fig. 2, A and B). Protein levels of IGFBP-2, -3, -4, and -5 were relatively stable across pubertal development. Levels of IGFBP-1 protein were highest between 4 and 6 wk of age at the onset of pubertal ductal growth (Fig. 2, A and B).
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). IGFBP-1, -2, and -3 were detectable in serum using 0.5 µl serum per lane at each age, whereas IGFBP-5 was undetectable in serum. Serum levels of IGFBP-2 and -3 showed no consistent variation from 4–10 wk of age, similar to what was seen for tissue levels of these proteins. However, serum IGFBP-1 levels increased from 4 wk through 10 wk of age, the reverse of what was seen at the tissue level (Fig. 2C). IGFBP-4 protein was detectable in serum only when 2.5 µl serum was analyzed and showed no difference in levels from 4–10 wk of age (data not shown).
IGFBP mRNA expression during pubertal development
To determine the local expression of IGFBPs 1–6 during postnatal mammary development, in situ hybridization was used to delineate mRNA expression patterns for the IGFBPs in sections from mammary glands at 4, 6, 8, 10, and 12 wk postnatal ages (Figs. 3 and 4; Table 1). Both IGFBP-1 and IGFBP-6 mRNA expression was low to undetectable in the mammary gland during postnatal development even after 12 wk exposure of the hybridized tissue to autoradiographic emulsion. Low levels of IGFBP-1 mRNA expression were observed in the epithelial cells, especially of the developing TEBs (data not shown). IGFBP-2, -3, and -5 mRNAs were the most prominent IGFBPs expressed in the TEBs; however, each had a distinct pattern of expression (Fig. 3). IGFBP-2 mRNA expression was detectable in the leading edge or cap cells of the TEB but was most prominent at the trailing edges of the TEB in cells flanking the TEB (Fig. 3A). IGFBP-3 mRNA was localized to the outer layer of epithelial cells consistent with expression in the cap cells in the TEBs (Fig. 3B). IGFBP-5 mRNA was localized to the epithelial cells within the body of the TEB (Fig. 3C).
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; Table 1). IGFBP-2 and -4 mRNAs were expressed predominantly in the stromal compartment, whereas IGFBP-3 and -5 were expressed in both the epithelial and stromal compartments. IGFBP-2 mRNA expression during postnatal development was seen in stromal cells immediately surrounding the ductal structures in a nonuniform pattern (Fig. 4, A–C). IGFBP-2 mRNA was seen in cells surrounding sites of branching (Fig. 4A) and in selected stromal cells along the ducts throughout pubertal ages (Fig. 4, A–C).
Expression of IGFBP-3 mRNA was localized to the outer layer of cells in the ductal epithelium (Fig. 4, D–F). This distinct pattern was noted at each time point investigated during pubertal development and was unique for IGFBP-3 in comparison with the other IGFBPs.
IGFBP-4 mRNA expression was localized to the stromal cells surrounding the epithelial structures during postnatal development (Fig. 4, G–I). IGFBP-4 expression appeared similar to that of IGFBP-2 at 4 wk of age (see Fig. 4, A and G); however, IGFBP-4 was less restricted than that of IGFBP-2 and appeared in cells throughout the stroma (Fig. 4G). By late pubertal stages, IGFBP-4 mRNA was less apparent in cells closely apposed to the ducts but remained prominent in cells throughout the stroma (Fig. 4I).
IGFBP-5 mRNA was expressed in the greatest abundance at all developmental ages investigated relative to the other IGFBPs based on intensity of the in situ hybridization signal after equivalent exposure times (Fig. 4, J–L). At the onset of puberty (4 wk of age, Fig. 4J), IGFBP-5 mRNA was highly expressed in the inner layer of luminal cells of the developing ducts. This pattern persisted at 8 (Fig. 4K) and 12 (Fig. 4L) wk of age. At the later ages, IGFBP-5 mRNA was seen in a nonuniform pattern along the luminal epithelium (Fig. 4, K and L).
IGFBP levels in mammary tissue during pregnancy, lactation, and involution
In contrast to the pubertal ages, protein expression of each IGFBP varied considerably during pregnancy, lactation, and forced involution (Fig. 5). IGFBP-2 and IGFBP-5 protein levels decreased by approximately 50% from late pregnancy ages to d 10 of lactation. Levels of both proteins increased during forced involution so that by d 6 of involution, levels of IGFBP-2 and -5 were nearly equivalent to the levels seen during pregnancy (Fig. 5, A and B). The decrease in protein levels for IGFBP-2 and -5 during lactation might be accounted for by the significant increase in milk protein expression at this time; however, it is interesting that the other IGFBPs examined did not show the same dramatic decrease in expression during lactation stages (Fig. 5). Although IGFBP-4 was decreased in lactation-stage glands compared with early and mid-pregnant glands, the decrease in this IGFBP occurred between d 13 and 18 of pregnancy, earlier than the decrease in IGFBP-2 and -5 (Fig. 5, A and B). In addition, IGFBP-4 levels did not increase during the 6 d of forced involution (Fig. 5, A and B). IGFBP-1 protein expression decreased progressively during pregnancy and lactation; the expression of IGFBP-1 at lactation d 10 was 50% of that at pregnancy d 8 (Fig. 5, A and B). However, from lactation d 2 through involution, IGFBP-1 protein levels gradually increased to the levels seen before pregnancy. In contrast to the other IGFBPs, IGFBP-3 protein expression remained relatively stable throughout pregnancy and lactation stages and then decreased by 50% during involution (Fig. 5, A and B).
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; Tables 2 and 3). IGFBP-2 mRNA was primarily located in the stroma surrounding the epithelial ductal structures during early pregnancy (pregnancy d 8; Fig. 6A) and then in specific cells around the alveoli during late pregnancy (pregnancy d 18; Figure 6B) and lactation (postpartum d 2; Fig. 6C). IGFBP-2 mRNA expression was not detectable above background during involution (6 d forced involution; Fig. 6D).
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, E and F). The lowest levels of IGFBP-3 mRNA expression were seen during lactation and involution when compared with the earlier pregnancy stages.
IGFBP-4 mRNA expression was detected in the stromal compartment surrounding both the primary ductal tree and also around the developing lobuloalveolar structures during pregnancy ages (Fig. 6, I and J). During lactation, IGFBP-4 mRNA expression remained in the stromal compartment but in a restricted pattern (Fig. 6K), similar to that observed for IGFBP-2 at this stage (compare with Fig. 6C). IGFBP-4 mRNA expression decreased during forced involution.
IGFBP-5 mRNA levels were the most abundant when compared with the other IGFBPs based on the in situ hybridization signal with equivalent exposure times. IGFBP-5 mRNA expression was located in the epithelial cells of both the primary ducts and the developing lobuloalveolar structures during pregnancy (Fig. 6, M and N). During pregnancy stages and again during forced involution, the intensity of the IGFBP-5 signal resulted in masking of the epithelial structures in histological sections, even after relatively short exposure times. IGFBP-5 mRNA levels decreased in the alveolar structures by lactation d 2 and remained low during lactation (Fig. 6O). IGFBP-5 mRNA expression increased during forced involution when it was apparent throughout the epithelial structures (Fig. 6P).
Consistent with the protein data, IGFBP-1 and IGFBP-6 mRNA expression was low during pregnancy, lactation, and involution in comparison with the other IGFBPs based on autoradiographic film and emulsion exposure times. IGFBP-1 mRNA expression was detectable only during the early stages of pregnancy in the stromal compartment (data not shown). IGFBP-6 mRNA expression was low to undetectable at each of the ages investigated.
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Discussion |
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Serum vs. local IGFBP expression
The levels of IGFBPs 1–5 were relatively stable throughout pubertal ages during ductal extension and branching. IGFBPs 2–5 mRNAs also were expressed at easily detectable levels in mammary tissue during pubertal stages, suggesting that the protein expression levels represent local tissue expression. IGFBP-5 was not detectable in serum during pubertal ages, consistent with previous data showing low to undetectable levels of this binding protein in rodent serum (for reviews see Refs. 29 and 30). Thus, IGFBP-5 levels were the result of mammary tissue expression. IGFBP-2, -3, and -4 were detected in serum during pubertal ages. Therefore, it is unclear from the protein analysis alone whether tissue expression represents the major source of these binding proteins in the developing mammary gland. However, IGFBP-2, -3, and -4 also were expressed locally during pubertal growth.
The only IGFBP for which there was significant serum levels and very limited local mRNA expression in mammary tissue was IGFBP-1. Thus, the limited local expression of IGFBP-1 does not preclude a more significant role for this IGFBP in mammary growth. However, whereas protein levels for IGFBPs 2–4 in mammary tissue and serum showed consistent levels throughout pubertal growth stages, the protein levels of IGFBP-1 in mammary tissue and serum were inversely related. IGFBP-1 levels in mammary tissue extracts were highest during early pubertal growth but, in serum, were highest in late pubertal stages. These results suggest that the local expression of IGFBP-1 in mammary tissue is distinctly regulated from serum IGFBP-1 and that serum IGFBP-1 is not the major source of IGFBP-1 tissue protein.
Endogenous expression of IGFBPs during pubertal and pregnancy growth stages
The mRNA expression patterns of the IGFBPs during pubertal and pregnancy-induced mammary development were distinct in their cell-type-specific expression and showed differences in their localization to epithelial and stromal compartments. Although IGFBP-3 and -5 were found in both epithelial and stromal compartments, IGFBP-2 and -4 were predominantly stromal in their mRNA expression patterns. However, even when IGFBPs were expressed in similar compartments, each IGFBP had a distinct mRNA expression pattern. The most abundant of the high-affinity IGFBPs was IGFBP-5. This was determined both by quantitative analysis at the protein level as well as by hybridization exposure times at the mRNA level. IGFBP-5 mRNA was most prominent in the epithelium throughout postnatal growth stages; however, IGFBP-5 mRNA also was observed in isolated cells in the stroma at these ages. Previous studies on IGFBP-5 in mammary tissue have focused on stages of involution when there is evidence that it has proapoptotic functions for tissue remodeling (25, 26, 28). It is interesting that IGFBP-5 mRNA was observed in the body of the TEB where significant apoptosis has been described as the ductal lumen is formed (41, 42). The expression of IGFBP-5 in the TEB body is consistent with a proapoptotic role in these cells. However, the high expression of IGFBP-5 throughout the ductal epithelium during pubertal and pregnancyinduced epithelial growth suggests additional roles for this IGFBP.
Similar to IGFBP-5, IGFBP-3 mRNA was expressed in both the epithelial and stromal compartments. However, in contrast to IGFBP-5, IGFBP-3 expression was detected in the outer epithelial cells in the TEB consistent with expression in the cap cells. In the ductal structures, IGFBP-3 was specifically localized to the outer layer of epithelial cells. IGFBP-3 is reported to have both IGF-dependent and IGF-independent actions on human breast epithelial cells, both of which appear to be predominantly growth inhibitory (43).
In contrast to IGFBP-3 and -5, IGFBP-2 and -4 mRNA expression was predominantly restricted to the stromal compartment. IGFBP-2 mRNA was localized primarily around the trailing edge of the TEB and in a nonuniform pattern in stromal cells along the ducts. Two populations of migrant stromal cells, macrophages and eosinophils, are recruited to areas around the TEBs during mammary development (44, 45). Specifically, macrophages are located along the trailing edge of the TEB in areas that are more differentiated, whereas eosinophils are located surrounding the undifferentiated cap cell layer at the leading edge of the TEB. The tissue-specific localization of the macrophages and eosinophils is necessary for proper mammary development because loss of these cells in mice carrying deletions of genes essential for their production or recruitment results in defects in TEB formation and ductal growth (44). It is of interest that mRNA production of IGFBP-2 correlates with the localization described for macrophages along the trailing edge or neck of the TEBs (44).
IGFBP-4 also was expressed in the stromal compartment but was more widespread than IGFBP-2. The widespread expression of IGFBP-4 in the stroma around the growing epithelial structures during both pubertal and pregnancy stages is of interest because this IGFBP is thought to have predominantly inhibitory effects on the IGFs. Taken together, these data suggest a potential role for IGFBP-4 in maintaining a boundary between stromal and epithelial IGF actions.
IGFBP expression during lactation and involution
Protein levels of the IGFBPs in mammary tissue varied greatly through lactation and involution stages. With the exception of IGFBP-3, IGFBP levels decreased during active milk secretion compared with pregnancy levels. The decrease in IGFBP during lactation also correlated with a low level of mRNA expression for IGFBPs 2–5 in the alveoli during lactation ages. These data suggest that several of the IGFBPs may be important for alveolar proliferation and/or differentiation during pregnancy and that decreased expression of these molecules is important for milk production or secretion. It is possible that the decrease in the IGFBPs during lactation allows for maximal effect of the IGFs during this time. This idea is consistent with our previous study showing an increase in IGF-I mRNA expression in the alveoli at the end of pregnancy (8). As mentioned previously, recent evidence suggests that IGFBP-5 is negatively regulated by prolactin (24, 34). Moreover, a recent study demonstrated that IGFBP-5 mRNA expression levels are decreased in mammary glands of mice carrying a null mutation in the transcription factor CAATT-enhancer binding protein-ß, which also results in induction of the prolactin receptor (21). These data suggest potential mechanisms for the decrease in IGFBP-5 during lactation. In contrast to IGFBP-5, IGF-II is induced by prolactin in mammary tissue (3, 22). Thus, the induction of IGF-II and repression of IGFBP-5 by prolactin suggests a coordinate regulation of IGF-II bioavailability during pregnancy and lactational stages.
Involution of the mammary gland is characterized by tissue remodeling such that the lobuloalveolar structures that were functioning for milk production during lactation regress, and the ductal tree more closely resembles that of the adult virgin state. Protein levels of IGFBP-1, -2, and -5 increased significantly during involution. For IGFBP-5, this increase in protein correlated with increased mRNA expression throughout the epithelial cells in the involuting gland. These data are consistent with previous data suggesting a role for IGFBP-5 in apoptosis and remodeling during involution (25, 26, 28). The increase in IGFBP-1 and -2 during involution suggests additional roles for these IGFBPs in the remodeling process. However, it is of interest that neither IGFBP-1 nor -2 mRNAs were obviously increased in the involuting gland, suggesting that serum levels may be the major source of these proteins at this stage. The function of the IGFBPs in involution may include inhibition of IGF survival actions on epithelial cells because involution is inhibited in transgenic mice overexpressing IGF-I or IGFBP-3 (16, 17). However, a recent report showing that IGF signaling pathways are dramatically down-regulated during forced, but not natural, involution, suggests that the increases in IGFBPs during forced involution are unlikely to have a major role in blocking IGF survival actions (46). Alternatively, additional evidence supports the hypothesis that IGFBP-5 produced by the epithelial cells during involution contributes to tissue remodeling through IGF-independent actions (25, 26, 28).
In conclusion, the data presented here demonstrate expression of the six high-affinity IGFBPs in the murine mammary gland during postnatal phases of rapid epithelial expansion and remodeling. Our results also demonstrate the localization of IGFBP mRNAs to specific cellular components of either stromal or epithelial structures, strengthening the idea that the IGFBPs have distinct functions in the mammary gland. Because most of the IGFBPs have the ability to bind either to the cell surface or to the extracellular matrix, it is likely that their prominent expression in and surrounding the epithelial structures positions them to modulate epithelial-stromal interactions that could include both IGF-dependent and IGF-independent actions.
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Acknowledgments |
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Footnotes |
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Abbreviations: HRP, Horseradish peroxidase; IGFBP, IGF binding protein; IGF-IR, primary IGF signaling receptor; TBS, Tris-buffered saline; TBST, TBS with Tween 20; TEB, terminal end bud.
Received December 2, 2003.
Accepted for publication January 21, 2004.
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