ARTICLE |
Correspondence to: Jonathan M. Shillingford, Lab. of Genetics and Physiology, NIDDK, NIH, Bethesda, MD 20892. E-mail: jonshi@helix.nih.gov
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Summary |
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Through the use of transgenic and gene knockout mice, several studies have identified specific genes required for the functional development of mammary epithelium. Although histological and milk protein gene analyses can provide useful information regarding functional differentiation, they are limited in their ability to precisely define the molecular lesions. For example, mice that carry a mutation in one of the subunits of the IB kinase, IKK
, cannot lactate despite the presence of histologically normal alveolar compartment and the expression of milk protein genes. To further define and understand such lesions on a molecular level, we sought evidence for proteins that are differentially expressed during mammary gland development with a view to generating a tissue proteotype. Using database screens and immunohistochemical analyses, we have identified three proteins that exhibit distinct profiles. Here, using mouse models as test biological systems, we demonstrate the development and application of mammary tissue proteotyping and its use in the elucidation of specific developmental lesions. We propose that the technique of proteotyping will have wide applications in the analyses of defects in other mouse models.
(J Histochem Cytochem 51:555565, 2003)
Key Words: proteotyping, mammary, development, immunohistochemistry, markers
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Introduction |
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The development of mammary epithelium is mediated by the action of peptide and steroid hormones (
Using knockout mouse models, others and we have defined an essential role for the prolactin receptor (PRLR), Jak2signal transducer and activator of transcription 5 (Stat5) pathway in pregnancy-mediated mammary gland development (
The most common techniques currently used in mouse mammary gland biology include analyses of proliferation, evaluation of histological sections, and determination of milk protein gene expression. Despite the usefulness of these methods, they are limited in their scope and application. In particular, the expression of some milk proteins is directly downstream of the PRLRJak2Stat5 pathway (B) pathway are not able to support their pups at parturition, they exhibit expanded alveoli, evidence of lipid droplets, and only a limited reduction in milk protein gene expression (
We have therefore developed a technique to which we refer as tissue proteotyping. This method uses antibodies that exhibit distinct expression profiles in different mammary epithelial cell types. Using an immunohistochemical approach and three defined antibodies, we have identified a mammary proteotype that can be used to monitor relative changes in epithelial cell type and to assess the attainment and maintenance of secretory cell function. Using mice defective in PRLRJak2Stat5, NFB, and inhibin ßB signaling, and mice with a conditional deletion of the Stat3 gene, we demonstrate the use of tissue proteotyping as a viable means to assess mammary gland development. We propose that the application of this defined mammary proteotype will prove useful in the characterization of mammary epithelial defects observed in other mouse models. Furthermore, with the use of defined antibody markers we believe that the concept of proteotyping can be used in the identification and development of additional tissue proteotypes to permit cell- and tissue-specific analysis of developmental lesions.
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Materials and Methods |
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Antibodies
The rabbit polyclonal antibody recognizing aquaporin 5 (AQP5) was purchased from Alpha Diagnostics (San Antonio, TX). The rabbit polyclonal antibodies recognizing NKCC1 and Npt2b were obtained from Dr. Jim Turner (NIDCR, NIH, Bethesda, MD) and Dr. Juerg Biber (University of Zurich, Switzerland), respectively. The mouse monoclonal antibodies (MAbs) recognizing E-cadherin and ß-cate-nin were obtained from BD Biosciences Pharmingen (San Diego, CA) and the mouse MAbs recognizing smooth muscle actin (SMA) were obtained from Sigma (St Louis, MO). Fluorescent-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).
Animals
All animals were treated according to animal protocols approved by ACAUC, NIH. Athymic nude (nu/nu) mice were used for all mammary gland transplantation experiments. Knockout mouse models lacking Jak2 ( locus (
Transplantation
Transplantation of adult and embryonic mammary glands has been described previously (
Immunohistochemistry and Imaging Analysis
Routinely, small pieces of isolated mammary gland were fixed in Tellyesniczky's fixative for 5 hr at room temperature (RT), followed by dehydration and paraffin embedding. Paraffin-embedded sections (5 µm) were cleared in xylene and rehydrated through an alcohol series. Sections were immersed in boiling antigen unmasking solution (Vector Laboratories; Burlingame, CA) for 3 min, allowed to cool, and placed in PBS containing 0.05% (v/v) Tween-20 (PBST). After blocking of the sections with 3% (v/v) normal horse serum, primary antibodies were applied (SMA, 1:1000; NKCC1, 1:1000; E-cadherin, 1:200; Npt2b, 1:100; AQP5, 1:100; ß-catenin, 1:200). Sections were incubated with primary antibody for 1 hr at 37C and washed in PBST. Fluorescent-conjugated secondary antibodies (1:400; Molecular Probes) were applied to the sections for 1 hr at RT, washed in PBST, and mounted with VectaShield (Vector Laboratories). Sections were viewed under an epifluorescence equipped Zeiss Axioscop (Carl Zeiss MicroImaging; Thornwood, NY) fitted with FITC, TRITC, and FITC/TRITC filter sets. Images were captured with a Sony DKC-5000 digital camera (Sony Medical Systems; Park Ridge, NJ).
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Results |
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Identification of Genes Preferentially Expressed in the Mammary Gland
To identify genes present primarily in cDNA libraries derived from mammary origin, we screened the mouse EST database with full-length cDNAs. Using this approach, we identified three cDNAs that exhibited preferential expression in mammary gland libraries (Table 1). Npt2b (accession no. NM_011402) was almost exclusively expressed in the cDNA libraries derived from lactating (NMLMG and Riken) mammary tissue and was absent from non-lactating (virgin) mammary tissue (NbMMG). In contrast, NKCC1 (accession no. NM_009194) and the water transporter aquaporin AQP5 (accession no. NM_009701) showed preferential expression in the mammary tumor libraries (Mam), with relatively equal expression in virgin and lactating mammary libraries, suggesting that these membrane transporters may prove useful as prognostic markers of tumor development.
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Immunohistochemical Analyses of NKCC1, AQP5, and Npt2b During Normal Mammary Gland Development
Because the mammary gland contains several cell types (predominantly adipocytes, myoepithelial cells, and ductal and secretory epithelial cells), whose ratio changes throughout mammary gland development, neither Northern nor Western analyses accurately reflect their relative contribution. Furthermore, during pregnancy, mammary epithelial cells undergo proliferation and differentiation to form alveolar structures composed of secretory cells. However, no reliable markers are currently able to discriminate between ductal cells and alveolar secretory cells. Although we have previously demonstrated high levels of NKCC1 in virgin ductal epithelial cells and reduced expression during pregnancy and lactation (
As shown in Fig 1A, AQP5 was apparent on the apical membrane of ductal epithelial cells during virgin development. However, analyses of additional time points revealed that it was absent in epithelial cells during pregnancy (Fig 1D and Fig 1G), at parturition (Fig 1J), and 2 days after the removal of pups (Fig 1M). Interestingly, AQP5 protein was not detected in ductal cells during pregnancy (Fig 1D and Fig 1G), suggesting that ductal epithelial cells in the pregnant animal are inherently different from those present in virgin mammary tissue. Further determination of AQP5 protein revealed expression in terminal end buds and at all stages of the estrous cycle (data not shown). No staining was observed in the absence of the AQP5 primary antibody (data not shown). Consistent with previous observations (
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Npt2b protein was first observed on the apical membrane of developing alveoli at day 15 of pregnancy (Fig 1I, yellow arrowheads) but not in ductal epithelium. Examination of later pregnancy time points established a gradual increase in the number of acini with apical Npt2b protein (data not shown), which was present in all secretory cells at parturition (Fig 1L) and throughout lactation (data not shown). Interestingly, by day 2 of involution Npt2b was not detected on the apical membrane (Fig 1O), although immunoreactive protein was present in the lumen (Fig 1O, yellow arrowhead). Further experiments revealed that the disappearance of apical Npt2b protein, which occurred between days 1 and 2 of involution, was a direct result of local milk accumulation within alveolar structures and was independent of circulating hormone levels (data not shown). No staining was observed in the absence of the Npt2b primary antibody (data not shown).
Taken together, these data demonstrate that the presence of AQP5 protein and high levels of NKCC1 protein are characteristic of virgin ductal mammary epithelial cells. During pregnancy there is a significant reduction of NKCC1 protein levels in the ductal epithelium and developing alveoli which is maintained throughout lactation and early involution. In contrast, Npt2b protein is a marker of alveolar cells, as evidenced by its induction in mid-pregnancy and its continued presence throughout lactation, followed by its loss during involution.
Mammary Proteotyping of Mouse Models that Possess a Defective PRLRJak2Stat5 Pathway
Using a transplantation approach, we have recently described the characterization of mammary development in the absence of the transcription factor Stat5 (both Stat5a and Stat5b) (
To extend these observations and to further understand the molecular lesions, we examined the overall pattern of AQP5 expression in these models. During virgin development, a time when the JakStat pathway is believed to be inactive, AQP5 protein was evident on the apical membrane in wild-type (Fig 2A, white arrowhead), Jak2-null (Fig 2B, white arrowhead), and Stat5-null (Fig 2C, white arrowhead) ductal epithelium. In contrast, whereas AQP5 was no longer evident on the apical membrane of mammary secretory epithelial cells in wild-type mice at parturition (Fig 2D), apical AQP5 was apparent in Jak2-null (Fig 2E, white arrowhead) and Stat5-null (Fig 2F, white arrowhead) epithelium at parturition. Taken together, the analysis of AQP5 protein in these mouse models demonstrates its usefulness as a marker of ductal epithelial cells and confirms previous data demonstrating the maintenance of a mammary ductal proteotype in postpartum mammary epithelium deficient in Stat5 (
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Proteotypic Analysis of Mammary Epithelium in Mice with Defective NFB Signaling
The replacement in mice of serine residues by alanine residues within the activation loop of IKK (IKK
AA/AA) results in reduced mammary epithelial cell proliferation and differentiation, and pups born to IKK
AA/AA female mice fail to thrive (
B and RANK signaling pathways. Despite these observations, many alveoli with expanded lumina were evident throughout the gland at parturition. Therefore, to gain more insight into the developmental and functional lesions observed in these mice, we examined the mammary proteotype. Analyses of NKCC1 and AQP5 protein revealed high levels of NKCC1 and AQP5 in virgin ductal epithelium and a subsequent reduction of NKCC1 during pregnancy and an absence of AQP5 similar to that observed in wild-type animals (Fig 1; and data not shown). Because IKK
AA/AA mice exhibit a defect during lactation, we hypothesized a lack of functional differentiation and examined the immunohistochemical localization of Npt2b protein to determine if the presence of Npt2b could be linked to alveolar function. In contrast to wild-type mice at lactation, in which all alveoli possessed apical Npt2b (Fig 3A), the majority of alveolar structures in IKK
AA/AA mice lacked detectable Npt2b (Fig 3B, white arrowhead) and apical Npt2b was evident in a few alveoli (Fig 3B, yellow arrowhead). Therefore, despite the manifestation of a mild mammary gland phenotype, the absence of Npt2b appears to indicate a lack of definitive alveolar function.
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To address the functional consequences of a reduction in cyclin D1 levels observed in the IKKAA/AA mice, an MMTV-cyclin D1 transgene was introduced. This resulted in an increase of cyclin D1 to levels seen in wild-type animals and a complete rescue of the lactational defect (
allele. Therefore, Npt2b was detected on the apical membrane of all alveoli in MMTVcyclin D1 transgenic mice at lactation (Fig 3C) similar to that observed in wild-type mice (Fig 3A). However, in contrast to a lack of apical Npt2b protein in most of the alveoli present in IKK
AA/AA mice (Fig 3B), the introduction of the MMTVcyclin D1 transgene and subsequent reestablishment of normal cyclin D1 levels in the IKK
mutant mice resulted in the detectable expression of apical Npt2b protein in all alveolar structures (Fig 3D). These data demonstrate that attainment of secretory function on restoration of cyclin D1 levels is associated with the induction of apical Npt2b protein and reestablishment of the secretory cell proteotype.
Proteotypic Analysis of Mammary Epithelium in Mice with a Loss of the Inhibin ßB-subunit
The functional disruption of the inhibin ßB-subunit in mice results in the birth of pups with open eyelids, and pups born to female inhibin ßB knockout mice fail to thrive (-lactalbumin was comparable to that of hemizygous and wild-type mice, suggestive of normal differentiation.
To further understand and define the molecular lesions in these mice, we determined the mammary proteotype. Analysis of virgin inhibin ßB-null mice revealed the presence of NKCC1 and AQP5 in developing ductal structures similar to that observed in wild-type mice (data not shown). Similarly, AQP5 was absent and NKCC1 protein levels were downregulated during pregnancy in inhibin ßB-null and wild-type mice (data not shown). Despite the inability of the mice to lactate, inhibin ßB-null mice demonstrated an appropriate induction of Npt2b protein on the apical membrane of developing alveolar cells and the induction of WAP protein at mid-pregnancy (data not shown), and Npt2b was observed at levels comparable to wild-type mice at parturition (cf. Fig 3A and Fig 3E). These data demonstrate that the mammary proteotype in inhibin ßB-null mice is normal, and further suggest that the defect in epithelial cell development is not associated with a lack of functional differentiation but may be a result of reduced proliferation and/or other cellular abnormalities independent of cell differentiation.
Delayed Involution in the Absence of Stat3 Is Associated with a Persistence of Apical Npt2b
Mammary gland involution is associated with a rapid loss of Stat5 phosphorylation and a concomitant increase in Stat1 and Stat3 phosphorylation (
We have recently examined (
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To broaden these observations and to further establish the lactational competency and mammary proteotype of Stat3 fl/fl;WC glands, we examined the status of Npt2b in Stat3 fl/fl;WC and wild-type mice after 6 days of involution followed by 5 days of re-suckling (
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Discussion |
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Many mouse models have been generated that exhibit defective mammary gland development and include altered activity of membrane receptors, tyrosine kinases, and transcription factors (
Using database screens and immunohistochemistry, we have identified three proteins that are regulated in a development-specific manner and are representative of distinct stages of mammary gland development. In this study, we have used these protein markers as a means to further understand mammary-associated developmental lesions in several mouse models (AA/AA (
AA/AA mice (
AA/AA mice by re-establishment of the cyclin D1 levels points to a role for cyclin D1 in mammary epithelial cell differentiation, in addition to its established role in mammary epithelial cell proliferation (
The localization, cell specificity, and developmental profile of NKCC1, AQP5, and Npt2b proteins in mammary tissue provide unique insight into the regulation of specialized transporter proteins (B kinase function, which results in the inability of mice to lactate, corresponds with a lack of Npt2b induction.
We have demonstrated that the IHC analysis of NKCC1, AQP5 and Npt2b proteins using specific antibodies provides a novel means to examine mammary-specific lesions observed in transgenic and gene knockout mouse models. The identification, localization, and developmental profile of these proteins and their defined application constitute what we refer to as proteotyping: tissue typing based on the analysis of cell type-specific changes in protein levels using IHC techniques. Although we have demonstrated the use of this method as it applies to mammary gland development, we believe that the concept of proteotyping is equally applicable to other tissues. In particular, we propose that proteotyping could be used to define changes in protein expression patterns that are associated with tumorigenesis and could thus serve as a prognostic tool. Such an application would probably involve a multidisciplinary approach, combining micro-array, protein array and tissue array analyses. One of the distinct advantages of using proteotyping is the ability to determine the localization of the protein of interest in specific cell types. For this reason, proteotyping offers a more sensitive method than the determination of changes in whole protein or mRNA levels, which would reflect the contribution of many different cell populations.
In conclusion, our results define the concept of a proteotype and further demonstrate the application of proteotyping in the analysis of developmental lesions observed during mammary epithelial cell development in transgenic and gene knockout mouse models. With the use of the mammary gland as a model system, we suggest that the identification and characterization of other tissue proteotypes using defined protein markers may represent a technique that can be applied to the analysis of normal tissue development and as a prognostic tool in the analysis of tumorigenesis.
Received for publication October 24, 2002; accepted November 27, 2002.
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