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Molecular Endocrinology, doi:10.1210/me.2005-0214
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Molecular Endocrinology 20 (9): 2187-2198
Copyright © 2006 by The Endocrine Society

Identification of a Basic Helix-Loop-Helix Transcription Factor Expressed in Mammary Gland Alveolar Cells and Required for Maintenance of the Differentiated State

Yan Zhao, Carina Johansson, Thai Tran, Ryan Bettencourt, Yoko Itahana, Pierre-Yves Desprez and Stephen F. Konieczny

Department of Biological Sciences and the Purdue Cancer Center (Y.Z., C.J., T.T., R.B., S.F.K.), Purdue University, West Lafayette, Indiana 47907-2064; and California Pacific Medical Center (Y.I., P.-Y.D.), Cancer Research Institute, San Francisco, California 94107

Address all correspondence and requests for reprints to: Stephen F. Konieczny, Department of Biological Sciences and the Purdue Cancer Center, Purdue University, Hansen Life Sciences Research Building, 201 South University Street, West Lafayette, Indiana 47907-2064. E-mail: sfk{at}bio.purdue.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The development of mammary glands relies on complicated signaling pathways that control cell proliferation, differentiation, and apoptotic events through transcriptional regulatory circuits. A key family of transcription factors used in mammary gland development is the helix-loop-helix/basic helix-loop-helix (HLH/bHLH) protein family. In this study, we identify Mist1 as a tissue-restricted Class II bHLH transcription factor expressed in lactating mammary glands. Mouse and human mammary glands accumulated Mist1 protein exclusively in secretory alveolar cells, and Mist1 transcripts were differentially expressed in mouse SCp2 cells induced to differentiate by addition of lactogenic hormones. Mist1 null (Mist1KO) lactating mammary glands were defective in normal lobuloalveolar organization, exhibiting shedding of cells into the alveolus lumen and premature activation of the signal transducer and activator of transcription 3 signaling pathway. These cells also failed to maintain expression of the gap junction proteins connexin26 and connexin32, leading to the loss of gap junctions. Our findings suggest that loss of Mist1 impairs the maintenance of the fully differentiated alveolar state and, for the first time, places Mist1 within the hierarchy of known HLH/bHLH proteins that control mammary epithelial cell development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMARY GLAND DEVELOPMENT is unique among most organ systems because it occurs primarily postnatally with terminal differentiation achieved only at the end of pregnancy. During pregnancy, mammary glands exhibit dramatic molecular and cellular changes that are characterized by extensive lobuloalveolar expansion and mammary epithelial cell maturation in preparation for milk secretion (1, 2, 3, 4, 5, 6). After weaning, the entire gland undergoes extensive remodeling and cellular apoptosis through a process known as involution. Multiple cycles of cell proliferation, differentiation, and apoptosis can occur in mammary glands throughout adult life, making this organ an excellent model system for studying aspects of tissue development.

Examination of the intracellular regulatory pathways that control each of these developmental decisions is critical to understanding the molecular processes involved in normal breast cell development, maintenance of stem cell populations, and the initiation of breast tumorigenesis. Signaling pathways transduced through the association of hormones and growth factors, interactions between epithelial cells and the extracellular matrix environment, and activation of specific transcriptional networks work in concert to regulate mammary gland development and cell function (5, 7, 8, 9, 10). One transcription factor network that is critical to these developmental stages is the evolutionarily conserved helix-loop-helix (HLH) protein family. More than 250 HLH proteins have been identified in eukaryotes ranging from plants to humans (11, 12). The HLH domain that defines members of this family functions as a protein dimerization motif to regulate an almost unlimited number of combinatorial dimer choices. In addition to the dimerization motif, most HLH factors utilize an adjacent basic domain (bHLH) that is required for interaction with DNA targets. Based on tissue distribution, dimerization properties, DNA-binding characteristics, and transcriptional activities, HLH proteins have been grouped into several distinct classes (11, 12, 13, 14). Class I bHLH factors (e.g. E12, E47, HEB) are widely expressed, whereas Class II bHLH proteins (e.g. MyoD, myogenin, Mash1, NeuroD, and neurogenin3) exhibit a tissue-restricted expression profile. Class I and II bHLH proteins function as heterodimer complexes to regulate expression of target genes by binding to E-box (-CANNTG-) DNA elements (12). The transcriptional activities of bHLH heterodimer complexes are often regulated by posttranslational modifications or by changes in dimerization partner preferences. In this regard, Class I and II bHLH proteins are subject to negative regulation through dimerization with the related Class V HLH proteins of the Id family. Id (inhibitor of differentiation/DNA binding) proteins retain the HLH dimerization motif but lack a DNA binding domain. The Id proteins function to repress bHLH-mediated transcription by forming DNA binding defective HLH-bHLH complexes (15).

Elegant studies from P.-Y. Desprez’s group and others have shown that the Id proteins Id1 and Id4 inhibit mammary epithelial cell differentiation and alter cellular proliferation properties, presumably by interacting with Class I and Class II bHLH factors and blocking activation of specific target genes (16, 17, 18, 19, 20, 21, 22, 23). Similarly, Id2 has been shown to be essential to mammary epithelial cell differentiation because mice lacking Id2 exhibit impaired lobuloalveolar development (21). The importance of these negative HLH regulators to mammary epithelial cell growth and differentiation suggests that a bHLH regulatory network controls key aspects of mammary gland development. Indeed, the ubiquitously expressed Class I bHLH protein, Ig transcription factor 2 (ITF-2), can interact with Id1 and block Id1-stimulated cell proliferation and apoptosis (22). However, despite the impressive progress that has been made in defining the HLH/bHLH regulatory network in mammary epithelial cells, these studies have failed to identify a tissue-restricted bHLH protein that is associated with differentiated lobuloalveolar cells. This is a critical gap in our understanding of this network because ITF-2 and the Id proteins function primarily by influencing the activity of Class II bHLH factors.

One candidate Class II bHLH factor that may be involved in mammary epithelial cell development is Mist1 (also called Bhlhb8) (24). Mist1 has been shown to be expressed in secretory epithelial cells of the pancreas, salivary gland, and stomach, and deletion of the Mist1 gene leads to general defects in these cell types (25, 26, 27). This factor is unique among most bHLH proteins because the preferred DNA binding complex consists of a Mist1 homodimer, although Mist1 can also form heterodimer complexes with Class I bHLH factors under certain conditions (27, 28, 29). Interestingly, Mist1/DNA interactions lead to either repression or activation of target genes in different cellular contexts (28, 29). Despite these observations, the mechanism(s) by which Mist1 regulates downstream gene expression profiles has not been established.

In this study, we report that Mist1 is also expressed in secretory mammary epithelial cells and in mammary epithelial cell lines that undergo alveolar differentiation. Analysis of Mist1 null (Mist1KO) mice revealed that Mist1 is critical to maintenance of the terminal differentiated mammary gland. In the absence of Mist1, lobuloalveolar cells lose their normal cellular organization, exhibit alterations in gap junction complexes, and prematurely activate the signal transducer and activator of transcription 3 (Stat3)-signaling pathway during lactation. These results identify Mist1 as the first known Class II bHLH transcription factor to be expressed exclusively in lactating mammary epithelial cells and demonstrate that Mist1 is a central player in the HLH/bHLH regulatory circuit that controls mammary gland development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mist1 Is Expressed during Mammary Gland Development
Mammary gland development involves a series of defined stages that utilize specific transcriptional circuits to control cell proliferation, terminal differentiation, and cell death decisions. Among the transcriptional regulators that are known to be critical to these events is the HLH/bHLH protein family (11, 12, 16, 22, 30). Although a number of studies have examined the importance of the widely expressed Class I bHLH and Class V HLH factors to mammary epithelial cell development (17, 18, 20, 21, 22, 31, 32, 33), it has not been possible to examine the role of tissue-restricted Class II bHLH factors because a mammary gland-restricted family member has not been identified. Because the major function of mammary tissue is to synthesize and secrete milk products, we reasoned that a mammary gland-restricted Class II bHLH factor might also function in other cell types that exhibit regulated exocytosis. One possible candidate is Mist1, a bHLH factor that is selectively expressed in acinar cell populations of the pancreas and salivary glands (25). To examine whether the Mist1 gene is transcriptionally active in mammary glands, we performed an RT-PCR expression analysis on RNA isolated from different mouse tissues. As reported previously (25), Mist1 transcripts were detected in the pancreas and salivary glands but not in isolates from kidney, muscle, liver, or lung (Fig. 1Go). Interestingly, Mist1 transcripts were also detected in mammary gland samples, whereas transcripts for the ubiquitously expressed Class I bHLH factor ITF-2 and the Class V HLH factor Id1 were found in all tissues (Fig. 1Go).


Figure 1
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  		Fig. 1. RT-PCR Expression Analysis of HLH/bHLH Transcription Factors in Adult Mouse Tissues

Whereas the Class I bHLH factor ITF-2 and the Class V HLH factor Id1 are expressed in all cell types, Mist1 expression is restricted to the secretory epithelial tissues pancreas, salivary gland, and mammary gland. Note that the mammary gland RNA was isolated from a lactating mouse. All other samples were isolated from adult male or adult (nonlactating) female mice.

 
The initial detection of Mist1 transcripts in mammary glands prompted us to examine the timing of Mist1 expression during mammary gland development and to establish whether a subset of HLH/bHLH factors exhibited similar expression profiles as Mist1. RT-PCR analysis revealed that Mist1 transcripts were abundant in d 15 lactating samples and very weakly detected in RNA isolated from pregnant (d 14) females (Fig. 2AGo). Mist1 transcripts were not detected in nulliparous or involuting mammary gland samples. Analysis of eight additional bHLH factors known to be expressed in mammary epithelial cells failed to reveal a similar lactation-restricted expression pattern (Fig. 2AGo).


Figure 2
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  		Fig. 2. Mist1 Expression during Mammary Gland Development

A, RT-PCR expression analysis of Mist1 and eight additional bHLH transcription factor genes in mammary gland samples from nulliparous (N), pregnant (P), lactating (L), and involuting (I) developmental stages (months and days are indicated by N3, P14, L15, and I4, respectively). Only Mist1 transcripts are detected exclusively in lactating mammary gland samples. B, RT-PCR analysis of Mist1 transcripts over an extended time course of mammary gland development. Mist1 gene expression is detected during late pregnancy and throughout all lactation stages. C, Immunoblot analysis for Mist1 protein in the same mammary gland samples tested in panel B. Mist1 protein is detected only in lactating samples. D, Immunoblot analysis of Mist1, Id1, and Id2 protein accumulation during the indicated mammary gland stages.

 
To extend this analysis further, we next isolated RNA and protein samples from mouse mammary glands derived from nulliparous, pregnant, lactating, and involuting females over a more comprehensive period of development. As shown in Fig. 2BGo, Mist1 transcripts were initially detected during late pregnancy, and this expression was maintained throughout lactation. In contrast, no Mist1 transcripts were detected in nulliparous, early pregnancy, or involuting samples. Immunoblots revealed the expected high levels of Mist1 protein in lactating samples, but no Mist1 protein was detected during pregnancy (Fig. 2CGo). The absence of Mist1 protein during late pregnancy likely reflects the difference in sensitivity between RT-PCR and immunoblot assays, although a differential posttranscriptional regulatory mechanism cannot be ruled out at this time. Analysis of other HLH proteins in these samples also revealed that the Class V HLH Id1 and Id2 proteins were uniquely modulated during development (Fig. 2DGo) (22). Id1 expression was restricted to nulliparous and involuting tissues whereas Id2 protein accumulated exclusively in lactating samples. Based on these results, and the known expression patterns of other HLH/bHLH factors (12, 14, 16, 30), we conclude that Mist1 represents the first known bHLH factor to exhibit a lactation-specific expression pattern in mammary glands.

Although RT-PCR and immunoblot assays confirmed a lactation-restricted Mist1 expression pattern, we wished to examine individual cells for the presence of Mist1 protein. Analysis of nulliparous, pregnant, and lactating mouse mammary gland sections revealed nuclear Mist1 staining only in cells from the lactating tissue (Fig. 3Go, A–C). A similar Mist1 staining pattern was observed in human lactating samples, although differences in the overall levels of Mist1 protein accumulation were detected among individual cells (Fig. 3Go, D and E). Some cells expressed high levels of human Mist1 whereas other cells exhibited low levels of protein. On rare occasions, a few cells within an alveolus remained Mist1 negative. The significance of this varied expression pattern in human alveolar cells is currently unknown.


Figure 3
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  		Fig. 3. Mist1 Protein Accumulates Exclusively in Lobuloalveolar Cells

A–C, Immunohistochemistry reveals that nuclear Mist1 protein (arrows) is detected only in lactating (L15) mouse mammary gland samples and not in glands isolated from nulliparous (N3) or pregnant (P5) females (DAPI, blue; Mist1, red). D and E, Human lactating mammary gland sections were processed for Mist1 immunohistochemistry. Mist1 protein is detected exclusively in lobuloalveolar cells. Note that the Mist1 protein levels are variable among different cells. Most alveolar cells express high levels of Mist1 (white arrows), whereas a subset of nuclei accumulate low levels of Mist1 (bracket). In this particular image, a single nucleus is Mist1 negative (yellow arrow). F–J, Lactating mouse mammary gland sections were stained with antibodies against smooth muscle actin (SMA) or Mist1 as indicated. Myoepithelial cells (arrows and inset) express smooth muscle actin but remain Mist1 negative. In contrast, all lobuloalveolar cells contain nuclear Mist1 protein. DAPI staining (blue) highlights the nuclei in these preparations. DAPI, 4',6-Diamidino-2-phenylindole.

 
Mammary epithelium is composed of two principal cell types: luminal epithelial cells and myoepithelial cells (34). The luminal epithelial cells (lobuloalveolar cells) synthesize and secret milk products whereas the elongated myoepithelial cells surrounding the acinus are highly contractile and play a central role in milk ejection (35). Because Mist1 is thought to be an exocrine-specific transcription factor, we speculated that Mist1 would be expressed exclusively in the secretory lobuloalveolar cells. To test this hypothesis, lactating mammary gland sections were costained with antibodies against Mist1 and smooth muscle actin, a marker for myoepithelial cells. As shown in Fig. 3Go, F–J, myoepithelial cells lined the individual acini structures and remained Mist1 negative. In contrast, most secretory lobuloalveolar cells present in lactating glands were Mist1 positive at all stages of lactation. These results confirm that the Class II bHLH factor Mist1 is expressed in fully differentiated lobuloalveoli in the lactating mammary gland of both human and mouse.

Finally, to determine whether Mist1 expression reflects a general property associated with lactating alveolar cells, we used the SCp2 mouse mammary epithelial cell line, which can be induced to produce milk products (17, 22, 32). SCp2 cells were grown in the absence or presence of lactogenic hormones and extracellular matrix, and individual RNA and protein samples were harvested at different time points. As expected, Mist1 transcripts and protein were not detected in undifferentiated SCp2 cells whereas the constitutively expressed bHLH factor ITF-2 was present in all samples (Fig. 4Go). However, by d 3 of lactogenic hormone addition, Mist1 and ß-casein transcripts and protein began to accumulate, and their levels increased throughout the duration of the experiment. Thus, even in this in vitro model, Mist1 expression correlated with the differentiated state of mammary epithelial cells.


Figure 4
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  		Fig. 4. Mist1 Expression Correlates with Differentiation of the Mouse Mammary Epithelial Cell Line SCp2

SCp2 cells were induced to differentiate as described in Materials and Methods. Cells were harvested for RNA and protein at different times after switching to differentiation medium on d 0. RT-PCR (panel A) and immunoblot assays (panel B) reveal that Mist1 and ß-casein transcripts and proteins accumulate to high levels after prolactin induction.

 
Mist1 Is Critical to Maintaining the Integrity of Mature Lobuloalveolar Structures
To study the function of Mist1 in lactating mammary glands, a targeted knock-in mouse was generated in which the coding region of the Mist1 locus was replaced by a bacterial LacZ gene encoding a nuclear localized ß-galactosidase (ß-gal) protein (26). Mist1 heterozygous (Mist1Het) and Mist1 null (Mist1KO) animals were viable and produced normal litter sizes (data not shown). Analysis of lactating mammary glands from Mist1KO animals confirmed the absence of Mist1 transcripts and protein in these samples (Fig. 5Go, A–D). Because LacZ was targeted to the Mist1 locus, we also were able to follow transcriptional expression of the Mist1 gene in Mist1Het and Mist1KO animals by examining ß-gal activity. As predicted, ß-gal was detected only in the lobuloalveolar cells of Mist1Het and Mist1KO lactating samples and was not detected in epithelial cells from nulliparous, pregnant, or involuting glands (Fig. 5Go, E and F, and data not shown). Interestingly, the ß-gal expression pattern in lactating d 2 Mist1KO lobuloalveoli was identical to that observed in Mist1Het alveoli (Fig. 5Go, E and F). Adjacent serial sections of a Mist1Het lactating d 5 sample confirmed that the same alveolar cells coexpressed Mist1 and ß-gal proteins whereas the myoepithelial cells remained Mist1 and ß-gal negative (Fig. 5Go, G and H). The comparable ß-gal staining in Mist1Het and Mist1KO mammary glands also demonstrates that Mist1 protein is not required to initiate or maintain expression of the Mist1 gene during mammary gland development.


Figure 5
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  		Fig. 5. Analysis of Mist1 Expression in Mist1KO Samples

RT-PCR (panel A) and immunoblot analysis (panel B) confirm that Mist1KO (KO) mammary gland (L2) samples do not express Mist1. C and D, Immunohistochemistry using anti-Mist1 reveals that L2 tissues from Mist1KO mice are devoid of Mist1 protein. E and F, Mist1Het and Mist1KO (L2) samples were stained with anti-ß-gal to follow expression of the Mist1 locus. As predicted, ß-gal is detected only in lobuloalveolar cells in both samples. G and H, Serial sections (5 µm) from an L5 Mist1Het mammary gland sample were processed for anti-ß-gal and anti-Mist1 immunohistochemistry. Identical cell nuclei coexpress both ß-gal and Mist1 in these animals (black arrows). As predicted, myoepithelial cells (red arrows) do not express ß-gal or Mist1. I and J, Standard H&E sections were obtained from L5 WT and Mist1KO samples showing normal alveoli formation at this early lactation stage. KO, Knockout.

 
To establish whether the absence of Mist1 protein affected early terminal differentiation events, wild-type (WT) and Mist1KO mammary glands were examined at different stages of mammary gland development. Whole-mount analyses failed to reveal significant differences during pregnancy (data not shown). During early lactation, Mist1KO mammary glands exhibited well-developed lobuloalveolar structures that were similar to WT glands (Fig. 5Go, I and J), confirming that Mist1 is not critical to the commitment and expansion of mammary epithelial cells during pregnancy. At these early lactation periods (L2–L5) both WT and Mist1KO glands expressed a normal complement of milk proteins (ß-casein, whey acidic protein) (data not shown). Analysis of individual litters also revealed no significant differences in the body weight or developmental progression of animals derived from WT and Mist1KO mothers, suggesting that Mist1KO mothers produce sufficient milk to support the normal growth of their offspring.

Although lobuloalveolar integrity at early lactation times was normal in Mist1KO tissues, significant morphological alterations were detected at later lactating stages. At L15, WT alveolar cells exhibited a typical cubical shape with very few variations among cells within an alveolus (Fig. 6Go, A and C). In contrast, L15 Mist1KO alveolar cells showed grossly irregular columnar morphologies that extended into the extracellular space (Fig. 6Go, B and D). These large extensions eventually pinched off and were shed into the alveolus lumen. The pinched off cells were clearly derived from Mist1 locus expressing lobuloalveolar cells as they remained ß-gal positive (Fig. 6FGo). In contrast, the shedding of cells into the lumen from WT or Mist1Het lactating mammary glands was seldom observed (Fig. 6EGo). Analysis of E-cadherin and ß-catenin expression failed to reveal major alterations in the cellular adhesion junctions associated with alveolar cells. WT, Mist1Het, and Mist1KO samples expressed normal levels of E-cadherin and ß-catenin, and both proteins preferentially localized to the basal and lateral membranes of the epithelial cells (Fig. 6Go, G–J). However, in the case of Mist1KO cells, the E-cadherin and ß-catenin lateral borders were significantly extended as the cells elongated into the acini lumens. Interestingly, many of the budding cells were binucleated (Fig. 6Go, H and J), suggesting that the structural defects associated with these cells negatively influenced cytokinesis events. Thus, in the absence of Mist1, the overall integrity of the lobuloalveolar cells within functional alveoli structures became disrupted.


Figure 6
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  		Fig. 6. Histological Characterization of Mist1KO Lactating Mammary Glands

WT (panels A and C) and Mist1KO L15 (panels B and D) mammary glands were processed for standard hematoxylin and eosin histology. Mist1KO lobuloalveolar cells assume a columnar morphology in which cells grossly extend into the lumen and eventually pinch off (arrows in B and D). E and F, Mist1Het (L15) and Mist1KO (L15) samples were stained with X-gal to detect nuclear ß-gal activity from the Mist1 locus. As expected, only lobuloalveolar cells are ß-gal positive (arrows). Note that Mist1KO cells found within the lumen express ß-gal. G–J, WT and Mist1KO L15 samples were stained for E-cadherin or ß-catenin as indicated. E-cadherin and ß-catenin primarily localize to the lateral borders of individual lobuloalveolar cells. Note that in Mist1KO samples the lateral borders significantly extend as the cells bud off into the lumens (yellow arrows). Many of the Mist1KO cells contain multiple nuclei (white arrows). ß-cat, ß-Catenin; E-cad, E-cadherin; DAPI, 4',6-diamidino-2-phenylindole; KO, knockout.

 
The shedding of cells in L15 Mist1KO alveoli was reminiscent of the normal transitions that occur during mammary gland involution when apoptotic epithelial cells are deposited into the lumen (36, 37). To determine whether late lactating Mist1KO lobuloalveolar cells prematurely become apoptotic, we performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assays on d 10–20 lactating mammary gland sections from WT and Mist1KO mice. Analysis of these samples revealed that the number of apoptotic cells was significantly elevated in Mist1KO mammary glands when compared with WT samples (Fig. 7AGo). As predicted, most of the Mist1KO apoptotic cells were present in the lumen of individual acini and were ß-gal positive (data not shown). Because Stat3 activation is known to be correlated with apoptotic events during involution (38, 39), we next examined whether Mist1KO cells showed alterations in Stat3 signaling. Cell extracts were isolated from WT and Mist1KO lactating samples and were subjected to immunoblot analyses using antibodies against activated Stat3 (Stat3-p). As shown in Fig. 7BGo, total Stat3 protein levels were equivalent in WT and Mist1KO samples. However, a large increase in phosphorylated Stat3 was observed in Mist1KO lactating tissues, demonstrating that the Stat3-signaling pathway was prematurely activated in Mist1KO cells. In contrast, there was no significant difference in phospho-Stat5 levels in these samples (our unpublished results). The increased epithelial cell apoptosis and precocious Stat3 activation suggested that Mist1KO mammary glands lose a fraction of luminal alveolar cells as lactation progresses. In support of this hypothesis, we consistently observed a lower epithelial cell density in Mist1KO mammary glands during late lactation events (L15–L20) (data not shown). These results confirm the importance of Mist1 in maintaining the integrity of alveolar cells and show that in the absence of Mist1, late lactating alveolar cells undergo early-stage apoptosis.


Figure 7
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  		Fig. 7. Mist1KO L15-L20 Mammary Glands Exhibit Precocious Activation of Stat3 and an Increase in the Number of Apoptotic Cells

A, TUNEL assays were performed on WT and Mist1KO sections prepared from L15–L20 mammary glands. The number of apoptotic cells is significantly elevated (P < 0.05) in Mist1KO samples. B, Immunoblot analysis of WT and Mist1KO (L15) samples reveals equivalent levels of Stat3 in all animals but a large increase in phospho-Stat3 (Stat3-p) in Mist1KO mice. KO, Knockout.

 
We next examined whether intercellular communication was altered in Mist1KO samples because previous reports showed that Mist1 was critical to maintaining gap junctions in the pancreas (29). Gap junction complexes in mouse mammary alveoli consist of connexin26 (Cx26) and connexin32 (Cx32) proteins (40, 41), both of which preferentially accumulate during lactation (42, 43). Analysis of Mist1KO lobuloalveolar cells revealed a progressive loss of Cx26 and Cx32. During early lactation periods (L1–L3) Cx26 was found associated with normal gap junction complexes positioned at cell borders (Fig. 8Go, A and B). However, during this same developmental period Cx32 was typically found only in diffuse cytoplasmic staining patterns that did not resemble the normal gap junction plaques (Fig. 8Go, C and D). As lactation progressed, gap junctions were no longer detected (Fig. 8Go, E–H). These results reveal that expression of Mist1 is critical to maintaining the integrity of functional gap junctions in mammary epithelial cells and suggest that Mist1KO lobuloalveolar cells are defective in intercellular communication. The lack of gap junctions in Mist1KO alveolar cells may contribute to the disorganization and luminal budding that occurs during late lactation.


Figure 8
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  		Fig. 8. Immunohistochemistry Using Antibodies against Cx26 and Cx32 Proteins in WT (A, C, E, and G) and Mist1KO (B, D, F, and H) L2 and L15 Mammary Gland Samples

At L2 the Cx32 protein accumulates in diffuse deposits (arrows in D) in Mist1KO samples. By L15 Cx26 and Cx32 are greatly reduced in the Mist1KO tissues. Arrows in A, B, C, E, and G highlight normal gap junction plaques. DAPI, 4',6-Diamidino-2-phenylindole; KO, knockout.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The HLH/bHLH transcription factor family has a critical role in the transcriptional circuitry that controls numerous developmental processes including myogenesis, hematopoiesis, neurogenesis, heart morphogenesis, and pancreas and mammary gland development (12, 22, 44, 45, 46, 47). In the case of mammary epithelial cells, the Class I bHLH factor, ITF-2, and the Class V inhibitor HLH factors, Id1 and Id2, are critical regulators (22). Id1 preferentially accumulates in undifferentiated breast epithelial cells (nulliparous, involution), whereas Id2 expression is restricted to differentiated lactating tissues (19, 22, 32). In contrast, ITF-2 expression remains constitutive throughout mammary gland development. Ectopic expression of Id1 in SCp2 mammary epithelial cells induces an increase in cell proliferation (19, 32), and inhibition of Id2 expression blocks terminal differentiation (22). The importance of the Id proteins to normal mammary gland development also has been suggested by the analysis of Id2 null mice, which acquire defects in lobuloalveolar cell expansion (21, 48). Recently, the widely expressed Class VI bHLH repressors, Stra13 and Sharp-1, also have been shown to be present during mammary gland involution (49), but the function of these proteins to mammary gland remodeling has not been determined. Despite the recent advancements in characterizing the role of HLH/bHLH factors in mammary gland development, studies have been hampered by the lack of a tissue-restricted Class II bHLH factor that could serve as the dimer partner to the Class I and Class V proteins.

Our identification of Mist1 as a bHLH protein expressed in the lactating mammary gland and in prolactin-treated SCp2 cells represents the first report of a tissue-restricted Class II bHLH factor that is found in differentiated mammary epithelial cells. The restricted expression of Mist1 in lobuloalveolar cells suggests that Mist1 has a role in regulating terminal differentiation events. During lactation, the lobuloalveolar cells synthesize and secrete milk products into the acinus lumen. To achieve these functions, epithelial cells establish a specific apical-to-basal polarity with the exocytotic vesicles aligned toward the apical pole (50). In addition to acquiring a columnar shape, Mist1KO mammary epithelial cells also exhibit a change in their overall polarity where secretory vesicles are no longer positioned within the apical portion of the cells (our unpublished data). The change in cellular polarity occurs despite normal basal-lateral localization of the E-cadherin adhesion complex. The molecular mechanism(s) responsible for this cellular disorganization is currently under investigation, but the loss of gap junctions likely plays a pivotal role. Previous studies have shown that pancreatic Mist1KO acinar cells also lack gap junctions, and their absence prevents intercellular communication (29). Although Mist1 is not required for the initial activation of the connexin genes, it is required to sustain Cx32 gene transcription in acinar cells (29). A similar response is observed in Mist1KO mammary epithelial cells where Cx32 expression is induced during the early stages of lactation, but the cells cannot maintain transcription of the Cx32 gene in the absence of Mist1. In this mouse model, the absence of Cx32 protein also leads to reduction in Cx26 protein. Ultimately, acinar and lobuloalveolar cells fail to form functional gap junction complexes and exhibit severe defects in cell-cell communication (29).

The absence of gap junctions in Mist1KO cells may also be responsible for the observed premature activation of the Stat3-signaling pathway. Activation of the Stat3 pathway is normally induced by the cytokines IL-6 and leukemia-inhibitory factor during the involution stage of mammary gland development (38, 39, 51, 52). However, Mist1KO mammary glands show a dramatic increase in phospho-Stat3 during lactation. These results suggest that uncoupled cell-cell communication impairs the normal signals that maintain terminal differentiation of luminal alveolar cells and, as a result, the cells precociously dedifferentiate. Although the consequence of this early Stat3 activity is unknown, it ultimately may be responsible for the increase in cell apoptosis observed in Mist1KO lactating mammary glands. Loss of gap junctions and/or activation of Stat3 are known to affect cell death decisions. For example, Bry et al. (40) have shown that inactivation of the Cx26 gene in mammary glands before puberty causes unscheduled cell death during pregnancy (40). The increased incidence of apoptosis in the Cx26 null cells is in agreement with our results where a similar increase in apoptosis is observed in lactating Mist1KO cells. Interestingly, loss of gap junction proteins and unscheduled activation of the Stat3/5 signaling pathways are also properties associated with the pathogenesis of breast cancer (53, 54, 55). The disorganization of Mist1KO lobuloalveolar cells could represent an initial defect in maintaining cells in a quiescent state. Whether loss of Mist1 eventually leads to hyperplasia and a predisposition to mammary tumor formation is an area of future research for our laboratory.

The lactation-specific expression of Mist1 positions it uniquely among the HLH/bHLH protein family. Although Mist1 preferentially forms homodimer complexes in vivo (27, 29), Mist1 also is capable of forming heterodimer complexes with other HLH factors (28). Indeed, in vitro protein-protein interaction studies have demonstrated that Mist1 and Id proteins form dimer complexes (our unpublished results), and it will be important to determine whether these proteins similarly interact in mammary epithelial cells. Although Id2 null mice have defects in lobuloalveolar cell expansion (21, 48), it remains unknown whether Mist1 is involved in this defect. However, because the Id2 null phenotype is significantly more severe than the Mist1KO phenotype, it seems likely that Mist1 and Id2 have unique functions within the lactating tissue. Nonetheless, the defects observed in Mist1KO mammary glands suggest that Mist1 is involved in controlling genes that are essential for maintaining the terminal differentiation state of the lobuloalveolar cell, possibly regulating genes that are involved in milk secretion. This idea is supported by the overall expression pattern of the Mist1 gene, which is transcriptionally active in differentiated mammary epithelial cells, and in other cell types (exocrine pancreas, salivary glands) that also exhibit regulated exocytotic events (25). In these tissues, defects in cellular organization and architecture are similarly observed (26, 27, 56). Future studies using Mist1KO mice and the SCp2 mammary epithelial cell model will permit us to identify specific Mist1 target genes and to determine whether alterations in gene expression patterns are similar between mammary lobuloalveolar cells and secretory cells from other exocrine tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
All mice used in this study were maintained on a C57BL/6 genetic background. Mist1 null mice were generated by standard embryonic stem cell homologous recombination procedures by replacing the entire Mist1 coding region with a bacterial LacZ gene engineered to encode a nuclear localized ß-gal protein (26). The genotypes of WT, heterozygous (Mist1Het), and Mist1 null (Mist1KO) mice were confirmed by standard PCR analyses of genomic DNA. Experimental and control female littermates were killed at different mammary gland developmental stages (nulliparous, pregnant, lactating, involuting), and inguinal glands 4 and 9 were isolated and used for histological and biochemical studies as described in the text. All animals used in this study were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Purdue Animal Care and Use Committee in accordance with National Institutes of Health (NIH) guidelines.

SCp2 Cell Cultures
Stock SCp2 cells were maintained at 37 C, 5% CO2 in DMEM/F12 (1:1) containing 5% fetal bovine serum, 10 µg/ml gentamicin, 5 µg/ml insulin. Differentiation was induced by plating cells at a concentration of 5 x 104/cm2 in DMEM/F12 lacking serum but supplemented with 5 µg/ml insulin, 1 µg/ml hydrocortisone, 3 µg/ml prolactin, 1% matrigel (BD Biosciences Pharmingen, San Diego, CA), as described previously (17). The culture medium was changed every other day, and the cells were harvested on d 0, d 1, d 3, d 5, and d 10 for protein or RNA isolation.

Histological and Immunofluorescence Analyses
Isolated mammary glands were fixed in 10% formalin, embedded in paraffin, and sectioned to 5 µm by standard procedures. Hematoxylin and eosin staining was performed as described previously (27). For ß-gal staining, tissues were fixed for 1 h in 10% formalin and incubated overnight at room temperature in X-gal staining buffer (2 mM MgCl2, 5 mM K-ferrocyanate, 5 mM K-ferricyanate, 0.01% Igepal CA 630, 0.1% Na-deoxycholate, 0.08% X-gal in PBS) before embedding in paraffin. Sections were deparaffinized, rehydrated, and counterstained with nuclear fast red.

For immunofluorescence studies, mammary gland tissues were fresh frozen in OCT, and 5 µm sections were prepared at –30 C. Sections were fixed in 4% paraformaldehyde, 0.1% Triton X-100 in PBS and blocked for 1 h with the Mouse on Mouse reagent (Vector Laboratories, Inc., Burlingame, CA). Individual sections were incubated with primary antibodies for 1 h at room temperature or 4 C overnight. Primary antibodies included rabbit polyclonal anti-Mist1 (1:1000) (25), mouse monoclonal anti-ß-gal (1:100; Developmental Studies Hybridoma Bank), mouse monoclonal antismooth muscle actin (1:10,000; Sigma Chemical Co., St. Louis, MO), rabbit polyclonal anticonnexin32 (1:100; Zymed Laboratories, Inc., South San Francisco, CA), rat monoclonal anti-E-cadherin (1:100; Zymed Laboratories), rabbit polyclonal anti-ß-catenin (1:1000; Sigma), and rabbit polyclonal anti-connexin26 (1:100; Zymed Laboratories). Sections were washed with PBS and incubated with either a biotinylated secondary antibody or fluorescein- or Texas Red-conjugated secondary antibodies (1:300; Vector Laboratories) for 20 min at room temperature. When biotinylated secondary antibodies were used, a streptavidin-conjugated Alexa 488 or 594 (Molecular Probes, Inc., Eugene, OR) tertiary antibody was added for 5 min following the instructions in the avidin/biotin blocking kit (Vector Laboratories). For some studies, the level of apoptosis was evaluated using standard TUNEL assays (Roche Applied Science, Indianapolis, IN) following the manufacturer’s instructions.

Immunoblot Analyses
Nuclear extracts were prepared from individual mammary glands by standard procedures. Briefly, tissues were homogenized and incubated in cell lysis buffer containing 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 85 mM KCl, 0.5% Nonidet P-40, 20 mM NaF, 5 mM Na orthovanadate containing protease and phosphatase inhibitor cocktails (Sigma) for 30 min at 4 C. The samples were then centrifuged at 5000 rpm for 20 min, and the pellets were suspended in nuclear extraction buffer (50 mM Tris, pH 8.1; 10 mM EDTA, 1% sodium dodecyl sulfate, 20 mM NaF, 5 mM sodium orthovanadate) containing protease and phosphatase inhibitor cocktails. After 10 min on ice, nuclei were sonicated, and the debris was removed by centrifugation for 15 min at 4 C. Nuclear extracts were then collected and stored at –80 C. Total cell lysates were prepared as described by Nevalainen et al. (57). Briefly, tissues were homogenized and incubated in lysis buffer (20 mM Tris, pH 7.4; 100 mM NaCl, 20 mM NaF, 10% glycerol, 5 mM sodium orthovanadate, 5 mM EDTA, 1% Nonidet P-40, plus protease and phosphatase inhibitor cocktails) for 30 min on ice. Protein extracts were cleared by centrifugation for 15 min at 4 C. Protein samples were quantified using the Pierce BCA protein assay reagent. Proteins (30–50 µg) were resolved by SDS-PAGE, transferred to polyvinylidine difluoride membranes, and incubated with primary antibodies. Id1, Id2, ß-actin, and Stat3 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas the phospho-Stat3 (Tyr705) antibody was obtained from Cell Signaling Technology (Danuers, MA). Antigen-antibody complexes were detected by incubating membranes with a horseradish peroxidase-coupled secondary antibody, followed by enhanced chemiluminescence substrate reactions using enhanced chemiluminescence Western blotting detection reagents (Pierce Chemical Co., Rockford, IL).

RNA Expression
RNA was isolated from mammary gland tissues or SCp2 cells using the TRIzol reagent (Invitrogen, San Diego, CA) following manufacturer’s instructions. Total RNA (2 µg) was reverse transcribed using Maloney murine leukemia virus reverse transcriptase (Invitrogen). cDNA reactions were subsequently amplified with gene-specific primers to mouse Mist1 (5'-GCGCGTACGGCCTCGAAT-3', 5'-CCAGCCCTAGAGAAGATG-3'); ß-actin (5'-ATTGTTACCAACTGGGACG-3', 5'-TCTCCTGCTCGAAGTCTAG-3'); ß-casein (5'-GTGCTACTT- GCTGCAGAAAGTACAG-3', 5'-ACTACATTTACTGTATCCTCTGAC-3'); and ITF-2 (5'-ACGACGACAAGAAGGATATC-3', 5'-ATAATACAGCTGTTAAGGAA-3'). PCR conditions for each primer set were 95 C/60 sec, 55 C/55 sec, 72 C/55 sec. A custom MultiGene-12 RT-PCR Profiling Kit (SuperArray Bioscience Corp., Frederick, MD) was used for the target genes Tcf3, Hes1, Tcf12, Hand2, Twist1, Ascl1, Bhlhb2, Bhlhb3, and Gapdhfollowing the manufacturer’s protocol and PCR conditions: 95 C/30 sec, 55 C/30 sec, and 72 C/30 sec.

Statistical Analysis
Quantitative data are expressed as the mean ± SE. Statistical analysis of the data was performed by one-way ANOVA and pair-wise comparisons using the SAS program. P values < 0.05 were considered statistically significant.


   ACKNOWLEDGMENTS
 
We wish to thank Judy Hallett and the Purdue Cancer Center Transgenic Mouse Core Facility for their assistance in generating the mice described in this report.


   FOOTNOTES
 
This work was supported by grants to S.F.K. from the Department of Defense Breast Cancer Research Program (BC043093), the NIH (DK55489), and from the Purdue University Cancer Center.

First Published Online April 27, 2006

Abbreviations: bHLH, Basic helix-loop-helix; Cx26, connexin 26; Cx32, connexin 32; ß-gal, ß-galactosidase; HLH, helix-loop-helix; Id, inhibitor of differentiation/DNA binding; ITF-2, Ig transcription factor 2; Stat, signal transducer and activator of transcription; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; WT, wild type.

Received for publication May 30, 2005. Accepted for publication April 17, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hennighausen L 2000 Mouse models for breast cancer. Breast Cancer Res 2:2–7[CrossRef][Medline]
  2. Hennighausen L, Robinson GW 2001 Signaling pathways in mammary gland development. Dev Cell 1:467–475[CrossRef][Medline]
  3. Hennighausen L, Robinson GW 1998 Think globally, act locally: the making of a mouse mammary gland. Genes Dev 12:449–455[Free Full Text]
  4. Hennighausen L, Robinson GW, Wagner KU, Liu X 1997 Developing a mammary gland is a stat affair. J Mammary Gland Biol Neoplasia 2:365–372[CrossRef][Medline]
  5. Hennighausen L, Robinson GW, Wagner KU, Liu W 1997 Prolactin signaling in mammary gland development. J Biol Chem 272:7567–7569[Free Full Text]
  6. Neville MC, Daniel CW 1987 The mammary gland. Development, regulation and function. New York and London: Plenum Press
  7. Mulac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM 2003 Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc Natl Acad Sci USA 100:9744–9749[Abstract/Free Full Text]
  8. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
  9. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, Warner M, Gustafsson JA 2002 Involvement of estrogen receptor ß in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 99:15578–15583[Abstract/Free Full Text]
  10. Streuli CH, Edwards GM, Delcommenne M, Whitelaw CB, Burdon TG, Schindler C, Watson CJ 1995 Stat5 as a target for regulation by extracellular matrix. J Biol Chem 270:21639–21644[Abstract/Free Full Text]
  11. Atchley WR, Fitch WM 1997 A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 94:5172–5176[Abstract/Free Full Text]
  12. Massari ME, Murre C 2000 Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol 20:429–440[Free Full Text]
  13. Fisher A, Caudy M 1998 The function of hairy-related bHLH repressor proteins in cell fate decisions. Bioessays 20:298–306[CrossRef][Medline]
  14. McLellan AS, Langlands K, Kealey T 2002 Exhaustive identification of human class II basic helix-loop-helix proteins by virtual library screening. Gene Expr Patterns 2:329–335[CrossRef][Medline]
  15. Norton JD 2000 ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci 113:3897–3905[Abstract]
  16. de Candia P, Benera R, Solit DB 2004 A role for Id proteins in mammary gland physiology and tumorigenesis. Adv Cancer Res 92:81–94[CrossRef][Medline]
  17. Desprez PY, Hara E, Bissell MJ, Campisi J 1995 Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1. Mol Cell Biol 15:3398–3404[Abstract]
  18. Itahana Y, Singh J, Sumida T, Coppe JP, Parrinello S, Bennington JL, Desprez PY 2003 Role of Id-2 in the maintenance of a differentiated and noninvasive phenotype in breast cancer cells. Cancer Res 63:7098–7105[Abstract/Free Full Text]
  19. Lin CQ, Parrinello S, Campisi J, Desprez PY 1999 Regulation of mammary epithelial cell phenotypes by the helix-loop-helix protein, Id-1. Endocr Relat Cancer 6:49–50[Abstract]
  20. Lin CQ, Singh J, Murata K, Itahana Y, Parrinello S, Liang SH, Gillett CE, Campisi J, Desprez PY 2000 A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells. Cancer Res 60:1332–1340[Abstract/Free Full Text]
  21. Mori S, Nishikawa SI, Yokota Y 2000 Lactation defect in mice lacking the helix-loop-helix inhibitor Id2. EMBO J 19:5772–5781[CrossRef][Medline]
  22. Parrinello S, Lin CQ, Murata K, Itahana Y, Singh J, Krtolica A, Campisi J, Desprez PY 2001 Id-1, ITF-2, and Id-2 comprise a network of helix-loop-helix proteins that regulate mammary epithelial cell proliferation, differentiation, and apoptosis. J Biol Chem 276:39213–39219[Abstract/Free Full Text]
  23. Shan L, Yu M, Qiu C, Snyderwine EG 2003 Id4 regulates mammary epithelial cell growth and differentiation and is overexpressed in rat mammary gland carcinomas. Am J Pathol 163:2495–2502[Abstract/Free Full Text]
  24. Lemercier C, To RQ, Swanson BJ, Lyons GE, Konieczny SF 1997 Mist1: a novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern. Dev Biol 182:101–113[CrossRef][Medline]
  25. Pin CL, Bonvissuto AC, Konieczny SF 2000 Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis. Anat Rec 259:157–167[CrossRef][Medline]
  26. Pin CL, Rukstalis JM, Johnson C, Konieczny SF 2001 The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity. J Cell Biol 155:519–530[Abstract/Free Full Text]
  27. Zhu L, Tran T, Rukstalis JM, Sun P, Damsz B, Konieczny SF 2004 Inhibition of Mist1 homodimer formation induces pancreatic acinar-to-ductal metaplasia. Mol Cell Biol 24:2673–2681[Abstract/Free Full Text]
  28. Lemercier C, To RQ, Carrasco RA, Konieczny SF 1998 The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of myoD. EMBO J 17:1412–1422[CrossRef][Medline]
  29. Rukstalis JM, Kowalik A, Zhu L, Lidington D, Pin CL, Konieczny SF 2003 Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1. J Cell Sci 116:3315–3325[Abstract/Free Full Text]
  30. Desprez PY, Sumida T, Coppe JP 2003 Helix-loop-helix proteins in mammary gland development and breast cancer. J Mammary Gland Biol Neoplasia 8:225–239[CrossRef][Medline]
  31. Coppe JP, Smith AP, Desprez PY 2003 Id proteins in epithelial cells. Exp Cell Res 285:131–145[CrossRef][Medline]
  32. Desprez PY, Lin CQ, Thomasset N, Sympson CJ, Bissell MJ, Campisi J 1998 A novel pathway for mammary epithelial cell invasion induced by the helix-loop-helix protein Id-1. Mol Cell Biol 18:4577–4588[Abstract/Free Full Text]
  33. Kolligs FT, Nieman MT, Winer I, Hu G, Van Mater D, Feng Y, Smith IM, Wu R, Zhai Y, Cho KR, Fearon ER 2002 ITF-2, a downstream target of the Wnt/TCF pathway, is activated in human cancers with ß-catenin defects and promotes neoplastic transformation. Cancer Cell 1:145–155[CrossRef][Medline]
  34. Tobon H, Salazar H 1975 Ultrastructure of the human mammary gland. II. Postpartum lactogenesis. J Clin Endocrinol Metab 40:834–844[Abstract/Free Full Text]
  35. Lazard D, Sastre X, Frid MG, Glukhova MA, Thiery JP, Koteliansky VE 1993 Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci USA 90:999–1003[Abstract/Free Full Text]
  36. Li M, Liu X, Robinson G, Bar-Peled U, Wagner KU, Young WS, Hennighausen L, Furth PA 1997 Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc Natl Acad Sci USA 94:3425–3430[Abstract/Free Full Text]
  37. Prince JM, Klinowska TC, Marshman E, Lowe ET, Mayer U, Miner J, Aberdam D, Vestweber D, Gusterson B, Streuli CH 2002 Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo. Dev Dyn 223:497–516[CrossRef][Medline]
  38. Chapman RS, Lourenco PC, Tonner E, Flint DJ, Selbert S, Takeda K, Akira S, Clarke AR, Watson CJ 1999 Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev 13:2604–2616[Abstract/Free Full Text]
  39. Chapman RS, Lourenco P, Tonner E, Flint D, Selbert S, Takeda K, Akira S, Clarke AR, Watson CJ 2000 The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3. Adv Exp Med Biol 480:129–138[Medline]
  40. Bry C, Maass K, Miyoshi K, Willecke K, Ott T, Robinson GW, Hennighausen L 2004 Loss of connexin 26 in mammary epithelium during early but not during late pregnancy results in unscheduled apoptosis and impaired development. Dev Biol 267:418–429[CrossRef][Medline]
  41. Carystinos GD, Bier A, Batist G 2001 The role of connexin-mediated cell-cell communication in breast cancer metastasis. J Mammary Gland Biol Neoplasia 6:431–440[CrossRef][Medline]
  42. Locke D, Perusinghe N, Newman T, Jayatilake H, Evans WH, Monaghan P 2000 Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland. J Cell Physiol 183:228–237[CrossRef][Medline]
  43. Pozzi A, Risek B, Kiang DT, Gilula NB, Kumar NM 1995 Analysis of multiple gap junction gene products in the rodent and human mammary gland. Exp Cell Res 220:212–219[CrossRef][Medline]
  44. Kim WY, Fritzsch B, Serls A, Bakel LA, Huang EJ, Reichardt LF, Barth DS, Lee JE 2001 NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development 128:417–426[Abstract]
  45. Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R 1993 MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75:1351–1359[CrossRef][Medline]
  46. Srivastava D, Cserjesi P, Olson EN 1995 A subclass of bHLH proteins required for cardiac morphogenesis. Science 270:1995–1999[Abstract/Free Full Text]
  47. Krapp A, Knofler M, Ledermann B, Burki K, Berney C, Zoerkler N, Hagenbuchle O, Wellauer PK 1998 The bHLH protein PTF1–p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev 12:3752–3763[Abstract/Free Full Text]
  48. Miyoshi K, Meyer B, Gruss P, Cui Y, Renou JP, Morgan FV, Smith GH, Reichenstein M, Shani M, Hennighausen L, Robinson GW 2002 Mammary epithelial cells are not able to undergo pregnancy-dependent differentiation in the absence of the helix-loop-helix inhibitor Id2. Mol Endocrinol 16:2892–2901[Abstract/Free Full Text]
  49. St-Pierre B, Cooper M, Jiang Z, Zacksenhaus E, Egan SE 2004 Dynamic regulation of the Stra13/Sharp/Dec bHLH repressors in mammary epithelium. Dev Dyn 230:124–130[CrossRef][Medline]
  50. Shennan DB, Peaker M 2000 Transport of milk constituents by the mammary gland. Physiol Rev 80:925–951[Abstract/Free Full Text]
  51. Kritikou EA, Sharkey A, Abell K, Came PJ, Anderson E, Clarkson RW, Watson CJ 2003 A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland. Development 130:3459–3468[Abstract/Free Full Text]
  52. Zhao L, Melenhorst JJ, Hennighausen L 2002 Loss of interleukin 6 results in delayed mammary gland involution: a possible role for mitogen-activated protein kinase and not signal transducer and activator of transcription 3. Mol Endocrinol 16:2902–2912[Abstract/Free Full Text]
  53. Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, Cox CE, Falcone R, Fairclough R, Parsons S, Laudano A, Gazit A, Levitzki A, Kraker A, Jove R 2001 Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 20:2499–2513[CrossRef][Medline]
  54. Yamashita H, Iwase H, Toyama T, Fujii Y 2003 Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells. Oncogene 22:1638–1652[CrossRef][Medline]
  55. Yamashita H, Nishio M, Fujii Y, Iwase H 2004 Dominant-negative Stat5 inhibits growth and induces apoptosis in T47D-derived tumors in nude mice. Cancer Sci 95:662–665[CrossRef][Medline]
  56. Johnson CL, Kowalik AS, Rajakumar N, Pin CL 2004 Mist1 is necessary for the establishment of granule organization in serous exocrine cells of the gastrointestinal tract. Mech Dev 121:261–272[CrossRef][Medline]
  57. Nevalainen MT, Xie J, Bubendorf L, Wagner KU, Rui H 2002 Basal activation of transcription factor signal transducer and activator of transcription (Stat5) in nonpregnant mouse and human breast epithelium. Mol Endocrinol 16:1108–1124[Abstract/Free Full Text]



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