Keratin 6 is not essential for mammary gland development
© Grimm et al.; licensee BioMed Central Ltd. 2006
Received: 7 February 2006
Accepted: 25 May 2006
Published: 21 June 2006
Keratin 6 (K6) has previously been identified as a marker of early mammary gland development and has also been proposed to be a marker of mammary gland progenitor cells. However, the function of K6 in the mammary gland was not known, so we examined the expression pattern of the protein during both embryonic and postnatal mammary development, as well as the mammary gland phenotype of mice that were null for both K6a and K6b isoforms.
Immunostaining was performed to determine the expression pattern of K6a throughout mammary gland development, from the embryonic mammary bud to lactation. Double immunofluorescence was used to co-localize K6 with known markers of mammary gland development. Wild-type and K6ab-null mammary tissues were transplanted into the cleared fat pads of nude mice and the outgrowths were analyzed for morphology by whole-mount staining and for markers of mammary epithelium by immunostaining. Finally, progesterone receptor (PR) and bromodeoxyuridine co-localization was quantified by double immunofluorescence in wild-type and K6ab-null mammary outgrowths.
Here we report that K6 is expressed earlier than described previously, by embryonic day 16.5. K6a is the predominant isoform expressed in the mammary gland, localized in the body cells and luminal epithelial cells but not in the cap cells or myoepithelial cells. Co-localization studies showed that most K6a-positive cells express steroid receptors but do not proliferate. When both the K6a and K6b genes are deleted, mammary gland development appears normal, with similar expression of most molecular markers examined in both the pubertal gland and the mature gland. Loss of K6a and K6b, however, leads to an increase in the number of steroid-receptor-positive cells, and increased co-localization of steroid receptor expression and proliferation was observed.
Although K6a was not essential for mammary gland development, loss of both K6a and K6b resulted in an increase in PR-positive mammary epithelial cells and decreased proliferation after exposure to steroid hormones. There was also increased co-localization of PR and bromodeoxyuridine, suggesting alterations in patterning events important for normal lobuloalveolar development.
The mammary gland is unique in that its development primarily occurs postnatally. However, the tissue is initially formed during embryonic development (reviewed in ). A milk line first appears at about embryonic day 10.5 (E10.5). At E11.5, five pairs of placodes have formed at specific positions along the milk line, and by E12.5 mammary buds invaginate from the ectoderm, surrounded by a specialized mammary mesenchyme. These mammary anlagen begin to form a lumen by E16.5 and sprout into the underlying fat pad. Branching morphogenesis then occurs, to give rise to a rudimentary ductal tree in the newborn pups.
Any piece of the mammary gland, from the embryonic mammary bud to the differentiated gland, can be transplanted into a cleared fat pat to generate another ductal structure containing all the epithelial cell types that make up the mammary gland, supporting the idea that progenitor cells are dispersed throughout the tissue . Although lineage markers have been identified in the hematopoietic system and epidermis, a clear picture of mammary lineage markers is still evolving [3–5]. However, a handful of putative markers have been identified, including keratin 6 (K6) [6, 7].
Cytokeratins, members of the intermediate filament superfamily, are the main structural components of most epithelial cells. There are more than 55 keratins, consisting of type I (K9 to K20) and type II (K1 to K8) filaments that partner to form coiled-coil heterodimers . Although multiple genes encoding K6 isoforms exist in both human and mouse, the mouse genes seem to have evolved after the species diverged . The mouse isoforms, K6a and K6b, are organized in tandem on chromosome 15 and although their coding sequences show 95% identity, the two genes are differentially regulated at the transcriptional level [10–12]. Germline deletion of both K6a and K6b genes led to the discovery of a third murine isoform, K6hf, expressed mainly in hair follicles . Expression of K6 in the skin is associated with hyperproliferative disorders and in response to stressful stimuli, such as wounding . After an injury, K6 is expressed at the wound site, where its expression is associated with activated keratinocytes . However, the function of K6 in mammary gland development is not known.
K6 is expressed in terminal end buds (TEBs) of the developing mammary gland [15, 16]. Ductal elongation is a highly proliferative phase of mammary gland development, but K6 expression is restricted to the body cells of the TEB and not the proliferative cap cell layer surrounding the tip of the TEB. However, expression of K6 is rare in the mature mammary gland [7, 16]. Additionally, K6 is misexpressed in mature mammary glands from mice that were null for CCAAT-enhancer binding protein-β (C/EBPβ), coinciding with an arrested state of differentiation and a block in cell fate .
The function of K6 in the mammary gland is not known, so we examined the expression pattern throughout mammary development, as well as the mammary gland phenotype of mice that are null for both K6a and K6b isoforms (K6ab-null). Here we report that K6 is expressed earlier than described previously, by E16.5. K6a is the predominant isoform expressed in the mammary gland, localized in the body cells and luminal epithelial cells but not in the cap cells or myoepithelial cells. Co-localization studies showed that most K6a-positive cells express steroid receptors but do not proliferate. When both the K6a and K6b genes are deleted, mammary gland development appears normal, with similar expression of most molecular markers examined in both the pubertal gland and the mature gland. However, an increase in the number of steroid-receptor-positive cells and increased co-localization of steroid receptor expression with proliferation were observed.
Materials and methods
Animals and tissue isolation
Animal care and procedures were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and were in accordance with the procedures detailed in the Guide for Care and Use of Laboratory Animals (NIH publication 85–23). Mice with targeted germline deletion of K6a and K6b have been described previously . Mammary glands were removed from intact 6-week-old wild-type (WT) and K6ab-null mice after an intraperitoneal injection with 5-bromo-2'-deoxyuridine (BrdU; 0.03 mg/g body weight; Sigma, St Louis, MO, USA) 2 hours before being killed. Mammary glands were also isolated from C57Bl/6 mice at various stages of development (embryonic days 14 and 16.5, day 5 pups, 6 and 12 weeks, days 10, 15, 18 of pregnancy, days 2 and 9 of lactation and day 3 of involution). After fixation for 2 hours in cold 4% paraformaldehyde, the tissues were embedded in paraffin. Mammary glands and skin samples were additionally collected from 6-week-old FVB females that were injected intraperitoneally with BrdU 2 hours before being killed. These tissues were either fixed overnight in 10% normal-buffered formalin (NBF) and embedded in paraffin or frozen in OCT (Optimal Cutting Temperature) for cryosectioning. Paraffin-embedded mammary glands and skin samples were sectioned (5 to 7 μm) onto Probe-On Plus charged slides (Fisher Scientific, Pittsburgh, PA, USA). Frozen sections were cut at 5 μm thickness and fixed in acetone for 10 minutes.
Mammary gland transplants
Mammary gland tissues from adult WT or K6ab-null mice were cut into small pieces (about 1 mm3) that were inserted into the cleared fat pads of 3-week-old athymic nu/nu mice . All clearings were subjected to whole-mount staining to verify that all epithelium was removed from the fat pad. Primary transplants were allowed to grow out for at least 16 weeks so that the fat pad was completely filled with epithelium. These outgrowths were removed, cut into small pieces, and slowly frozen in RPMI-1640 medium containing 2% fetal bovine serum with 7% dimethylsulphoxide and used to perform secondary transplants. Outgrowths were taken at 4 and 10 weeks after transplantation. At 10 weeks after transplantation, the host mice were treated for 48 hours with a single interscapular subcutaneous injection of 17β-estradiol benzoate (1 μg) and progesterone (1 mg) in 100 μl of sesame oil (all from Sigma). At 2 hours before being killed, animals were injected with BrdU. After fixation for 2 hours in cold 4% paraformaldehyde, the glands were cut in half lengthways. One half was embedded in paraffin and the other was subjected to hematoxylin whole-mount staining. Whole-mount images were captured with an Olympus SZ40 dissecting microscope connected to a QCapture digital camera.
Antibodies and immunostaining
Paraffin-embedded tissue sections were deparaffinized in xylene, then rehydrated through a graded ethanol series. Immunostaining was performed after microwave antigen retrieval (20 minutes) in 10 mM sodium citrate and blocking in 5% bovine serum albumin in phosphate-buffered saline containing 0.5% Tween 20. For immunohistochemistry, sections were incubated overnight with the following primary antibodies at room temperature: K6 rabbit polyclonal at a dilution of 1:5000 (Covance, Richmond, CA, USA), keratin 5 (K5) rabbit polyclonal at 1:5000 (Covance), keratin 8 (K8) rat monoclonal at 1:5000 (University of Iowa Developmental Studies Hybridoma Bank), estrogen receptor α (ERα) rabbit polyclonal at 1:5000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and biotinylated BrdU mouse monoclonal at 1:50 (BD Biosciences, San Jose, CA, USA). Immunoperoxidase staining was detected using the appropriate biotinylated secondary antibodies and Vectastain Elite ABC and diaminobenzidine substrate kits in accordance with manufacturer's instructions (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence, sections were incubated overnight with the following primary antibodies at room temperature: K6a rabbit polyclonal at 1:100 (Covance), K6b/hf guinea pig polyclonal at 1:100 , smooth muscle α-actin (SMA) mouse monoclonal at 1:100 (Dako, Carpinteria, CA, USA), K8 rat monoclonal at 1:100 (University of Iowa Developmental Studies Hybridoma Bank), ERα rabbit polyclonal at 1:1000 (Santa Cruz Biotechnology), fluorescein isothiocyanate-conjugated BrdU at 1:10 (BD Biosciences), and progesterone receptor (PR) rabbit polyclonal at 1:50 (Dako). Immunofluorescence staining was detected with the appropriate secondary antibodies conjugated with Texas Red, Alexa 568, or Alexa 488 (Molecular Probes, Eugene, OR, USA), and nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; Vector Laboratories).
Image capture, cell counting and statistical analysis
An Olympus BX40 light microscope connected to a MagnaFire digital camera was used to capture images from immunohistochemical staining. A Zeiss Axioskop2 Plus fluorescence microscope connected to an AxioCamMR digital camera was used to capture images from immunofluorescence staining with Axiovision Rel. 4.2 software. At least eight individual 40× fields per group were captured for counting. K6a-positive cells were scored and then the number of cells expressing ERα or BrdU in that subset was counted. Fluorescent images of PR and BrdU immunostaining were captured digitally with an Olympus BX50 microscope connected to a Hamamatsu C5810 charge-coupled device camera. At least eight individual 60× fields per sample were captured for counting. The number of positively stained mammary epithelial cell (MEC) nuclei was expressed as a percentage of the total number of luminal epithelial cells. Statistical significance was determined by Student's t test (two-sample assuming unequal variance).
This study shows that the expression of K6 is highest during the initial development of the gland and becomes sporadic in the mature gland. K6a expression was confined to cells destined to become the luminal epithelial cells of the mammary gland. Even as early as E16.5, K6a expression was detected in the innermost cells of the mammary bud, as opposed to the outer ring of epithelial cells that are p63-positive (SLG and JMR, unpublished observation). Similarly, the body cells of TEBs, as opposed to the cap cell layer, expressed K6a. K6 expression in the epidermis has previously been shown to be regulated by epidermal growth factor and tumor necrosis factor-α signaling pathways [22–24], but nothing is known about what regulates its expression in subpopulations of cells in the mammary anlage or TEBs.
Our original observation of an increased number of K6a-positive cells in the mature mammary glands from C/EBPβ-null mice correlated with a block in development, suggesting an accumulation of more primitive cells . Several other groups have also observed K6-positive cells in their mouse models of mammary gland development, supporting the idea that K6 may indeed be a putative marker of progenitor cells in the gland. For example, Stingl and colleagues have recently described a population of mouse mammary cells called 'colony-forming cells' or Ma-CFCs that were isolated by fluorescence-activated cell sorting based on a CD24highCD49flow profile and occurred with a frequency of about 1 in 63 cells . Although these cells did not display outgrowth potential in fat pad transplantation experiments, Ma-CFCs were defined by Stingl and colleagues as progenitor cells that are able to grow discrete colonies on low-density adherent cultures. Both mRNA and protein levels of K6 were enriched in these Ma-CFC cells, which also had increased expression of other luminal cell markers, such as K8, K18, and K19.
Notch signaling has been implicated in stem cell self-renewal . Recombination signal binding protein, J-type (RBP)-J is the common transcriptional mediator of Notch receptors. Targeted deletion of RBP-J in the mammary gland resulted in a transient increase in K6 expression in luminal epithelial cells during early pregnancy . These results suggested an arrest at an immature stage of mammary gland development, similar to that in the C/EBPβ-null mice.
In addition to being a marker of early mammary gland development and putative progenitor cells, K6 expression has been observed in a subpopulation of cells in both mammary hyperplasias and tumors induced by transgenic expression of Wnt-1, β-catenin, or Myc . However, hyperplasias and tumors induced by polyoma middle T antigen, Neu or H-Ras are more homogeneous and do not express K6. Li and colleagues therefore suggested that some tumors might arise from the amplification of progenitor cells, whereas the others might promote differentiation of the progenitors, thereby depleting the population.
Lisanti and colleagues have reported increased K6 expression in the hyperplastic mammary ducts of caveolin-1-null mice, along with increased β-catenin expression . They proposed that activation of the Wnt/β-catenin pathway led to the accumulation of mammary progenitor cell accumulation. Finally, overexpression of the proto-oncogene Met, a receptor tyrosine kinase, under the control of the MSCV (mouse stem cell virus), resulted in non-progressive mammary neoplasms . These lesions contained large numbers of K6-positive cells, again suggesting that this might represent the expansion of a progenitor cell population. However, when Met was overexpressed under the control of MMTV (mouse mammary tumor virus), no neoplasms were detected and K6 expression was not observed.
Keratinocytes activated by injury or stress express K6 and migrate to the site of wound healing . Although K6 expression has been associated with hyperproliferation, expression of K6 in the epidermis does not overlap with the incorporation of [3H]thymidine , supporting our observation that K6-positive cells are mostly quiescent. Instead, it is possible that K6 regulates a migratory function required in progenitor cells so as to permit their dispersal throughout the mammary gland. However, deletion of K6a and K6b did not seem to have any detectable effect on ductal elongation in mammary glands from intact animals or in outgrowths from tissue transplants.
The lack of an overt mammary gland phenotype in the K6a/b double knockout mice was not unexpected, because it has been difficult to demonstrate the functional properties of most markers used to isolate and characterize stem and progenitor cells by means of gene deletion in genetically engineered mice. For example, deletion of one of the best-characterized mammary stem/progenitor cell markers, CD49f (α6-integrin), did not have any reported mammary gland developmental phenotypes in transplants of the null mammary anlage . This unexpected result might be considered surprising, because integrins have been suggested to have an essential function in adhesion in the stem cell niche . Furthermore, conditional deletion of CD29 (β1-integrin) resulted in impaired alveolar development and lactation , with no reported effect on ductal morphogenesis . Although the triple knockout mouse for Brcp1 (Abcg2), Mdr1a, and Mdr1b resulted in the loss of side population (SP) cells, a phenotype defined by the ability of these cells to efflux Hoechst 33342 dye associated with stem/progenitor cells, there was no mention of a mammary gland development phenotype in these mice .
A key experiment to test the functional effect of loss of progenitor cell markers requires limiting dilution serial transplantation to distinguish long-term versus short-term engraftment of stem/progenitor cells deleted for specific genes. This has only recently been accomplished for CD49f and CD29 . It is most likely that many of these markers may be only just markers, and may not have critical functions. However, notwithstanding this caveat, it is crucial to identify lineage markers as tools for the characterization of different mammary cell types. K6 seems to be one such useful lineage marker.
In the present study, an increase in the number of proliferating PR-positive cells was observed after the loss of K6a/b. K6a-positive cells co-localized with steroid receptor expression, but rarely with BrdU incorporation, a marker of proliferation. Normally, steroid receptor expression does not overlap with proliferation [19–21]. However, inappropriate co-localization of steroid receptors with proliferative markers is often found in pre-neoplastic disease [34–36]. A recent study suggests that activated TGF-β acts in an autocrine manner to prevent ERα-positive mammary epithelial cells from proliferating . In addition to an increased number of K6a-positive MECs, C/EBPβ-null mice have increased levels of activated TGF-β as well as increased expression of PR and decreased proliferation in the mature mammary gland, further supporting this hypothesis [6, 38]. Interestingly, ERα and PR have also been proposed to be markers of mammary progenitor cells, representing a quiescent population scattered throughout the gland hypothesized to self-renew through asymmetric division [39, 40]. Human mammary epithelial cells sorted for expression of p21 or Msi-1, suggested to be putative stem cell markers, also had enriched expression of steroid receptors. Additionally, SP cells had sixfold enrichment of ERα-positive cells in this model system; however, K6 expression was not analyzed . Alternatively, recent studies with a xenograft model of T47D human breast cancer cells expressing ER plus different PR isoforms demonstrated increased K6 mRNA expression in tumors in response to treatment with E + P that was dependent on the expression of PR . Thus, the precise relationship between steroid receptor and K6 expression in normal mammary epithelial cells, and how this might be altered in breast cancer, remains to be established, but it is interesting to speculate that K6 might have a function in this interaction.
Although K6a may not be essential for mammary gland development, the loss of both K6a and K6b resulted in an increased number of PR-positive MECs and decreased proliferation after exposure to steroid hormones. There was also increased co-localization of PR and BrdU, suggesting alterations in patterning events important for lobuloalveolar development. Although K6 does not seem to have a function in progenitor cells, it may still provide a useful lineage marker for studies of mammary gland development and tumorigenesis.
Note added in proof
Interestingly, an unexpected function for keratin 17, a potential keratin 6 partner, in regulating protein synthesis and epithelial cell growth has been reported recently .
CCAAT-enhancer binding protein
- E + P:
estrogen and progesterone
estrogen receptor α
mammary epithelial cell
smooth muscle α-actin
terminal end bud
We would like to thank Maria Gonzalez-Rimbau for histology support and Shirley Small for animal handling support. This work was supported partly by funds from the National Cancer Institute (CA16303 to JMR), the Department of Defense USAMRMC (BC030755 to YL), and the National Institutes of Health (AR052263, AR47898, CA52607 and CA105491 to DRR).
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