PTEN overexpression suppresses proliferation and differentiation and enhances apoptosis of the mouse mammary epithelium (original) (raw)
Generation of transgenic mice overexpressing PTEN specifically in the mammary gland. To determine the role of PTEN in the developing mammary gland, we overexpressed the human PTEN cDNA transgene (hPTEN) specifically in the mammary epithelium using the MMTV-LTR. The transgenic construct used is shown in Figure 1a. Six founders with transgene integration were obtained. A representative Southern blot and PCR derived from BamHI-digested genomic DNA are shown for two founders in Figure 1b. Of these six founders, five transmitted the transgene through the germline. These founders were then bred with WT mice to generate females to test the expression of PTEN.
(a) Schematic of the construct used to generate MK-PTEN mice. (b) Southern blot and PCR show transmission of the transgene to the germline. Left: the presence of the 2.1-kb transgene is indicated by the arrow. Right: the 536-bp product corresponds to PTEN. (c) mRNA and (d) protein profile expression of PTEN throughout the development of the mammary gland in MK-PTEN and WT mice. (c) Northern blot analysis was performed using total RNA isolated from mammary gland of WT and MK-PTEN mice at various stages of development. The blot was first hybridized with the full-length hPTEN probe followed by a control 18S probe (data not shown). The ratio of MK-PTEN expression levels to that in WT mice, as normalized to 18S RNA levels, is shown on the graph. Results represent mean ± SD for four MK-PTEN and four WT mice at each stage. (d) Protein extracts from mammary glands of MK-PTEN and WT mice at various stages were subjected to immunoblotting for PTEN. Samples contained equal levels of protein, as confirmed by reprobing each membrane with an anti–α-actin antibody (data not shown). The ratio of PTEN protein level between MK-PTEN and WT mice at various stages of mammary development is represented as the mean ± SD for four MK-PTEN and four WT mice at each stage. V-5 and V-6, virgin 5 and 6 weeks; P-9, P-14, and P-20, pregnancy 9, 14, and 20 days; L-2 and L-10, lactation 2 and 10 days; I-2 and I-6, involution 2 and 6 days.
Expression of PTEN throughout the development of the mammary gland in MK-PTEN and WT mice. We next analyzed each of the transgenic lines for the expression of PTEN. Total RNA was isolated from biopsies of inguinal mammary glands (number 4) from transgenic and WT littermates 10 days after the onset of lactation, when the MMTV-LTR has been shown to be highly active. In the mammary glands, we detected PTEN as a major transcript of 2.5 kb and two other mRNA species of 5 and 3 kb, the latter two being weakly expressed (data not shown). Two founders, F7011 and F7044, highly expressed PTEN at day 10 of lactation, as compared with the WT mice. The profile of PTEN mRNA expression during the development of the mammary gland was determined in the founder F7011 (Figure 1c). In MK-PTEN mice, the level of PTEN mRNA progressively increased, as compared with WT littermates, from the age of 6 weeks (virgins), through the beginning of puberty and throughout pregnancy, peaking at day 10 of lactation and then decreasing during involution of the mammary gland (Figure 1c). These findings are in agreement with the hormonally regulated expression profile of the MMTV-LTR (23).
To determine whether the differences in mRNA were also reflected at the level of protein, we performed Western blot analyses on mammary gland extracts prepared from MK-PTEN and WT female mice at different stages of development. In 3- and 5-week-old virgins, two bands of approximately 54 and 58 kDa in size were detected in both WT and MK-PTEN mice. These two bands are also detected when using two other PTEN antibodies, suggesting that they are specific and correspond to two isoforms of the PTEN protein (data not shown). At the age of 6 weeks, the level of PTEN protein is about twofold higher in MK-PTEN than in WT mice (Figure 1d). This difference between transgenic and WT mice progressively increases throughout all stages of pregnancy and becomes maximal at day 10 of lactation, then decreases during involution (Figure 1d). These results show that PTEN mRNA and protein expression profiles closely correlate throughout development of the mammary gland (Figure 1, compare c and d). To determine whether the transgene was expressed in the expected tissue-specific manner, protein extracts from different tissues of MK-PTEN mice at day 20 of pregnancy and day 10 of lactation were immuno-blotted for PTEN. PTEN expression was observed only in the mammary gland (data not shown). This protein expression profile is in agreement with that of other MMTV-driven transgenes (21).
We used immunohistochemistry to localize PTEN within the mammary gland of WT and transgenic mice (Figure 2). In WT mice, PTEN immunostaining is very low in virgin, pregnant, and lactating mice. In 7-week-old virgins, higher PTEN staining is observed in ductal epithelial cells in MK-PTEN as compared with WT littermates (Figure 2). Despite the higher expression of PTEN within the virgin MK-PTEN mammary gland after 6 weeks (Figure 1d), we did not detect any changes in ductal growth or in cellular morphology within the nulliparous mammary gland at the ages of 7 or 9 weeks, as determined by whole mounts and histologically stained paraffin-embedded sections (data not shown). During early pregnancy, late pregnancy, and lactation, about 70%, 80%, and 95% of the lobuloalveoli in MK-PTEN mice were immunoreactive for PTEN, respectively. PTEN staining is very weak in the ductal epithelial cells at each of these stages, as shown in Figure 2, suggesting that overexpression of PTEN in the mammary gland of MK-PTEN mice is more specific to the lobuloalveoli. Thus, the immunohistochemical results are in close agreement with those obtained from PTEN Northern and Western blots.
Immunohistochemical localization of PTEN in the mammary gland of MK-PTEN and WT mice. Sections from mammary glands removed from virgin, 9-day-pregnant, 14-day-pregnant, and 2-day-lactating mice were analyzed by immunohistochemistry as described in Methods. PTEN-expressing lobuloalveoli and ductal epithelial cells are indicated by black arrows and white arrows, respectively.
PTEN phosphatase activity and phosphorylation of Akt and MAPK in the mammary gland of MK-PTEN mice. To determine the functionality of PTEN in the mammary gland of MK-PTEN mice, we measured the PTEN phosphatase activity. We also determined the phosphorylation state of Akt and Erk1/2, which are two known targets of PTEN. PTEN was immunoprecipitated from mammary gland extracts at different stages, and phosphatase activity was measured using PIP3 as substrate. As shown in Figure 3, PTEN phosphatase activity levels remain relatively constant throughout mammary gland development in WT mice. In striking contrast, PTEN phosphatase activity progressively increases throughout development, pregnancy, and lactation in MK-PTEN mice and decreases after involution (Figure 3a). We also found that during early and middle pregnancy (9 and 14 days), the phosphorylation state of Akt and FKHR was decreased by about 50% in MK-PTEN mice, as compared with WT mice (Figure 3b). However, the level of Erk1/2 phosphorylation was similar in WT and MK-PTEN mice (Figure 3b). Thus, these results suggest that PTEN functions through the PI3K/Akt pathway in the mammary gland of MK-PTEN mice.
PTEN phosphatase activity and phosphorylation state of Akt and MAPK in mammary glands of MK-PTEN and WT mice. (a) Mammary gland protein extracts from MK-PTEN and WT mice (500 μg protein per sample) were subjected to immunoprecipitation with the PTEN polyclonal C20 antibody, and the phosphatase activity was measured as described in the Methods. The phosphatase activity in MK-PTEN and WT is represented as mean ± SD in the graph (n = 4 MK-PTEN and WT mice for each group except in virgin and involution, where n = 2 mice in each group). *P < 0.05, ***P < 0.001. (b) Akt and Erk1/2 activation in mammary gland extracts from MK-PTEN and WT mice at 9 and 14 days of pregnancy. Extracts containing 120 μg of protein were analyzed by immunoblotting with anti-Akt and anti-Erk1/2 antibodies that either recognize the phosphorylated form (pAkt or pErk1/2) or equally recognize the phosphorylated and dephosphorylated forms, i.e., total levels (Akt and Erk1/2). The blots were also analyzed for phospho-FKHR immunoreactivity. These results are representative of at least two identical experiments.
Decreases in epithelial cell number and milk protein expression in the mammary gland of MK-PTEN mice are associated with a defect in lactation. The primary biologic function of the mammary gland is to provide nourishment to suckling young in the form of milk. To determine whether PTEN is involved in the functionality of the mammary gland, we examined the ability of MK-PTEN mice to adequately nourish pups (measured by pup weight). We observed that 2 days after the onset of lactation, 30% of the pups from MK-PTEN females died (data not shown), and the survivors exhibited significant growth retardation until 9 days old (Figure 4a). However, if, immediately after the delivery, WT female mice nursed MK-PTEN pups, the pups survived and grew normally (Figure 4a). These findings suggest that the MK-PTEN newborns are viable but die of malnutrition during nursing. To understand the origin of these physiological perturbations, we performed histological examinations on the mammary glands from MK-PTEN mice. We found that the number of lobuloalveoli in MK-PTEN mice was reduced by 30–50% in pregnant MK-PTEN mice, as compared with the WT littermates (Figure 2). We also investigated whether these lobuloalveoli were functional by measuring the expression levels of various milk mRNAs during early and middle pregnancy (Figure 4b). At 9 days of pregnancy, WDNM1 and β-casein mRNA levels were reduced by about 40% in MK-PTEN as compared with WT littermates, as normalized to 18S RNA levels. Similar results were observed for the mRNA expression of Stat5a and 5b, lactalbumin, and WAP after 14 days of pregnancy (Figure 4b). Keratin 18 serves as a marker of the epithelial cells. No differences in litter size were observed between MK-PTEN and WT mice (data not shown), although MK-PTEN male mice expressed higher levels of PTEN in certain reproductive organs (data not shown).
(a) Growth rate of pups from WT and MK-PTEN mice. Newborns from WT and MK-PTEN mothers were weighed each day for 2 weeks. Data are expressed as the mean ± SD of 16 MK-PTEN pups nursing with either an MK-PTEN or a WT mother, as indicated. (b) Milk protein mRNA expression in mammary glands of MK-PTEN and WT mice. Total RNA was isolated from mammary glands of WT and MK-PTEN at days 9 and 14 of pregnancy. Northern blot analysis was performed using probes corresponding to cDNAs of various milk proteins, as indicated. Blots were stripped and reprobed with an 18S probe as a loading control and with K18 as a marker of epithelial cells. Each lane represents a single animal. Similar results were obtained with a total of four WT and four MK-PTEN mice.
Mammary epithelium overexpressing PTEN exhibits decreased proliferation and increased apoptosis. The observed decrease in the epithelial content of mammary glands harboring PTEN-overexpressing epithelium may reflect a decrease in the rate of epithelial proliferation and/or an increase in the level of epithelial apoptosis. We tested these two possibilities by quantifying BrdU incorporation and by TUNEL assays, respectively. To assess the levels of epithelial cell proliferation in mice overexpressing PTEN specifically in the mammary gland, mice were labeled with the BrdU. Based on immunohistochemical analysis, the number of BrdU-positive nuclei in mammary epithelium overexpressing PTEN appeared to be lower than that in mammary epithelium of WT mice (Figure 5). To quantify this decrease, the percentage of BrdU-positive nuclei relative to the total number of nuclei was calculated at three different stages of pregnancy (P-9, P-14, and P-20; Figure 5b, left). The percentage of BrdU-positive epithelial nuclei was decreased by approximately 50% in mammary epithelium overexpressing PTEN compared with that in WT mice. In accordance with these results, we also found that cyclin D1 immunoreactivity was significantly reduced in mammary epithelium of MK-PTEN as compared with WT mice (data not shown). These data demonstrate that the mammary epithelium of MK-PTEN mice is indeed hypoplastic at all stages of pregnancy.
Cell proliferation is decreased and apoptosis is increased in mammary epithelium from MK-PTEN and WT mice during pregnancy. At the indicated stages, pregnant MK-PTEN and WT mice were injected with BrdU and sacrificed 2 hours later, as described in Methods. (a) BrdU-labeled nuclei were detected by immunostaining, and apoptotic nuclei were stained by TUNEL. (b) About 3,000 nuclei were counted, and the percentage of labeled epithelial cells was determined at 9 (P-9), 14 (P-14), and 20 (P-20) days of pregnancy. Data are expressed as the percentage of labeled cells ± SD. Averages were compared by t test. Averages were compared by t test. *P < 0.05, ***P < 0.001. WT, black bars; MK-PTEN, white bars.
To assess the level of apoptosis in mammary epithelium of MK-PTEN mice, TUNEL analysis was performed on sections prepared from MK-PTEN and WT mice at different stages of pregnancy. TUNEL-positive nuclei were detected by immunohistochemistry. Based on immunohistochemical analysis, the percentage of TUNEL-positive nuclei in mammary epithelium of MK-PTEN mice was approximately two- to threefold greater than that observed in the mammary epithelium of WT mice (Figure 5b, right). Thus, these data suggest that the lower cell density in mammary epithelium of MK-PTEN mice is due to both a decrease in cell proliferation and a concomitant increase in apoptosis.
Profiling gene expression in MK-PTEN transgenic mice. To further understand the developmental defects in the MK-PTEN transgenic mice, we profiled the gene expression pattern in mammary tissue 2 days after parturition. Since most genes expressed in mammary tissue are absent from commercial cDNA arrays, we generated an array, the so-called mammochip, that contains 3,600 cDNAs selected from different cDNAs of mammary origin. While 11 genes were expressed preferentially in mammary tissue from MK-PTEN mice, nine genes were preferentially expressed in WT tissue (Table 1). Out of the 11 genes overexpressed in MK-PTEN mammary tissue, six corresponded to known genes. Most notably, the expression of the IGFBP-5 gene was 26 times higher in MK-PTEN mammary tissue. Interestingly, a database search demonstrated that expression of IGFBP-5 is preferentially seen in mammary tumors of transgenic mice. The expression of the carbonic anhydrase 3 was also increased fivefold in MK-PTEN mammary epithelium. The higher expression of both IGFBP-5 and carbonic anhydrase 3 genes in MK-PTEN than in WT mice was confirmed by RT-PCR (Figure 6a). We used immunohistochemistry to localize IGFBP-5 within the mammary gland of WT and MK-PTEN mice (Figure 6b). IGFBP-5 immunostaining is very high in epithelial cells of MK-PTEN mice at day 2 of lactation as compared with the WT littermates (Figure 6b).
(a) mRNA expression determined by RT-PCR of some genes up- or downregulated in mammary tissue of MK-PTEN mice as compared with that of WT mice. These results are representative of two WT and two MK-PTEN mice different from those used to perform the microarray experiment. (b) Immunohistochemical localization of IGFBP-5 in the mammary gland of MK-PTEN and WT mice. Sections from mammary glands removed from mice lactating for 2 days were analyzed by immunohistochemistry as described in Methods. (c) An IGFBP-5–blocking peptide reduces the level of the apoptotic mammary epithelial cells of MK-PTEN mice. Following incubation of mammary epithelial cells from WT and MK-PTEN mice in DMEM with serum for 48 hours, the cells were untreated or treated with 5 or 10 μg/ml of an IGFBP-5–blocking peptide. Apoptotic cells were detected by TUNEL assay and caspase-3 activity was determined by the ApoAlert colorimetric assay as described in Methods. The data are representative of three independent experiments performed in triplicates.
Genes differentially regulated in mammary tissue of MK-PTEN and WTmice
Expressed sequence tag database searches demonstrated that the genes overexpressed in MK-PTEN tissue were also highly expressed in mammary tumors of transgenic mice. Three known genes, those encoding δ-casein, JNK/SAPK-1, and WAP, were expressed 27-, 15-, and 7-fold more highly, respectively, in WT tissue (Table 1). Again, these results were confirmed by RT-PCR (Figure 6a). The genes and gene products corresponding to the differentially expressed sequence tags have not been investigated. We further monitored the expression of 2,600 known genes, including many oncogenes, on a commercial oncochip. While six genes were expressed preferentially in mammary tissue from MK-PTEN mice, six other genes were preferentially expressed in WT tissue (Table 1). Most of the identified genes were involved in the cell proliferation or apoptosis. Indeed, the cdc2, complement component 3, thymosin β 10, and cell cycle G0/G1 genes were expressed 11-, 6-, 5-, and 3-fold more highly, respectively, in MK-PTEN mammary tissue, whereas the high-mobility group protein I isoform C (HMG-IC), glucocorticoid-induced leucine zipper (GILZ), and receptor her3 genes were expressed 13-, 4-, and 4-fold more highly, respectively, in WT mammary tissue (Table 1).
IGFBP-5 is involved in the apoptosis observed in the MK-PTEN mammary epithelial cells. IGFBP-5 is an IGF-binding protein involved in vivo in mammary apoptosis during the involution stage. To determine the role of IGFBP-5 in the apoptosis observed in MK-PTEN mammary epithelium, we isolated and cultured mammary epithelial cells from WT and MK-PTEN mice. After 48 hours of culture, cells were incubated for 24 hours in the presence or absence of an IGFBP-5–blocking peptide, and we determined the apoptosis level by tunnel assay and measured the caspase-3 activity. The percentage of apoptotic cells and the caspase-3 activity were about three and four times higher in MK-PTEN mammary epithelial cells than in WT, respectively (Figure 6c). Interestingly, the IGFBP-5–blocking peptide (5 or 10 μg/ml) reduced by about 40% (P < 0.05) the higher apoptosis level observed in the MK-PTEN mammary epithelial cells (Figure 6c). Thus, these results demonstrate that the apoptosis observed in mammary MK-PTEN epithelial cells is due in part to IGFBP-5.