p190-B RhoGAP Regulates Mammary Ductal Morphogenesis
Geetika Chakravarty,
Darryl Hadsell,
William Buitrago,
Jeffrey Settleman and
Jeffrey M. Rosen
Department of Molecular & Cellular Biology (G.C., W.B., J.M.R.), Baylor College of Medicine, and Department of Pediatrics (D.H.), Childrens Nutrition Research Center, Houston, Texas 77030; and Massachusetts General Hospital Cancer Center and Harvard Medical School (J.S.), Charlestown, Massachusetts 02129
Address all correspondence and requests for reprints to: Dr. Jeffrey M. Rosen, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: jrosen{at}bcm.tmc.edu.
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ABSTRACT
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Previous studies from our laboratory have demonstrated that p190-B RhoGAP (p190-B) is differentially expressed in the Cap cells of terminal end buds (TEBs) and poorly differentiated rodent mammary tumors. Based on these observations we hypothesized that p190-B might play an essential role in invasion of the TEBs into the surrounding fat pad during ductal morphogenesis. To test this hypothesis, mammary development was studied in p190-B-deficient mice. A haploinsufficiency phenotype was observed in p190-B heterozygous mice as indicated by decreased number and rate of ductal outgrowth(s) at 3, 4, and 5 wk of age when compared with their wild-type littermates. This appeared to result from decreased proliferation in the Cap cells of the TEBs, a phenotype remarkably similar to that observed previously in IGF-I receptor null mammary epithelium. Furthermore, decreased expression of insulin receptor substrates 1 and 2 were observed in TEBs of p190-B heterozygous mice. These findings are consistent with decreased IGF signaling observed previously in p190-B-/- mouse embryo fibroblasts. To further assess if this defect was cell autonomous or due to systemic endocrine effects, the mammary anlagen from p190-B+/+, p190-B+/-, and p190-B-/- mice was rescued by transplantation into the cleared fat pad of recipient Rag1-/- mice. Surprisingly, as opposed to 7580% outgrowths observed using wild-type donor epithelium, only 40% of the heterozygous and none of the p190-B-/- epithelial transplants displayed any outgrowths. Together, these results suggest that p190-B regulates ductal morphogenesis, at least in part, by modulating the IGF signaling axis.
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INTRODUCTION
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EPITHELIAL-STROMAL INTERACTIONS PLAY a critical role in ductal morphogenesis, lobuloalveolar development, and tissue remodeling during involution (1). In the virgin mammary gland, extracellular matrix (ECM) modifications influence ductal branching, end bud development, and epithelial proliferation. The signals propagated by ECM interactions with integrin receptors result in the activation of a number of signaling pathways, among which the activation of Rho family of small GTP-binding proteins play an important role in regulating the formation of focal adhesions and the organization of the actin cytoskeleton (2, 3, 4).
ECM interactions directly facilitate invasion of a highly proliferative structure called the terminal end bud (TEB) into the surrounding fat pad. They are composed of an outer layer of highly proliferative cells called the Cap cells, and a multilayered inner mass containing body cells, some of which undergo apoptosis to give rise to the hollow mammary ducts (5). The Cap cell layer contains a pleuripotent stem cell population that can give rise to intermediate, luminal, and myoepithelial cells of the advancing duct (6, 7, 8, 9). Because of their high proliferative potential, these cells are also believed to be the targets of carcinogenesis (10). Although morphological changes in TEBs that lead to the arborization of the ductal system have been well documented, the genes that facilitate invasion of the TEBs remain elusive. In recent years, it has been demonstrated that both IGF-I and IGF-I receptor (IGF-IR) play a major role in TEB formation and elongation (11, 12).
To identify genes that might be preferentially expressed in TEBs, differential-display PCR was originally employed to distinguish genes that were preferentially expressed in TEBs as compared with the midgland and stromal tissue fractions of the nulliparous Wistar-Furth rats. Interestingly, one of the clones identified in this screen was a new member of the RhoGAP family of proteins called p190-B. Because RhoGAPs have been shown to transduce signals from the ECM to the nucleus, it appeared to be a likely candidate to play a role in TEB invasion. Accordingly, using in situ hybridization, p190-B expression was observed to be highest in the highly proliferative, outer cap cell layer of the TEB. Ducts, alveolar buds, and stroma all expressed p190-B but at much reduced levels. These findings were corroborated by Northern analysis, which detected the highest level of p190-B expression in the virgin mammary gland declining with pregnancy and lactation, suggesting again that p190-B might play a critical role in ductal morphogenesis during virgin mammary gland development (13).
p190-B is a member of RhoGAP family of proteins and is recruited to the sites of integrin clustering. It encodes a protein with an N-terminal GTPase domain and a C-terminal RhoGAP domain that stimulates the intrinsic GTPase activity of Rho, Rac, and cdc-42, thereby functioning as a negative regulator of their signal-transducing activity. These proteins are conserved from flies to humans. p190-B shares some features in common with several members of the Ras, Rab, Ral, and Rho family of GTPases, but it is most closely related to p190-A, sharing 51% amino acid identity (14). These two genes have been mapped to two different chromosomes, suggesting they diverged early in evolution and may have distinct functions (15).
Rho GTPases are key regulators of a wide range of physiological processes (16, 17). Hence, their spatio-temporal expression is tightly regulated through the opposing actions of guanine nucleotide exchange factors, and the GTPase-activating proteins (GAPs) like the p190RhoGAPs. Studies with p190-A and p190-B null mice have revealed that maintaining a tight spatio-temporal regulation of these genes is critical for normal embryonic development. Loss of p190-RhoGAP results in perinatal lethality due to pleural defects, e.g. p190-A null mice die of severe defects in eye development, formation of the corpus callosum, and neural tube closure (18), whereas p190-B null mice exhibit differentiation defects in the brain and thymus (19).
Despite convincing evidence supporting their role in normal embryonic development and its expression in the Cap cells of the TEBs, the exact function of p190-B signaling in postnatal mammary gland development has not been investigated. Accordingly, the role of p190-B was studied in prepubescent and pubescent mice that were either heterozygous or wild type at the p190-B locus. A haploinsufficiency phenotype was observed in p190-B heterozygous mice as indicated by decreased number and rate of ductal outgrowth(s) at 3, 4, and 5 wk of age when compared with their wild-type littermates. This appeared to result from decreased proliferation in the Cap cells of the TEBs, a phenotype remarkably similar to that observed previously in IGF-IR null mammary epithelium. Accordingly, decreased expression of insulin receptor substrate (IRS)-1 and IRS-2 was observed in TEBs of p190-B heterozygous mice. This was consistent with decreased IGF signaling observed previously in p190-B knockout mouse embryo fibroblasts (MEFs) (19). To further assess whether this defect was cell autonomous or due to systemic endocrine effects, the mammary anlagen from p190-B+/+, p190-B+/-, and p190-B-/- mice was rescued by transplantation into the cleared fat pad of recipient Rag1-/- mice. Surprisingly, as opposed to 7580% outgrowths observed using wild-type donor epithelium, only 40% of the heterozygous and none of the p190-B-/- epithelial transplants displayed outgrowths. Together, these results suggest that p190-B regulates ductal morphogenesis, at least in part, by modulating the IGF-signaling axis.
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RESULTS
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Deletion of a Single Functional Allele of p190-B Is Sufficient to Retard Ductal Morphogenesis
Examination of mature p190-B heterozygous females revealed no apparent defects at most stages of mammary gland development. The heterozygous females were viable, gave birth to normal-sized litters, and nursed their young. Examination of whole mounts of the mammary gland from mature virgin, midpregnant, and lactating mice did not reveal any apparent differences in wild-type and heterozygous mice (data not shown). However, because differential expression of p190-B was observed previously in the Cap cell layer of the TEBs, we hypothesized that deletion of one p190-B allele might influence the rate of ductal outgrowth into the surrounding fat pad in prepubescent and/or early pubescent mice. To explore this possibility, whole-mount analysis was employed to compare the extent of ductal outgrowth in sister pairs of wild-type and heterozygous virgin females at prepubertal and early pubertal stages of development. As seen in Fig. 1A
, p190-B heterozygous females exhibited significantly retarded ductal growth at 3, 4, and 5 wk of age when compared with their wild-type littermates. To obtain a quantitative measure of the defect, morphometric analysis was performed on the whole-mount images captured using a charge-coupled device camera. Both the extent of outgrowth and the ratio of the area occupied by the ducts to that of the entire fat pad were analyzed. Two-way ANOVA was used to measure the effect of age, genotype, and the interaction between the two on the extent of ductal outgrowth. Using the lymph node as the reference, both age (P < 0.001) and genotype (P < 0.001) revealed statistically significant differences on ductal outgrowth, whereas the interaction between age and genotype did not reach statistical significance (P > 0.1), suggesting that the genotype had an independent influence on extent of ductal outgrowth in the heterozygous group of mice. When individual time points were analyzed for differences in ductal outgrowth, a dramatic reduction in growth was noted in 3-, 4-, and 5-wk-old mice [P < 0.05 (adjusted), t test with equal variance, Fig. 1B
]. Next, two-way ANOVA was used to measure the effect of age and genotype (and interaction between the two) on the capacity of wild-type and heterozygous epithelium to fill the fat pad. These analyses also revealed a statistically significant influence of age (P < 0.001) and the genotype of the mice, i.e. heterozygous mice had significantly reduced capacity to fill the fat pad when compared with wild-type mice (P < 0.001). This trend continued to be statistically significant at all ages when the data were analyzed for individual time points [P < 0.002 (adjusted) at 3, 4, 5, and 6 wk of age, respectively; t test with equal variance, Fig. 1C
]. All the above results clearly indicate that p190-B is essential for ductal morphogenesis.

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Figure 1. Haploinsuficiency at the p190-B Locus Delays Early Postnatal Development
A, Mammary glands from sister pairs of 3-, 4-, 5-, and 6-wk-old wild-type (a, b, c, and d, respectively) and p190-B heterozygous (e, f, g, and h, respectively) mice were whole mounted to analyze the extent of ductal outgrowth. This defect was partially rescued in 6-wk-old heterozygotes (compare panels d and h). B, Using lymph node as the reference, length of the ductal outgrowths was recorded in centimeters for 3-, 4-, 5-, and 6-wk-old wild-type (black bar) and heterozygous (white bars) females from 5 x 7 prints of the photomicrographs (see Materials and Methods). Plot indicates significantly reduced outgrowth at 3-, 4-, and 5-wk time points [P = 0.04, P = 0.04, and P = 0.002 (adjusted), respectively, t test with equal variance]. Values presented are the mean and 95% confidence interval of the mean from five mice per genotype. C, Quantitative measure of the ability of p190-B heterozygous epithelium to fill the fat pad when compared with wild-type epithelium. Statistically significant differences were observed in the reduced ability of heterozygous epithelium to grow out in the 3-wk- (P 0.002), 4-wk- (P 0.000), 5-wk- (P 0.0001), and 6-wk-old (P 0.0001) virgins [t test with equal variance]. When compared with wild type (black bar), heterozygous epithelium (white bars) exhibited a 3.0-fold lower ability to grow out in 4-wk-old females. By 5 wk, this difference was reduced to 1.5-fold, and by 6 wk, no major visual differences were apparent among the two genotypes. Values presented are the mean and 95% confidence interval of the mean from five mice per genotype.
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Loss of One Allele of p190-B Phenocopies the Proliferation Defect in the Cap Cell Layer of IGF-IR-/- Mice
To determine whether the retarded ductal development of p190-B+/- was due to differences in the rate of proliferation in TEBs among the two genotypes, both the overall level of proliferation in the TEBs and proliferation in the two outer most cell layers were quantitated by analyzing the number of cells labeled with bromodeoxyuridine (BrdU) during a 2-h pulse. Cells in S phase were detected by IHC using a peroxidase-labeled anti-BrdU antibody. The average level of proliferation in the TEBs at 45 wk of age varied considerably, and ranged from 2227% in both the heterozygous and wild-type mice, similar to values reported previously (5, 11). It was, therefore, difficult to obtain statistically significant differences in the overall level of proliferation in TEBs among the two genotypes. However, as evident from Fig. 2A
(compare panel a and c with b and d), proliferation in the Cap cell layer of p190-B+/- mice was dramatically reduced. Although the difference in rate of proliferation in the two outer most cell layers of the TEBs of heterozygous (30.48% ± 6.96, mean ± SEM) and the wild type (35.35% ± 5.52, mean ± SEM) mice at 4 wk of age was not significant (P > 0.55), this difference was highly statistically significant [P = 0.009 (unadjusted), P = 0.017 (adjusted), t test with equal variance, Fig. 2B
] between 5-wk-old heterozygous (5.21% ± 1.75, mean ± SEM) and wild-type mice (29.09% ± 6.77, mean ± SEM). This difference was also apparent in ANOVA where both age (P < 0.03) and genotype (P < 0.02) had statistically significant influences on rate of proliferation in the Cap cell layer, while the interaction among the two was not (P > 0.1). This was similar to what has been observed previously in mammary transplants from IGF-IR null as compared with wild-type mice, which also displayed a marked decrease in the level of proliferation in the cap cells of TEBs (Fig. 2A
, compare panels e and f) (11). These results are also consistent with recent studies (19) suggesting a possible role of p190-B in mediating the cross-talk between IGF and ECM signaling.

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Figure 2. Delayed Postnatal Development in p190-B Heterozygous Mice Results from Reduced Levels of Proliferation in the Cap Cell Layer of TEBs
A, Representative fields from 4-wk- and 5-wk-old virgin mammary glands of p190-B+/+ (a and c), p190-B+/-(b and d) IGF-IR+/+ (e), and IGF-IR-/- (f) showing immunohistochemical localization of BrdU-positive cells in TEBs. These panels are depicted here to highlight the differences in the levels of proliferation in the outer cell layers of TEBs of both p190-B+/- and IGF-IR-/- mice. Bars, 50 µm. B, BrdU-positive cells for both genotypes (p190-B+/+ and p190-B+/-) were counted and expressed as a ratio of positive nuclei to the total number of nuclei counted in percent. Each slide was scanned to randomly select 1012 TEBs to count the positive as well as the total number of nuclei in the two outer most cell layers of TEBs. Values represent mean and 95% confidence interval of the mean from five mice per genotype.
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Reduced IRS-1 and IRS-2 Expression in p190-B +/- Mice
The reduction in Cap cell proliferation in both the p190-B+/- mice and the IGF-IR null transplants prompted an investigation of the immediate downstream targets of the IGF-I signaling pathway, i.e. insulin receptor substrate proteins, IRS-1 and IRS-2, in p190-B+/- mice. IRS-1 has been shown previously to be targeted for proteosome-mediated degradation as a function of Rho-kinase-mediated phosphorylation in p190-B null MEFs (19). Using IHC, the expression levels of IRS-1 and IRS-2 were compared between the two genotypes. As has been observed previously (20), IRS-1 expression was detected as diffuse, predominantly cytoplasmic staining only in the body cells of the TEBs both in p190-B+/- mice and p190-B+/+ mice. No such staining was observed in the TEBs in IRS-1 null mice (20). In contrast, IRS-2 expression was detected in both body cells and in the Cap cell layer with a characteristic ring-like staining pattern around the cell periphery. Both IRS-1 and IRS-2 levels were reduced in p190-B+/- mice as compared with their wild-type littermates. In addition, the ring-like staining pattern of IRS-2 was not apparent in both the body cells and the Cap cell layer of p190-B+/- females (as indicated by arrows in Fig. 3
). These results further support a possible interaction between these two pathways that regulates ductal morphogenesis.

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Figure 3. Reduced IRS-1 and IRS-2 Expression in p190-B Heterozygous Mice
IHC for IRS-1 (top panels) and IRS-2 (bottom panels) in 4-wk-old p190-B+/+and p190-B+/- mice. Thin arrows indicate intense IRS-1 or IRS-2 staining in wild-type mice. Please note the dramatic reduction in IRS-1 and IRS-2 staining in p190-B+/- mice as indicated here with thick arrows. These photomicrographs are representative of images collected from two each of 4-wk- and 5-wk old wild-type and heterozygous mice. Bars, 50 µm.
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Reduced IRS-2 Levels May Account for Lack of Proliferation in the Cap Cell Layer
Because reduced levels of either IRS-1or IRS-2, or both, may influence proliferation in mammary epithelial cells, we next examined whether they colocalized with the proliferating cells and might account for the lack of proliferation in the Cap cell layer of p190-B+/- mice. For these experiments, IRS-1 and IRS-2 were detected using indirect Immunofluorescence (IF) with a secondary antibody conjugated to Texas Red, and proliferation was assessed with a fluorescein isothiocyanate (FITC)-tagged anti-BrdU antibody. The immunofluorescence (IF) results depicted in Fig. 4
are in agreement with those obtained previously by IHC: a reduction of both IRS-1 and IRS-2 expression was observed in p190-B+/- as compared with wild-type mice. However, most of the IRS-1-positive cells did not colocalize with BrdU-positive cells (see Fig. 4A
, intense red staining cells marked with a white arrow that is completely lacking in panel B), and IRS-1 expression was not detected in the Cap cells. On the other hand, most IRS-2-positive cells, especially the ones in the Cap cell layer, also stained with the FITC-BrdU antibody, giving rise to yellow (marked with *) or green nuclei with a red border (see Fig. 4C
, intense red, ring-like membrane staining all around the green fluorescent BrdU-labeled nuclei, marked with a white arrow). Once again, this staining pattern was lost in the heterozygous mice. Thus, it appears that the decreased expression of IRS-2 in p190-B+/- females correlates best with the decreased proliferation in the Cap cell layer (Fig. 4
, B and D).

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Figure 4. IRS-2 But Not IRS-1 Staining Correlates with Proliferation in the Cap Cell Layer
Immunofluorescence staining for IRS-1: BrdU (top panels: A and B) and IRS-2: BrdU (bottom panels: C and D) was performed on sections of wild-type (A and C) or p190-B+/- (B and D) glands taken at 4 wk of age. In the wild-type mice, most of the IRS-1 staining was confined to body cells (indicated with white arrows in A) and did not colocalize with BrdU-labeled cells. However, most proliferating cells were positive for IRS-2 expression (cells appear yellow) as indicated here with * in panel B or show a thin red ring-like staining around the green nuclei shown here with arrows. On the other hand, both IRS-1 and IRS-2 levels were significantly reduced in the p190-B+/- mice (B and D) and the correlation with BrdU-positive cells was lost. These photomicrographs are representative of images collected from two each of 4-wk- and 5-wk-old wild-type and heterozygous mice. Bar, 50 µm.
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The Ductal Defect in p190-B Heterozygous Mice Is Cell Autonomous
Because mammary development is controlled by both systemic hormones and locally acting growth factors, we next asked whether the observed effects on ductal morphogenesis in p190-deficient mice were cell autonomous. However, because p190-B-/- mice are perinatal lethal, embryonic mammary bud transplantation studies were required to address this question. This approach has been employed successfully to rescue the mammary anlagen from a number of gene-targeted mice, such as the pRb-deficient mice, which die as early as embryonic d 12.5 (E12.5) (21). For this assay, both the no. 4 inguinal mammary buds from E16 embryos of the 129Sv/C57 donor mice were rescued by transplantation into the cleared fat pad of Rag1-/- recipient mice. In all three genotypes, mammary anlagen were present at E16, although, as expected, they were smaller in the p190-B null embryos (19). A detailed description of the effects of p190-B on embryonic mammary gland development will be presented elsewhere.
Six weeks after transplantation, the grafts were removed and whole mounted to check the extent of outgrowth. The donor mice were genotyped by PCR after transplantation to ascertain the sex and p190-B status of the embryos. However, to avoid any bias, the mammary outgrowths from all female donors were scored for the extent of outgrowth before evaluating their p190-B status. As seen in representative transplants shown in Fig. 5A
, the epithelium from wild-type donors completely filled the fat pad. However, the epithelium transplanted from heterozygous donor mice only partially filled the fat pad, and the epithelium derived from p190-B null donors failed to grow out. To quantify the severity of the phenotype, we monitored the take rate (percentage of outgrowths to total tissue grafts) in each of the three subgroups (Fig. 5B
). Approximately 75% of the wild-type transplants grew out and filled the fat pad completely, while only 40% of the heterozygous transplants grew out, suggesting that the take rate and extent of outgrowth were dependent on the genotype and not experimental variables. Furthermore, a complete failure of outgrowth was observed in the p190-B null transplants. These results indicate that the mammary defect is cell autonomous. Furthermore, even haploinsufficiency exerted a profound effect, and the loss of both alleles of p190-B severely affected mammary ductal morphogenesis.

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Figure 5. p190-B RhoGAP Is Essential for Mammary Morphogenesis
A, Whole-mount analysis of mammary glands from RAG1-/- mice harboring E16 mammary anlagen transplants. Samples shown here are representative of the phenotype of the respective genotype. Note the difference in the extent of mammary outgrowth in p190-B (+/-) and p190-B (-/-) transplants, as compared with p190-B (+/+) epithelium. Bar, 1 mm. B, Complete failure of p190-B-null embryonic epithelium to give rise to the mammary ductal tree. Bars represent the percentage of embryonic buds that successfully repopulate the host fat pad out of the total number of grafts. n = 16 for p190-B (+/+), n = 15 for p190-B (+/-), and n = 5 for p190-B (-/-).
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DISCUSSION
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Mammary gland development occurs in distinct phases: embryonic, prepubertal, pubertal, pregnancy, lactation, and involution (22). Each of these phases is characterized by distinct morphological changes (23). Because ductal morphogenesis is unique to virgin animals and is the period when they are most susceptible to carcinogenesis, there has been considerable interest in isolating genes that were expressed in this particular window of development. Using differential display and in situ hybridization, a novel RhoGAP protein p190-B was identified previously in our laboratory (13). It was differentially expressed in TEBs and some rodent mammary tumors. RhoGAPs negatively regulate Rho and promote cell motility and invasion (13, 16, 24). Thus, it was hypothesized that p190-B might play a role in TEB invasion into the surrounding fat pad, thereby facilitating ductal morphogenesis. This hypothesis is supported by the observation that the arborated ductal system of virgin mammary gland is formed as a result of reciprocal communication between the extracellular matrix and the invading TEBs (9). To test our hypothesis, p190-B knockout mice were used to examine its functional role in mammary morphogenesis. Mice lacking one allele of p190-B exhibited retarded ductal morphogenesis. This appears to result from reduced levels of proliferation in the Cap cell layer, in turn resulting, at least in part, from reduced IGF signaling. Surprisingly, deletion of both alleles completely prevented outgrowth of the transplanted mammary anlagen. Thus, p190-B may facilitate ductal morphogenesis by modulating the insulin-signaling pathway through Rho proteins.
Mammary morphogenesis ensues as early as d 1011 of embryonic development when five pairs of placodes appear on the ventral skin. The growth of the placodes is regulated mainly through reciprocal communication between epithelial cells and the surrounding mesenchyme. Once the epithelial bud is initiated, it induces the formation of the mammary mesenchyme (25). Later, postnatal remodeling of the gland continues in response to systemic and local cues. Although the genetic interactions and signaling pathways regulating postnatal mammary development are starting to be elucidated (21), much less is known about the genes that facilitate invasion of TEBs into the fat pad. Interactions between the estrogen receptor, GH, IGF, and IGF-IR have been shown to be involved in TEB growth and morphogenesis.
In this respect, it was intriguing to see that p190-B was differentially expressed during mammary development and that loss of one allele of p190-B retarded mammary development as visualized in whole mounts (Fig. 1A
). However, as evident from the data presented in Fig. 1
, B and C, the phenotype is partially rescued by 6 wk of age. One interpretation of these data is that the heterozygous mice have less epithelium at birth, so they grow out more slowly as compared with the wild-type mice, similar to what was observed in the E16 transplants (Fig. 5A
). However, at 56 wk after birth when they have attained sexual maturity under the influence of increasing estrogen, progesterone, and local IGF-I, a burst of epithelial proliferation may rescue the phenotype so that by 6 wk of age the extent of outgrowth is more or less similar in the heterozygotes and wild-type mice.
Postnatal ductal development of the mammary gland is a unique process in which ductal morphogenesis and ductal elongation go hand in hand and require extensive remodeling of the ECM in response to systemic and local cues. As has been noted in previous studies, the level of proliferation during ductal elongation is considerably higher in the outer Cap cell layer in the TEB, as compared with the cells closest to the lumen that are undergoing apoptosis (5), suggesting that reciprocal communication between the ECM and the Cap cell layer of TEBs may regulate levels of proliferation. Because p190-B is recruited to the sites of integrin clustering and is tyrosine phosphorylated, one can speculate that p190-B may modulate ductal morphogenesis by transducing signals from ECM through Rho proteins. This notion is supported by our observation that loss of one allele of p190-B significantly reduced the level of proliferation (Fig. 2
) in the Cap cell layers in the p190-B heterozygous mice when compared with wild-type mice. This hypothesis is further supported by the observation that loss of IRS-2 also correlates with reduced BrdU-labeled cells in the Cap cell layer of the heterozygous mice. Further indirect evidence comes from studies in Drosophila. Billuart et al. (26) have shown that p190RhoGAP is regulated by integrins and is essential for axon branch stability in Drosophila. Additionally, in the mammary gland, laminin has been shown to transduce signals through integrin receptors to the actin cytoskeleton that result in changes in mammary cell shape (27) and affect TEB function. It is tempting to speculate that p190-B RhoGAP may be a critical component of this signaling pathway.
p190-B may also influence ductal morphogenesis by interacting with the IGF-I signaling pathway. Both IHC and IF analyses (Figs. 3
and 4
) demonstrated decreased expression of both IRS-1 and IRS-2 in p190-B+/- mice. However, it appears that the decreased expression of IRS-2 in p190-B+/- females correlated best with the decreased proliferation in the Cap cell layer (Fig. 4
, B and D). Because IRS-2 appears to be reduced in both the body and the Cap cells, whereas BrdU incorporation was significantly different only in the Cap cells, it is apparent that reduction in IRS-2 levels alone may not be sufficient to directly regulate proliferation. Most likely the Cap cells may respond to the reduction of IRS-2 differently than the body cells, and other factors such as cross-talk with integrin-mediated signal transduction pathways may play a role in regulating TEB proliferation. The later possibility that Cap cells may utilize different signaling mechanisms than the body cells is supported by a recent report from the laboratory of Barcellos-Hoff (28) showing that TGF-ß is differentially expressed and activated in the luminal cells of TEBs but is absent from the Cap cells. However, it also cannot be ruled out that IGF-I-regulated signaling through IRS-1 in the body cells may somehow regulate proliferation in the Cap cells via a paracrine mechanism. In either case, it remains to be determined whether reduction, but not complete elimination, of IRS-1 and IRS-2 expression in the TEBs is sufficient to disrupt IGF signaling in the heterozygous mice. This question can be addressed in the future by analyzing ductal morphogenesis in individual and double IRS-1 and IRS-2 knockout mice. However, at present, with a haploinsufficiency phenotype, and not a complete loss of function, it is not feasible to determine whether there is altered IGF signaling from a small population of Cap cells in the TEB. Attempts to quantitate the response of IGF signaling on downstream targets such as AKT and ERK in situ using currently existing phospho-specific antibodies in the Cap and body cells of the TEBs have not provided definitive results. Notwithstanding these technical difficulties, the results presented in this study are consistent with recently published observations of Sordella et al. (19), who demonstrated that p190-B plays a critical role in regulating the IGF-I signaling pathway via the modulation of Rho kinase and IRS-I levels (19) in MEFs derived from p190-B null mice. Thus, loss of p190-B resulted in elevated levels of Rho kinase and increased threonine phosphorylation of IRS-1, leading to proteosome-mediated degradation, decreased IGF-I signaling, and a decrease in phospho-cAMP response element binding protein. Furthermore, cell size also appeared to be affected in the p190-B-deficient embryos, consistent with the smaller size observed in our studies for the mammary anlage. A second connection with IGF signaling is that defects similar to those observed in Cap cell proliferation in IGF-IR-null mammary epithelial outgrowths (11) were seen in the p190-B heterozygotes, suggesting, once again, that p190-B may interact with the IGF-I signaling pathway to regulate ductal morphogenesis. Together, these data suggest that RhoGAP p190-B plays an essential role in ductal morphogenesis and that it might influence Cap cell proliferation and invasion through interaction with both the integrin and IGF-I signaling pathways.
Finally, although the transplantation of the embryonic mammary anlagen was an effective way 1) to circumvent the problem associated with embryonic or perinatal lethal phenotype observed in p190-B mice and 2) to determine the epithelial cell autonomous nature of the decreased rate of ductal outgrowth, this approach still precluded the analysis of postnatal mammary morphogenesis in mice deficient in both alleles of p190-B. Both gain- and loss-of-function experiments will need to be performed using inducible systems to either overexpress p190-B in the mammary gland or floxed alleles with inducible Cre recombinase to permit conditional deletion of this essential gene at different stages of development in a spatio-temporally defined manner. These approaches should help provide additional insight on the role of p190-B in postnatal development. In addition, these models should permit a functional evaluation of the importance of p190-B in mammary tumorigenesis and metastasis.
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MATERIALS AND METHODS
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Mouse Strains
p190-B-null mice, generated in the laboratory of Dr. Jeffrey Settleman (Harvard Medical School, Cambridge, MA), were maintained on a C57Bl/6 x 129SV background. A heterozygous breeding pair was used to establish a colony in the animal facility at Baylor College of Medicine (Houston, TX). Mice were fed a conventional diet ad libitum and maintained at 21-22 C with a 12-h light, 12-h dark cycle. Animal protocols were approved by the Animal Care and Use Committee of Baylor College of Medicine and were conducted in accordance with NIH guidelines. All animals were maintained in accordance with the provisions of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act.
Whole Mounts
Whole-mount hematoxylin staining was performed as described by Medina et al. (29). Whole mounts were analyzed for growth by measuring the percent fat pad filled by the ductal system of the outgrowth. This was estimated by measuring the area occupied by the ducts divided by the total area of the fat pad, expressed in percent. The analyses were performed using Adobe Photoshop (Adobe Systems, San Jose, CA) and Scion Image (Scion Corp., Frederick, MD) image processing and analysis software. Grayscale TIFF images of whole mounts were captured with a Sony video camera (DXC-151A) and Scion LG3 frame grabber (Scion Corp.) at a resolution of 72 pixels/inch. To quantitate the area occupied by the ductal system and the fat pad, images of whole mounts (magnification, x6.7) were viewed in Adobe Photoshop and were layered with the grid. The total number of squares overlying these structures provided an approximate measure of the area occupied by them and was used to calculate the ratios. To determine the extent of outgrowth, the whole-mount images were captured as above and printed as 5 x 7 prints. Using lymph node as the reference, the farthest tip of each outgrowth was measured in centimeters, and the mean and 95% confidence interval of the mean were plotted against each genotype.
Immunohistochemistry
Dr. Adrian V. Lee generously provided us the IRS-1 and IRS-2 antibodies for IHC and IF studies. Tissues were fixed with 4% paraformaldehyde, dehydrated through the ethanol series, and paraffin embedded. Five-micrometer sections were baked overnight at 37 C, deparaffinized with xylene, and rehydrated with ethanols. Heat-induced antigen retrieval was performed in 0.1 M Tris-HCl, pH 9.0, for 5 min. Endogenous peroxidase activity was blocked by incubating the sections in 5% hydrogen peroxide solution for 5 min. All incubations were preformed at room temperature, and all washing was performed with TBST (0.15 M NaCl; 0.01 M Tris-HCl, pH 7.4; 0.05% Tween 20) unless otherwise stated. Endogenous biotin was blocked using the Avidin/Biotin blocking kit according to the manufacturers instructions (Vector Laboratories, Inc., Burlingame, CA). Slides were then incubated with IRS-1 antibody (1:800 dilution in Tris-buffered saline + 1% BSA) or IRS-2 antibody (1:800 dilution in Tris-buffered saline + 1% BSA) for 1 h, biotinylated secondary antibody (1:250) for 30 min, and then horseradish peroxidase-labeled avidin (1:200) for 30 min. As a negative control, slides were incubated with purified rabbit immunoglobulin (The Jackson Laboratory, Bar Harbor, ME). Detection was achieved by incubation with diaminobenzidine (DAKO Corp., Carpinteria, CA) for 2 min. Slides were counterstained with 0.05% methylene green for 30 sec, dehydrated, and mounted using Permount (Sigma, St. Louis, MO). Hadsell et al. (30) have shown previously that IRS-1 IHC on lactating mammary glands reveals a specific cytoplasmic staining that was absent in control IgG incubations. Both antibodies have been shown to be very specific by immunoblotting, giving a single band at 175 and 185 kDa, respectively. In addition, IRS-1 antibody did not show any staining on IRS-1 null mammary tissue (Dr. A. V. Lee, personal communication). BrdU IHC was performed essentially using the BrdU in situ detection kit (catalog no. 550803) as per the manufacturers instruction (BD PharMingen, San Diego, CA).
Immunofluorescent Detection of IRS-1, IRS-2, and BrdU
Sections were dewaxed and subjected to microwave antigen retrieval in 10 mM citrate buffer, pH 6.0. After blocking in 5% BSA/0.5% Tween-20 for 4 h at room temperature (RT), sections were incubated overnight with IRS-1 or IRS-2 and anti-BrdU-FITC-conjugated antibody (1:5; Becton Dickinson and Co., Franklin Lakes, NJ) in blocking solution at RT. Slides were washed in PBS and incubated for 30 min to 1 h in Texas red-conjugated goat antirabbit polyclonal antiserum (1:1000, catalog no. T-6391, Molecular Probes, Inc., Eugene, OR) at RT. After washing off the secondary antibody, slides were mounted in Vectashield + 4',6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories, Inc.). All procedures were carried out in the dark to prevent fluorochrome quenching.
Mammary Tissue Transplantation
Heterozygous males and females were bred to generate wild-type (p190-B+/+), heterozygous (p190-B+/-), and null (p190-B-/-) donor embryos for mammary transplants. The appearance of a vaginal plug was considered to mark d 0 of gestation. On d 16 of pregnancy, embryos were harvested by cesarean section and kept on ice in Hanks balanced salt solution until dissection. As p190-B heterozygotes were in a mixed background, immunocompromised RAG1-/- mice were employed as hosts to avoid graft rejection. In addition, unlike nude or skid mice, RAG1-/- mice have a more normal hormonal milieu, making them an ideal host for mammary transplant studies that heavily depend on systemic hormones as well as local growth factors. Three-week-old RAG1-/- mice were directly purchased from The Jackson Laboratory. Both the no. 4 mammary glands of the 3-wk-old RAG1-/- recipients were cleared of any endogenous epithelium (31). The no. 4 inguinal mammary buds from each donor embryo were located under a dissecting scope, surgically removed using a fine clipping forceps, and implanted into an incision in the cleared fat pad of the recipient gland. The mammary buds were transplanted along with the overlying skin. Mammary gland outgrowths were analyzed 6 wk after transplantation by whole-mount staining (25). Embryos were genotyped by PCR to ascertain their sex and p190-B status. The following primer sets (forward, 5'-GGT TCTTCACTTAGAACGG-3'; reverse, 5'-TAATGATAGGCGGATCCC-3'; neo reverse, 5'-CGGTGGATGTGGAATGTG-3') were used to amplify p190-B wild-type and mutant alleles. The sex of the embryos was established by PCR on embryo DNA by amplification of the sex-determining region of the Y chromosome (SRY gene). The forward SRY primer was 5'-CGCCCCATGAATGCATTTATG-3', and the reverse primer was 5'-CCTCCGATGAGGCTGATAT-3'. PCR cycling was for 1 min at 94 C, 2 min at 55 C, and 2 min at 72 C, for 30 cycles. Mouse ß-casein primers from exon 7 (MBC7F: 5'-GATGTGCTCCAGGCTAAAGTT-3'; MBC7R: 5'-AGAAACGGAATGTTGTGGAGT-3') were used as internal positive controls for the latter PCRs.
Statistical Analysis
Data for extent of outgrowth, percent fat pad filled, and BrdU incorporation were summarized with means, SEMs, and 95% confidence intervals. Two-way ANOVA was used to test for main effects of age, genotype, and interaction. In addition, two sample t tests were used to compare genotypes at individual time points. P values were adjusted for multiple comparisons using the step-down approach to the Sidak method (32). For the BrdU incorporation experiments, the test for homogeneity of variances was significant, and the data were also analyzed using the arcsin square root transform to equalize variances. The results were essentially unchanged (data not shown), and only the untransformed results are reported. Data were analyzed using the software SAS 8.02 (SAS Institute, Inc., Cary, NC).
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ACKNOWLEDGMENTS
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We thank the following persons from the Breast Center, Baylor College of Medicine: Dr. Susan Galloway Hilsenbeck for all the statistical analyses; Dr. Adrian Lee for sharing IRS-1 and IRS-2 antibodies; Dr. Allred and Dr. Mohsin for help with IHC studies; and Nicole Lawrence for technical assistance with immunoblotting and phospho-specific antibody staining.
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FOOTNOTES
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This work was supported by United States Army Medical Research and Materiel Command DAMD-17-99-1-9073 and R01-CA-64255 postdoctoral support (to G.C.) and R01-DK-052197-06A1 (to D.H.).
Abbreviations: Brdu, Bromodeoxyuridine; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; GAP, GTPase-activating protein; IF, immunofluorescence; IGF-IR, IGF-I receptor; IHC, immunohistochemistry; IRS-1, insulin receptor substrate 1; MEF, mouse embryo fibroblast; RT, room temperature; TEB, terminal end bud.
Received for publication December 18, 2002.
Accepted for publication March 5, 2003.
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