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Department of Molecular and Cellular Biology (T.V.-G., B.M.H., J.M.R.), Baylor College of Medicine, Houston, Texas 77030; Jake Gittlen Cancer Foundation (E.J.G.), Penn State College of Medicine, Hershey, Pennsylvania 17033; and Department of Cancer Biology (L.A.C.), Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Jeffrey M. Rosen, Ph.D., C.C. Bell Professor of Molecular and Cellular Biology and Medicine, DeBakey Building, M638a, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3498. E-mail: jrosen{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Precise regulation of Rho GTPase activity is critically important, and several families of proteins including the Rho GTPase-activating proteins (GAPs) are capable of modulating their activity (6). RhoGAPs function as negative regulators of Rho activity by enhancing the intrinsic GTPase activity of the Rho proteins to rapidly convert active GTP-bound Rho to inactive GDP-bound Rho (7). The role of the Rho-signaling pathway in mammary gland development and breast cancer progression is not well understood. Several studies have reported overexpression of Rho family members in human breast cancers (8, 9, 10), and a number of reports have delineated functions of the Rho pathway by introducing dominant-negative or active forms of Rho into breast cancer cell lines (11).
Until recently, Rho signaling in normal mammary gland development had not been examined. P190-B RhoGAP, an important negative regulator of the Rho pathway, was identified in a screen for genes showing enriched expression in TEBs (12). P190-B is highly expressed throughout virgin mammary gland development in both the body and cap cell layers of the TEBs and in the mature ducts. Expression of p190-B decreases during late pregnancy and remains low, but detectable, during lactation. Homozygous deletion of this RhoGAP gene completely inhibits ductal outgrowth (12, 13). Loss of one allele of p190-B results in decreased proliferation within the TEBs, causing a transient delay in ductal morphogenesis. Thus, mammary gland development is critically dependent on p190-B RhoGAP.
To further elucidate the role of p190-B in mammary gland development and tumor progression, a tetracycline (tet)-regulatable p190-B-overexpressing mouse model was developed. This inducible system was chosen because it allows for manipulation of p190-B expression during distinct stages of mammary gland development and function. Using this approach, p190-B overexpression during ductal morphogenesis is shown to drastically alter TEB architecture. As a result, ductal elongation is delayed, branching is increased, and organization of the ductal tree is disrupted. Overexpression of p190-B during pregnancy results in hyperplastic lesions, which persist after postlactational involution. These studies demonstrate, for the first time, that overexpression of a RhoGAP is sufficient to disrupt mammary gland architecture and promote hyperplasia, confirming our previous findings that precise regulation of p190-B RhoGAP is critically important in the developing mammary gland.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) (14). The presence of the IRES-luciferase allows for rapid identification of transgene expression in the mammary gland after induction. Injection of this construct into the pro-nuclei of fertilized FVB oocytes yielded nine founder lines, as determined by Southern blot and PCR analyses (Fig. 1 and data not shown).
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) (15). Bigenic mice (5 wk old) from each line and MTB control mice were treated with the tet analog doxycycline (Dox) at 2 mg/ml in their drinking water for 3–7 d to induce transgene expression. After treatment, mammary glands were dissected from the mice and analyzed for luciferase activity, p190-B transgene expression by RT-PCR, and morphological changes by whole-mount mammary gland analysis (Fig. 1C). Three of the lines (6667, 6671, and 6674, denoted with asterisks in Fig. 1B) showed inducible p190-B transgene expression as determined by luciferase activity and RT-PCR (Fig. 1, D and E). To determine which cell types express the p190-B transgene, immunofluorescence for the HA-tagged p190-B was performed on mammary gland tissue sections from Dox-treated midpregnant bigenic and MTB control mice. As expected, the HA-tagged p190-B transgene is localized to the mammary epithelial cells, which is consistent with the expression of the MMTV-rtTA within this compartment of the mammary gland (Fig. 1F) (15).
Because p190-B was originally identified in a screen for genes showing enriched expression in the TEBs, the effects of acute overexpression of p190-B on TEB morphology were examined. Strikingly, within 3 d of p190-B transgene induction, aberrant TEBs with extensive budding off the neck region were apparent in the whole-mounted mammary glands (Fig. 1C). Histological analysis of hematoxylin and eosin (H&E)-stained tissue sections from these glands further demonstrated the extent of disruption of the TEB architecture (Fig. 2, A and B). The TEBs exhibited extensive budding, abnormal morphologies, and disorganized and thickened stroma that, in some cases, encompassed the leading edge of the TEB. To quantify the extent of disruption of the TEB structures in the p190-B-overexpressing mice, the percentage of normal TEBs was determined after 3 d of transgene induction. Structures were designated normal if they did not exhibit budding off the neck region of the TEB. In comparison with the Dox-treated control mice (n = 6; 57 TEBs analyzed), the percentage of normal TEBs was significantly decreased in the p190-B-overexpressing mice [n = 5 (67 TEBs analyzed), 98.2 ± 1.85 vs. 34.8 ± 12.2 (P < 0.0003), respectively (Fig. 2E)]. Despite the pronounced TEB anomalies seen after short-term p190-B overexpression, acute overexpression of p190-B did not have any apparent effects on the morphology of the mature ducts in these mice (Fig. 2, C and D). These phenotypes were not observed in the Dox-treated MTB control mice. All three inducible lines showed the TEB phenotype, and subsequent studies were performed on two of the lines, 6667 and 6671. These results indicate that tight regulation of p190-B expression is critical to maintain normal TEB architecture.
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). Total ROK expression, however, was equivalent between the MTB and p190-B-overexpressing mice. Similarly, pPAK-2 expression was lower, but this likely reflects decreased total PAK-2 expression that was detected in the p190-B-overexpressing as compared with the MTB control mammary glands (Fig. 2F). Recently, coordination of Rho and ERK signaling was shown to control tissue architecture (16). Thus, the pronounced decrease in Rho signaling that was detected in the p190-B-overexpressing mammary glands might alter ERK activity. To investigate this possibility, Western blotting for phosphorylated ERK was performed. This analysis showed a marked decrease in phosphorylation of ERK, whereas total ERK levels were similar between the p190-B-overexpressing and MTB controls (Fig. 2F). Taken together, these results demonstrate that overexpression of p190-B in the mammary gland inhibits signaling downstream of the Rho family proteins.
To assess whether the newly formed buds off the neck region of the TEBs will persist or regress, cell proliferation and apoptosis were evaluated. To detect proliferation within the buds, immunohistochemical staining for the proliferation marker Ki67 was performed. As seen in Fig. 3B, the aberrant budding structures extending from the TEBs in the p190-B-overexpressing mice are highly proliferative. Apoptosis was assessed by immunohistochemical staining for the apoptotic marker, cleaved caspase-3, and few cells within the aberrant buds are undergoing apoptosis (Fig. 3D). These data suggest that the newly formed buds will grow out to form branches because the cells within the buds are proliferating and undergoing apoptosis similarly to cells within the control TEBs. Taken together, these data demonstrate that short-term p190-B overexpression disrupts TEB morphology and may lead to aberrant branching off of the neck of the TEB.
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, E–H). This analysis demonstrated that both cell types are present within the aberrant TEBs. However, p63 immunostaining revealed that the cap/myoepithelial cell layer surrounding the aberrant TEBs is discontinuous along the neck region both at sites of aberrant budding and in areas not associated with aberrant budding (Fig. 3H). This disruption was not observed in the control TEBs, which show a continuous myoepithelial cell layer including at sites of initiating side branches distal to the TEBs (Fig. 3G, arrowhead; shown at higher magnification in the inset). Immunostaining for the myoepithelial marker smooth muscle actin also show discontinuity in this cell layer (data not shown). Thus, the alteration in the myoepithelial layer surrounding the abnormal TEBs does not appear to reflect a normal phenomenon associated with side branching. These data suggest that p190-B overexpression alters cell-cell or cell-extracellular matrix (ECM) interactions to impact the myoepithelial cell layer surrounding the TEBs.
P190-B Overexpression Results in Abnormal Stroma Surrounding the TEBs
Histological analysis of the H&E-stained TEBs demonstrated that the stroma surrounding the TEBs was altered in the p190-B-overexpressing mice. The stroma in the p190-B-overexpressing glands appeared disorganized, thicker, and more cellular. The degree of stromal disorganization correlated with the extent of TEB disruption such that the TEBs with drastically altered morphologies had more pronounced stromal anomalies. To further examine the stromal changes occurring in the p190-B-overexpressing mice, Masson’s trichrome staining was performed as it allows for visualization of aniline blue-stained collagen fibers. As seen in Fig. 4B, the stroma surrounding the aberrant TEB from the p190-B-overexpressing mammary gland is highly enriched in collagen fibers as compared with the control TEB in which the collagen fibers are localized primarily to the neck region of the TEB (Fig. 4A). This result suggests that p190-B overexpression results in altered stromal-epithelial interactions during TEB outgrowth.
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, C and D). Taken together, these data suggest that p190-B overexpression influences immune cell infiltration within the stroma adjacent to the TEBs, which ultimately affects TEB architecture.
Downstream of p190-B RhoGAP are the insulin receptor substrate (IRS) proteins 1 and 2 (19). Deficiency of p190-B leads to increased ROK activity and phosphorylation of the IRS proteins, which targets them for degradation. As a result, IGF receptor (IGFR) signaling is diminished. Signaling through the IGFR pathway has also been shown to play an important role in mammary gland ductal morphogenesis because IGF-IR deficiency impairs take rate and ductal outgrowth in mammary gland transplantation studies (20). Constitutive activation of IGF-IR increases ductal side branching, delays ductal outgrowth, and results in rapid formation of adenocarcinomas (21). To determine whether signaling through the IGFR pathway is altered in the aberrant TEBs in the p190-B-overexpressing mice, immunohistochemistry for IRS-1, IRS-2, and a downstream target of the IGFR-signaling pathway, phosphorylated Akt (pAKT) was performed. Interestingly, this analysis revealed a reduction in IRS-1 and IRS-2 expression levels (Fig. 5, A–D) as well as a reduction in pAkt in the aberrant TEBs as compared with control TEBs (Fig. 5, E and F). Western blotting of mammary gland extracts confirmed decreased expression of IRS-1, IRS-2, and total AKT in the p190-B-overexpressing as compared with the Dox-treated control mammary glands (Fig. 5G). Although a decrease in expression of these proteins in the aberrant TEBs was not predicted, these results indicate that p190-B overexpression impacts signaling through the IGFR pathway.
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, analysis of proliferation and apoptosis within the aberrant TEBs suggested that these buds may persist and form new branches. To determine whether the TEB abnormalities would ultimately affect ductal outgrowth and formation of the arborized ductal tree, long-term p190-B overexpression studies were performed in which p190-B expression was induced throughout postnatal mammary gland development. For these studies, bigenic (n = 6; three mice from each line) and wild-type (n = 4) littermate control mice were treated continuously with Dox beginning at 5.5 wk of age until 9.5 wk of age, at which time the growing ducts normally reach the end of the fat pad. In comparison with the Dox-treated wild-type littermate control mammary glands, which showed normal architecture within the ductal tree, long-term p190-B overexpression resulted in disorganization of the ductal tree and increased branching, as seen in the whole-mounted mammary glands (Fig. 6B). Luciferase assays and RT-PCR were performed to confirm expression of the p190-B transgene (Fig. 6E and data not shown).
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). Histological examination of H&E-stained tissue sections demonstrated that long-term p190-B overexpression throughout ductal morphogenesis results in the presence of abnormal TEBs and thickened stroma surrounding the mature ducts (Fig. 6, C and D). Normally at 9.5 wk of age, the TEBs have reached the end of the fat pad and begin to regress. TEBs were still detected in the most disorganized p190-B-overexpressing mammary glands in which the ducts had not yet reached the end of the fat pad. Proliferation rates within the mature ducts were similar between the p190-B-overexpressing and Dox-treated wild-type littermate control mice as determined by quantification of Ki67-positive cells (2.6 ± 0.97 vs. 2.1±1.1; P > 0.07). Taken together, these data demonstrate that the aberrant budding off the TEBs in the p190-B-overexpressing mice results in a disorganized ductal tree with increased branching and altered stroma surrounding the mature ducts.
Upon examination of the long-term p190-B-overexpressing mammary glands, it was noted that the severity of the aberrant branching and disorganization within the growing ductal tree correlated with a delay in ductal outgrowth. To further investigate the effects of p190-B overexpression on ductal elongation, p190-B-overexpressing mammary tissue was transplanted into the cleared fat pads of 3-wk-old female mice. As a control, MTB tissue was transplanted into the contralateral cleared no. 4 fat pads. The transplants were allowed to grow out for 8 wk, at which time the mice were bred to wild-type FVB males. Mammary glands were collected 3 d after parturition and analyzed by H&E staining. Luciferase assays were performed to confirm transgene expression (data not shown). Interestingly, p190-B overexpression resulted in a dramatic delay in ductal outgrowth as compared with the MTB controls. This analysis revealed that 100% (six of six) of the MTB control transplants completely filled the fat pad, whereas only 33% (two of six) of the p190-B-overexpressing transplants filled the fat pad (n = 6; P < 0.01) (Fig. 6G). The remaining four p190-B-overexpressing transplants filled the fat pad 50% or less (data not shown). These data demonstrate that p190-B overexpression delays ductal morphogenesis, and pregnancy does not rescue this defect.
Overexpression of p190-B during Pregnancy Results in Hyperplastic Lesions
To examine the affects of p190-B overexpression during pregnancy and lactation, 12-wk-old bigenic (n = 6) and MTB (n = 3) and wild-type littermate (n = 3) control mice were bred to wild-type male mice. To induce p190-B transgene expression, Dox treatment was started when the males were placed with the females and continued throughout pregnancy and lactation. MTB and wild-type control mice were also Dox treated. During late pregnancy (d 16–18) 3- to 5-mm biopsy samples were collected from the bigenic (n = 2) and wild-type littermate (n =2) control mice. Interestingly, histological examination of H&E-stained sections of biopsy samples from both bigenic mice showed hyperplastic lesions that were readily detectable within the small samples that were collected (Fig. 7, C and D). Neither of the wild-type controls contained hyperplastic lesions. Furthermore, hyperplastic lesions were detected in involuted mammary glands from the p190-B-overexpressing mice, but not in the controls (Fig. 7B). Overexpression of p190-B, however, did not inhibit lactation because all six bigenic mice were able to support their litters (six or more pups) to weaning age.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Acute overexpression of p190-B during ductal morphogenesis dramatically altered the architecture of the TEBs and the adjacent microenvironment. The abnormal TEBs were characterized by extensive budding off the neck region, disruption of the myoepithelial cell layer, and pronounced stromal alterations. Overexpression of p190-B throughout ductal morphogenesis resulted in delayed ductal elongation, disorganization of the ductal tree, and increased side branching. Previously, loss of p190-B was shown to completely inhibit ductal morphogenesis (13). Haploinsufficiency of p190-B was shown to transiently delay ductal morphogenesis, due to decreased proliferation in the cap cell layer of the TEB, possibly resulting from diminished expression of IRS proteins (13). The current study complements the loss of function studies and demonstrates that precise regulation of p190-B in the developing mammary gland is required for normal TEB structure, ductal elongation, and organization of the ductal tree.
p190-B overexpression resulted in several cellular and molecular changes within the TEBs and surrounding microenvironment, all of which are likely to contribute to the aberrant TEB architecture. Interestingly, the myoepithelial layer was found to be discontinuous along the neck region and at sites of aberrant budding in the p190-B-overexpressing TEBs. In contrast, alterations in the myoepithelial cell layer were not detected at sites of lateral budding or at any point along the neck region of the control TEBs. This result suggests that disruption of the myoepithelial cell layer in the aberrant TEBs is not reflective of a normal phenomenon associated with lateral branch points. However, the role of the myoepithelial cell layer in the formation of lateral branches is not clear. One model in the literature suggests that the myoepithelial cell layer is normally absent at branch points, although the data supporting this model are unclear (24). In contrast to this model, it has also been suggested that the myoepithelial cells reform the cap cell layer during the initiation of lateral branches (25). One possible role for the myoepithelial cells, which secrete a number of proteases, is that they may contribute to maintenance and remodeling of the ECM underlying the ductal epithelium during lateral branching (26). Furthermore, myoepithelial cells may have tumor suppressor roles because they have been shown to inhibit proliferation, induce apoptosis, and block invasion of breast cancer cells (27, 28, 29). Mice overexpressing an inducible form of fibroblast growth factor receptor 1 (MMTV-iFGFR1) also had a noncontiguous myoepithelial cell layer at sites of aberrant branching (30). Thus, disruption of the myoepithelial cell layer in the p190-B-overexpressing TEBs may play an important role in the abnormal TEB architecture and aberrant budding off the neck region of the TEBs. The molecular mechanisms by which overexpression of p190-B contributes to alterations in the myoepithelial cell layer remain unclear. Future studies examining the interactions between primary myoepithelial and luminal epithelial cells isolated from the tet-regulatable p190-B-overexpressing mice in a three-dimensional culture system will help to elucidate the molecular signaling pathways involved in the cross-talk between the myoepithelial and luminal epithelial cells.
Another phenotype observed in the aberrant TEBs was a pronounced alteration in the adjacent microenvironment. The stroma was thicker, more cellular, and contained more collagen as determined by Masson’s trichrome staining. Recently, elegant studies by Dr. Valerie Weaver and colleagues (16) demonstrated that matrix rigidity plays a critical role in epithelial morphogenesis. Rho-dependent cytoskeletal tension and ERK activity are increased in epithelial cells grown on a stiff stroma, thereby altering cell-cell/cell-matrix adhesion and polarity to ultimately disrupt morphogenesis. These studies demonstrated that even small increases in matrix stiffness are sufficient to increase cell proliferation and compromise tissue architecture. Furthermore, ROK-mediated contractility is required for breast epithelial cells to sense the rigidity of their environment, and down-regulation of Rho activity is necessary for epithelial cell differentiation (31). Thus, the increase in collagen and stromal thickness adjacent to the aberrant TEBs may result in a more rigid stroma, leading to disrupted TEB architecture in the p190-B-overexpressing mice.
Interestingly, ROK and PAK activity is decreased in the p190-B-overexpressing mammary glands. The Rho pathway plays an essential role in regulation of actin cytoskeletal dynamics, and remodeling of the actin cytoskeleton is required for a number of cellular processes (32). Continual inhibition of Rho signaling by overexpression of p190-B RhoGAP, therefore, is not likely to be tolerated within normal mammary epithelial cells. Thus, it is probable that a compensatory up-regulation of other signaling pathways that contribute to cytoskeletal regulation occurs in response to p190-B overexpression. The stromal response in the p190-B-overexpressing mice may occur to compensate for the chronically depressed ROK and PAK activity, thereby allowing for modulation of Rho-dependent cytoskeletal tension.
In addition to the changes in matrix deposition, a significant increase in the number of immune cells was detected by immunostaining for the macrophage and eosinophil marker F4/80. Whereas disruption of the myoepithelial cell layer may contribute to alterations in the stromal environment as discussed above, it is likely that p190-B overexpression modulates inside-out signaling pathways that influence immune cell infiltration and ECM deposition. Macrophages and eosinophils have been shown to play an essential role in branching morphogenesis of the mammary gland because depletion of these cells inhibited ductal branching and elongation (17). Their ability to promote ECM remodeling and aid in the release of growth factors may contribute to the disruption in ductal morphogenesis seen in the p190-B-overexpressing mice. Thus, the aberrant budding off the TEBs and increased branching observed after long-term p190-B overexpression is likely to be influenced by the marked increase in F4/80-positive immune cells observed in association with the aberrant TEBs. The molecular mechanisms by which overexpression of p190-B contributes to the recruitment of immune cells remain unclear. In breast cancer cells, the Rho-signaling pathway was shown recently to be important for production of colony-stimulating factor 1 (33), which is a major regulator of macrophage activation (34). Thus, it is possible that p190-B overexpression leads to alterations in Rho signaling that impact expression of colony-stimulating factor 1 expression, resulting in the recruitment of macrophages.
Similar to p190-B RhoGAP, IGF-IR is critical for normal mammary gland ductal morphogenesis. In transplant studies, embryonic mammary buds deficient for IGF-IR show a significant reduction in take rate, and ductal outgrowth is severely impaired (20). Overexpression of a constitutively active IGF-IR in the developing mammary gland increased side branching, delayed ductal elongation, and resulted in the rapid formation of adenocarcinomas (21). p190-B RhoGAP was recently shown to interact with the IGF-signaling axis in vivo. Deficiency of p190-B resulted in increased activity of ROK, which phosphorylates the IRS proteins and targets them for degradation (19). In addition, IGF-IR activation positively regulates p190-B activity through phosphorylation events that alter the subcellular location of p190-B (35). Furthermore, decreased expression of IRS-1 and IRS-2 was detected in the TEBs of p190-B heterozygous mice (13). Thus, interactions between IGF-IR and p190-B signaling are likely to play an important role in the developing mammary gland.
In the current report, expression of the IRS proteins and activation of the downstream effector AKT were significantly reduced in the aberrant TEBs. This result was initially unexpected because p190-B deficiency also results in reduced IGFR signaling. Although it may seem surprising that IGFR signaling is decreased as a result of both p190-B deficiency and overexpression, this finding may not be unexpected when considering that normal regulation of Rho signaling is highly dynamic (32). Chronic suppression of a pathway that normally undergoes transient fluctuations may have unanticipated effects on interacting pathways. Furthermore, overexpression of p190-B should not necessarily lead to an increase in IRS-1/2 expression just because the reciprocal experiment was shown to lead to increased ROK activity and IRS degradation (19). There may be a finite level of IRS gene expression, and the steady-state level does not, therefore, necessarily have to increase when p190-B is overexpressed. The reduced IGFR signaling in response to p190-B overexpression may account for the delayed ductal elongation that was detected in the p190-B-overexpressing mice, whereas the increased branching may result from changes in the stromal compartment and cap/myoepithelial cell layer as discussed above.
The ability to temporally regulate expression of the p190-B transgene allowed for examination of the role of p190-B overexpression during distinct stages of mammary gland development and function. To investigate the consequences of p190-B overexpression during pregnancy and lactation, the transgene was induced on the first day of pregnancy and continued throughout lactation. p190-B overexpression did not have any apparent effect on lactation. Interestingly, hyperplastic lesions were detected in biopsies from p190-B-overexpressing pregnant and involuted mammary glands. To our knowledge, this is the first report in which overexpression of a RhoGAP was shown to have neoplastic activities in vivo. One potential explanation for the development of these lesions is that there may also be perturbations in the myoepithelial cell layer when p190-B is overexpressed during pregnancy, which could lead to a loss of growth control as discussed above. Alternatively, increased rigidity of the stroma may lead to loss of growth control and tissue architecture (16). Analysis of multiparous mice in which p190-B is chronically overexpressed is necessary to determine whether these hyperplastic lesions will progress. This study, as well as transplantation of the hyperplastic lesions, is currently ongoing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bigenic mice were obtained by breeding TetO-p190-B-IRES-luciferase mice to MTB mice, which contain the reverse tet transactivator under the control of the MMTV promoter (15). For genotyping, PCR amplification of the MTB and p190-B transgenes was performed on genomic DNA prepared from tail cuts using the following oligonucleotide pairs: for TetO-p190-B, 5'-CCTCAAAAAAGTCATGGGGAACGGAGC-3' and 5'-CGCTGACACGGTAGAGTCCTTCGG-3'; for MTB, 5'-TCCAAGGGCATCGGTAAACA-3' and 5'-GCATCAAGTCGCTAAAGAAG-3'. The p190-B oligonucleotide pair is specific for the human p190-B transgene and does not cross-react with endogenous murine p190-B. Reaction conditions were 94 C for 3 min followed by 30 cycles of 94 C for 30 sec, 60 C for 45 sec, 72 C for 45 sec, followed by a 5-min extension at 72 C. To induce transgene expression, bigenic mice were treated with Dox (CLONTECH, Mountain View, CA) at 2 mg/ml in their drinking water containing 5% sucrose. Fresh Dox water was given twice weekly. Animal care and procedures were approved by the Institutional Animal Care and Use committee at Baylor College of Medicine and were in accordance with the procedures outlined in the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication 85–23).
For mammary gland transplantation, the inguinal no. 4 mammary glands from 21-d-old FVB/N female mice were cleared of the mammary epithelium as previously described (36). Small pieces of tissue approximately 1 mm (3) in size were transplanted into the cleared fat pads. Transplants were allowed to grow out for 8 wk. Mammary gland tissue isolation and whole-mount preparation were performed as previously described (37). Analysis of branch points and TEB morphology were performed blindly by examining whole-mounted mammary glands. For branch point analysis, the primary duct was identified starting at the nipple, and the average number of secondary and tertiary branch points off the primary duct was determined. The unpaired Student’s t test was used to determine statistical significance.
Luciferase Assay
Snap-frozen mammary tissue was ground using a mortar and pestle. Tissue extracts were prepared in Passive Lysis Buffer (Promega Corp., Madison, WI) and cleared by centrifugation. Luciferase activity was measured using Promega’s Luciferase Assay System according to the manufacturer’s instructions. Protein concentrations in the tissue extracts were determined using the BCA Protein Quantitation Assay (Pierce Chemical Co., Rockford, IL).
Southern Hybridization
A random primed (DNA labeling kit, Roche, Indianapolis IN) cDNA probe recognizing the first 1.3 kb of the human p190-B coding region was used to probe Southern blots containing EcoRI-digested genomic DNA prepared from tail cuts as previously described (38). The digested DNA was transferred to Zetaprobe (Bio-Rad Laboratories, Inc., Hercules, CA).
Immunohistochemical Staining
Paraffin-embedded tissue sections (5 µm) were deparaffinized in xylenes and rehydrated through a series of graded ethanols. Tissue sections were then stained with hematoxylin and eosin, Accustain (Masson’s) Trichrome stain (Sigma-Aldrich, St. Louis, MO), or antibodies to detect specific proteins. Antigen retrieval was performed by microwaving slides in 10 mM citrate, pH 6, for 20 min. For immunostaining with mouse monoclonal antibodies, the M.O.M kit (Vector Laboratories, Burlingame, CA) was used to block nonspecific binding and for dilution of primary antibodies. For primary polyclonal antibodies, the tissue sections were blocked and primary antibodies were diluted in a 5% solution of BSA in PBS + 0.5% Tween-20. Sections were incubated with primary antibody overnight at room temperature. The following antibodies and dilutions were used: E-cadherin, 1:250 (BD Transduction Laboratories, San Jose, CA); Ki67, 1:5000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); cleaved caspase-3 (Asp175), 1:1000 (Cell Signaling, Beverly MA); p63, 1:500 (Lab Vision Neomarkers, Fremont, CA); IRS-1, 1:800 (Upstate Biotechnology, Inc., Lake Placid, NY); IRS-2 1:800 (Upstate Biotechnology); pAKT (Ser473), 1:50 (Cell Signaling); F4/80, 1:50 with no antigen retrieval (Caltag Laboratories, Burlingame, CA). Biotinylated antirat (Molecular Probes, Eugene, OR), antirabbit (Oncogene Research, Darmstadt, Germany), and antimouse (Oncogene Research) secondary antibodies were diluted 1:200 in PBS and were incubated on the tissue sections for 1 h at room temperature. Vectastain Elite ABC and diaminobenzidine substrate kits were used to detect immunoperoxidase staining according to the manufacturer’s instructions (Vector Laboratories). To detect the HA-tagged p190-B by immunofluorescence, tissue sections from 18-d pregnant mice were subjected to antigen retrieval as described above. To detect the HA tag, a monoclonal antibody against HA 1:200 (Covance Laboratories, Inc., Denver, PA) was used with the M.O.M kit. Fluorescent-tagged antimouse Alexa 594 secondary antibody (Molecular Probes) was used at 1:1000. Nuclei were visualized by staining with 4',6-diamidino-2-phenylindole.
RT-PCR
RNA was prepared from mammary glands using Trizol Reagent according to the manufacturer’s recommendations. To prepare cDNA, 1 µg of RNA was first DNase treated, primed with oligo-dT, and reverse transcribed using MMLV-reverse transcriptase (RT) (all reagents for RT were purchased from Invitrogen, Carlsbad CA). The human p190-B-specific oligonucleotides that were used for genotyping PCR were also used for RT-PCR. As a negative control, reactions were also performed in the absence of RT (data not shown). Amplification of L19 served as a control for the RT reaction. The following oligonucleotides and conditions were used: 5'-AGTATCCTCAGGCTTCAGAA-3' and 5'-TTCCTTGGTCTTAGACCTGC-3'. Reaction conditions were 94 C for 3 min followed by 30 cycles of 94 C for 30 sec, 60 C for 45 sec, 72 C for 45 sec, followed by 5 min at 72 C.
Western Blotting
To examine expression and phosphorylation of proteins by Western analysis, mammary gland extracts were first prepared by pulverizing snap-frozen tissues followed by lysis in Passive Lysis Buffer (Promega) containing a protease inhibitor cocktail (Roche) and clearing by centrifugation. Protein concentrations in the tissue extracts were determined using the BCA Protein Quantitation Assay (Pierce). Mammary gland extracts were prepared from p190-B-overexpressing (n = 4) or MTB control glands (n = 4) at d 3 of involution that had been treated continuously with Dox throughout pregnancy and involution. Extracts were pooled (20 µg of each), electrophoresed on 6% or 12% SDS-PAGE gels, and transferred to polyvinylidine difluoride membrane (Millipore Corp., Bedford MA). Membranes were blocked in 5% milk/Tris-buffered saline followed by incubation with pROKII (thr396) 1:1000 or total ROKII 1:1000 antibodies (AnaSpec, Inc., San Jose, CA), IRS-1 and IRS-2 1:1000 (Upstate Biotechnology), AKT 1:1000 (Cell Signaling), pPAK-2 (thr402) and total PAK-2 1:1000 (Cell Signaling), focal adhesion kinase 1:1000 (Cell Signaling), and phosphorylated ERK and ERK 1:1000 (Cell Signaling) in 5% milk/Tris-buffered saline-Tween 20. Peroxidase-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:5000, and signal was detected with Supersignal West Pico Solutions (Pierce). Membranes were stripped and reprobed whenever possible. Fast green stain was also used to confirm equal loading of proteins on the membranes (data not shown).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure summary: T.V-G., B.H., E.G., and J.R. have nothing to declare. L.C. has received lecture fees from Amgen, Inc., Bristol-Meyers Squibb, Merck & Co., Inc., Eli Lilly & Co., and Genentech, Inc.
First Published Online February 9, 2006
Abbreviations: AKT, Protein kinase B; Dox, doxycycline; ECM, extracellular matrix; GAP, GTPase-activating protein; HA, hemagglutinin; H&E, hematoxylin and eosin; IGF-IR, IGF receptor I; IRES, interribosomal entry site; IRS, insulin receptor substrate; MTB, MMTV-rtTA transgenic mice; ROK, Rho kinase; RT, reverse transcriptase; rtTA, reverse tet transactivator; PAK, p21-activated kinase; pAKT, phosphorylate AKT; pPAK, phosphorylated PAK; pROK, phosphorylated ROK; TEB, terminal end bud; tet, tetracycline; TetO, tet operator.
Received for publication October 25, 2005. Accepted for publication February 2, 2006.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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  D. Vaught, J. Chen, and D. M. Brantley-Sieders Regulation of Mammary Gland Branching Morphogenesis by EphA2 Receptor Tyrosine Kinase Mol. Biol. Cell, May 15, 2009; 20(10): 2572 - 2581. [Abstract] [Full Text] [PDF]
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 D. L. Kleinberg, T. L. Wood, P. A. Furth, and A. V. Lee Growth Hormone and Insulin-Like Growth Factor-I in the Transition from Normal Mammary Development to Preneoplastic Mammary Lesions Endocr. Rev., February 1, 2009; 30(1): 51 - 74. [Abstract] [Full Text] [PDF]
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 C.-H. Shen, H.-Y. Chen, M.-S. Lin, F.-Y. Li, C.-C. Chang, M.-L. Kuo, J. Settleman, and R.-H. Chen Breast Tumor Kinase Phosphorylates p190RhoGAP to Regulate Rho and Ras and Promote Breast Carcinoma Growth, Migration, and Invasion Cancer Res., October 1, 2008; 68(19): 7779 - 7787. [Abstract] [Full Text] [PDF]
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 F. Guegan, F. Tatin, T. Leste-Lasserre, G. Drutel, E. Genot, and V. Moreau p190B RhoGAP regulates endothelial-cell-associated proteolysis through MT1-MMP and MMP2 J. Cell Sci., June 15, 2008; 121(12): 2054 - 2061. [Abstract] [Full Text] [PDF]
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