Article |
Address correspondence to Rakesh Kumar, M.D. Anderson Cancer Center-108, Rm. Y4.6032, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: (713) 745-3558. Fax: (713) 745-3792. E-mail: rkumar{at}mdanderson.org
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Abstract |
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Key Words: mammary gland; morphogenesis; Pak1; Stat5; milk proteins
* Abbreviations used in this paper: BLG, ß-lactoglobulin; DIP, dexamethasone, insulin, and prolactin; DN, dominant negative; H-E, hematoxylin and eosin; Pak1, p21-activated kinase; PCNA, proliferative cell nuclear antigen; WAP, whey acidic protein.
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Introduction |
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Stat5 was originally isolated from the nuclear extracts of lactating mammary gland and designated as mammary growth factor (Wakao et al., 1992). There are two separately coded isoforms of Stat5, Stat5a and Stat5b, which are highly homologous and provide functional redundancy to each other (Hennighausen and Robinson, 2001). Upon phosphorylation, Stat5 translocates to the nucleus and activates the expression of milk proteins such as ß-casein (Hennighausen and Robinson, 2001). Stat5 could be activated by prolactin and its receptor, and by peptide growth factors and their receptors, e.g., HER4 has also been shown to activate Stat5 (Ruff-Jamison et al., 1995; David et al., 1996; Olayioye et al., 1999). However, the signaling pathway connecting growth factors to Stat5a in mammary epithelium cells remains poorly understood.
One of the major protein kinases downstream of heregulin and the EGF family of receptors is the p21-activated kinase (Pak)1,* a serine/threonine kinase (Adam et al., 1998). The Pak1 is an effector of the small GTPases Cdc42 and Rac1 (Manser et al., 1994; Nicolas and Hall, 1997) and phosphatidylinositol-3 kinase and mediates the cellular effects of polypeptide growth factors in breast cancer cells. Recent studies have shown that Pak1 regulates motility, invasiveness, anchorage-independent growth, cell survival, and angiogenesis in human breast cancer cells (Bagrodia and Cerione, 1999; Kumar and Vadlamudi, 2002). Pak1 also modulates the activation status of MAPK and p38MAPK, and thus, influences nuclear signaling (Adam et al., 2000; Vadlamudi et al., 2000a). In spite of a large number of studies in tissue culture model systems, the role of Pak1 signaling in the normal mammary development in a physiological relevant whole animal setting remains unknown and is investigated in the present study.
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Results |
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The kinetics of DN-Pak1 expression in the various stages of mammary gland development was determined using a semiquantitative RT-PCR followed by Southern blot hybridization. DN-Pak1 expression began on the tenth day of pregnancy, peaked around the second day of lactation, and returned to near the basal level, albeit detectable, at the seventh day after weaning (Fig. 1 E). This profile is in accordance with the previously reported BLG-MDM2 mice (Lundgren et al., 1997). As expected, DN-Pak1 expression inhibited the endogenous Pak1 activity in lactating mammary glands (Fig. 1 F).
DN-Pak1 expression impairs morphogenesis during pregnancy
To determine the effect of Pak1 inactivation on the morphogenesis of the mammary gland, whole-mount and section hematoxylin and eosin (H-E) staining were performed. Normally, during pregnancy the mammary glands grow quickly and the ducts branch out and form lobules and alveoli. On the second day of lactation, the glandular tissues filled up the fat pad and the epithelial cells fully differentiate. During the virgin and early pregnant stages, the mammary glands of DN-Pak1-TG mice were not obviously different from those of the wild-type mice (unpublished data). However, at the late stages of pregnancy, the transgenic mammary glands showed marked dystrophy with fewer visible branches and poorly developed alveoli compared with the widespread branches and flourishing alveoli from the age-matched wild-type mammary glands (Fig. 2). Furthermore, on the 12th day of lactation, although the glandular tissues filled up the fat pad in the wild-type mammary glands, the transgenic mammary glands had distinct spaces in the absence of glandular tissues (Fig. 2, C and F). The H-Estained sections also demonstrated poor lobular alveolar development on the 18th day of pregnancy and moderate dystrophy during lactation (Fig. 3, B, C, E, and F).
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Pak1 stimulates ß-casein promoter activity through phosphorylation of Stat5a at Ser 779 with the establishment of Ser 779 of Stat5a as the major phosphorylation site by Pak1. We next evaluated if mutation of this site will also block Pak1-mediated stimulation of ß-casein promoter activity similar to the inhibition obtained by a truncated Stat5a mutant in Fig. 8 A. Not surprisingly, S779A mutation of Stat5a also totally blocked the Pak1 stimulation of ß-casein promoter activity with or without DIP treatment (Fig. 10, A and B).
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Discussion |
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Mammary gland development can be divided into phases of ductal development and lobuloalveolar development. Ductal development is achieved after puberty without pregnancy. Usually, in 12-wk-old virgin mice ductal branches fill the whole mammary pad. And the lobuloalveolar development is dependent on pregnancy and requires pregnancy-linked signals. Among a bunch of signals, prolactin-Jak2-Stat 5 pathways are believed to play key roles in alveolar development and differentiation. Null mutation of any one of these three molecules has been found to result in almost complete loss of alveolar development (Miyoshi et al., 2001; Shillingford et al., 2002). However, several other molecules, including growth factors and protein kinases, are also up-regulated during pregnancy and lactation and have been shown to be involved in alveolar development or milking (Chodosh et al., 2000). For example, Stat5 cannot only be activated through the prolactin receptor but also by growth hormones and EGF receptors in mammary epithelia (Gallego et al., 2001). Moreover, the prolactin receptor also stimulates phosphatidylinositol 3-kinase pathway, a known upstream activator of Pak1 signaling (Adam et al., 1998). Pak1-mediated signal in alveolar development may not be related to the phosphorylation of Stat5a, since the null mutation of Stat5a did not display a defect in alveolar development but rather in the functional differentiation (Liu et al., 1997). The mechanism of Pak1 regulation of the alveolar morphogenesis might involve its role in supporting cell survival (Schurmann et al., 2000) and promoting cell proliferation (Howe and Juliano, 2000; Vadlamudi et al., 2000a), since we noticed an increased apoptosis and a decreased proliferation rate of epithelial cells in the pregnant DN-Pak1-TG mammary glands.
We also considered the possibility of whether Pak1 inhibition-associated disruption of the cytoskeleton might contribute to the noticed phenotypes. However, our data does not support this possibility, since there was no significant difference in the status of actin staining in mammary glands from transgenic and wild-type mice (Fig. S1, available at http://www.jcb.org/content/full/jcb.200212066/DC1). It is possible that the lack of effect of Pak1 on cytoskeletal reorganization in the transgenic mice could be due to the fact that Pak1 affects cytoskeleton via kinase-dependent and -independent functions (Sells et al., 1997; Adam et al., 2000; Vadlamudi et al., 2000a). Kinase-dependent functions are implicated in dissolution of stress fibers, whereas kinase-independent functions are implicated in generation of lamellipodia and ruffling via proteinprotein interactions. In addition, Pak2 and Pak3 whose kinase domain is highly conserved to Pak1 is highly expressed in mammary gland and may compensate the Pak1 kinase activity, which is required for reorganization of cytoskeleton.
Stat5a was originally isolated from pregnant mammary gland (Wakao et al., 1992), named mammary gland growth factor, and is shown to play a role in the normal morphogenesis of mammary gland by supporting cell survival and stimulating epithelial cell proliferation. Indeed, the Stat5a-null mutation in mice on a large scale blocked the alveolar development of mammary gland during pregnancy, with a marked reduction in cell proliferation index of alveolar cells. Therefore, a full functional activation of Stat5a might be required for the normal mammary gland development. In brief, our findings of a close phenotypic resemblance between DN-Pak1-TG mutant Stat5a mice and that of Pak1 phosphorylated Stat 5a, reduction in the Stat5a phosphorylation in Pak1 mutant mice, and Pak1 regulation of transcriptional functions of Stat 5a strongly suggest that Pak1 regulates Stat5a pathway, and in principle, Stat 5a might act as a downstream target of upstream activators of Pak1. Therefore, Pak1 regulation of Stat5a might play a role in morphogenesis and differentiation of the mouse mammary gland.
The process of mammary gland differentiation is profoundly influenced by Stat5 transactivation functions (Hennighausen et al., 1997). Tyrosine phosphorylation of Stat5, both a/b isoforms at siteY694/699, is critical for the milking function of mammary epithelia. In the present study, the inability of transgenic lactating DN-Pak1-TG mice to nurse pups is partly due to the impaired alveolar development. However, drastic reduction in ß-casein and WAP expression indicated an impairment of the functional differentiation of existing alveolar epithelia. In fact, the morphology of DN-Pak1-TG mammary alveoli was distinctly less differentiated with a reduced level of Stat5 expression and its phosphorylation at Y694/699. Our data from normal mammary epithelial HC11 cells also suggested that Pak1 stimulates ß-casein promoter activity in a lactating hormone-independent manner. Moreover, K299R Pak1 blocks the basal ß-casein promoter activity, highlighting the significance of Pak1 activity in the functions of mammary glands. Immunohistochemical staining and Western blotting showed that DN-Pak1-TG mammary glands exhibited both reduced expression and decreased tyrosine phosphorylation of Stat5. Although these data help us understand the impairment of mammary gland functions, the mechanism of this regulation is still yet to be determined.
Aside from tyrosine phosphorylation, Stat5 is also phosphorylated on two other serine sites, Ser 725 (Stat5a)/Ser 730 (Stat5b) and Ser 779, which only exist in Stat5a (Yamashita et al., 1998, 2001; Beuvink et al., 2000); however, the kinase which phosphorylates these two sites are not known to date. Here we provided evidence that Pak1 directly interacts and phosphorylates Stat5a at Ser 779. We also demonstrated that mutation of Ser 779 to Ala blocks the ability of Pak1 to phosphorylate Stat5a, and consequently, stimulation of ß-casein promoter activity in physiological relevant murine normal mammary epithelial HC11 cells. These findings are significant, sine earlier studies using COS or MCF-7 cells did not notice any inhibitory effect of Stat5a Ser 779 Ala on the stimulation of ß-casein promoter activity by prolactin (Beuvink et al., 2000; Yamashita et al., 2001). It is also possible that prolactin signaling does not target Ser 779 phosphorylation, since prolactin is not an effective inducer of Pak1 activity in HC11 cells (unpublished data). In summary, results presented here have shown a role of Pak1 signaling in the Stat5a Ser 779 phosphorylation and transactivation of ß-casein promoter and have shown that targeted inactivation of Pak1 in mammary epithelial cells leads to severe impairment of normal mammary gland development.
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Materials and methods |
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Whole-mount and H-E staining
The whole left no. 4 mammary gland of each animal was removed and mounted on slides. After drying for 1030 min, the tissues were fixed with acetic acid/ethanol (1:3) for 1 h. Then, the tissues were rinsed with water briefly and stained with 0.2% carmine/0.5% aluminum potassium sulfate overnight. Tissues were then dehydrated through graded ethanol, defatted through acetone, cleared, and preserved in methyl salicylate. The right no. 4 mammary glands were fixed with 10% formaldehyde, processed to paraffin sections, and stained with H-E.
Kinase assay
Pak1 assay was performed as reported previously (Adam et al., 1998). 1 mg of total protein lysate was immunoprecipitated with an anti-Pak1 antibody (sc-882; Santa Cruz Biotechnology, Inc.) and subjected to an in vitro complex kinase assay using myelin basic protein as a substrate for 30 min at 30°C. The reaction products were resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was exposed to a phosphoscreen overnight and scanned with Storm PhosphorImager.
Immunohistochemistry and TUNEL staining
Immunohistochemical staining of PCNA (P8825; Sigma-Aldrich), Y694-phospho-Stat5 (1:50; 716900; Zymed Laboratories) was done with the indirect enzyme labeling method as described previously (Wang et al., 1998). Antigen retrieval was performed by boiling the sections for 10 min and gradually cooling them down for 30 min in 0.01 M, pH 6.0, citrate buffer before incubating with the antibody. TUNEL staining was also performed as described previously (Wang et al., 1998).
RT-PCR, Northern blot, and immunoblotting
RT-PCR was performed using the Access RT-PCR system (Promega) per the manufacturer's instructions. Before performing RT-PCR, the total RNA was first digested with Q1 DNase for 10 min to get rid of possible genomic DNA contamination. For Northern blot analysis, 20 µg of total RNA was resolved on a 1% formaldehyde agarose gel, transferred to a nylon membrane, and probed with appropriate probes and exposed to phosphoscreen. For immunoblot analysis, 200 µg of the total protein lysate was resolved on a 10% SDSpolyacrylamide gel, transferred to nitrocellulose membrane, and probed with appropriate antibodies.
Expression vectors
Wild-type Pak1, K299R-Pak1 and T423E-Pak1 (Adam et al., 1998), and GSTS779A-Stat5a (Beuvink et al., 2000) plasmids have been described previously. ß-casein promoter-luciferase vector was obtained from Dr. J. Rosen (Baylor College of Medicine, Houston, TX), DNp38-MAPK was from Dr. S. Ludwig (University of Wurzburg, Wurzburg, Germany).
Cell culture, transfection, and luciferase assay
HC11 mouse epithelial cells were maintained in RPMI-1640 medium supplemented with 8% FCS, 10 ng EGF/ml, and 5 µg insulin/ml as described previously (Vadlamudi et al., 2000b). Cells were plated in six-well culture plates, and transfection was performed using the Fugene (Roche) method. Luciferase assay was performed according to the manufacturer's instructions as described previously (Mazumdar et al., 2001) and results were standardized against ß-gal luciferase of internal control. Each experimental group includes three triplicate plates.
GST pull-down assays
In vitro transcription and translation of the Pak1 were performed using the TNT transcriptiontranslation system (Promega) as described (Mazumdar et al., 2001). In brief, the appropriate cDNAs (1 µg) were translated in vitro in the presence of 35S-methionine in a reaction volume of 50 µl using a T7-TNT kit (Promega). The reaction mixture was diluted to 1 ml with NP-40 lysis buffer (25 mM Tris, 50 mM NaCl, 1% Nonidet P40). A 250-liter aliquot was used for each GST pull-down assay. Translation and product size were verified by analyzing 2 µl of the reaction mixture with SDS-PAGE and autoradiography. GST pull-down assays were performed by incubating equal amounts of GST and GST immobilized on GST beads (Amersham Biosciences) with in vitrotranslated 35S-labeled Pak1. After incubating for 2 h at 4°C, the beads were washed five times with NP-40 lysis buffer, eluted with 2x SDS buffer, and resolved on SDS-PAGE and revealed by autoradiography.
In vitro and in vivo phosphorylation assays
In vitro and in vivo phosphorylation assay was done as described previously (Vadlamudi et al., 2002). Briefly, for in vitro assay the GSTStat5a fusion protein was incubated in 50 mM Hepes, 10 mM MgCl2, 2 mM MnCl2, and 1 mM dithiothreitol containing 1 µg of purified bacterially expressed GSTPak1 enzyme, 10 µCi of [-32P]ATP and 25 M cold ATP. The reaction was performed in a volume of 30 µl for 30 min at 30°C and then stopped by adding 10 µg of 4x SDS sample buffer, resolved on SDS-PAGE gel, and revealed by autoradiography. For in vivo phosphorylation assay, HC 11 cells were transfected with wild-type or S779A mutant Stat5a, wild-type Pak1 or Pak1 83149 inhibitory peptide, or K299R Pak1. After 36 h, the cells were labeled with 32P-orthophosphate overnight (0.2 mci/ml) and treated with DIP for 30 min. Cells were lysed, and equal amounts of the protein were immunoprecipitated with an antibody specific for HA tag to immunoprecipitate HA-STAT or HA S779A Stat. An aliquot of total lysate was run as a separate gene gel to analyze the expression of GSTPak inhibitory fragment aa 83149 and myc-tagged Pak constructs.
In situ hybridization
In situ hybridization was done as we described previously (Wang et al., 2002). Mouse mammary glands were fixed with 4% PFA, and frozen sections were cut. A 419 bp of mouse WAP cDNA was amplified by RT-PCR, subcloned into TOPO II vector (In Vitrogen), and used for riboprobe synthesis under the control of T7 promoter. Primers used are forward, 5'-CCTGACACCGGTACCATGCGTTGC-3' and reverse, 5'-CACTGAAGGGTTATCACTGGCACT-3'; RNA probes were labeled with digoxigenin (Roche) and hybridized for 1620 h in buffer containing 0.1 µg/ml riboprobes, 50% formamide, 300 mM NaCl, 10 mM Tris (pH 7.4), 10 mM NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8.0), 0.2% Ficoll 400, 0.2% polyvinyl pyrolidone, 10% dextran sulfate, 200 µg/ml yeast total RNA, and 50 mM dithiothreitol. Alkaline phosphatase-labeled sheep antidigoxigenin antibody was applied, and signals were visualized by NBT-BCIP. Hybridization with sense probe was used as background control.
Quantitation and statistical analysis
Counting of PCNA and TUNEL-positive cells was done manually on the computer screen for five fields of each case. Statistical analysis for pups weight, and PCNA and TUNEL staining and ß-casein promoter luciferase assay were done by Student's t test. Semiquantitation of Western blotting was performed by using the SigmaGel software.
Online supplemental material
Fig. S1 showing phalloidin staining of actin in mammary glands from wild-type and DN-Pak1-TG mice is available at http://www.jcb.org/content/full/jcb.200212066/DC1.
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Acknowledgments |
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This work was supported by the National Institutes of Health grant CA90970, Cancer Center Core grant CA16672, and a grant from the Breast Cancer Research Program of the University of Texas M.D. Anderson Cancer Center (to R. Kumar).
Submitted: 11 December 2002
Revised: 3 March 2003
Accepted: 25 March 2003
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References |
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Adam, L., R. Vadlamudi, S.B. Kondapaka, J. Chernoff, J. Mendelsohn, and R. Kumar. 1998. Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase. J. Biol. Chem. 273:2823828246.
Adam, L., R. Vadlamudi, M. Mandal, J. Chernoff, and R. Kumar. 2000. Regulation of microfilament reorganization and invasiveness of breast cancer cells by p21-activated kinase-1 K299R. J. Biol. Chem. 275:1204112050.
Baeckstrom, D., D. Alford, and J. Taylor-Papadimitriou. 2000. Morphogenetic and proliferative responses to heregulin of mammary epithelial cells in vitro are dependent on HER2 and HER3 and differ from the responses to HER2 homodimerisation or hepatocyte growth factor. Int. J. Oncol. 16:10811090.[Medline]
Bagheri-Yarmand, R., M. Mandal, A.H. Taludker, R.A. Wang, R.K. Vadlamudi, H.J. Kung, and R. Kumar. 2001. Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells. J. Biol. Chem. 276:2940329409.
Bagrodia, S., and R.A. Cerione. 1999. Pak to the future. Trends Cell Biol. 9:350355.[CrossRef][Medline]
Beuvink, I., D. Hess, H. Flotow, J. Hosteenge, B. Groner, and N.E. Hynes. 2000. Stat5a serine phophsphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity. J. Biol. Chem. 275:1024710255.
Chodosh, L.A., H.P. Gardner, J.V. Rajan, D.B, Stairs, S.T. Marquis, and P.A. Leder. 2000. Protein kinase expression during murine mammary development. Dev. Biol. 219:259276.[CrossRef][Medline]
David, M., L. Wong, R. Flavell, S.A. Thompson, A. Wells, A.C. Larner, and G.R. Johnson. 1996. STAT activation by epidermal growth factor (EGF) and amphiregulin. Requirement for the EGF receptor kinase but not for tyrosine phosphorylation sites or JAK1. J. Biol. Chem. 271:91859188.
Fowler, K.J., F. Walker, W. Alexander, M.L. Hibbs, E.C. Nice, R.M. Bohmer, G.B. Mann, C. Thumwood, R. Maglitto, J.A. Danks, et al. 1995. A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation. Proc. Natl. Acad. Sci. USA. 92:14651469.[Abstract]
Gallego, M.I., N. Binart, G.W. Robinson, R. Okagaki, K.T. Coschigano, J. Perry, J.J. Kopchick, T. Oka, P.A. Kelly, and L. Hennighausen. 2001. Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev. Biol. 229:163175.[CrossRef][Medline]
Hennighausen, L., G.W. Robinson, K.U. Wagner, and X. Liu. 1997. Developing a mammary gland is a stat affair. J. Mammary Gland Biol. Neoplasia. 2:365372.[Medline]
Hennighausen, L., and G.W. Robinson. 2001. Signaling pathways in mammary gland development. Dev. Cell. 1:467475.[Medline]
Howe, A.K., and R.L. Juliano. 2000. Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nat. Cell Biol. 2:593600.[CrossRef][Medline]
Jones, F.E., and D.F. Stern. 1999. Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation. Oncogene. 18:34813490.[CrossRef][Medline]
Jones, F.E., D.J. Jerry, B.C. Guarino, G.C. Andrews, and D.F. Stern. 1996. Heregulin induces in vivo proliferation and differentiation of mammary epithelium into secretory lobuloalveoli. Cell Growth Differ. 7:10311038.[Abstract]
Jones, F.E., T. Welte, X.Y. Fu, and D.V. Stern. 1999. Erb4 signaling in the mammary gland is required for lobulo-alveolar development and Stat5 activation during lactation. J. Cell Biol. 147:7787.
Kazansky, A.V., E.B. Kabotyanski, S.L. Wyszomierski, M.A. Mancini, and J.M. Rosen. 1999. Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5b and Stat5a. J. Biol. Chem. 274:2248422492.
Kumar, R., and R.A. Wang. 2002. Protein kinases in mammary gland development and cancer. Microsc. Res. Tech. 59:4957.[CrossRef][Medline]
Kumar, R., and R.K. Vadlamudi. 2002. Emerging functions of p21-activated kinases in human cancer cells. J. Cell Physiol. 190:189199.[CrossRef][Medline]
Liu, B., M. Fang, Y. Lu, G.B. Mills, and Z. Fan. 2001. Involvement of JNK-mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in SiHa human cervical cancer cells. Br. J. Cancer. 85:303311.[CrossRef][Medline]
Liu, X., G.W. Robinson, K.U. Wagner, L. Garrett, A. Wynshaw-Boris, and L. Hennighausen. 1997. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11:179186.[Abstract]
Luetteke, N,C., T.H. Qiu, S.E. Fenton, K.L. Troyer, R.F. Riedel, A. Chang, and D.C. Lee. 1999. Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126:27392750.
Lundgren, K., R. Montes de Oca Luna, Y.B. McNeill, E.P. Emerick, B. Spencer, C.R. Barfield, G. Lozano, M.P. Rosenberg, and C.A. Finlay. 1997. Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53. Genes Dev. 11:714725.[Abstract]
Manser, E., T. Leung, H. Salihuddin, Z.S. Zhao, and L. Lim. 1994. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 367:4046.[CrossRef][Medline]
Marte, B.M., M. Jeschke, D. Graus-Porta, D. Taverna, P. Hofer, B. Groner, Y. Yarden, and N.E. Hynes. 1995. Neu differentiation factor/heregulin modulates growth and differentiation of HC11 mammary epithelial cells. Mol. Endocrinol. 9:1423.[Abstract]
Mazumdar, A., R.A. Wang, S.K. Mishra, L. Adam, R. Bagheri-Yarmand, M. Mandal, R.K. Vadlamudi, and R. Kumar. 2001. Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor. Nat. Cell Biol. 3:3037.[CrossRef][Medline]
Medina, D. 1996. The mammary gland: a unique organ for the study of development and tumorigenesis. J. Mammary Gland Biol. Neoplasia. 1:519.[Medline]
Miyoshi, K., J.M. Shillingford, G.H. Smith, S.L. Grimm, K.U. Wagner, T. Oka, J.M. Rosen, G.W. Robinson, and L. Hennighausen. 2001. Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium. J. Cell Biol. 155:531542.
Mui, A.L., H. Wakao, T. Kinoshita, T. Kitamura, and A. Miyajima. 1996. Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation. EMBO J. 15:24252433.[Abstract]
Nicolas, T., and A. Hall. 1997. Rho, Rac, CDC42 regulate organization of the cytoskeleton. Curr. Opin. Cell Biol. 9:8692.[CrossRef][Medline]
Niemann, C., V. Brinkmann, E. Spitzer, G. Hartmann, M. Sachs, H. Naundorf, and W. Birchmeier. 1998. Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis. J. Cell Biol. 143:533545.
Olayioye, M.A., I. Beuvink, K. Horsch, J.M. Daly, and N.E. Hynes. 1999. ErbB receptor-induced activation of Stat transcription factors is mediated by Src Tyrosine kinases. J. Biol. Chem. 274:1720917218.
Ruff-Jamison, S., K. Chen, and S. Cohen. 1995. Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat5 in mouse liver. Proc. Natl. Acad. Sci. USA. 92:42154218.[Abstract]
Schurmann, A., A.F. Mooney, L.C. Sanders, M.A. Sells, H.G. Wang, J.C. Reed, and G.M. Bokoch. 2000. p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol. Cell. Biol. 20:453461.
Sells, M.A., U.G. Knaus, S. Bagrodia, D.M. Ambrose, G.M. Bokoch, and J. Chernoff. 1997. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7:202210.[Medline]
Shillingford, J.M., K. Miyoshi, G.W. Robinson, S.L. Grimm, J.M. Rosen, H. Neubauer, K. Pfeffer, and L. Hennighausen. 2002. Jak2 is an essential tyrosine kinase involved in pregnancy-mediated development of mammary secretory epithelium. Mol. Endocrinol. 16:563570.
Vadlamudi, R.K., L. Adam, R.A. Wang, M. Mandal, D. Nguyen, A. Sahin, J. Chernoff, M.C. Hung, and R. Kumar. 2000a. Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells. J. Biol. Chem. 275:3623836244.
Vadlamudi, R., R.A. Wang, A.H. Talukder, L. Adam, R. Johnson, and R. Kumar. 2000b. Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells. Mol. Cell. Biol. 20:90929101.
Vadlamudi, R.K., F. Li, L. Adam, D. Nguyen, Y. Ohta, T.P. Stossel, and R. Kumar. 2002. Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase. Nat. Cell Biol. 4:681690.[CrossRef][Medline]
Wakao, H., N.M. Schmitt, and B. Groner. 1992. Mammary gland specific nuclear factor is present in lactating rodent and bovine mammary tissue and composed of a single polypeptide of 89 kDa. J. Biol. Chem. 267:1636516370.
Wang, R.A., P.K. Nakane, and T. Koji. 1998. Autonomous cell death of mouse male germ cells during fetal and postnatal period. Biol. Reprod. 58:12501256.[Abstract]
Wang, R.A., R.A. Mazumdar, R.K. Vadlamudi, and R. Kumar. 2002. P21-activated kinase-1 phosphorylates and transactivates estrogen receptor- and promotes hyperplasia in mammary epithelium. EMBO J. 21:54375447.
Xie, W., A.J. Paterson, E. Chin, L.M. Nabell, and J.E. Kudlow. 1997. Targeted expression of a dominant negative epidermal growth factor receptor in the mammary gland of transgenic mice inhibits pubertal mammary duct development. Mol. Endocrinol. 11:17661781.
Yamashita, H., J. Xu, R.A. Erwin, W.L. Farrar, R.A. Kirken, and H. Rui. 1998. Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells. J. Biol. Chem. 273:3021830224.
Yamashita, H., M.T. Nevalainen, J. Xu, M.J. LeBaron, K.U. Wagner, R.A. Erwin, J.M. Harmon, L. Hennighausen, R.A. Kirken, and H. Rui. 2001. Role of serine phosphorylation of Stat5a in prolactin-stimulated ß-casein gene expression. Mol. Cell. Endocrinol. 183:151163.[CrossRef][Medline]
Yang, Y., E. Spitzer, D. Meyer, M. Sachs, C. Niemann, G. Hartmann, K.M. Weidner, C. Birchmeier, and W. Birchmeier. 1995. Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J. Cell Biol. 131:215226.[Abstract]