Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M308545200 on September 2, 2003

J. Biol. Chem., Vol. 278, Issue 46, 46171-46178, November 14, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/46/46171    most recent
M308545200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Srivastava, S.
Right arrow Articles by Horseman, N. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srivastava, S.
Right arrow Articles by Horseman, N. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Receptor Activator of NF-{kappa}B Ligand Induction via Jak2 and Stat5a in Mammary Epithelial Cells*

Sunil Srivastava{ddagger}, Manabu Matsuda{ddagger}§, Zhaoyuan Hou{ddagger}, Jason P. Bailey{ddagger}, Riko Kitazawa¶, Matthew P. Herbst{ddagger}, and Nelson D. Horseman{ddagger}||

From the {ddagger}Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576, the§Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-003, Japan, and the Department of Pathology, Kobe University, Kobe 650-0017, Japan

Received for publication, August 4, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-{kappa}B ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-{kappa}B) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a {gamma}-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammary gland development is a postnatal and discontinuous process, with two important phases: puberty and pregnancy. Each phase is controlled by the actions of reproductive hormones derived from the pituitary gland, ovaries, and placenta. At the onset of puberty estrogen and progesterone stimulate ductal growth and branching in the mammary fat pad. The epithelial ducts become decorated with subordinate branches and alveolar buds especially during pregnancy or repeated estrous cycles. The final development of mammary tissue into a functional lactating gland is primarily regulated by the pituitary hormone, prolactin (PRL),1 and occurs during post-partum (1-4).

The actions of peptide and steroid hormones in mammary gland development are overlapping. However, classic hormone ablation and replacement studies identified the critical roles of progesterone (P4) and PRL in the later stages of mammary gland development (4-6). Although these experiments identified P4 and PRL as two master regulators of lobuloalveolar development, the results failed to identify their individual roles and their target genes under normal physiological conditions. With the advent of gene targeting in the mouse germ line, combined with tissue transplantation techniques, it has been possible to identify the functional roles of each hormone. Mice with targeted disruptions of PRL or its receptor have mammary glands with a fully developed tubular epithelial system but absence of alveolar buds (7, 8). In contrast, P4 receptor knockout mice lack terminal buds and side branching (9, 10). These results provided genetic opportunities to study PRL signaling during development of the mammary alveolar bud system and milk synthesis.

Previous studies have confirmed that PRL, its target genes, and its signaling molecules are essential for lobuloalveolar morphogenesis during pregnancy (1, 11, 12). The primary pathway by which PRL transduces its signal in epithelial cells has been identified (13, 14). Binding of PRL to its receptor activates Janus kinase2 (Jak2) a member of the protein-tyrosine kinase superfamily, which phosphorylates the latent transcription factor Stat5 (signal transducer and activator of transcription 5) (8). Phosphorylated Stat5 dimerizes, translocates to the nucleus, and binds to the PRL response elements (TTCTTGGAA), which conforms to the consensus {gamma}-interferon-activating sequence GAS (TTCXXXGAA) (15). At present four Jak and seven Stat family members are known in mammals (16). Among them Jak2, Stat5a, and Stat5b have been shown definitively to be involved in PRL signaling (13). Stat5a and 5b are encoded by two distinct genes, yet are highly similar. They bind to the same GAS site and show redundant functions except in the mammary gland, where only Stat5a is necessary for alveolar morphogenesis (16). Consistent with this model, mice null for genes that encode PRL, PRL-receptor, and Stat5a exhibit similar phenotypes (8). All these mice failed to lactate due to undeveloped lobuloalveolar structures during pregnancy.

RANKL knockout (RANKL-/-) mice exhibit severe osteopetrosis and failure of tooth eruption because of a complete absence of osteoclasts and defective bone remodeling. In addition, these mice show undeveloped lobuloalveolar buds and fail to express the milk protein {beta}-casein, resulting in the death of pups. This phenotype has similarities to those earlier reported in mice lacking expression of genes related to PRL signaling. Taken together these studies suggest that RANKL may be one of the downstream targets of PRL (14, 20).

RANKL, its biological receptor RANK, and decoy receptor osteoprotegerin (OPG) are the members of tumor necrosis factor and tumor necrosis factor receptor families. RANKL and RANK were first identified in activated T-cells and dendritic cells and subsequently in bone-forming osteoblast and bone-resorbing osteoclast cells (17). The main function of RANKL is to increase the survival of dendritic cells and regulate Ca2+ homeostasis by regulating bone resorption in physiological and pathological conditions. RANKL signaling has been extensively studied in osteoclasts. Binding of RANK initiates a signaling cascade that activates NF-{kappa}B, mitogen-activated protein kinases, and protein kinase B/AKT (17-19). In subsequent studies it was noted that mice with a mutant version of the NF-{kappa}B regulatory kinase inhibitor of {kappa}B kinase {alpha} (IKK{alpha}) exhibited a similar mammary gland phenotype to that observed in RANKL-/- mice. IKK{alpha}-/- mice failed to activate NF-{kappa}B and up-regulate cyclin D1, a protein essential for epithelial cell proliferation. Interestingly, the IKK{alpha}-defective phenotype was rescued by overexpression of cyclin D1 in mammary epithelial cells, suggesting that NF-{kappa}B signaling, in addition to Stat signaling, plays a critical role in alveolar morphogenesis by inducing the cell cycle protein cyclin D1 (21).

Previous studies have provided conflicting evidence regarding whether RANKL is one of the targets of PRL (20-22). Therefore, this study has been carried out to test the hypothesis that RANKL expression is PRL-dependent and to identify a signaling pathway by which PRL may induce RANKL expression in mammary gland. Here we show that RANKL expression is regulated during the course of mammary gland development. Using PRL gene-disrupted mice and in vitro studies, we show that PRL and P4 induce the expression of RANKL and that PRL acts through a Jak-Stat pathway that preferentially uses Stat5a.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Hormone Treatment—Mice were maintained on a 14-h, 10-h daily light/dark cycle with food and water available ad libitum. An ovariectomy (ovx) was done via a bilateral incision in the flanks. Surgeries were performed under general anesthesia and aseptic conditions. All experiments were conducted under Institutional Animal Care and Use Committee approved protocols. Sixteen-week-old female prolactin-deficient (PRL-/-) mice were treated by grafting an anterior pituitary gland from normal PRL+/- mice under the kidney capsule, implanted subcutaneously with 21-day release progesterone and estrogen tablets for 18 days, or were sham-operated essentially as described (23). Lactating and virgin wt C57Bl/6J mice were purchased from the Harlan Laboratory.

Cell Culture—Primary mouse mammary epithelial cells (PMECs) were prepared from virgin or pregnant mice by enzymatic digestion and differential trypsinization, according to the methods of Imagawa with modifications (24). In brief, excised mammary glands were minced with a razor blade, transferred into 50-ml conical tubes, and digested in M199 medium (medium 199, 1x, Invitrogen catalog number 12350-39) containing bovine serum albumin (205 mg/ml, fraction V, Sigma) and 0.1% collagenase type III (Worthington Biochemical Corp., Freehold, NJ), penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin (100 µg/ml, Sigma-Aldrich, St. Louis, MO) for 3 h at 37 °C. Organoids were allowed to settle in rat tail collagen-coated dishes (BD Biosciences), washed twice with PBS, and incubated at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium/F-12 (1:1) containing 1% bovine serum albumin, insulin (5 mg/ml), epidermal growth factor (10 ng/ml), and 1x pen/strep (100 µg/ml penicillin and 100 µg/ml streptomycin). Resulting epithelial cells were plated (2 x 105) on conventional plastic tissue culture plates and treated with oPRL (5 µg/ml) (25).

CHOD6 cells, a cell line that constitutively expresses pigeon PRL receptor, were maintained in Ham's F-12 medium (Invitrogen, Rockville MD), supplemented with 10% fetal bovine serum, 1% L-glutamine, 1x penicillin/streptomycin, and 0.5 mg/ml Geneticin (Sigma-Aldrich, St. Louis. MO). COS7 cells were purchased from ATCC and were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1x penicillin/streptomycin.

Preparation of Total RNA and RT-PCR—Total RNA was isolated from mammary gland or primary epithelial cells using TRI reagent (Molecular Research Center Inc., Cincinnati, OH) according to manufacturer's instructions. Total RNA from each sample was treated with DNase I following the manufacturer's protocol (Promega, Madison WI). Two micrograms of the treated total RNA was reversed-transcribed in a 20-µl reaction using Superscript II RT and oligo(dT) (Invitrogen, Carlsbad, CA). The reverse-transcribed reaction (2 µl) was amplified by PCR (PCRExpress thermal cycler, Hybaid, Middlesex, UK) using TaqDNA polymerase (Promega) in a final reaction volume of 50 µl with 35 cycles (15 s at 94 °C, 30s at 55 °C, and 1 min at 72 °C). Amplified products (10 µl) were resolved by electrophoresis on 1% agarose gels, and bands were visualized by ethidium bromide staining. Sense and antisense primers for RANKL, RANK, and OPG used were as follows: RANKL, Sense 5'-CGCTCTGTTCCTGTACTTTCGAGCG-3' and Antisense 5'-TCGTGCTCCCTCCTTTCATCAGGTT-3'; RANK, sense 5'-TTAAGCCAGTGCTTCACGGG-3' and antisense 5'-ACGTAGACCACGATGATGTCGC-3'; and OPG, sense 5'-GTGGTGCAAGCTGGAACCCCAG-3' and antisense 5'-AGGCCCTTCAAGGTGTCTTGGTC-3'. GAPDH, 28-S, WAP, and {beta}-casein primers were used as described earlier (23).

Transient Transfection—CHOD6 cells were grown at a density of 1.5 x 106 cells in 12-well plates 24 h prior to the experiments. Transfections were conducted using either FuGENE (Roche Applied Science, Indianapolis, IN) or LipofectAMINE (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. One microgram of total plasmid was transiently transfected containing 0.5 µg of reporter gene, and 100 ng each of {beta}-galactosidase expression vector or pcDNA empty vector. In some experiments RANK and PRL-receptor and wild type Stat5 expression vectors were also used at a concentration of 100 ng/well. CHOD6 and PMEC cells were treated with 1 µg or 5 µg/ml ovine prolactin (oPRL) as noted and harvested after 24 h. Luciferase activities were measured using a Luciferase (Luc) reporter assay kit (Roche Applied Science), and {beta}-galactosidase activities were measured using the Galacto-Light PlusTM Chemiluminescent reporter assay (Tropix, Bedford, MA) according to the manufacturer's instructions in a single cell luminometer. Each well was corrected with {beta}-galactosidase activity, and results were expressed as Luc activity.

Site-directed Mutagenesis and Plasmid Constructs—Site-directed mutagenesis was conducted using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and mutants were confirmed by DNA sequencing. The GAS sequence present in the RANKL promoter TACAGAGAA was changed to TACAGAcAg. Dn JAk2KD (double mutation in C-terminal kinase domain) and Jak2{Delta}829 (deletion of kinase domain after amino acid 829) were described previously (26). Full-length and Dn Stat5a and Stat5b constructs were as described (27).

Electrophoretic Mobility Shift Assay—CHOD6 cells were grown in 100-mm culture plates and were either treated with 1 µg/ml oPRL for 30 min or remained untreated. Wild type and mutant probes were synthesized, annealed in 50 mM Tris-HCl (pH 7.9), 150 mM NaCl, and 1 mM EDTA and radiolabeled with [{gamma}-32P]deoxy-CTP purchased from PerkinElmer Life Sciences (Boston, MA) using Klenow Large Fragment DNA polymerase (Promega). Stat5a antibody #sc 1081 and Stat binding sequence #sc 2565(GAS) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nuclear extracts and EMSA were conducted as previously described (28).

Statistics and Experimental Replications—Each experiment was done in triplicate and was repeated at least three times. One representative experiment has been shown. Differences between means were tested by Student's t test (two-tailed). Differences among groups were tested by one-way analysis of variance with the Tukey's test for pairwise multiple comparisons. Significance was accepted for p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mammary RANKL Was Induced during Pregnancy and by Elevated PRL—The mammary gland is regulated by a variety of hormones and growth factors at specific stages of development. We first investigated whether RANKL expression was developmentally regulated and determined the effects of PRL, P4, and estrogen (E2) on RANKL, its receptor RANK, and its decoy receptor OPG. Total RNA was isolated from 4th mammary glands at various developmental stages in PRL normal+/- and PRL-/- mice. Total RNA (2 µg) was reversed-transcribed and subjected to PCR using gene-specific primers for RANKL, its receptor RANK, and osteoprotegerin (OPG). GAPDH was used as an internal control. As shown in Fig. 1A, RANKL expression was undetectable in normal virgin+/- and PRL-/- mice, and it was markedly increased with pregnancy and in PRL-/- mice exposed to high PRL levels by pituitary grafting (23). In contrast, RANK and OPG were expressed similarly in all conditions examined. We also examined RANKL expression at various stages of pregnancy and lactation. RANKL expression was undetectable in virgin mice, gradually increased during pregnancy, peaked at day 17.5 during pregnancy, and decreased to a barely detectable level in late pregnancy, during lactation and involution. Gene expression for {beta}-casein and whey acidic protein (WAP) was high from mid-pregnancy through lactation, and remained detectable during involution (Fig. 1B)



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 1.
RANKL mRNA expression in mammary tissue. Total RNA of PRL-/- and normal mice PRL+/- was isolated at various stages of mammary gland development, reverse-transcribed, and amplified using gene-specific primers for RANKL, RANK, OPG, WAP, and {beta}-casein. GAPDH and 28 S RNA were used as internal controls. Amplified PCR products were visualized by ethidium bromide on 1% agarose gels. A, steady-state mRNA expression of RANKL, RANK, and OPG in mammary tissue of virgin PRL+/- mice (lane 1), PRL-/- mice (lane 2), PRL-/- grafted mice (lane 3), and pregnant PRL+/- mice (lane 4). B, steady-state mRNA expression of {beta}-casein, WAP, and RANKL of normal mice (PRL+/-), lane 2; virgin mice PRL+/-, lanes 3-7: days 6.5, 10.5, 14.5, 17.5, and 19.5 pregnant+/-; lanes 8 and 9 (days 2 and 10 of lactation) and lanes 10-12 (days 2, 5, and 10 of involution).

 
Prolactin, Progesterone, and Estrogen Regulate RANKL Expression in Mammary Epithelial Cells—PRL, P4, and E2 are key hormones that coordinate the development of the mammary gland during early pregnancy. Therefore, we examined their effects on RANKL mRNA expression in PRL-/- mice using RT-PCR. 3-week-old mice were ovariectomized (ovx) or sham-operated and given 2 weeks for recovery. At the end of 2 weeks mice were pituitary-grafted to provide a high level of PRL stimulation (23). In some mice E2 and P4 slow release capsules were implanted. After 9 days mice were sacrificed, and total RNA was extracted from the mammary gland and subjected to RT-PCR (diagrammatic representation Fig. 2A). As shown in Fig. 2B, normal+/- and PRL-/- virgin mice controls did not express detectable levels of RANKL mRNA (lanes 1 and 2), and P4 treatment alone did not induce RANKL mRNA expression. Pituitary grafted mice with intact ovaries (lane 4) had markedly induced RANKL expression. In ovariectomized mice E2 and P4 together induced RANKL mRNA expression to a barely detectable level (lane 5). Pituitary grafted ovx mice expressed RANKL at high levels only when treated with E2 and P4 together (lane 8). These results demonstrate that PRL, E2, and progesterone are required to induce RANKL expression and alveolar bud formation in vivo.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 2.
Effects of PRL, estrogen (E2), and progesterone (P4) treatments on the RANKL mRNA expression in PRL-/- mice. A, diagrammatic representation of treatments. 6-week-old PRL mice-/- were ovariectomized (ovx) or sham-operated, and 2 weeks later, implanted with slow release pellets of P4, E2, or pituitary grafted (pitgr). Mice were sacrificed at day 9 of treatment and RANKL, WAP, and {beta}-casein mRNA expressions from mammary tissue were examined by RT-PCR. B, lanes 1 and 2 are virgin mice PRL+/- and PRL-/- mice. GAPDH was used as an internal control. ovx PRL-/- mice were treated with pituitary grafted, E2, or P4. Pituitary grafted alone induced RANKL and {beta}-casein expression (lane 4), which was further induced in presence of E2 and P4 (lane 8). P4 alone or E2 and P4 together did not induce RANKL (lanes 3 and 5). Pituitary grafted alone or pituitary grafted ovx mice in the presence of steroid-restored alveolar bud formation (not shown). The WAP, {beta}-casein, GAPDH, and 28 S RNA control data in Figs. 1 and 2 were shown in a previous publication (Matsuda et al. (23)) and are shown here (with permission) to facilitate the readers' interpretation.

 
RANKL Is Expressed in Mammary Epithelial Cells but Not in Stromal Cells—Because whole mammary glands contain cells from both epithelial and stromal compartments, we could not conclude whether RANKL expression was restricted to one type or expressed in both cell types. To investigate this issue we isolated and cultured mammary epithelial cells and stromal cells from mammary glands of normal PRL+/- virgin mice for treatments in vitro. PRL treatment significantly induced RANKL mRNA expression in epithelial cells, whereas stromal cells failed to express RANKL under basal conditions or after stimulation with PRL (Fig. 3). RANK expression was also restricted to mammary epithelial cells and was not induced by PRL. In contrast, OPG was expressed in both stromal and epithelial cells. These observations suggest that an autocrine/paracrine mode of RANK/RANKL signaling in the mammary gland, with potential modulation by stromally derived OPG.



View larger version (63K):
[in this window]
[in a new window]
  		FIG. 3.
mRNA expression of RANKL in mammary epithelial but not in stromal cells. Primary epithelial and stromal cells isolated from mammary glands from normal virgin PRL+/- mice were treated with 5 µg/ml ovine PRL for 24 h, after which total RNA was prepared and subjected to RT-PCR using specific primers for RANKL, RANK, and OPG. Primers for GAPDH were used as internal control. A, epithelial cell cultures, in which PRL induced RANKL expression; B, stromal cells, which did not respond to PRL, and expressed neither RANKL nor RANK.

 
Overexpression of Stat5a Induces RANKL Expression in Primary Epithelial Cells—To determine whether PRL and/or P4 alone can also induce RANKL expression in vitro, PMEC were treated with PRL or P4 (10-6 M) in the presence or absence of E2 (10-7 M). Epithelial cells treated with PRL and mammary tissues from pregnant mice were used as positive controls. P4 induced RANKL expression only in the presence of E2, to a degree similar to that of PRL-treated cells (Fig. 4A). These observations suggest that P4 and PRL both induce RANKL expression in vitro, but that E2 is a necessary factor for P4 response.



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 4.
mRNA expression of RANKL in mammary epithelial treated with PRL and P4. A, mammary epithelial cells from normal (PRL+/-) virgin mice were treated with 5 µg/ml oPRL (lane 2), or with P4 (10-6 M) in the absence (lane 3) or presence (lane 4) of E2. Pregnant mouse mammary tissue was used as a positive control (lane 5). B, effects of Stat5a and Stat5b overexpression on the RANKL mRNA expression in mammary epithelial cells. Mammary epithelial cells were transfected with control, Stat5a, or Stat5b expression vectors, as indicated, and cells were induced with 5 µg/ml oPRL or remained untreated for 24 h. Total RNA was extracted and subjected to RT-PCR. Stat5a significantly enhanced RANKL expression in a dose-dependent manner, whereas Stat5b did not.

 
Previous studies have established that Stat5a-/- and RANKL-/- mice share similar mammary gland phenotypes (14, 20). To determine whether Stat5 expression is critical for RANKL expression and determine the relative effectiveness of Stat5a and Stat5b in RANKL induction, PMEC were transiently transfected with 1 and 2 µg/well of wt Stat5a or Stat5b expression vectors. PMEC transfection efficiency was >30% (27). Empty vector was used as control. Cells were treated with or without 5 µg/ml oPRL for 24 h. RANKL expression was induced by PRL alone, and the PRL response was enhanced in a dose-dependent manner by overexpression of Stat5a. In contrast, Stat5b overexpression did not cause a dose-dependent increase in RANKL expression. Basal RANKL expression was quieted by overexpression of Stat5, perhaps as a result of quenching of endogenous co-regulators (Fig. 4B). These results suggested that RANKL induction by PRL is mediated preferentially by Stat5a and offered a potential link by which Stat5a mediates alveolar morphogenesis.

Prolactin Increases RANKL-Luciferase Activity in a Dose-dependent Manner in CHO-D6 Cells—The RANKL promoter has been extensively characterized, but there are no prior data regarding PRL regulation of the RANKL promoter or a mechanism of PRL action (20, 29). To address these questions, we transiently transfected a mouse RANKL-luciferase (RANKL-Luc) reporter construct containing residues to -965 from the transcription start site into CHOD6 cells, which have been previously used to study PRL-induced signaling (26, 30). The reporter construct is diagrammatically shown in Fig. 5A. After transfection the cells were treated for 24 h with various concentrations of PRL as shown in Fig. 5B. PRL treatment induced RANKL-Luc activity in a dose-dependent manner. To determine the sequences responsible for induction by PRL, we transiently transfected different deletion constructs of the RANKL reporter into CHOD6 cells and found that the region between -965 and -725 residues conveyed all the response to PRL (Fig. 5C). Sequence analysis of this region revealed that it contained a GAS-like site that previously had not been reported (Fig. 5A). To test whether this GAS site is essential for PRL response we mutated the site in the context of the RANKL promoter and tested the normal and mutated promoters in CHOD6 cells. And as shown in Fig. 5D, mutation of the GAS site in the RANKL promoter eliminated the PRL response.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 5.
PRL increased RANKL reporter activity in a dose-dependent manner. A, the RANKL promoter containing -965 nucleotides cloned into PGL3 basic vector. The region from -890 to -881 contains a potential GAS site with one mismatch shown in italics. B, RANKL reporter construct (-965) was transiently co-transfected into CHOD6 cells with {beta}-galactosidase expression vector. Luciferase activities were measured after 24 h of treatment with various doses of oPRL. Transfection efficiency was normalized for each well with {beta}-galactosidase activity and expressed as Luc activity. C, RANKL-Luc reporter full-length construct (-965) and deletion construct (-723) were transiently transfected in CHOD6 cells that were treated with or without PRL for 24 h. The Luciferase response to PRL was abrogated by the deletion mutant, because only the -965 promoter construct responded to PRL. D, wild type (965nt) and mutated (965mut) RANKL promoter constructs were transiently transfected into CHOD6 cells. The mutant construct contained a mutation in the putative GAS element as shown in A. The mutation in the putative GAS site abolished the PRL response.

 
To further confirm that the RANKL GAS site binds to Stat5 protein, we conducted an electrophoretic mobility shift assay (EMSA) using the normal and mutated GAS sequence with nuclear extracts of PRL-stimulated and unstimulated CHOD6 cells. As shown in Fig. 6, the PRL-induced nuclear extract bound strongly with the normal GAS sequence (lane 2), whereas mutated GAS sequence failed to bind to the PRL induced nuclear protein (lane 5). The specificity of the binding was confirmed by competition with cold probe (lane 4) and Stat5 antibody (lane 3). The {beta}-casein GAS sequence, which binds to Stat5 protein strongly, was used as a positive control (lanes 7 and 8). These data suggested that PRL could directly activate RANKL expression by activating the Jak2/Stat5 pathway.



View larger version (80K):
[in this window]
[in a new window]
  		FIG. 6.
Electrophoretic mobility shift assay. PRL-treated or untreated CHOD6 cell nuclear extracts were incubated with 32P-radiolabeled oligonucleotide sequences corresponding to the putative GAS sequence in the RANKL promoter, the mutated RANKL GAS sequence, or control {beta}-casein PRL-response element probe. DNA-binding activities were examined in 4% polyacrylamide gels. The {beta}-casein GAS sequence (Santa Cruz Sc# 2565) used a positive control (lane 7) identifies the position of the Stat5 complex band, and the reaction of the complex to incubation with Stat5a antibody.

 
Dominant Negative Mutants of Jak2 and Stat5a Block PRL Induction of RANKL Expression—PRL induces its signal via Jak2, which binds to PRL receptor and phosphorylates multiple substrates, including Stat proteins. To investigate signaling involved in PRL induction of RANKL, CHOD6 cells were transiently cotransfected with RANKL-Luc reporter with Dn Jak2 mutants, which have been used previously and have been shown to effectively block Jak2 activation in our laboratory (26). Results in Fig. 7A showed that the PRL effect on the RANKL reporter construct activity was completely attenuated in cells cotransfected with either Dn Jak2{Delta}859 or Jak2KD, confirming that PRL induction of RANKL transcription depends upon Jak2 activation.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 7.
Jak2 and Stat5a mediation of PRL-induced RANKL expression. A, CHOD6 cells were transiently co-transfected with the RANKL promoter reporter construct (-965) and dominant-negative Jak2 mutants (Jak2KD or Jak2{Delta}829), either of which blocks induction of RANKL transcription. B, CHOD6 cells were co-transfected with Stat5a or Stat5b or empty vector (EV) constructs and remained untreated or treated with PRL. PRL induced RANKL-Luc activity in cells cotransfected with EV. Stat5a (wt) increased basal Luc activity as well as PRL-induced activity. In contrast, Stat5b (wt) failed to induce RANKL-Luc activity and interfered with PRL induction of RANKL-Luc activity. Overexpression of mutant Stat5 constructs resulted in suppression of the PRL response of the RANKL promoter and suppression of basal activity. C, COS7 cells were co-transfected with PRL receptor and either Stat5a or Stat5b expression vectors along with RANKL-Luc. Cells were treated with PRL for 24 h. Stat5a, but not Stat5b, reconstituted RANKL induction by PRL in the COS7 cells.

 
Because one of the likely downstream targets of Jak2 is Stat5, we next examined the contribution of Stat5a and Stat5b by cotransfecting RANKL-Luc with either wild type (wt) or mutant Stat5a and Stat5b in CHOD6 cells. As shown in Fig. 7B, expression of wt Stat5a enhanced both the basal and PRL-induced RANKL-Luc reporter activity, whereas wt Stat5b overexpression interfered with the PRL response, without affecting basal expression. Overexpression of mutated Stat5 constructs (either 5a or 5b) attenuated the PRL response of the RANKL promoter as well as basal activity.

Because mutated Stat5a and 5b could compete for upstream factors (such as Jak2 or PRL receptor binding), the dominant-negative approach was not informative regarding the relative effectiveness of Stat5a and Stat5b in the induction of a particular downstream event (RANKL promoter activation). Consequently, we reconstituted the PRL-Jak2-Stat5 system in COS7 cells, which express low endogenous Stat protein activity and fail to induce genes that engage Stat protein for their signaling (26, 30) to test the roles of Stat5a and 5b. To reconstitute the PRL response, COS7 cells were cotransfected with constructs expressing wt Stat5a or Stat5b, and RANKL-Luc, along with PRL receptor. As shown in Fig. 7C, PRL induced RANKL reporter activity in cells expressing Stat5a, whereas Stat5b expression failed to mediate an induction of RANKL reporter activity. None of the constructs induced RANKL-Luc activity in the absence of PRL receptor (data not shown). In contrast to the RANKL reporter, which showed a strong preference for Stat5a reporter, a construct containing the PRL response element from the {beta}-casein promoter responded equally well to Stat5a and 5b when stimulated with PRL (data not shown). These results suggest that transcriptional induction of RANKL expression by PRL, as well as endogenous RANKL expression (Fig. 4B) preferentially utilizes a Jak2-Stat5a pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism by which prolactin (PRL) regulates milk proteins has been extensively studied. However, very little is known about PRL-regulated genes involved in mammary gland morphogenesis. Epithelial cells present in lobuloalveolar buds begin proliferating during early pregnancy under the influence of progesterone and PRL, giving rise to the lobuloalveolar units (5, 16). Based on similarities with the mammary gland phenotypes of PRL and PRLR knockout mice, Stat5a, IKK{alpha}, cyclin D1, and RANKL have been suggested as likely targets of PRL that participate in lobuloalveolar morphogenesis. Each of these gene-disrupted mouse strains failed to lactate due to impaired alveolar development caused by lack of proliferation and/or increased apoptosis during pregnancy (13, 20, 21, 31, 32). Although these mice show apparently similar phenotypes, there are subtle differences in {beta}-casein expression, a marker used to identify the differentiated state of epithelial cells, suggesting that a hierarchy exists among the genes that are expressed in response to PRL during alveolar development. The developmental lesions in RANK or RANKL -/- mice are histologically similar to that observed in PRL-/- or PRLR-/- mice, suggesting that these two pathways are dependent upon each other (14). Furthermore, Stat5a activity precedes RANKL expression by approximately 1 day during pregnancy, and the Stat5a activity is preserved in the mammary gland of RANKL-/- mice, thus excluding RANK signaling as an activator of Stat5a (13, 14). Taken together these observations clearly suggest that mammary gland development is not controlled exclusively by systemic hormones but also by hormones and growth factors produced locally by the stromal and/or epithelial compartments.

Although PRL is the primary hormone that initiates a signaling cascade leading to proliferation and differentiation of alveolar mammary epithelial cells, it is conceivable that PRL does not directly regulate proliferation. Consequently, PRL may trigger the expression of systemic and local factors that regulate and amplify the process of proliferation. Here we have reported that RANKL is a potential target of PRL and P4, two endocrine factors that must collaborate to cause appropriate mammary gland development. In the present study we have shown both in vivo and in vitro that RANKL expression is PRL-dependent and is developmentally regulated. In mice its expression is confined to early pregnancy and precipitously decreases to an undetectable level and stay lows during late pregnancy, lactation, and involution. Therefore, RANKL expression is very distinct from WAP and {beta}-casein milk genes, which are also regulated by PRL. One interesting finding in these studies is that either P4 or PRL were sufficient to induce RANKL expression in vitro, but in vivo both hormones were necessary. This result contrasts with a previous study that suggested that P4 alone was sufficient to induce RANKL (20, 22). One explanation for this discrepancy is that the study by Brisken et al. (22) was done in mice with an intact PRL gene, whereas our studies were done in PRL-deficient mice. Therefore, endogenously secreted PRL may have provided the permissive environment for P4 induction of RANKL in previous studies, whereas our studies uncovered an obligatory role for PRL during mammary RANKL expression.

PRL (or placental lactogens) along with P4 are each necessary for lobuloalveolar development (5, 6). Both are secreted at high levels during early and middle pregnancy, when lobuloalveolar growth is maximal. This is the time during which we show that RANKL expression is highest (Fig. 1B). Although P4 activity is sufficient alone to induce subordinate branching of the ductal systems, lobuloalveolar growth requires both PRL and P4 (33, 34). This collaboration between PRL and P4 appears from our data to require RANKL signaling in vivo. In addition, our results clearly show for the first time that RANKL expression as well as expression of the RANKL receptor, RANK is confined to epithelial cells and that mammary stromal cells express only the decoy receptor OPG. Because stromal and epithelial cell interplay is essential for alveolar morphogenesis, this finding raises the possibility that stromal cell-derived OPG may also regulate RANKL biological functions and thus provide another level of regulation. Together these results suggest that RANKL expression in vivo is tightly regulated by steroid hormones, PRL, and possibly by factors from stromal cells.

The results of our studies using primary mammary epithelial cells and RANKL promoter activity strongly suggest that RANKL gene induction is preferentially mediated by Stat5a. To our knowledge this is the first evidence of preferential regulation of a developmentally important gene by Stat5a, providing a vital link between PRL signaling, Stat5a expression, and mammary gland morphogenesis. The distinct morphogenetic defects in Stat5a-deficient mice, without loss of milk protein induction (35), suggested that Stat5a mediated the induction of one or more PRL-dependent genes that were important for lobuloalveologenesis. Our results provide a basis for hypothesizing that RANKL is a local morphogenetic factor that responds specifically through a PRL-Jak2-Stat5a pathway. The mechanisms that allow the RANKL promoter to distinguish Stat5a and Stat5b are unknown at this time.

IKK{alpha}-/- and RANKL-/- mice share similar phenotypes of poor mammary development, whereas mice overexpressing NF-{kappa}B in mammary epithelial cells exhibit proliferation and secondary branching of the ducts (36). In contrast, hyperactivation of Jak2 induces differentiation of mammary epithelial cells (37). Furthermore, increased NF-{kappa}B activity was detected during mid-pregnancy, corresponding to the time period when we found the highest level of RANKL expression (38). These results together suggest that cross-talk exists between the Jak2/Stat5 and NF-{kappa}B pathways and that NF-{kappa}B expression is essential for mammary gland development during pregnancy, but not for lactation. Selective expression of Stat5a, which mediates RANKL induction, may be one of the key events necessary for rapid proliferation during early pregnancy. Our working hypothesis is that PRL induces RANKL expression and that this induction requires the presence of P4 in vivo. RANKL expression initiates a cascade that activates cyclin D1, which is the direct target of NF-{kappa}B (39). In other cell systems, Stat5a has been described as a target of NF-{kappa}B (40). Therefore, it is possible that RANKL induces Stat5a expression via NF-{kappa}B, whereas Stat5a selectively activates RANKL expression, setting up a positive loop that ensures maximal epithelial cell proliferation.

We also showed for the first time that RANK and OPG expression are independent of PRL and the stage of mammary gland development. Expression of RANKL, RANK, and OPG in epithelial cells suggests that RANKL expression in mammary epithelial cells may be subjected to similar regulation as previously described in bone and the immune system (41). The role of RANKL in cell proliferation and differentiation is widely accepted. In addition RANKL signaling have been implicated in the development of lymph node. Expression of RANKL on epithelial cells has any consequence on bone resorption or immune system is speculative with our present knowledge. We do not expect, that RANKL expression, which acts as a local factor can affect either immune system or bone resorption.

Having established that PRL-induces RANKL expression in vivo and in vitro, we analyzed the RANKL promoter and identified a GAS-like site, which was not previously reported (29). Furthermore, in this study we showed that RANKL transcription is regulated by a Jak2/Stat5 pathway in mammary epithelial cells. Although we showed that PRL induced RANKL promoter-reporter activity in a dose-dependent manner with a maximum induction of 3-fold, in vivo RANKL expression was regulated over a much greater extent. The endogenous promoter of mouse (-2300 bp from the transcription start site, GenBankTM accession number AF332141 [GenBank] ) contains several GAS sites, including two tandem repeats that resemble other promoters whose genes are activated by PRL (42). The promoter used in our studies contained one GAS site (29). This difference in RANKL expression in vivo and in vitro may be due to the fact that the endogenous promoter contains several GAS sites and therefore responded better than our RANKL-Luc reporter to PRL.

Studies with mice deficient in RANKL or its cognate receptor, RANK, support roles for these proteins in alveolar development. However, the molecular mechanisms by which RANK signaling supports alveologenesis are not known. Studies by Feta et al. (20) have demonstrated that RANKL acts as survival factor by activating AKT-mediated pathways. In addition, studies by Kim et al. (25) have shown that RANKL regulates {beta}-casein gene expression independent of the Jak/Stat pathway by inducing the transcription factor CCAAT-enhancer binding protein C/EBP. This pathway has been extensively studied in mammary epithelial cells and lack of this signal prevents proliferation and differentiation of epithelial cells, causing defective alveologenesis (43). Studies by Cao et al. (21) suggested that RANKL induces cell proliferation by activating the NF-kB pathway and targeting cyclin D1 gene, which is essential for G1/S transition during cell cycle (21, 39). Taken together these studies demonstrate that, although PRL and Jak2/Stat pathways are key regulators of mammary gland development, these factors require other signals to maintain alveolar morphogenesis during pregnancy. RANKL acts as a local effector to facilitate PRL action by activating pathways other than Jak2/Stat such as NF-{kappa}B, AKT, and C/EBP.

Recently IGF-2 has been identified as a potential downstream target of PRL. IGF-2 is involved in lobuloalveolar proliferation (22, 44), and ectopic overexpression of IGF-2 in PMEC of PRLR-/- mice led to the development of multiple out-pouchings of the mammary epithelial cells resembling normal alveoli at mid-pregnancy. The facts that IGF-2-/- mice breed normally and feed their pups and do not exhibit an alveolar developmental defect suggest that its function in alveolar morphogenesis is redundant (22). RANKL-/- mice, in contrast, have an intact Jak2/Stat system and showed related mammary developmental defects to those observed in PRL-/- mice. In addition to similar histological defects, the molecular consequences of inactivation of PRLR and RANK pathways are also related (14). Together these observations provide a novel and informative link between the RANKL induction by reproductive hormones P4, PRL, and E2.

In summary, we have shown that PRL induces RANKL expression preferentially via a Jak2/Stat5a pathway by binding to a previously unidentified GAS-like site present in the RANKL promoter.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK-53124 and grants from the Shriners Hospital for Children (to N. D. H.) and National Osteoporosis Foundation grant (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, 231 Albert Sabin Way, Cincinnati, OH 45267-0576. Tel.: 513-558-3019; Fax: 513-558-5738; E-mail: nelson.horseman{at}uc.edu.

1 The abbreviations used are: PRL, prolactin; oPRL, ovine PRL; Jak2, Janice kinase 2; Stat, signal transducers and activators of transcription; OPG, osteoprotegerin; RANK, receptor activator of NF-{kappa}B ligand; RANKL, receptor activator of NF-{kappa}B ligand; IKK{alpha}, inhibitor of {kappa}B kinase {alpha}; ovx, ovariectomy; wt, wild type; PMEC, primary mouse mammary epithelial cell; CHO, Chinese hamster ovary; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; WAP, whey acidic protein; IGF-2, insulin-like growth factor-2; GAS, {gamma} interferon activating sequence. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical support provided by Meenakshi J. Mistry and Kathryn M. Nieport, and Marilyn C. Paolo for her secretarial help.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Horseman, N. D. (1999) J. Mammary Gland Biol. Neoplasia 4, 79-88[CrossRef][Medline] [Order article via Infotrieve]
  2. Shyamala, G. (1999) J. Mammary Gland Biol. Neoplasia 4, 89-104[CrossRef][Medline] [Order article via Infotrieve]
  3. Nandi, S. (1958) J. Natl. Cancer Inst. 21, 1039-1063[Medline] [Order article via Infotrieve]
  4. Topper, Y. J., and Freeman, C. S. (1980) Physiol. Rev. 60, 1049-1106[Free Full Text]
  5. Brisken, C. (2002) J. Mammary Gland Biol. Neoplasia 7, 39-48[CrossRef][Medline] [Order article via Infotrieve]
  6. Neville, M. C., McFadder, T. B., and Forsythe, I. (2002) J. Mammary Gland Biol. Neoplasia 7, 49-66[CrossRef][Medline] [Order article via Infotrieve]
  7. Horseman, N. D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S. J., Smith, F., Markoff, E., and Dorshkind, K. (1997) EMBO J. 16, 6926-6935[CrossRef][Medline] [Order article via Infotrieve]
  8. Kelly, P. A., Bachelot, A., Kedzia, C., Hennighausen, L., Ormandy, C. J., Kopchick, J. J., and Binart, N. (2002) Mol. Cell Endocrinol. 197, 127-131[CrossRef][Medline] [Order article via Infotrieve]
  9. Ormandy, C. J., Binart, N., and Kelly, P. A. (1997) J. Mammary Gland Biol. Neoplasia 2, 355-364[CrossRef][Medline] [Order article via Infotrieve]
  10. Lydon, J. P., DeMayo, F. J., Funk, C. R., Mani, S. K., Hughes, A. R., Montgomery, C. A., Jr., Shyamala, G., Conneely, O. M., and O'Malley, B. W. (1995) Genes Dev. 9, 2266-2278[Abstract/Free Full Text]
  11. Silberstein, G. B., Van Horn, K., Shyamala, G., and Daniel, C. W. (1996) Cell Growth Differ. 7, 945-952[Abstract]
  12. Wakao, H., Gouilleux, F., and Groner, B. (1994) EMBO J. 13, 2182-2191[Medline] [Order article via Infotrieve]
  13. Hennighausen, L., and Robinson, G. W. (1998) Genes Dev. 12, 449-455[Free Full Text]
  14. Hennighausen, L., and Robinson, G. W. (2001) Dev. Cell 1, 467-475[CrossRef][Medline] [Order article via Infotrieve]
  15. Ihle, J. N. (2001) Curr. Opin. Cell Biol. 13, 211-217[CrossRef][Medline] [Order article via Infotrieve]
  16. Watson, C. J. (2001). J Mammary Gland Biol Neoplasia 6, 115-127[CrossRef][Medline] [Order article via Infotrieve]
  17. Khosla, S. (2001) Endocrinology 142, 5050-5055[Abstract/Free Full Text]
  18. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y. X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., and Boyle, W. J. (1998) Cell 93, 165-176[CrossRef][Medline] [Order article via Infotrieve]
  19. Anderson, D. M., Maraskovsky, E., Billingsley, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F., Cosman, D., and Galibert, L. (1997) Nature 390, 175-179[CrossRef][Medline] [Order article via Infotrieve]
  20. Fata, J. E., Kong, Y. Y., Li, J., Sasaki, T., Irie-Sasaki, J., Moorehead, R. A., Elliott, R., Scully, S., Voura, E. B., Lacey, D. L., Boyle, W. J., Khokha, R., and Penninger, J. M. (2000) Cell 103, 41-50[CrossRef][Medline] [Order article via Infotrieve]
  21. Cao, Y., Bonizzi, G., Seagroves, T. N., Greten, F. R., Johnson, R., Schmidt, E. V., and Karin, M. (2001) Cell 107, 763-775[CrossRef][Medline] [Order article via Infotrieve]
  22. Brisken, C., Ayyannan, A., Nguyen, C., Heineman, A., Reinhardt, F., Tan, J., Dey, S. K., Dotto, G. P., Weinberg, R. A., and Jan, T. (2002) Dev. Cell 3, 877-887[CrossRef][Medline] [Order article via Infotrieve]
  23. Matsuda, M. (2003) Endocrine 20, 91-100[CrossRef][Medline] [Order article via Infotrieve]
  24. Imagawa, W., Tomooka, Y., and Nandi, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4074-4077[Abstract/Free Full Text]
  25. Kim, H. J., Yoon, M. J., Lee, J., Penninger, J. M., and Kong, Y. Y. (2000) J. Biol. Chem. 277, 5339-5344
  26. Gao, J., Hughes, J. P., Auperin, B., Buteau, H., Edery, M., Zhuang, H., Wojchowski, D. M., and Horseman, N. D. (1996) Mol. Endocrinol. 10, 847-856[Abstract/Free Full Text]
  27. Hou, Z., Srivastava, S., Mistry, M. J., Herbst, M. P., Bailey, J. P., and Horseman, N. D. (2003) Mol. Endocrinol. 17, 1910-1920[Abstract/Free Full Text]
  28. Srivastava, S., Weitzmann, M. N., Cenci, S., Ross, F. P., Adler, S., and Pacifici, R. (1999) J. Clin. Invest. 104, 503-513[Medline] [Order article via Infotrieve]
  29. Kitazawa, R., Kitazawa, S., and Maeda, S. (1999) Biochim. Biophys. Acta 1445, 134-141[Medline] [Order article via Infotrieve]
  30. Brockman, J. L., Schroeder, M. D., and Schuler, L. A. (2002) Mol. Endocrinol. 16, 774- 784
  31. Fantl, V., Edwards, P. A., Steel, J. H., Vonderhaar, B. K., and Dickson, C. (1999) Dev. Biol. 212, 1-11[CrossRef][Medline] [Order article via Infotrieve]
  32. Liu, X., Robinson, G. W., Wagner, K. U., Garrett, L., Wynshaw-Boris, A., and Hennighausen, L. (1997) Genes Dev. 11, 179-186[Abstract/Free Full Text]
  33. Vomachka, A. J., Pratt, S. L., Lockefeer, J. A., and Horseman, N. D. (2000) Oncogene 19, 1077-1087[CrossRef][Medline] [Order article via Infotrieve]
  34. Brisken, C., Park, S., Vass, T., Lydon, J. P., O'Malley, B. W., and Weinberg, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5076-5081[Abstract/Free Full Text]
  35. Miyoshi, K., Shillingford, J. M., Smith, G. H., Grimm, S. L., Wagner, K. U., Oka, T., Rosen, J. M., Robinson, G. W., and Hennighausen, L. (2001) J. Cell Biol. 155, 531-542[Abstract/Free Full Text]
  36. Brantley, D. M., Chen, C. L., Muraoka, R. S., Bushdid, P. B., Bradberry, J. L., Kittrell, F., Medina, D., Matrisian, L. M., Kerr, L. D., and Yull, F. E. (2001) Mol. Biol. Cell 12, 1445-1455[Abstract/Free Full Text]
  37. Xie, J., LeBaron, M. J., Nevalainen, M. T., and Rui, H. (2002) J. Biol. Chem. 277, 14020-14030[Abstract/Free Full Text]
  38. Clarkson, R. W. E., and Watson, C. J. (1999) J. Mammary Gland Biol. Neoplasia 4, 165-173[CrossRef][Medline] [Order article via Infotrieve]
  39. Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C., and Strauss, M. (1999) Mol. Cell. Biol. 19, 2690-2698[Abstract/Free Full Text]
  40. Hinz, M., Lemke, P., Anagnostopoulos, I., Hacker, C., Krappmann, D., Mathas, S., Dorken, B., Zenke, M., Stein, H., and Scheidereit, C. (2002) J. Exp. Med. 196, 605-617[Abstract/Free Full Text]
  41. Theill, L. E., Boyle, W. J., and Penninger, J. M. (2002) Annu. Rev. Immunol. 20, 795-823[CrossRef][Medline] [Order article via Infotrieve]
  42. Verdier, F., Rabionet, R., Gouilleux, F., Beisenherz-Huss, C., Varlet, P., Muller, O., Mayeux, P., Lacombe, C., Gisselbrecht, S., and Chretien, S. (1998). Mol. Cell. Biol. 18, 5852-5860[Abstract/Free Full Text]
  43. Seagroves, T. N., Krnacik, S., Raught, B., Gay, J., Burgess-Beusse, B., Darlington, G. J., and Rosen, J. M. (1998) Genes Dev. 12, 1917-1928[Abstract/Free Full Text]
  44. Hovey, R. C., Harris, J., Hadsell, D. L., Lee, A. V., Ormandy, C. J., and Vonderhaar, B. K. (2003) Mol. Endocrinol. 17, 460-471[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
E. Jang, S.-H. Cho, H. Park, D.-J. Paik, J. M. Kim, and J. Youn
A Positive Feedback Loop of IL-21 Signaling Provoked by Homeostatic CD4+CD25- T Cell Expansion Is Essential for the Development of Arthritis in Autoimmune K/BxN Mice
J. Immunol., April 15, 2009; 182(8): 4649 - 4656.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. D. Aupperlee, A. A. Drolet, S. Durairaj, W. Wang, R. C. Schwartz, and S. Z. Haslam
Strain-Specific Differences in the Mechanisms of Progesterone Regulation of Murine Mammary Gland Development
Endocrinology, March 1, 2009; 150(3): 1485 - 1494.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
J. E Morabito, J. F Trott, D. M Korz, H. E Fairfield, S. H Buck, and R. C Hovey
A 5' distal palindrome within the mouse mammary tumor virus-long terminal repeat recruits a mammary gland-specific complex and is required for a synergistic response to progesterone plus prolactin
J. Mol. Endocrinol., August 1, 2008; 41(2): 75 - 90.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. J. Santos, S. Z. Haslam, and S. E. Conrad
Estrogen and Progesterone are Critical Regulators of Stat5a Expression in the Mouse Mammary Gland
Endocrinology, January 1, 2008; 149(1): 329 - 338.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
K. C. Horner, Y. Cazals, R. Guieu, M. Lenoir, and N. Sauze
Experimental estrogen-induced hyperprolactinemia results in bone-related hearing loss in the guinea pig
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1224 - E1232.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. M. Gharavi, J. A. Alva, K. P. Mouillesseaux, C. Lai, M. Yeh, W. Yeung, J. Johnson, W. L. Szeto, L. Hong, M. Fishbein, et al.
Role of the JAK/STAT Pathway in the Regulation of Interleukin-8 Transcription by Oxidized Phospholipids in Vitro and in Atherosclerosis in Vivo
J. Biol. Chem., October 26, 2007; 282(43): 31460 - 31468.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
E. Gonzalez-Suarez, D. Branstetter, A. Armstrong, H. Dinh, H. Blumberg, and W. C. Dougall
RANK Overexpression in Transgenic Mice with Mouse Mammary Tumor Virus Promoter-Controlled RANK Increases Proliferation and Impairs Alveolar Differentiation in the Mammary Epithelia and Disrupts Lumen Formation in Cultured Epithelial Acini
Mol. Cell. Biol., February 15, 2007; 27(4): 1442 - 1454.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
Q. Fu, S. C. Manolagas, and C. A. O'Brien
Parathyroid Hormone Controls Receptor Activator of NF-{kappa}B Ligand Gene Expression via a Distant Transcriptional Enhancer.
Mol. Cell. Biol., September 1, 2006; 26(17): 6453 - 6468.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
F. Zhang, Q. Zhang, A. Tengholm, and A. Sjoholm
Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion
Am J Physiol Cell Physiol, September 1, 2006; 291(3): C466 - C475.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
E. M. Johansson, M. Kannius-Janson, A. Gritli-Linde, G. Bjursell, and J. Nilsson
Nuclear Factor 1-C2 Is Regulated by Prolactin and Shows a Distinct Expression Pattern in the Mouse Mammary Epithelial Cells during Development
Mol. Endocrinol., April 1, 2005; 19(4): 992 - 1003.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/46/46171    most recent
M308545200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Srivastava, S.
Right arrow Articles by Horseman, N. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srivastava, S.
Right arrow Articles by Horseman, N. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement