[Expression of amphiregulin in different types of nasal polyps and its correlation with tissue remodeling].

Objective: To explore the expression of amphiregulin (AREG) in nasal polyps patients with different degrees of eosinophil infiltration, and to analyze the correlation between AREG and tissue remodeling. Methods: Forty-eight patients underwent endoscopic sinus surgery in the Department of Otorhinolaryngology Head and Neck Surgery, Remin Hospital, Wuhan University from July 2017 to August 2018 were recruited, including 40 males and 8 females, aged from 16 to 60 years old. The subjects were divided into three groups: control group (n=14), eosinophilic chronic sinusitis with nasal polyps (ECRSwNP) group (n=19) and noneosinophilic chronic rhinosinusitis with nasal polyps (non-ECRSwNP) group (n=15). The relative expression of AREG in nasal mucosa was detected by Western blot assay and immunohistochemical staining. Tissue remodeling was detected by HE staining, AB-PAS staining and Masson staining. Kruskal-Wallis test was used for comparison among multiple groups, and Spearman correlation analysis was conducted between the expression level of AREG and the related indexes of tissue remodeling. Results: The expression of AREG in ECRSwNP group was significantly higher than that in non-ECRSwNP group and control group (median protein expression of Western blot was 1.592 vs 0.617 vs0.582, all P<0.05). The degree of epithelial injury and goblet cell metaplasia in ECRSwNP group was significantly higher than that in control group (all P<0.05), the percentage of collagen fibrosis area in ECRSwNP group was significantly lower than that in control group (P=0.01). In chronic rhinosinusitis with nasal polyps (CRSwNP) patients, the area of mucous glands was negatively correlated with the expression of AREG (r=-0.616, P<0.05), and the percentage of collagen fibrosis area was negatively correlated with the expression of AREG (r=-0.738, P<0.05). Conclusion: The expression of AREG is higher in ECRSwNP patients, which is related to the process of tissue remodeling.

Prognostic role of Amphiregulin and the correlation with androgen receptor in invasive breast cancer.

BACKGROUND: In androgen-sensitive prostate cancer, androgenic stimulation induces the synthesis of amphiregulin (AREG). Research is lacking on the role of AREG in invasive breast cancer and the co-expression with androgen receptor (AR) status. MATERIALS AND METHODS: The present study investigated the prognostic role of AREG in invasive breast cancer cases (N = 298) and the co-expression with the AR status as analysed by immunohistochemistry (IHC). RESULTS: The samples were divided into groups according to AREG expression levels: low/no expression (AREGlow/no) and high expression (AREGhigh). As shown by cytoplasmic immunostaining, 46.0% (137/298) of invasive breast cancers were AREGhigh, and 54.0% (161/298) of cases were AREGlow/no. Co-expression of the AR and AREG accounted for 62.4% (186/298) of cases. A Kaplan-Meier analysis revealed that AREGhigh and AR+/AREGhigh decreased patients' overall survival (OS) (P = 0.002 and P = 0.006, respectively) and disease-free survival (DFS) (P < 0.001 and P < 0.001, respectively). In Cox models, AR+/AREGhigh remained an independent prognostic indicator of OS and DFS in invasive breast cancer (hazard ratio [HR], 0.591, 95% confidence interval [CI], 0.407-0.859, P = 0.006; HR, 0.449, 95% CI, 0.236-0.853, P = 0.014, respectively). AREGhigh remained an independent prognostic indicator of OS and DFS in estrogen receptor (ER)-negative tumours (P < 0.05). CONCLUSIONS: This study suggested that AREG and the AR were co-expressed in invasive breast cancer. Thus, AREG and the AR may be valuable prognostic biomarkers in invasive breast cancer and promising therapeutic targets, especially in ER-negative breast cancer.

The immunopathology of lung fibrosis: amphiregulin-producing pathogenic memory T helper-2 cells control the airway fibrotic responses by inducing eosinophils to secrete osteopontin.

Fibrosis is defined as excessive deposition of the extracellular matrix (ECM) in the parenchyma of various organs, and sometimes leads to irreversible organ malfunction such as idiopathic pulmonary fibrosis (IPF), a fatal disorder of the lung. Chronic inflammatory stimuli induce fibrotic responses in various organs. Various immune cells, including T helper (Th) cells in the lung, protect the host from different harmful particles, including pathogenic microorganisms. However, the dysregulation of the function of these immune cells in the lung sometimes causes inflammatory diseases, such as lung fibrosis. In this review, we will introduce an outline of the cellular and molecular mechanisms underlying the pathogenic fibrotic responses in the lung. We will also introduce the concept of the "Pathogenic Th population disease induction model," in which unique subpopulations of certain Th cell subsets control the pathology of immune-mediated inflammatory diseases. Finally, we introduce our recent findings, which demonstrate that amphiregulin-producing pathogenic memory Th2 cells control airway fibrosis through the osteopontin produced by inflammatory eosinophils. The identification of this new pathogenic Th cell population supports the concept of "Pathogenic Th population disease induction model", and will provide novel strategies for treating intractable diseases, including lung fibrosis.

Epigenetic regulation of Amphiregulin and Epiregulin in colorectal cancer.

Expression of the epidermal growth factor ligands amphiregulin (AREG) and epiregulin (EREG) is positively correlated with a response to EGFR-targeted therapies in colorectal cancer. Gene-body methylation sites, which show a strong inverse correlation with AREG and EREG gene expression, were identified in cell lines using targeted 454 FLX-bisulfite sequencing and SIRPH analyses for AREG/EREG promoters and intragenic CpGs. Upon treatment of colorectal cancer cells with 5-aza-2'-desoxycytidine, methylation decreases at specific intragenic CpGs accompanied by upregulation of AREG and EREG gene expression. The same AREG gene-body methylation was also found in human colorectal cancer samples and is independent of KRAS and NRAS mutations. Methylation is specifically decreased in the tumor epithelial compartment as compared to stromal tissue and normal epithelium. Investigation of a promoter/enhancer function of the AREG exon 2 region revealed a potential promoter function in reverse orientation. Retrospective comparison of the predictive power of AREG gene-body methylation versus AREG gene expression using samples from colorectal cancer patients treated with anti-EGFR inhibitors with complete clinical follow-up revealed that AREG expression is superior to AREG gene methylation. AREG and EREG genes undergo a complex regulation involving both intragenic methylation and promoter-dependent control.

Triiodothyronine Promotes Cell Proliferation of Breast Cancer via Modulating miR-204/Amphiregulin.

Breast cancer (BC) severely threatens women's life, and Triiodothyronine (T3) shows a positive role on BC cell proliferation, while the potential mechanism underlying it is still unclear. T3 was used to stimulate BC cell lines MCF-7 and T47-D. Real-time PCR was performed to determine the expression of miRNAs, while western blot was used to measure protein expression of Amphiregulin (AREG), AKT and p-AKT. The interaction between miR-204 and AREG was determined using luciferase reporter assay. MTT was performed to detect cell viability. The expression of miR-204 was decreased, while AREG and p-AKT was increased in T3 stimulated BC cell lines. T3 stimulation promoted cell viability. miR-204 targets AREG to regulate its expression. T3 promoted expression of AREG and p-AKT, while miR-204 overexpression reversed the effect of T3, however, pcDNA-AREG transfection abolished the effect of miR-204 mimic. T3 promoted cell viability of BC cells via modulating the AKT signaling pathway. The detailed mechanism was that the down-regulated miR-204 that induced by T3 stimulation promoted the expression of AREG, the up-regulated AREG activated AKT signaling pathway, while the activated AKT signaling promoted cell proliferation.

Amphiregulin in intestinal acute graft-versus-host disease: a possible diagnostic and prognostic aid.

Amphiregulin, a weak epidermal growth factor receptor agonist, is elevated, while epidermal growth factor, a strong epidermal growth factor receptor agonist, is low in the blood of patients with severe acute graft-versus-host disease. However, the tissue expression and function of these epidermal growth factor receptor ligands in acute graft-versus-host disease target organs is unknown. We compared by immunohistochemistry expression of amphiregulin and epidermal growth factor in archived, formalin-fixed, paraffin-embedded intestinal tissues of 48 patients with biopsy-proven gastrointestinal acute graft-versus-host disease to 3 groups: (1) 10 non-hematopoietic cell transplant normal controls, (2) 11 patients with newly diagnosed ulcerative colitis (ulcerative colitis), (3) 8 patients with a clinical diagnosis of acute graft-versus-host disease despite pathologically non-diagnostic biopsies, (4) and 10 cases of cytomegalovirus colitis. We used a semi-quantitative score of 0 (absent) through 3 (strong) to describe the intensity of immunohistochemical staining. We correlated serum and tissue amphiregulin and epidermal growth factor in patients with acute graft-versus-host disease. Gastrointestinal amphiregulin was significantly lower in acute graft-versus-host disease biopsies (median score 1), ulcerative colitis (median score 1.5), and cytomegalovirus colitis (median score 1) than in normal colon (median score 2, p = 0.004, p = 0.03, p = 0.009 respectively). Amphiregulin expression in was low in 74% of acute graft-versus-host disease cases with or without significant apoptosis. Patients with acute graft-versus-host disease exhibiting the pattern of high gastrointestinal amphiregulin but low serum amphiregulin (n = 14) had the best 1-year survival at 71%, but patients with high serum amphiregulin had poorer survival (<30%) regardless of gastrointestinal amphiregulin expression. Overall, our results lead to the hypothesis that amphiregulin is released into the circulation from damaged intestinal epithelium and stroma, although contributions from other cellular sources are likely. Low gastrointestinal amphiregulin expression by immunohistochemistry may be further studied for its utility in the pathologic acute graft-versus-host disease diagnosis without classic apoptotic changes.

CRB3 and the FERM protein EPB41L4B regulate proliferation of mammary epithelial cells through the release of amphiregulin.

Numerous observations have suggested a connection between the maintenance of cell polarity and control of cell proliferation; however, the mechanisms underlying these connections remain poorly understood. Here we found that ectopic expression of CRB3, which was previously shown to restore tight junctions and membrane polarity in MCF-10A cells, induced a hyperproliferative phenotype, with significantly enlarged acini in basement membrane culture, similar to structures induced by expression of proliferative oncogenes such as cyclinD1. We found that CRB3-induced proliferation is epidermal growth factor (EGF)-independent and occurs through a mechanism that involves secretion of the EGF-family ligand, amphiregulin (AREG). The increase in AREG secretion is associated with an increase in the number and size of both early and late endosomes. Both the proliferative and endocytic phenotypes associated with CRB3 expression require the FERM-binding domain (FBD) but not the PDZ-binding domain of CRB3, arguing that this proliferative phenotype is independent of the PDZ-dependent polarity signaling by CRB3. We identified the FBD-containing protein, EPB41L4B, as an essential mediator of CRB3-driven proliferation and observed that the CRB3-dependent changes in endocytic trafficking were also dependent on EPB41L4B. Taken together, these data reveal a previously uncharacterized role for CRB3 in regulating proliferation in mammalian cells that is associated with changes in the endocytic trafficking machinery.

Amphiregulin-Producing Pathogenic Memory T Helper 2 Cells Instruct Eosinophils to Secrete Osteopontin and Facilitate Airway Fibrosis.

Memory T cells provide long-lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin-33 (IL-33) enhanced amphiregulin production by the IL-33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin-epidermal growth factor receptor (EGFR)-mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL-5-producing memory Th2 cells and amphiregulin-producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin-producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin-producing eosinophils. Thus, the IL-33-amphiregulin-osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders.

Cutting Edge: Low-Affinity TCRs Support Regulatory T Cell Function in Autoimmunity.

Regulatory T cells (Tregs) use a distinct TCR repertoire and are more self-reactive compared with conventional T cells. However, the extent to which TCR affinity regulates the function of self-reactive Tregs is largely unknown. In this study, we used a two-TCR model to assess the role of TCR affinity in Treg function during autoimmunity. We observed that high- and low-affinity Tregs were recruited to the pancreas and contributed to protection from autoimmune diabetes. Interestingly, high-affinity cells preferentially upregulated the TCR-dependent Treg functional mediators IL-10, TIGIT, GITR, and CTLA4, whereas low-affinity cells displayed increased transcripts for Areg and Ebi3, suggesting distinct functional profiles. The results of this study suggest mechanistically distinct and potentially nonredundant roles for high- and low-affinity Tregs in controlling autoimmunity.

Development of an in vitro model to study uterine functions and early implantation using rat uterine explants.

This study was conducted to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2 mm) were isolated, cultured and further characterized. Steroid hormone treatment of cultured explants showed that both Muc1 and Pr were significantly up-regulated (P < 0.05) by E2. Areg was significantly up-regulated (P < 0.05) by P4 and Igfbp1 was significantly up-regulated (P < 0.05) by the combination of E2 and P4, although, in rat, Igfbp1 is E2-dependent. In vitro decidualization of cultured explants was induced and two potential markers of decidualization, Prl8a2 and Bmp2, were examined. Real-time quantitative PCR data revealed that both Prl8a2 and Bmp2 were significantly up-regulated (P < 0.05) in MPA- and db-cAMP-treated explants compared to the control group of explants. Then, an individual hatched blastocyst and cultured explant was placed in a 96-well (round-bottom U-shaped) plate. Co-culture results showed that stable attachments were observed after 48 h, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. The rates of attachment of embryos to the explants were increased significantly in the P4-treated group (63.6%) compared to the control group (35.5%), after steroid hormone treatment. The rates of attachment were reduced significantly in the E2-treated group (0.0%), where no stable attachments were observed. Despite the necessity of comprehensive investigation, our results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation.

YAP, TAZ and AREG expression in eighth cranial nerve schwannoma.

BACKGROUND: Although the diagnosis and treatment of eighth cranial nerve (VIII CN) schwannoma (acoustic neuroma) has improved over the years, no factors capable of predicting tumor growth have been identified as yet. This study is a preliminary investigation of the expression in sporadic VIII CN schwannomas of Yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), and amphiregulin (AREG), a direct target gene of YAP and TAZ. The expression of YAP, TAZ and AREG was correlated with the volumetric dimensions of tumors on contrast-enhanced magnetic resonance imaging (ceMRI). METHODS: YAP, TAZ and AREG expression was assessed immunohistochemically in surgical specimens of 36 consecutive sporadic VIII CN schwannomas. 3D reconstructions of the tumors and their corresponding volumes in cm3 were obtained from measurements on ceMRI images using the OsiriX® software. RESULTS: We found a significant direct correlation between TAZ expression and VIII CN schwannoma volumes on latest preoperative ceMRI (p<0.0003). Mean TAZ expression was also significantly higher in VIII CN schwannomas with a volume ≥2.1 cm3 than in those with a volume <2.1 cm3(p<0.0018). No significant correlations emerged for YAP or AREG expression and VIII CN schwannoma volume. CONCLUSIONS: The immunohistochemical expression of TAZ (but not YAP or AREG) correlated significantly with schwannoma volume measured on ceMRI. Further investigations are needed to identify the biological factors influencing tumor proliferation (ideally secreted proteins like AREG) that might be detected using non-invasive approaches (i.e., blood samples).

Amphiregulin impairs apoptosis-stimulating protein 2 of p53 overexpression-induced apoptosis in hepatoma cells.

Overexpression of apoptosis-stimulating protein 2 of p53 (ASPP2) induces apoptotic cell death in hepatoma cells (e.g. HepG2 cells) by enhancing the transactivation activity of p53, but long-term ASPP2 overexpression fails to induce more apoptosis since activation of the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway impairs the pro-apoptotic role of ASPP2. In this study, in recombinant adenovirus-ASPP2-infected HepG2 cells, ASPP2 overexpression induces amphiregulin expression in a p53-dependent manner. Although amphiregulin initially contributes to ASPP2-induced apoptosis, it eventually impairs the pro-apoptotic function of ASPP2 by activating the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway, leading to apoptosis resistance. Moreover, blocking soluble amphiregulin with a neutralizing antibody also significantly increased apoptotic cell death of HepG2 cells due to treatment with methyl methanesulfonate, cisplatin, or a recombinant p53 adenovirus, suggesting that the function of amphiregulin involved in inhibiting apoptosis may be a common mechanism by which hepatoma cells escape from stimulus-induced apoptosis. Thus, our data elucidate an apoptosis-evasion mechanism in hepatocellular carcinoma and have potential implications for hepatocellular carcinoma therapy.

Foxp3+ Regulatory T Cell Expression of Keratinocyte Growth Factor Enhances Lung Epithelial Proliferation.

Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3+ regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.

An early innate response underlies severe influenza-induced exacerbations of asthma in a novel steroid-insensitive and anti-IL-5-responsive mouse model.

BACKGROUND: Acute worsening of asthma symptoms (exacerbation) is predominantly triggered by respiratory viruses, with influenza causing the most severe exacerbations. The lack of an adequate animal model hampers mechanistic insight and the development of new therapeutics. AIM: We developed and characterized a robust, consistent, and reproducible mouse model of severe exacerbation of chronic allergic asthma. METHODS: Chronic allergic airway inflammation was induced following a house dust mite (HDM) sensitization protocol. HDM-sensitized mice and controls were infected with influenza virus A/X31 H3N2 and either or not treated with inhaled fluticasone propionate (FP), systemic corticosteroids (Pred), or anti-IL-5. Mice were killed at different time points after infection: Cellular accumulation and cytokines levels in the airways, PenH as a measure of airway hyper-responsiveness (AHR), and lung histology and viral replication were assessed. RESULTS: Infection with low-dose A/X31 H3N2 led to prolonged deterioration of lung function, aggravated mucus production, peri-vascular, peri-bronchial, and allergic inflammation that was unresponsive to inhaled corticosteroids, but responsive to systemic corticosteroids. The exacerbation was preceded at 14 h after virus exposure by a marked innate, but no Th2 and Th1 response subsequently followed by enhanced numbers of eosinophils, neutrophils, dendritic, and T cells into the lung lumen, parenchyma, and draining lymph nodes in HDM-sensitized mice. Anti-IL-5 treatment attenuated eosinophils and prevented the X31-induced exacerbation. CONCLUSIONS: Together, these findings indicate that an early innate response that involves eosinophils underlies the exacerbation. This model recapitulates all major features of severe asthma exacerbations and can serve to discern driving mechanisms and promote the development of novel therapeutics.

Association of CpG island methylator phenotype and EREG/AREG methylation and expression in colorectal cancer.

BACKGROUND: High EREG and AREG expression, and left-sided primary tumours are associated with superior efficacy of anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (CRC), but a unifying explanation of these findings is lacking. METHODS: RNA-seq, gene expression arrays, and DNA methylation profiling were completed on 179 CRC tumours. Results were validated using independent The Cancer Genome Atlas data sets. An independent cohort of 198 KRAS wild-type metastatic CRC tumours was tested for CpG island methylator phenotype (CIMP) status, and progression-free survival (PFS) with the first anti-EGFR regimen was retrospectively determined. RESULTS: EREG and AREG expression was highly inversely correlated with methylation and was inversely associated with right-sided primary tumour, BRAF mutation, and CIMP-high status. Treatment of CRC cell lines with hypomethylating agents decreased methylation and increased expression of EREG. Inferior PFS with anti-EGFR therapy was associated with CIMP-high status, BRAF mutation, NRAS mutation, and right-sided primary tumour on univariate analysis. Among known BRAF/NRAS wild-type tumours, inferior PFS remained associated with CIMP-high status (median PFS 5.6 vs 9.0 mo, P=0.023). CONCLUSIONS: EREG and AREG are strongly regulated by methylation, and their expression is associated with CIMP status and primary tumour site, which may explain the association of primary tumour site and EREG/AREG expression with anti-EGFR therapy efficacy.

Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice.

The epidermal growth factor receptor ligand amphiregulin (AREG) has been implicated in bone physiology and in bone anabolism mediated by intermittent parathyroid hormone treatment. However, the functions of AREG in bone have been only incipiently evaluated in vivo. Here, we generated transgenic mice overexpressing AREG specifically in osteoblasts (Col1-Areg). pQCT analysis of the femoral metaphysis revealed increased trabecular bone mass at 4, 8, and 10weeks of age in Col1-Areg mice compared to control littermates. However, the high bone mass phenotype was transient and disappeared in older animals. Micro-CT analysis of the secondary spongiosa confirmed increased trabecular bone volume and trabecular number in the distal femur of 4-week-old AREG-tg mice compared to control littermates. Furthermore, µ-CT analysis of the primary spongiosa revealed unaltered production of new bone trabeculae in distal femora of Col1-Areg mice. Histomorphometric analysis revealed a reduced number of osteoclasts in 4-week-old Col1-Areg mice, but not at later time points. Cancellous bone formation rate remained unchanged in Col1-Areg mice at all time points. In addition, bone mass and bone turnover in lumbar vertebral bodies were similar in Col1-Areg and control mice at all ages examined. Proliferation and differentiation of osteoblasts isolated from neonatal calvariae did not differ between Col1-Areg and control mice. Taken together, these data suggest that AREG overexpression in osteoblasts induces a transient high bone mass phenotype in the trabecular compartment of the appendicular skeleton by a growth-related, non-cell autonomous mechanism, leading to a positive bone balance with unchanged bone formation and lowered bone resorption.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty.

INTRODUCTION: Puberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined. METHODS: Pubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL). RESULTS: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression. CONCLUSIONS: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.