The Wnt Receptor, Lrp5, Is Expressed by Mouse Mammary Stem Cells and Is Required to Maintain the Basal Lineage Nisha M. Badders1, Shruti Goel1, Rod J. Clark1, Kristine S. Klos1, Soyoung Kim1, Anna Bafico2, Charlotta Lindvall3, Bart O. Williams 3 , Caroline M. Alexander1* 1 McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 2 Department of Pathology, Cardiff University, Cardiff, Wales, United Kingdom, 3 Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan, United States of America Abstract Background: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. Methodology/Principal Findings: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by .200 fold (over 80% of activity), based on high Lrp5 expression alone. Though Lrp5 glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22%) and Lrp52/2 mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16Ink4a and TA-p63. Conclusions/Significance: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function. Citation: Badders NM, Goel S, Clark RJ, Klos KS, Kim S, et al. (2009) The Wnt Receptor, Lrp5, Is Expressed by Mouse Mammary Stem Cells and Is Required to Maintain the Basal Lineage. PLoS ONE 4(8): e6594. doi:10.1371/journal.pone.0006594 Editor: Joseph Najbauer, City of Hope Medical Center, United States of America Received April 20, 2009; Accepted July 9, 2009; Published August 12, 2009 Copyright: 2009 Badders et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Era of Hope Scholars award to CMA (W81XWH-06-1-0491) and a post-doctoral award from the American Cancer Society to NMB (PF-07-294-01-CNE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexander@oncology.wisc.edu Introduction The Wnt signaling pathway is required for normal development, but when ectopically expressed, is highly oncogenic for human epithelia [1]. Wnt signaling is used at many different developmental stages, as an effector of pathways involved in processes as distinct as planar cell polarity, neuronal axon guidance and the activation and regulation of somatic epithelial stem/progenitor cell compartments [2,3,4,5]. This latter function appears to be key to the oncogenic role of Wnt signaling. In gut, the mutation of key tumor suppressor molecules in the Wnt signaling pathway leads to amplification of stem/progenitor compartments, followed by the appearance of differentiated adenomas and tumors [6,7,8]. Our previous data has shown that gain of function of Wnt signaling in mammary glands also induces an increase in the stem/progenitor cell activity in the preneoplastic condition [9]. In order to understand the normal function of Wnt signaling in mammary glands, we chose to study how loss of function of Wnt signaling affected mammary development. There are many Wnt-dependent signaling events, but only one pathway has so far been associated with the stem cell functions and oncogenic properties. This so-called canonical pathway is mediated by the interaction of Wnt ligands with a pair of cell surface receptors, comprising a Frizzled receptor and an Lrp5 or - 6 receptor [10,11]. Binding of the Wnt ligand to the Frizzled and Lrp5/6 receptor is followed by the recruitment of axin from the b- catenin destruction complex, stabilization of b-catenin and, transactivation of specific target genes via a b-catenin/TCF complex [12]. There are many members of the Wnt family of secreted lipoglycoprotein ligands [13], and several (Wnt-2,-4,-5a,-5b,6,7b) are expressed during mammary gland development [14,15,16,17,18]. Similarly, there are 10 known Frizzled homo- logues, of which Frizzled 1–8 are known to be expressed in mammary epithelial cells [19]. However, there is an absolute requirement for either Lrp5 or Lrp6 for canonical Wnt signaling [20]. Lrp5 and -6 belong to the LDL receptor related protein family of single-span transmembrane receptors, which mediate binding and internalization of various lipoprotein particles [21]. Current studies have not addressed whether Lrp5 and Lrp6 have distinct molecular properties. Ablation of Lrp5 and Lrp6 produce entirely different phenotypes in mice. Lrp6 expression PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6594