an autocrine Wnt signaling loop (Bafico et al 2004). Recently, a splice variant of Lrp5, DLrp5, was found in the majority (85%) of breast tumors, and was required for their continued growth. The deletion of the variant exons by splicing was associated with resistance to inhibition by Dkk1 [41]. These data suggest that ectopic Wnt signaling could be an important source of growth dysregulation in breast tumors. These tumors are not all basaloid, they include tumors of many classes, which suggests that the Lrp5- dependent growth pathway could become viable in many candidate tumor precursor cell types. Lrp5 and -6 are co-expressed in the majority of basal cells. Lrp6 co-expression is high enough that the absence of Lrp5 has no effect on Wnt transactivation in response to Wnt3A (with no gross transcriptional compensation of Lrp6 mRNA). However, there is a very specific effect of the loss of Lrp5 on the maintenance of adult somatic stem cell activity. This corresponds to the lack of tumorigenicity of Wnt1 in Lrp52/2 mammary glands (Lindvall et al, 2006). This could be explained by the following scenarios - 1) Lrp5 has a distinct function in mammary stem cell biology compared to Lrp6, 2) Lrp5 expression augments Lrp6 expression to push the total expression over a critical threshold for growth promotion, 3) both Lrp5 and -6 are required for the stem cell function, 4) the ligand for Lrp5/Fzd is not Wnt1/Wnt3A (whereas Wnt3A binds and stimulates Lrp6/Fzd), or 4) Lrp5 has a different subcellular presentation from Lrp6. Often, Lrp5/6 are used in experiments interchangeably, since they exhibit high sequence homology. Though they show similar expression patterns in embryonic and adult tissues [42,43,44], they have distinct functions. For example, an allelic series of mutations in Lrp5 and 6 in mouse embryos revealed that the Lrp5 loss in combination with Lrp6 loss produces a more severe phenotype than Lrp6 loss alone, and that Lrp6 loss is more severe than Lrp5 loss alone [22,45]. The loss of Lrp5 tends to produce a subset of the phenotypes typical of Lrp6 mice. One possibility is relative ligand specificity, and this may be influenced by the presence of LDLR repeat sequences in the extracellular domains. Although the extracellular domains are highly conserved between the two receptors (73%), the LDLR repeats demonstrate significantly lower sequence homology [44]. In addition, the extraceullar domain of Lrp6 contains 10 putative glycosylation sites, whereas Lrp5 only contains 5, which may also play a role in directing ligand specificity. Another possible mechanism leading to the divergent functions of Lrp5 and 6 may be cell surface presentation and intracellular processing of the receptors. Internalization of Lrp6 has been found to be regulated by clathrin, caveolin, ligand interaction (including R- spondin) and c-secretase-dependent mechanisms [46,47,48,49,50], indicating Lrp6 (and possibly Lrp5) activity can be regulated by different internalization patterns. Although the internalization mechanism of Lrp5 is unknown, it likely differs from that of Lrp6, since the intracellular domains are much less conserved [44]. It is likely that Wnt ligands have specific cognate Frizzled receptors. This specificity has been demonstrated for Wnt5A, which can induce canonical signaling only when Fzd4 and Lrp5 (not Lrp6) are present [51]. Wnt5A is known to be expressed and bioactive during mouse mammary gland development; a gain of function inhibits ductal outgrowth, and loss promotes hyperpro- liferation [52]. Most Fzd mRNAs are expressed in virgin ductal mammary glands, except for Fzd4. If Fzd4 is expressed, it is a very low level, or in a very small subpopulation (unpublished data, Y C Kim and C M Alexander). The overall output of canonical and other responses depends upon the relative amount of receptors and ligands. Non-canonical and canonical pathways can inhibit each other, and even non- Lrp5 in Mammary Stem Cells productive interactions (for example Wnt5A:Lrp6) can compete with normal signaling activities (for example, activation of the non- canonical target Rac1) [53,54]. It is possible that Lrp5 binds Wnt5A during normal development (with no canonical signaling effect), but the absence of Lrp5 enhances the inhibitory effects of Wnt5A on ductal outgrowth (and more specifically, stem cell self- renewal). Although the embryonic outgrowth of mammary rudiments is Wnt-dependent [15,16], the phenotype of early mammary development of Lrp52/2 mice is only marginally affected. This suggests that only the adult somatic ductal stem cells require Lrp5, for either 1) stem cell specification during tree outgrowth, or 2) survival in adult glands. Prior modeling has predicted that if most cells in the mammary rudiment have at least 30 division cycles, there is a reserve of growth potential [55]. In fact, the challenge of development appears to be to control and attenuate the growth potential to enable functional differentiation. In other words, there is no need to invoke stem cells to explain the growth associated with ductal outgrowth, estrus cycling, or pregnancy. The Lrp5 mouse is an example of this, as is the b1 integrin mouse [56,57,58]. Both these strains show approximately normal ductal extension, but neither ductal tree has significant regenerative capacity. Yet, we report that the stem cell-deficient gland is affected in a predictable way. When mammary epithelial cell populations are transferred to culture, there is increased expression of senescence-associated markers, such as p16Ink4a and TAp63. (Our data suggests that senescence markers are transient in vivo, unless glands are actively growing; presumably cells are removed by apoptosis or autophagy). By separating the luminal and basal cells for independent culture, we show that the effect of the Lrp5 mutation is evident not only in the basal cell population (where we would anticipate the effect of this mutation), but also in the luminal cells. We propose that this is consistent with the stem cell origin of this effect. Cellular senescence is described as a natural mechanism of tumor suppression [59]. The mechanism of several tumor suppressors has been demonstrated to be the induction of senescence or apoptosis. More specifically, it has been proposed that tumor suppressors may act by reducing the stem/progenitor cell pool, since overexpression often leads to a reduction in the regenerative capacity of a tissue [59]. For example, the tumor suppressor, p16Ink4a, is thought to act this way. It is deleted or inactivated in numerous tumors, whereas overexpression results in senescence and an aged phenotype [60]. Indeed, ectopic p16Ink4a expression has been shown to deplete stem cell activity in a number of tissues (brain, pancreas, and hematopoetic system) [61,62]. Similar to p16Ink4a, p53, is also a widely recognized tumor suppressor, where loss of function mutations are associated with tumorigenesis and gain of function mutations result in aging and senescence [59]. p63 is a closely related family member to p53, yet very little is known about the function of this protein. It has been shown to be required for mammary gland development [63] and is frequently up-regulated (although, not mutated) in several epithelial cancers [38]. The TAp63 isoform has been shown to be pro-apoptotic [64] and can bind to p53 response elements, driving transcription of p53 target genes. The DNp63 isoform, however, acts as a dominant-negative competitor for TAp63 and p53 [65]. The DNp63 isoform is expressed at higher levels than TAp63 during development and at lower levels during differen- tiation [66,67]. Consequently, it has been suggested that the ratio of DNp63 to TAp63 isoform expression may dictate whether a cell follows its normal differentiation program, becomes senescent, or undergoes oncogenic transformation [38]. It is, therefore, not PLoS ONE | www.plosone.org 10 August 2009 | Volume 4 | Issue 8 | e6594