Faculty Bibliography

Hair follicle morphogenesis and cycling were examined in transgenic mice that overexpress the bone morphogenetic protein (BMP) inhibitor Noggin under the control of the neuron-specific enolase promoter. The Noggin transgene was misexpressed in the proximal portion of the hair follicle, primarily the matrix cells, apart from the usual expression in neurons. Transgene expression appeared only after induction of both the primary (tylotrich) and secondary (nontylotrich) pelage hair follicles had already occurred, thus allowing examination of the role of BMP signaling in follicles that had been induced normally in the presence of BMPs. The overexpression of Noggin in these animals resulted in a dramatic loss of hair postnatally. There was an apparently normal, but shortened period of postnatal hair follicle morphogenesis, followed by premature initiation of hair follicle cycling via entry into the first catagen transformation. This resulted in a complete loss of hair shafts from the nontylotrich hair follicles in these mice while the tylotrich hair follicles were normal. The onset of anagen of the first postnatal hair follicle cycle was also accelerated in the transgenic mice. Our results show that BMP signaling is specifically required for proper proliferation and differentiation during late morphogenesis of nontylotrich hair follicles and that inhibition of this signaling pathway may be one of the triggers for the onset of catagen when the follicles are in anagen and the onset of anagen when the follicles are in telogen. Ectopic sebocyte differentiation was another hallmark of the phenotype of these transgenic mice suggesting that BMP signaling may be an important determinant of lineage selection by common progenitor cells in the skin. BMPs likely promote a hair follicle-type differentiation pathway of keratinocytes while suppressing the sebaceous differentiation pathway of skin epithelium

Beta-catenin and cyclin D1 have attracted considerable attention due to their proto-oncogenic roles in human cancer. The finding of cyclin D1 as a direct target gene of beta-catenin in colon cancer cells led to the assumption that cyclin D1 upregulation is pivotal to beta-catenin's oncogenicity. Our recent paper shows that this is not the case; cyclin D1 dampens the oncogenicity of activated beta-catenin (MMTV-DN89beta-catenin). The relationships and dependencies of beta-catenin and cyclin D1 point to distinct, essential and sequential roles during alveologenesis. These results support the concept that both beta-catenin's and cyclin D1's actions are more sophisticated than simple acceleration of the cell cycle clock. These proteins are employed at critical junctures involving cell fate decisions that we speculate require specific types of cell cycle to traverse

Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast

Mice with a null mutation in the cell surface heparan sulfate (HS) proteoglycan, syndecan-1 (Sdc1), develop almost normally, but resist mammary tumor development in response to Wnt-1. Here, we test the hypothesis that Sdc1 promotes Wnt-1-induced tumor development by interacting with the Wnt cell surface signaling complex. Thus, the response of Sdc1-/- mammary epithelial cells (mecs) to the intracellular, activated Wnt signal transducer, DeltaNbeta-catenin, was assayed both in vitro and in vivo, to test whether beta-catenin/TCF transactivation was Sdc1-independent. Surprisingly, we found that the expression of a canonical Wnt pathway reporter, TOP-FLASH, was reduced by 50% in both unstimulated Sdc1-/- mecs and in stimulated cells responding to Wnt1 or DeltaNbeta-catenin. Tumor development in response to DeltaNbeta-catenin was also significantly delayed on a Sdc1-/- background. Furthermore, the average beta-catenin/TCF transactivation per cell was normal in Sdc1-/- mec cultures, but the number of responsive cells was reduced by 50%. Sdc1-/- mecs show compensatory changes that maintain the number of HS chains, hence these experiments cannot test the coreceptor activity of HS for Wnt signaling. We propose that TCF-dependent transactivational activity is suppressed in 50% of cells in Sdc1-/- glands, and conclude that the major effect of Sdc1 does not map to the activity of the Wnt signaling complex, but to another pathway to create or stabilize the beta-catenin/TCF-responsive tumor precursor cells in mouse mammary gland

A considerable body of circumstantial data suggests that cyclin D1 is an attractive candidate to mediate the effects of beta-catenin in mammary tissue. To test the functional significance of these correlative findings, we investigated the genetic interaction between transcriptionally active beta-catenin (DeltaN89beta-catenin) and its target gene cyclin D1 in the mouse mammary gland during pubertal development, pregnancy, and tumorigenesis. Our data demonstrate that cyclin D1 is dispensable for the DeltaN89beta-catenin-stimulated initiation of alveologenesis in virgin females, for the de novo induction of alveoli in males, and for the formation of tumors. Indeed, lack of cyclin D1 accentuates and enhances these hyperplastic and tumorigenic DeltaN89beta-catenin phenotypes. Although alveologenesis is initiated by DeltaN89beta-catenin in a cyclin D1-independent fashion, up-regulation of cyclin D1 occurs in DeltaN89beta-catenin mice and its expression remains essential for the completion of alveolar development during the later stages of pregnancy. Thus, alveologenesis is a two-step process, and cyclin D1 activity during late alveologenesis cannot be replaced by the activity of other beta-catenin target genes that successfully drive proliferation at earlier stages

Plakoglobin provides a key linkage in protein chains that connect desmosomal and classical cadherins to the cytoskeleton. It is also present in a significant cytosolic pool that has the capacity to impact on canonical Wnt signaling by competing for interaction with partner proteins of beta-catenin. The closely related protein, beta-catenin, is rapidly targeted for proteasomal degradation by phosphorylation of a 'destruction box' within the N-terminal domain. Inhibition of this process forms the basis of Wnt signaling. This destruction box is also found in the N-terminal domain of plakoglobin. We report that plakoglobin is modified by the addition of O-GlcNAc at a single site in close proximity to the destruction box. O-GlcNAc modification has been proposed to counteract phosphorylation, provide protection from proteasomal degradation, mediate signal transduction, silence transcription, and regulate multimolecular protein assembly. This finding has potential implications for understanding the roles of plakoglobin

Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor beta (TGF-beta) complex (TGF-beta plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with alpha(IV)beta(1) and alpha(9)beta(1) integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1-3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene

Beta-catenin regulates cell-cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that beta-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/beta-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized deltaN89beta-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous beta-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized beta-catenin induces hyperplasia and mammary tumors in mice. Each of the beta-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion

A striking immunologic abnormality of normal and SCID Tgfb1(-/-) mice is the total absence of Langerhans cells in their epidermis. Here we show that transfer of Tgfb1(+/-) SCID bone marrow causes, within a few weeks, the appearance of Langerhans cells in the epidermis of gamma-irradiated and unirradiated Tgfb1(-/-) SCID recipients. In addition, local injection of 2 x 10(5) latent transforming growth factor-beta1 cDNA-transduced cloned CD4+ T lymphocytes causes the appearance of Langerhans cells in the ear epidermis of Tgfb1(-/-) SCID mice. This effect is enhanced by antigen-specific activation of these T cells. Injection of recombinant active transforming growth factor-beta 2 into the ear of Tgfb1(-/-) SCID mice also results in the migration of Langerhans cells into the epidermis locally, but no epidermal Langerhans cells are seen after systemic injections of transforming growth factor-beta 2. Our results suggest that transforming growth factor-beta can act in paracrine as well as autocrine fashion to induce the differentiation of precursors into Langerhans cells. Furthermore, these results indicate that the relative roles of different transforming growth factor-beta isoforms in vivo may be influenced by their local availability and/or the regulation of their conversion from latent into active form