Timothy F. Lane

Overexpression of Cyclooxygenase-2 Is Sufficient to Induce Tumorigenesis in Transgenic Mice

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene inMin mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the β-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-xL and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Type I Interferon Production Enhances Susceptibility to Listeria monocytogenes Infection

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes–induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.

Toll-like Receptors Induce a Phagocytic Gene Program through p38

Toll-like receptor (TLR) signaling and phagocytosis are hallmarks of macrophage-mediated innate immune responses to bacterial infection. However, the relationship between these two processes is not well established. Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program. Importantly, TLR-induced phagocytosis of bacteria was found to be reliant on myeloid differentiation factor 88–dependent signaling through interleukin-1 receptor–associated kinase-4 and p38 leading to the up-regulation of scavenger receptors. Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process. We also demonstrate that TLR ligands not only amplify the percentage of phagocytes uptaking Escherichia coli, but also increase the number of bacteria phagocytosed by individual macrophages. Taken together, our data describe an evolutionarily conserved mechanism by which TLRs can specifically promote phagocytic clearance of bacteria during infection.

Compartment Syndrome Following Robotic Pelvic Urological Surgery: Diagnosis and Management

with a similar mechanism of injury in each location. Increasing its content or decreasing the size of the compartment can raise compartment pressure. This triggers a vicious cycle whereby increased venous pressure causes an increased capillary pressure and the increase in exudate increases the content of the compartment further. De-creasing tissue perfusion by reducing arteriolar pressure even-tually increases capillary permeability and causes a further in-crease in exudate. Muscle and nerve necrosis follows, causing the earliest clinical signs of compartment syndrome. Figure 1

Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone‐forming osteoblasts and bone‐resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen‐induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre‐osteoclasts. We have characterized a cell‐type‐specific hormone‐inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor‐alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre‐osteoclasts by the same osteoblast‐dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre‐osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

β1 Integrin Establishes Endothelial Cell Polarity and Arteriolar Lumen Formation via a Par3-Dependent Mechanism

Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of beta1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). beta1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in beta1-deficient vessels. Combined, our findings demonstrate that beta1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.

Wnt-10b directs hypermorphic development and transformation in mammary glands of male and female mice.

Wnt-10b is expressed during the formation of the mammary rudiment in mouse embryos and its expression continues through puberty when the mammary ductal pattern is established under control of ovarian steroids. Recently, viral activation of the Wnt-10b locus has linked its overexpression to mammary tumor formation, suggesting a role for Wnt-10b in patterning and growth-regulation of the mammary gland. To test this notion, we created lines of transgenic mice that express elevated levels of Wnt-10b under the control of the MMTV promoter/enhancer. Overexpression of this gene resulted in profound developmental alterations in the mammary gland, including expanded glandular development and the precocious appearance of alveoli in virgin females. Moreover, transgenic male mice also exhibited dramatic mammary development involving highly branched mammary ducts and gynecomastia. Aberrant expression of Wnt-10b in the mammary rudiments of males evidently bypasses the normal requirement for ovarian hormonal control in stimulating mammary ductal growth and the repressive effects of androgens. In addition to these developmental effects, transgenic mice of both sexes were highly susceptible to the development of mammary adenocarcinomas. Such tumors arose in a solitary manner indicating that Wnt-10b is a proto-oncogene which provides a necessary, but insufficient signal for oncogenesis. Relevant to this, there was no evidence of amplified expression of FGF mRNAs in these tumors though the Fgfs are a class of genes often implicated as collaborators in Wnt-mediated tumor formation. Indeed, co-expression of MMTV-Wnt-10b and MMTV-FGF-3/int-2 resulted in sterile offspring with highly disorganized mammary epithelium, demonstrating a potent interaction between their respective developmental pathways. These results suggest that Wnt-10b, or other Wnt genes expressed early in mammary development, play a role in regulating sexual dimorphism and show potent transforming activity when overexpressed.

VE-cadherin-CreERT2 transgenic mouse: A model for inducible recombination in the endothelium

To introduce temporal control in genetic experiments targeting the endothelium, we established a mouse line expressing tamoxifen‐inducible Cre‐recombinase (Cre‐ERT2) under the regulation of the vascular endothelial cadherin promoter (VECad). Specificity and efficiency of Cre activity was documented by crossing VECad‐Cre‐ERT2 with the ROSA26R reporter mouse, in which a floxed‐stop cassette has been placed upstream of the β‐galactosidase gene. We found that tamoxifen specifically induced widespread recombination in the endothelium of embryonic, neonatal, and adult tissues. Recombination was also documented in tumor‐associated vascular beds and in postnatal angiogenesis assays. Furthermore, injection of tamoxifen in adult animals resulted in negligible excision (lower than 0.4%) in the hematopoietic lineage. The VECad‐Cre‐ERT2 mouse is likely to be a valuable tool to study the function of genes involved in vascular development, homeostasis, and in complex processes involving neoangiogenesis, such as tumor growth. Developmental Dynamics 235:3413–3422, 2006.

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

Wnt signaling interacts with Shh to regulate taste papilla development

Wnt and Shh signaling pathways are critical for the development and maturation of many epithelial tissues. Both pathways have roles in stem cell maintenance, tissue development, and tumorigenesis. However, linkage between these pathways in mammalian systems had not been well established. Here, we report that Shh expression in fungiform papillae and formation of normal mature fungiform papillae depend on signaling through Wnt and β-catenin. We observed that during fungiform papilla formation in mice, Shh and components of the Wnt/β-catenin signaling pathway are expressed together in the developing placode. The elimination of Wnt/β-catenin signaling in either Lef1 or Wnt10b knockout mice resulted in down-regulation of Shh expression. In addition, the size and number of fungiform papillae were greatly reduced in Lef1 knockout mice. By examining embryonic mouse tongues in culture we determined that activation of Wnt/β-catenin signaling up-regulates Shh expression. We observed that blocking Shh signaling in cultured tongue explants enhanced papillae formation and was accompanied by an up-regulation of Wnt/β-catenin signaling, indicating that Shh inhibits the Wnt/β-catenin pathway. Exogenously added Shh suppressed expression of endogenous Shh and inhibited Wnt/β-catenin signaling (assessed in TOPGAL mice), further implicating Shh as an inhibitor of the Wnt/β-catenin pathway. Our observations indicate that Wnt/β-catenin signaling and interactions between the Wnt and Shh pathways play essential roles in the development of fungiform papillae.