Faculty Bibliography

The mammalian telomere-binding protein Rap1 was recently found to have additional nontelomeric functions, acting as a transcriptional cofactor and a regulator of the NF-kappaB pathway. Here, we assess the effect of disrupting mouse Rap1 in vivo and report on its unanticipated role in metabolic regulation and body-weight homeostasis. Rap1 inhibition causes dysregulation in hepatic as well as adipose function, leading to glucose intolerance, insulin resistance, liver steatosis, and excess fat accumulation. Furthermore, Rap1 appears to play a pivotal role in the transcriptional cascade that controls adipocyte differentiation in vitro. Using a separation-of-function allele, we show that the metabolic function of Rap1 is independent of its recruitment to TTAGGG binding elements found at telomeres and at other interstitial loci. In conclusion, our study underscores an additional function for the most conserved telomere-binding protein, forging a link between telomere biology and metabolic signaling.

The concept of the tumour microenvironment recognizes that the interplay between cancer cells and stromal cells is a crucial determinant of cancer growth. In this Perspectives article, we propose the novel concept that the tumour microenvironment is built through rate-limiting steps during multistage carcinogenesis. Construction of a 'precancer niche' is a necessary and early step that is required for initiated cells to survive and evolve; subsequent niche expansion and maturation accompany tumour promotion and progression, respectively. As such, cancer niches represent an emergent property of a tumour that could be a robust target for cancer prevention and therapy.

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

PGRN and its derived engineered protein, Atsttrin, were reported to antagonize TNFalpha and protect against inflammatory arthritis [Tang, W. et al. (2011) The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332 (6028) 478-484]. Here we found that PGRN level was also significantly elevated in skin inflammation. PGRN-/- mice exhibited more severe inflammation following induction of oxazolone (OXA). In contrast, recombinant Atsttrin protein effectively attenuated inflammation in mice dermatitis model. In addition, the protective role of PGRN and Atsttrin in dermatitis was probably due to their inhibition on NF-kappaB signaling. Collectively, PGRN, especially its derived engineered protein, Atsttrin, may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.

Endochondral ossification plays a key role in the bone healing process, which requires normal cartilage callus formation. Progranulin (PGRN) growth factor is known to enhance chondrocyte differentiation and endochondral ossification during development, yet whether PGRN also plays a role in bone regeneration remains unknown. In this study we established surgically-induced bone defect and ectopic bone formation models based on genetically-modified mice. Thereafter, the bone healing process of those mice was analyzed through radiological assays including X-ray and micro CT, and morphological analysis including histology and immunohistochemistry. PGRN deficiency delayed bone healing, while recombinant PGRN enhanced bone regeneration. Moreover, PGRN was required for BMP-2 induction of osteoblastogenesis and ectopic bone formation. Furthermore, the role of PGRN in bone repair was mediated, at least in part, through interacting with TNF-alpha signaling pathway. PGRN-mediated bone formation depends on TNFR2 but not TNFR1, as PGRN promoted bone regeneration in deficiency of TNFR1 but lost such effect in TNFR2 deficient mice. PGRN blocked TNF-alpha-induced inflammatory osteoclastogenesis and protected BMP-2-mediated ectopic bone formation in TNF-alpha transgenic mice. Collectively, PGRN acts as a critical mediator of the bone healing process by constituting an interplay network with BMP-2 and TNF-alpha signaling, and this represents a potential molecular target for treatment of fractures, especially under inflammatory conditions.

Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.

PURPOSE: Our aim in this study was to apply three-dimensional MRI methods to analyze early postnatal morphological phenotypes in a Gbx2 conditional knockout (Gbx2-CKO) mouse that has variable midline deletions in the central cerebellum, reminiscent of many human cerebellar hypoplasia syndromes. METHODS: In vivo three-dimensional manganese-enhanced MRI at 100-microm isotropic resolution was used to visualize mouse brains between postnatal days 3 and 11, when cerebellum morphology undergoes dramatic changes. Deformation-based morphometry and volumetric analysis of manganese-enhanced MRI images were used to, respectively, detect and quantify morphological phenotypes in Gbx2-CKO mice. Ex vivo micro-MRI was performed after perfusion-fixation with supplemented gadolinium for higher resolution (50-microm) analysis. RESULTS: In vivo manganese-enhanced MRI and deformation-based morphometry correctly identified known cerebellar defects in Gbx2-CKO mice, and novel phenotypes were discovered in the deep cerebellar nuclei and the vestibulo-cerebellum, both validated using histology. Ex vivo micro-MRI revealed subtle phenotypes in both the vestibulo-cerebellum and the vestibulo-cochlear organ, providing an interesting example of complementary phenotypes in a sensory organ and its associated brain region. CONCLUSION: These results show the potential of three-dimensional MRI for detecting and analyzing developmental defects in mouse models of neurodevelopmental diseases. Magn Reson Med, 2013. (c) 2013 Wiley Periodicals, Inc.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

Current treatment of hemophilia consists of the administration of recombinant clotting factors, such as factor VIII (FVIII). However, patients with severe hemophilia can mount immune responses targeting therapeutically administered FVIII through inhibitory immunoglobulins that limit treatment efficacy. Induction of immune tolerance to FVIII in hemophilia has been extensively studied but remains an unmet need. We found that nondepleting anti-CD4 monoclonal antibodies (mAbs) are effective in inducing long-term tolerance to FVIII in different strains of hemophilic mice. Tolerance induction was facilitated when anti-CD4 mAbs were administered together with FVIII adsorbed in an adjuvant (alum). The observed state of tolerance was antigen specific, with mice remaining immune competent to respond to different antigens. Importantly, we found that following immunization with FVIII, the primed cells remained susceptible to tolerance induction. Studies with Foxp3-deficient and interleukin 10 (IL-10)-deficient mice demonstrated that the underlying tolerance mechanism is Foxp3 independent but requires IL-10. Our data show that an adjuvant, when administered together with a tolerizing agent such as nondepleting anti-CD4, can facilitate the induction of long-term tolerance to recombinant proteins, possibly not only in hemophilia but also in other diseases that are treated with potentially immunogenic therapeutics.

Avian malaria is of significant ecological importance and serves as a model system to study broad patterns of host switching and host specificity. The erythrocyte invasion mechanism of the malaria parasite Plasmodium is mediated, in large part, by proteins of the erythrocyte-binding-like (ebl) family of genes. However, little is known about how these genes are conserved across different species of Plasmodium, especially those that infect birds. Using bioinformatical methods in conjunction with polymerase chain reaction (PCR) and genetic sequencing, we identified and annotated one member of the ebl family, merozoite apical erythrocyte-binding ligand (maebl), from the chicken parasite Plasmodium gallinaceum. We then detected the expression of maebl in P. gallinaceum by PCR analysis of cDNA isolated from the blood of infected chickens. We found that maebl is a conserved orthologous gene in avian, mammalian, and rodent Plasmodium species. The duplicate extracellular binding domains of MAEBL, responsible for erythrocyte binding, are the most conserved regions. Our combined data corroborate the conservation of maebl throughout the Plasmodium genus and may help elucidate the mechanisms of erythrocyte invasion in P. gallinaceum and the host specificity of Plasmodium parasites.