Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

A Field Evaluation of an External and Neutrally Buoyant Acoustic Transmitter for Juvenile Salmon: Implications for Estimating Hydroturbine Passage Survival

Turbine-passed fish are exposed to rapid decreases in pressure which can cause barotrauma. The presence of an implanted telemetry tag increases the likelihood of injury or death from exposure to pressure changes, thus potentially biasing studies evaluating survival of turbine-passed fish. Therefore, a neutrally buoyant externally attached tag was developed to eliminate this bias in turbine passage studies. This new tag was designed not to add excess mass in water or take up space in the coelom, having an effective tag burden of zero with the goal of reducing pressure related biases to turbine survival studies. To determine if this new tag affects fish performance or susceptibility to predation, it was evaluated in the field relative to internally implanted acoustic transmitters (JSATS; Juvenile Salmon Acoustic Telemetry System) used widely for survival studies of juvenile salmonids. Survival and travel time through the study reach was compared between fish with either tag type in an area of high predation in the Snake and Columbia rivers, Washington. An additional group of fish affixed with neutrally-buoyant dummy external tags were implanted with passive integrated transponder (PIT) tags and recovered further downstream to assess external tag retention and injury. There were no significant differences in survival to the first detection site, 12 river kilometers (rkm) downstream of release. Travel times were also similar between groups. Conversely, externally-tagged fish had reduced survival (or elevated tag loss) to the second detection site, 65 rkm downstream. In addition, the retention study revealed that tag loss was first observed in fish recaptured approximately 9 days after release. Results suggest that this new tag may be viable for short term (<8 days) single-dam turbine-passage studies and under these situations, may alleviate the turbine passage-related bias encountered when using internal tags, however further research is needed to confirm this.     

Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast

Background

The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/C^Ama1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.

Findings

Here we show that Ama1p is a substrate of APC/C^Cdc20 but not APC/C^Cdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/C^Cdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/C^Ama1-mediated Cdc20p degradation.

Conclusions

Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.

     

The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations

The remarkable success of immune therapies emphasizes the need for immune‐competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well‐defined human‐relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype‐specific cancer biology.     

Self-Generated Chemoattractant Gradients: Attractant Depletion Extends the Range and Robustness of Chemotaxis

Chemotaxis is fundamentally important, but the sources of gradients in vivo are rarely well understood. Here, we analyse self-generated chemotaxis, in which cells respond to gradients they have made themselves by breaking down globally available attractants, using both computational simulations and experiments. We show that chemoattractant degradation creates steep local gradients. This leads to surprising results, in particular the existence of a leading population of cells that moves highly directionally, while cells behind this group are undirected. This leading cell population is denser than those following, especially at high attractant concentrations. The local gradient moves with the leading cells as they interact with their surroundings, giving directed movement that is unusually robust and can operate over long distances. Even when gradients are applied from external sources, attractant breakdown greatly changes cells'' responses and increases robustness. We also consider alternative mechanisms for directional decision-making and show that they do not predict the features of population migration we observe experimentally. Our findings provide useful diagnostics to allow identification of self-generated gradients and suggest that self-generated chemotaxis is unexpectedly universal in biology and medicine.