EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Sclerostin antibody improves alveolar bone quality in the Hyp mouse model of X-Linked Hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and poor mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases bone mass, strength and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg/kg of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (non-phosphorylated) ß-catenin stained alveolar osteocytes. Scl-Ab had no effect on mineralized tissues of the tooth - dentin, enamel, acellular and cellular cementum. There was a non-significant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fibral structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in the Hyp mouse model of XLH.

Wnt regulation of hematopoietic stem cell development and disease.

Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all cells of the blood and most immune cells. Due to their capacity for unlimited self-renewal, long-term HSCs replenish the blood and immune cells of an organism throughout its life. HSC development, maintenance, and differentiation are all tightly regulated by cell signaling pathways, including the Wnt pathway. Wnt signaling is initiated extracellularly by secreted ligands which bind to cell surface receptors and give rise to several different downstream signaling cascades. These are classically categorized either ß-catenin dependent (BCD) or ß-catenin independent (BCI) signaling, depending on their reliance on the ß-catenin transcriptional activator. HSC development, homeostasis, and differentiation is influenced by both BCD and BCI, with a high degree of sensitivity to the timing and dosage of Wnt signaling. Importantly, dysregulated Wnt signals can result in hematological malignancies such as leukemia, lymphoma, and myeloma. Here, we review how Wnt signaling impacts HSCs during development and in disease.

Thermoneutral Housing Temperature Improves Survival in a Murine Model of Polymicrobial Peritonitis.

Regulatory guidelines mandate housing for laboratory mice at temperatures below their thermoneutral zone, creating chronic cold stress. However, increases in housing temperature could alter immune responses. We hypothesized housing mice at temperatures within their thermoneutral zone would improve sepsis survival and alter immune responses. Male C57BL/6 mice were housed at 22°C or 30°C after cecal ligation and puncture (CLP) for 10 days. Survival of mice housed at 30°C (78%) after CLP was significantly increased compared with mice housed at 22°C (40%). Experimental groups were repeated with mice euthanized at 0, 12, 24, and 48 h post-surgery to examine select immune parameters. Raising housing temperature minimally altered systemic, peritoneal, or splenic cell counts. However, IL-6 levels in plasma and peritoneal lavage fluid were significantly lower at 12 h post-surgery in mice housed at 30°C compared with 22°C. Bacterial colony counts from peritoneal lavage fluid were significantly lower in mice housed at 30°C and in vivo studies suggested this was the result of increased phagocytosis by neutrophils. As previously demonstrated, adoptive transfer of fibrocytes significantly increased sepsis survival compared with saline at 22°C. However, there was no additive effect when adoptive transfer was performed at 30°C. Overall, the results demonstrated that thermoneutral housing improves survival after CLP by increasing local phagocytic activity and technical revisions may be necessary to standardize the severity of the model across different housing temperatures. These findings stress the pronounced impact housing temperature has on the CLP model and the importance of reporting housing temperature.