Heather Whetstone

Side Population Cells Isolated from Mesenchymal Neoplasms Have Tumor Initiating Potential

Although many cancers are maintained by tumor-initiating cells, this has not been shown for mesenchymal tumors, in part due to the lack of unique surface markers that identify mesenchymal progenitors. An alternative technique to isolate stem-like cells is to isolate side population (SP) cells based on efflux of Hoechst 33342 dye. We examined 29 mesenchymal tumors ranging from benign to high-grade sarcomas and identified SP cells in all but six samples. There was a positive correlation between the percentage of SP cells and the grade of the tumor. SP cells preferentially formed tumors when grafted into immunodeficient mice, and only cells from tumors that developed from the SP cells had the ability to initiate tumor formation upon serial transplantation. Although SP cells are able to efflux rhodamine dye in addition to Hoechst 33342, we found that the ability to efflux rhodamine dye did not identify a population of cells enriched for tumor-initiating capacity. Here, we identify a subpopulation of cells within a broad range of benign and malignant mesenchymal tumors with tumor-initiating capacity. In addition, our data suggest that the proportion of SP cells could be used as a prognostic factor and that therapeutically targeting this subpopulation of cells could be used to improve patient outcome.

Modulating hedgehog signaling can attenuate the severity of osteoarthritis

Osteoarthritis is associated with the irreversible degeneration of articular cartilage. Notably, in this condition, articular cartilage chondrocytes undergo phenotypic and gene expression changes that are reminiscent of their end-stage differentiation in the growth plate during skeletal development. Hedgehog (Hh) signaling regulates normal chondrocyte growth and differentiation; however, the role of Hh signaling in chondrocytes in osteoarthritis is unknown. Here we examine human osteoarthritic samples and mice in which osteoarthritis was surgically induced and find that Hh signaling is activated in osteoarthritis. Using several genetically modified mice, we found that higher levels of Hh signaling in chondrocytes cause a more severe osteoarthritic phenotype. Furthermore, we show in mice and in human cartilage explants that pharmacological or genetic inhibition of Hh signaling reduces the severity of osteoarthritis and that runt-related transcription factor-2 (RUNX2) potentially mediates this process by regulating a disintegrin and metalloproteinase with thrombospondin type 1 motif-5 (ADAMTS5) expression. Together, these findings raise the possibility that Hh blockade can be used as a therapeutic approach to inhibit articular cartilage degeneration.

β-Catenin Signaling Pathway Is Crucial for Bone Morphogenetic Protein 2 to Induce New Bone Formation

Endochondral ossification is recapitulated during bone morphogenetic protein (BMP)-induced ectopic bone formation. Although BMP and β-catenin have been investigated in bone development and in mesenchymal cells, how they interact in this process is not clear. We implanted recombinant BMP-2 into the muscle of mice to investigate the effect of β-catenin signaling on BMP-induced in vivo endochondral bone formation. BMP-2 induced expression of several Wnt ligands and their receptors and also activated β-catenin-mediated T cell factor-dependent transcriptional activity. An adenovirus expressing Dickkopf-1 (Dkk-1, an inhibitor of canonical Wnt pathway) inhibited β-catenin signaling and endochondral bone formation. Interestingly, Dkk-1 inhibited both chondrogenesis and osteogenesis. Likewise, mice expressing conditional β-catenin null alleles also displayed an inhibition of BMP-induced chondrogenesis and osteogenesis. This is in contrast to studies of embryonic skeletogenesis, which demonstrate that β-catenin is required for osteogenesis but is dispensable for chondrogenesis. These findings suggest that embryonic development pathways are not always recapitulated during post-natal regenerative processes, and the biochemical pathways utilized to regulate cell differentiation may be different. During in vivo ectopic bone formation, BMP-2 induces β-catenin-mediated signaling through Wnt ligands, and β-catenin is required for both chondrogenesis and osteogenesis.

Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing

After cutaneous injury, a variety of cell types are activated to reconstitute the epithelial and dermal components of the skin. β‐Catenin plays disparate roles in keratinocytes and fibroblasts, inhibiting keratinocyte migration and activating fibroblast proliferation, suggesting that β‐catenin could either inhibit or enhance the healing process. How β‐catenin functions in concert with other signaling pathways important in the healing process is unknown. Wound size was examined in mice expressing conditional null or conditional stabilized alleles of β‐catenin, regulated by an adenovirus expressing cre‐recombinase. The size of the wounds in the mice correlated with the protein level of β‐catenin. Using mice expressing these conditional alleles, we found that the wound phenotype imparted by Smad3 deficiency and by the injection of TGF+ before wounding is mediated in part by β‐catenin. TGF+ was not able to regulate proliferation in β‐catenin null fibroblasts, whereas keratinocyte proliferation rate was independent of β‐catenin. When mice are treated with lithium, β‐catenin‐mediated signaling was activated in cutaneous wounds, which healed with a larger size. These results demonstrate a crucial role for β‐catenin in regulating cutaneous wound size. Furthermore, these data implicate mesenchymal cells as playing a critical role regulating wound size.‐Cheon, S. S., Wei, Q., Gurung, A., Youn, A., Bright, T., Poon, R., Whetstone, H., Guha, A., Alman, B. A. Beta‐catenin regulates wound size and mediates the effect of TGF‐beta in cutaneous healing. FASEB J. 20, 692–701 (2006)

T-Lymphocytes Enable Osteoblast Maturation via IL-17F during the Early Phase of Fracture Repair

While it is well known that the presence of lymphocytes and cytokines are important for fracture healing, the exact role of the various cytokines expressed by cells of the immune system on osteoblast biology remains unclear. To study the role of inflammatory cytokines in fracture repair, we studied tibial bone healing in wild-type and Rag1−/− mice. Histological analysis, µCT stereology, biomechanical testing, calcein staining and quantitative RNA gene expression studies were performed on healing tibial fractures. These data provide support for Rag1−/− mice as a model of impaired fracture healing compared to wild-type. Moreover, the pro-inflammatory cytokine, IL-17F, was found to be a key mediator in the cellular response of the immune system in osteogenesis. In vitro studies showed that IL-17F alone stimulated osteoblast maturation. We propose a model in which the Th17 subset of T-lymphocytes produces IL-17F to stimulate bone healing. This is a pivotal link in advancing our current understanding of the molecular and cellular basis of fracture healing, which in turn may aid in optimizing fracture management and in the treatment of impaired bone healing.

Gli2 and p53 Cooperate to Regulate IGFBP-3- Mediated Chondrocyte Apoptosis in the Progression from Benign to Malignant Cartilage Tumors

Clinical evidence suggests that benign cartilage lesions can progress to malignant chondrosarcoma, but the molecular events in this progression are unknown. Mice that develop benign cartilage lesions due to overexpression of Gli2 in chondrocytes developed lesions similar to chondrosarcomas when they were also deficient in p53. Gli2 overexpression and p53 deficiency had opposing effects on chondrocyte differentiation, but had additive effects negatively regulating apoptosis. Regulation of Igfbp3 expression and insulin-like growth factor (IGF) signaling by Gli and p53 integrated their effect on apoptosis. Treatment of human chondrosarcomas or fetal mouse limb explants with IGFBP3 or by blocking IGF increased the apoptosis rate, and mice expressing Gli2 developed substantially fewer tumors when they were also deficient for Igf2. IGF signaling-meditated apoptosis regulates the progression to malignant chondrosarcoma.

Macrophages Promote Osteoblastic Differentiation In Vivo: Implications in Fracture Repair and Bone Homeostasis

Macrophages are activated in inflammation and during early phases of repair processes. Interestingly, they are also present in bone during development, but their function during this process is unclear. Here, we explore the function of macrophages in bone development, growth, and repair using transgenic mice to constitutively or conditionally deplete macrophages. Depletion of macrophages led to early skeletal growth retardation and progressive osteoporosis. By 3 months of age, macrophage‐deficient mice displayed a 25% reduction in bone mineral density and a 70% reduction in the number of trabecular bone compared to control littermates. Despite depletion of macrophages, functional osteoclasts were still present in bones, lining trabecular bone and the endosteal surface of the cortical bone. Furthermore, ablation of macrophages led to a 60% reduction in the number of bone marrow mesenchymal progenitor cells and a decrease in the ability of these cells to differentiate to osteoblasts. When macrophages were depleted during fracture repair, bone union was impaired. Calluses from macrophage‐deficient animals were smaller, and contained less bone and more fibrotic tissue deposition. Taken together, this shows that macrophages are crucial for maintaining bone homeostasis and promoting fracture repair by enhancing the differentiation of mesenchymal progenitors.

β-Catenin–regulated myeloid cell adhesion and migration determine wound healing

A β-catenin/T cell factor-dependent transcriptional program is critical during cutaneous wound repair for the regulation of scar size; however, the relative contribution of β-catenin activity and function in specific cell types in the granulation tissue during the healing process is unknown. Here, cell lineage tracing revealed that cells in which β-catenin is transcriptionally active express a gene profile that is characteristic of the myeloid lineage. Mice harboring a macrophage-specific deletion of the gene encoding β-catenin exhibited insufficient skin wound healing due to macrophage-specific defects in migration, adhesion to fibroblasts, and ability to produce TGF-β1. In irradiated mice, only macrophages expressing β-catenin were able to rescue wound-healing deficiency. Evaluation of scar tissue collected from patients with hypertrophic and normal scars revealed a correlation between the number of macrophages within the wound, β-catenin levels, and cellularity. Our data indicate that β-catenin regulates myeloid cell motility and adhesion and that β-catenin-mediated macrophage motility contributes to the number of mesenchymal cells and ultimate scar size following cutaneous injury.

Erratum: Exposure to a youthful circulation rejuvenates bone repair through modulation of β-catenin.

The capacity for tissues to repair and regenerate diminishes with age. We sought to determine the age-dependent contribution of native mesenchymal cells and circulating factors on in vivo bone repair. Here we show that exposure to youthful circulation by heterochronic parabiosis reverses the aged fracture repair phenotype and the diminished osteoblastic differentiation capacity of old animals. This rejuvenation effect is recapitulated by engraftment of young haematopoietic cells into old animals. During rejuvenation, β-catenin signalling, a pathway important in osteoblast differentiation, is modulated in the early repair process and required for rejuvenation of the aged phenotype. Temporal reduction of β-catenin signalling during early fracture repair improves bone healing in old mice. Our data indicate that young haematopoietic cells have the capacity to rejuvenate bone repair and this is mediated at least in part through β-catenin, raising the possibility that agents that modulate β-catenin can improve the pace or quality of fracture repair in the ageing population.

Fibronectin and β-Catenin Act in a Regulatory Loop in Dermal Fibroblasts to Modulate Cutaneous Healing

β-Catenin is an important regulator of dermal fibroblasts during cutaneous wound repair. However, the factors that modulate β-catenin activity in this process are not completely understood. We investigated the role of the extracellular matrix in regulating β-catenin and found an increase in β-catenin-mediated Tcf-dependent transcriptional activity in fibroblasts exposed to various extracellular matrix components. This occurs through an integrin-mediated GSK3β-dependent pathway. The physiologic role of this mechanism was demonstrated during wound repair in extra domain A-fibronectin-deficient mice, which exhibited decreased β-catenin-mediated signaling during the proliferative phase of healing. Extra domain A-fibronectin-deficient mice have wounds that fail at a lower tensile strength and contain fewer fibroblasts compared with wild type mice. This phenotype was rescued by genetic or pharmacologic activation of β-catenin signaling. Because fibronectin is a transcriptional target of β-catenin, this suggests the existence of a feedback loop between these two molecules that regulates dermal fibroblast cell behavior during wound repair.