Raymond Poon

β-Catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds

Fibroproliferative processes are a group of disorders in which there is excessive proliferation of spindle (mesenchymal fibroblast-like) cells. They range from hypertrophic scars to neoplasms such as aggressive fibromatosis. Cells from these disorders share cytologic similarity with fibroblasts present during the proliferative phase of wound healing, suggesting that they represent a prolonged wounding response. A critical role for β-catenin in mesenchymal cells in fibroproliferative processes is suggested by its high rate of somatic mutation in aggressive fibromatosis. Using a Tcf-reporter mouse we found that β-catenin protein level and Tcf-transcriptional activity are elevated in fibroblasts during the proliferative phase of healing. We generated a transgenic mouse in which stabilized β-catenin is expressed in mesenchymal cells under control of a tetracycline-regulated promoter. Fibroblasts from the transgenic mice exhibited increased proliferation, motility, and invasiveness when expressing stabilized β-catenin and induced tumors after induction of the transgene when grafted into nude mice. Mice developed aggressive fibromatoses and hyperplastic gastrointestinal polyps after 3 months of transgene induction and healed with hyperplastic cutaneous wounds compared with control mice, which demonstrates an important function for β-catenin in mesenchymal cells and shows a central role for β-catenin in wound healing and fibroproliferative disorders.

A mutant PTH/PTHrP type I receptor in enchondromatosis

Enchondromas are common benign cartilage tumors of bone. They can occur as solitary lesions or as multiple lesions in enchondromatosis (Ollier and Maffucci diseases). Clinical problems caused by enchondromas include skeletal deformity and the potential for malignant change to chondrosarcoma. The extent of skeletal involvement is variable in enchondromatosis and may include dysplasia that is not directly attributable to enchondromas. Enchondromatosis is rare, obvious inheritance of the condition is unusual and no candidate loci have been identified. Enchondromas are usually in close proximity to, or in continuity with, growth-plate cartilage. Consequently, they may result from abnormal regulation of proliferation and terminal differentiation of chondrocytes in the adjoining growth plate. In normal growth plates, differentiation of proliferative chondrocytes to post-mitotic hypertrophic chondrocytes is regulated in part by a tightly coupled signaling relay involving parathyroid hormone related protein (PTHrP) and Indian hedgehog (IHH). PTHrP delays the hypertrophic differentiation of proliferating chondrocytes, whereas IHH promotes chondrocyte proliferation. We identified a mutant PTH/PTHrP type I receptor (PTHR1) in human enchondromatosis that signals abnormally in vitro and causes enchondroma-like lesions in transgenic mice. The mutant receptor constitutively activates Hedgehog signaling, and excessive Hedgehog signaling is sufficient to cause formation of enchondroma-like lesions.

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)

Cyclooxygenase-two (COX-2) modulates proliferation in aggressive fibromatosis (desmoid tumor)

Aggressive fibromatosis is a locally invasive soft tissue lesion. Seventy-five per cent of cases harbor a somatic mutation in either the APC or β-catenin genes, resulting in β-catenin protein stabilization. Cyclooxygenase-2 (COX-2) is an enzyme involved in prostaglandin synthesis that modulates the formation of colonic neoplasia, especially in cases due to mutations resulting in β-catenin stabilization. Human aggressive fibromatoses and lesions from the Apc+/Apc1638N mouse (a murine model for Apc-driven fibromatosis) demonstrated elevated COX-2 levels. COX-2 blockade either by the selective agent DFU or by non-selective COX blocking agents results in reduced proliferation in human tumor cell cultures. Breeding mice with Cox-2−/− mice resulted in no difference in number of aggressive fibromatoses formed, but in a smaller tumor size, while there was a decrease in number of GI lesions by 50%. Mice fed various COX blocking agents also showed a decline in tumor size. COX-2 expression was regulated by tcf-dependent transcription in this lesion. COX-2 partially regulates proliferation due to β-catenin stabilization in aggressive fibromatosis. Although COX blockade alone does not cause tumor regression, this data suggests that it may have a role as an adjuvant therapy to slow tumor growth in this lesion.

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.

Prolonged β-catenin stabilization and tcf-dependent transcriptional activation in hyperplastic cutaneous wounds

Mesenchymal cells that accumulate during the proliferative phase of wound healing and that are present in hyperplastic wounds share cytologic similarities with the cells from fibroproliferative lesions in which there is activation of β-catenin-mediated transcription. Re-excision wounds from a previous biopsy and samples from hyperplastic cutaneous wounds were studied along with normal tissues. During normal wound healing, there was an increase in β-catenin protein level, peaking 4 weeks following the insult and returning towards baseline level by 12 weeks. Hyperplastic wounds exhibited a prolonged duration of elevated β-catenin, lasting more than 2 years following the initial injury. The level of expression of genes known to be upregulated in the proliferative phase of wound healing (α-smooth muscle actin and type three collagen), correlated with β-catenin protein level. The phosphorylation level of glycogen synthase kinase-3-β, a kinase important for β-catenin protein destabilization, correlated with β-catenin protein level. β-Catenin was transcriptionally active in these wounds as demonstrated by the expression of the β-catenin target genes (MMP-7 and FN) and by activation of a tcf-reporter in primary cell cultures. β-catenin stabilization increases cell proliferation and motility in fibroblasts in vitro, and likely has a similar function during its transient elevation in the proliferative phase of normal wound healing. In hyperplastic wounds, there is dysregulation of β-catenin, maintaining the mesenchymal cells in a prolonged proliferative state. As such, β-catenin likely plays a central role in mesenchymal cells during the healing process, and is an appealing therapeutic target for disorders of wound healing.

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.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Significance Current genomic and biochemical analysis revealed mutations in isocitrate dehydrogenase (IDH) genes associated with several neoplasms and a novel enzymatic activity of IDH mutations to catalyze α-ketoglutarate to d-2-hydroxyglutarate, contributing to tumorigenesis. We identified a broad range of IDH1 mutations, including a previously unidentified IDH1-R132Q mutation, in cartilage tumors. Cartilage-specific Col2a1-Cre/ERT2;Idh1-R132 mutant knock-in mice developed multiple enchondroma-like lesions. These data show that mutant Idh in growth-plate cells causes persistence of chondrocytes, giving rise to enchondromas adjacent to the growth cartilage in bone. Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Matrix Metalloproteinase Activity Modulates Tumor Size, Cell Motility, and Cell Invasiveness in Murine Aggressive Fibromatosis

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) regulate the degradation of extracellular matrix components and play important roles in the progression of select neoplastic processes. The locally invasive soft tissue tumor, aggressive fibromatosis (also called desmoid tumor), is caused by mutations resulting in β-catenin-mediated T-cell factor (tcf)-dependent transcriptional activity. Because β-catenin can regulate MMP expression, we investigated the expression of several MMPs and TIMPs in aggressive fibromatosis tumors that develop in Apc+/Apc1638N mice. Mmp-3 and Timp-1 were differentially regulated (5-fold and 0.5-fold, respectively) in tumors compared with normal fibrous tissue. Conditioned media from tumor cells showed an increased ability to degrade collagen, and inhibition of MMPs using GM6001 decreased the ability of the tumor cells to invade through Matrigel. Both the treatment of Apc/Apc1638N mice with GM6001 or crossing with a transgenic mouse that overexpresses Timp-1 resulted in a significant reduction in tumor volume. Surprisingly, overexpression of Timp-1 also resulted in a 50% increase in tumor number. Although TIMP-1 can induce growth stimulatory effects in some cell types, we found no difference in proliferation or apoptosis rate in cells from tumors that developed in the Timp-1–transgenic mice compared with mice that did not express the Timp-1 transgene, suggesting that TIMP-1 promotes aggressive fibromatosis tumor formation through an alternate mechanism. These data suggest that MMPs play a crucial role in regulating the invasiveness of mesenchymal cells and in modulating aggressive fibromatosis tumor progression. Because this is a locally invasive tumor, MMP inhibition could slow tumor growth and may prove to be an effective adjuvant therapy.