Tomoyo Sasaki

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

BackgroundWnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.ResultsHere we report that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.ConclusionThe current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.

1α,25‐Dihydroxyvitamin D3 Promotes Vascularization of the Chondro‐osseous Junction by Stimulating Expression of Vascular Endothelial Growth Factor and Matrix Metalloproteinase 9

Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro‐osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] directly stimulates transcription of mRNAs encoding VEGF121 and −165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1α,25(OH)2D3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro‐osseous junction. The alterations in VEGF and MMP‐9 expression were accompanied by enhanced vascular invasion of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1α,25(OH)2D3 signaling stimulates VEGF and MMP‐9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.

TGFβ-mediated FGF signaling is crucial for regulating cranial neural crest cell proliferation during frontal bone development

The murine frontal bone derives entirely from the cranial neural crest (CNC) and consists of the calvarial (lateral) aspect that covers the frontal lobe of brain and the orbital aspect that forms the roof of bony orbit. TGFβ and FGF signaling have important regulatory roles in postnatal calvarial development. Our previous study has demonstrated that conditional inactivation of Tgfbr2 in the neural crest results in severe defects in calvarial development, although the cellular and molecular mechanisms by which TGFβ signaling regulates the fate of CNC cells during frontal bone development remain unknown. Here, we show that TGFβ IIR is required for proliferation of osteoprogenitor cells in the CNC-derived frontal bone anlagen. FGF acts downstream of TGFβ signaling in regulating CNC cell proliferation, and exogenous FGF2 rescues the cell proliferation defect in the frontal primordium of Tgfbr2 mutant. Furthermore, the CNC-derived frontal primordium requires TGFβ IIR to undergo terminal differentiation. However, this requirement is restricted to the developing calvarial aspect of the frontal bone, whereas the orbital aspect forms despite the ablation of Tgfbr2 gene, implying a differential requirement for TGFβ signaling during the development of various regions of the frontal bone. This study demonstrates the biological significance of TGFβ-mediated FGF signaling cascade in regulating frontal bone development, suggests that TGFβ functions as a morphogen in regulating the fate of the CNC-derived osteoblast and provides a model for investigating abnormal craniofacial development.

Cell autonomous requirement for TGF-β signaling during odontoblast differentiation and dentin matrix formation

TGF-beta subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-beta signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-beta signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2(fl/fl);Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-beta2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-beta signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c‐MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo‐oxygenase‐2 (COX‐2), an inducible rate‐limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX‐2 inhibitor, JTE‐522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose‐dependent manner. Northern blotting showed a very high level of COX‐2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c‐MC, but it increased when BALB/c‐MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX‐2 protein in respective cultures were compatible with the results of COX‐2 mRNA. In vivo, BALB/c‐MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX‐2 protein. These findings suggest that COX‐2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.

The small molecule Wnt signaling modulator ICG-001 improves contractile function in chronically infarcted rat myocardium.

The adult mammalian heart has limited capability for self-repair after myocardial infarction. Therefore, therapeutic strategies that improve post-infarct cardiac function are critically needed. The small molecule ICG-001 modulates Wnt signaling and increased the expression of genes beneficial for cardiac regeneration in epicardial cells. Lineage tracing experiments, demonstrated the importance of β-catenin/p300 mediated transcription for epicardial progenitor contribution to the myocardium. Female rats given ICG-001 for 10 days post-occlusion significantly improved ejection fraction by 8.4%, compared to controls (P<0.05). Taken together, Wnt modulation via β-catenin/CBP inhibition offers a promising therapeutic strategy towards restoration of myocardial tissues and an enhancement of cardiac functions following infarction.

Lef1 Haploinsufficient Mice Display a Low Turnover and Low Bone Mass Phenotype in a Gender- and Age-Specific Manner

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

Inhibition of β-catenin/p300 interaction proximalizes mouse embryonic lung epithelium

BackgroundWnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown.MethodsWe examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization.ResultsInhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media.ConclusionsThe results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium.