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Yasuhiro Minami - One of the best experts on this subject based on the ideXlab platform.
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e2f1 ROR2 signaling mediates coordinated transcriptional regulation to promote g1 s phase transition in bfgf stimulated nih 3t3 fibroblasts
The FASEB Journal, 2020Co-Authors: Mitsuharu Endo, Yuki Tanaka, Mako Otsuka, Yasuhiro MinamiAbstract:ROR2 signaling has been shown to regulate the cell cycle progression in normal and cancer cells. However, the molecular mechanism of the cell cycle progression upon activation of ROR2 signaling still remains unknown. Here, we found that the expression levels of ROR2 in G1-arrested NIH/3T3 fibroblasts are low and are rapidly increased following the cell cycle progression induced by basic fibroblast growth factor (bFGF) stimulation. By expressing wild-type or a dominant negative mutant of E2F1, we show that E2F1 mediates bFGF-induced expression of ROR2, and that E2F1 binds to the promoter of the ROR2 gene to activate its expression. We also found that G1/S phase transition of bFGF-stimulated NIH/3T3 cells is delayed by the suppressed expression of ROR2. RNA-seq analysis revealed that the suppressed expression of ROR2 results in the decreased expression of various E2F target genes concomitantly with increased expression of Forkhead box O (FoxO) target genes, including p21Cip1 , and p27Kip1 . Moreover, the inhibitory effect of ROR2 knockdown on the cell cycle progression can be restored by suppressed expression of p21Cip1 , p27Kip1 ,or FoxO3a. Collectively, these findings indicate that E2F1-ROR2 signaling mediates the transcriptional activation and inhibition of E2F1-driven and FoxO3a-driven cell cycle-regulated genes, respectively, thereby promoting G1/S phase transition of bFGF-stimulated NIH/3T3 cells.
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Genetic interactions between ROR2 and Wnt9a, Ror1 and Wnt9a and ROR2 and Ror1: Phenotypic analysis of the limb skeleton and palate in compound mutants.
Genes to cells : devoted to molecular & cellular mechanisms, 2019Co-Authors: Martina Weissenböck, Michiru Nishita, Yasuhiro Minami, Richard Latham, Lena Ingeborg Wolff, Christine HartmannAbstract:Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in ROR2-/- mutant mice. In contrast, Ror1-/- mutant mice are viable and show no limb phenotype. Ror1-/- ;ROR2-/- double mutants are embryonic lethal, whereas double mutants containing a hypomorphic Ror1 allele (Ror1hyp ) survive up to birth and display a more severe shortened limb phenotype. Both orphan receptors have been shown to act as possible Wnt coreceptors and to mediate the Wnt5a signal. Here, we analyzed genetic interactions between the Wnt ligand, Wnt9a, and ROR2 or Ror1, as Wnt9a has also been implicated in skeletal development. Wnt9a-/- single mutants display a mild shortening of the long bones, whereas these are severely shortened in ROR2-/- mutants. ROR2-/- ;Wnt9a-/- double mutants displayed even more severely shortened long bones, and intermediate phenotypes were observed in compound ROR2;Wnt9a mutants. Long bones were also shorter in Ror1hyp/hyp ;Wnt9a-/- double mutants. In addition, Ror1hyp/hyp ;Wnt9a-/- double mutants displayed a secondary palate cleft phenotype, which was not present in the respective single mutants. Interestingly, 50% of compound mutant pups heterozygous for ROR2 and homozygous mutant for Ror1 also developed a secondary palate cleft phenotype.
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Expression of ROR2 associated with fibrosis of the submandibular gland
Cell structure and function, 2017Co-Authors: Daiki Takahashi, Yasuhiro Minami, Hiroaki Suzuki, Takahide Komori, Yasumasa Kakei, Kimi Yamakoshi, Michiru NishitaAbstract:The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person's quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and ROR2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, ROR2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1, TNF-α, IL-1β, and MMP-2. Increased immunostaining signal of ROR2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that ROR2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.Key words: submandibular gland, ROR2, Wnt5a, fibrosis, inflammation.
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the ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle
Journal of Biological Chemistry, 2017Co-Authors: Koki Kamizaki, Mitsuharu Endo, Ryosuke Doi, Makoto Hayashi, Takeshi Saji, Motoi Kanagawa, Tatsushi Toda, Soichiro Fukada, Michael E Greenberg, Yasuhiro MinamiAbstract:The Ror family receptor tyrosine kinases, Ror1 and ROR2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and ROR2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and ROR2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and ROR2 in injured muscles. Importantly, expression of Ror1, but not ROR2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.
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critical role of ROR2 receptor tyrosine kinase in regulating cell cycle progression of reactive astrocytes following brain injury
Glia, 2017Co-Authors: Mitsuharu Endo, Guljahan Ubulkasim, Chiho Kobayashi, Reiko Onishi, Atsu Aiba, Yasuhiro MinamiAbstract:ROR2 receptor tyrosine kinase plays crucial roles in developmental morphogenesis and tissue-/organo-genesis. In the developing brain, ROR2 is expressed in neural stem/progenitor cells (NPCs) and involved in the regulation of their stemness. However, it remains largely unknown about its role in the adult brain. In this study, we show that ROR2 is up-regulated in reactive astrocytes in the neocortices within 3 days following stab-wound injury. Intriguingly, ROR2-expressing astrocytes were detected primarily at the area surrounding the injury site, where astrocytes express Nestin, a marker of NPCs, and proliferate in response to injury. Furthermore, we show by using astrocyte-specific ROR2 knockout (KO) mice that a loss of ROR2 in astrocytes attenuates injury-induced proliferation of reactive astrocytes. It was also found that basic fibroblast growth factor (bFGF) is strongly up-regulated at 1 day post injury in the neocortices, and that stimulation of cultured quiescent astrocytes with bFGF restarts their cell cycle and induces expression of ROR2 during the G1 phase predominantly in proliferating cells. By using this culture method, we further show that the proportions of ROR2-expressing astrocytes increase following treatment with the histone deacetylases inhibitors including valproic acid, and that bFGF stimulation increases the levels of ROR2 expression within the respective cells. Moreover, we show that bFGF-induced cell cycle progression into S phase is inhibited or promoted in astrocytes from ROR2 KO mice or NPCs stably expressing ROR2-GFP, respectively. Collectively, these findings indicate that ROR2 plays a critical role in regulating the cell cycle progression of reactive astrocytes following brain injury, GLIA 2016. GLIA 2017;65:182-197.
Michiru Nishita - One of the best experts on this subject based on the ideXlab platform.
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Genetic interactions between ROR2 and Wnt9a, Ror1 and Wnt9a and ROR2 and Ror1: Phenotypic analysis of the limb skeleton and palate in compound mutants.
Genes to cells : devoted to molecular & cellular mechanisms, 2019Co-Authors: Martina Weissenböck, Michiru Nishita, Yasuhiro Minami, Richard Latham, Lena Ingeborg Wolff, Christine HartmannAbstract:Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in ROR2-/- mutant mice. In contrast, Ror1-/- mutant mice are viable and show no limb phenotype. Ror1-/- ;ROR2-/- double mutants are embryonic lethal, whereas double mutants containing a hypomorphic Ror1 allele (Ror1hyp ) survive up to birth and display a more severe shortened limb phenotype. Both orphan receptors have been shown to act as possible Wnt coreceptors and to mediate the Wnt5a signal. Here, we analyzed genetic interactions between the Wnt ligand, Wnt9a, and ROR2 or Ror1, as Wnt9a has also been implicated in skeletal development. Wnt9a-/- single mutants display a mild shortening of the long bones, whereas these are severely shortened in ROR2-/- mutants. ROR2-/- ;Wnt9a-/- double mutants displayed even more severely shortened long bones, and intermediate phenotypes were observed in compound ROR2;Wnt9a mutants. Long bones were also shorter in Ror1hyp/hyp ;Wnt9a-/- double mutants. In addition, Ror1hyp/hyp ;Wnt9a-/- double mutants displayed a secondary palate cleft phenotype, which was not present in the respective single mutants. Interestingly, 50% of compound mutant pups heterozygous for ROR2 and homozygous mutant for Ror1 also developed a secondary palate cleft phenotype.
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Expression of ROR2 associated with fibrosis of the submandibular gland
Cell structure and function, 2017Co-Authors: Daiki Takahashi, Yasuhiro Minami, Hiroaki Suzuki, Takahide Komori, Yasumasa Kakei, Kimi Yamakoshi, Michiru NishitaAbstract:The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person's quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a, Ror1 and ROR2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1, ROR2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1, TNF-α, IL-1β, and MMP-2. Increased immunostaining signal of ROR2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that ROR2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.Key words: submandibular gland, ROR2, Wnt5a, fibrosis, inflammation.
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Essential role of Wnt5a-Ror1/ROR2 signaling in metanephric mesenchyme and ureteric bud formation.
Genes to cells : devoted to molecular & cellular mechanisms, 2016Co-Authors: Yuka Okinaka, Michiru Nishita, Yasuhiro MinamiAbstract:Spatiotemporally regulated interaction between the metanephric mesenchyme (MM) and Wolffian duct (WD) is essential for the induction of a single ureteric bud (UB). The MM then interacts with the tip of the UB to induce outgrowth and branching of the UB, which in turn promotes growth of the adjacent MM. The Ror family receptor tyrosine kinases, Ror1 and ROR2, have been shown to act as receptors for Wnt5a to mediate noncanonical Wnt signaling. Previous studies have shown that ROR2-mutant mice exhibit ectopic formation of the UB, due to abnormal juxtaposition of the MM to the WD. We show here that both Ror1 and ROR2 are expressed in the mesenchyme between the MM and WD during UB formation. Although Ror1-mutant mice show no apparent defects in UB formation, Ror1;ROR2-double-mutant mice exhibit either defects in UB outgrowth and branching morphogenesis, associated with the loss of the MM from the UB domain, or ectopic formation of the UB. We also show genetic interactions between Ror1 and Wnt5a during UB formation. These findings suggest that Wnt5a-Ror1/ROR2 signaling regulates cooperatively the formation of the MM at the proper position to ensure normal development of the UB.
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activation of wnt5a ROR2 signaling associated with epithelial to mesenchymal transition of tubular epithelial cells during renal fibrosis
Genes to Cells, 2013Co-Authors: Kaoru Yamagata, Michiru Nishita, Mitsuharu Endo, Nur Arfian, Yoshiyuki Rikitake, Noriaki Emoto, Ken-ichi Hirata, Yoshiya Tanaka, Yasuhiro MinamiAbstract:Activation of Wnt5a-ROR2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-ROR2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and ROR2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that ROR2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these ROR2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that ROR2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at ROR2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from ROR2(+/-) mice compared with those from wild-type ROR2(+/+) mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from ROR2(+/-) mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-ROR2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.
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Activation of Wnt5a‐ROR2 signaling associated with epithelial‐to‐mesenchymal transition of tubular epithelial cells during renal fibrosis
Genes to cells : devoted to molecular & cellular mechanisms, 2013Co-Authors: Kaoru Yamagata, Michiru Nishita, Mitsuharu Endo, Nur Arfian, Yoshiyuki Rikitake, Noriaki Emoto, Ken-ichi Hirata, Yoshiya Tanaka, Yasuhiro MinamiAbstract:Activation of Wnt5a-ROR2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-ROR2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and ROR2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that ROR2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these ROR2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that ROR2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at ROR2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from ROR2(+/-) mice compared with those from wild-type ROR2(+/+) mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from ROR2(+/-) mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-ROR2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.
Alexandra Schambony - One of the best experts on this subject based on the ideXlab platform.
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ROR2 signaling is required for local upregulation of GDF6 and activation of BMP signaling at the neural plate border.
Development (Cambridge England), 2016Co-Authors: Carolin Schille, Michaela Bayerlová, Annalen Bleckmann, Alexandra SchambonyAbstract:The receptor tyrosine kinase ROR2 is a major Wnt receptor that activates β-catenin-independent signaling and plays a conserved role in the regulation of convergent extension movements and planar cell polarity in vertebrates. Mutations in the ROR2 gene cause recessive Robinow syndrome in humans, a short-limbed dwarfism associated with craniofacial malformations. Here, we show that ROR2 is required for local upregulation of gdf6 at the neural plate border in Xenopus embryos. ROR2 morphant embryos fail to upregulate neural plate border genes and show defects in the induction of neural crest cell fate. These embryos lack the spatially restricted activation of BMP signaling at the neural plate border at early neurula stages, which is required for neural crest induction. ROR2-dependent planar cell polarity signaling is required in the dorsolateral marginal zone during gastrulation indirectly to upregulate the BMP ligand Gdf6 at the neural plate border and Gdf6 is sufficient to rescue neural plate border specification in ROR2 morphant embryos. Thereby, ROR2 links Wnt/planar cell polarity signaling to BMP signaling in neural plate border specification and neural crest induction.
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wnt5a ROR2 induced upregulation of xpapc requires xshca
Biochemical and Biophysical Research Communications, 2010Co-Authors: Ann Caroline Feike, Klara Rachor, Marc Gentzel, Alexandra SchambonyAbstract:Ror receptor-tyrosine kinases act as Wnt-5a receptors in beta-catenin independent Wnt-signaling pathways. In Xenopus, expression of xPAPC is regulated by a Wnt-5a/ROR2 pathway, which resembles typical signaling cascades downstream of receptor-tyrosine kinases. Here, we have identified the phospho-tyrosine binding protein ShcA as an intracellular binding partner of ROR2. ShcA binds to a conserved motif in ROR2 via its SH2-domain. Wnt-5a induces clustering of ROR2 in the cell membrane and recruitment of ShcA to the ROR2 receptor complex. We further show that ShcA is co-expressed with ROR2 in developing Xenopus embryos and ShcA is required for Wnt-5a/ROR2 mediated upregulation of xPAPC, demonstrating the functional relevance of this interaction.
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Wnt5a/ROR2-induced upregulation of xPAPC requires xShcA.
Biochemical and biophysical research communications, 2010Co-Authors: Ann Caroline Feike, Klara Rachor, Marc Gentzel, Alexandra SchambonyAbstract:Ror receptor-tyrosine kinases act as Wnt-5a receptors in beta-catenin independent Wnt-signaling pathways. In Xenopus, expression of xPAPC is regulated by a Wnt-5a/ROR2 pathway, which resembles typical signaling cascades downstream of receptor-tyrosine kinases. Here, we have identified the phospho-tyrosine binding protein ShcA as an intracellular binding partner of ROR2. ShcA binds to a conserved motif in ROR2 via its SH2-domain. Wnt-5a induces clustering of ROR2 in the cell membrane and recruitment of ShcA to the ROR2 receptor complex. We further show that ShcA is co-expressed with ROR2 in developing Xenopus embryos and ShcA is required for Wnt-5a/ROR2 mediated upregulation of xPAPC, demonstrating the functional relevance of this interaction.
Caroline E. Ford - One of the best experts on this subject based on the ideXlab platform.
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ROR2 Is Epigenetically Regulated in Endometrial Cancer.
Cancers, 2021Co-Authors: Dongli Liu, Luis Enriquez, Caroline E. FordAbstract:The Wnt signalling receptor ROR2 has been identified as a possible therapeutic target in numerous cancers; however, its exact role remains unclear. The aim of this study was to investigate the role of ROR2 in endometrial cancer (EC) and the potential mechanism associated with its altered expression. The association between ROR2 mRNA expression levels and clinicopathological parameters, including overall survival (OS), in EC was analysed in The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) cohort and GEO dataset GSE17025. Four EC cell lines (KLE, MFE-296, Ishikawa and ARK-1) and eight clinical EC samples were analysed for ROR2 methylation via Combined Bisulphite Restriction Analysis (COBRA) and bisulphite genomic sequencing (BGS). In addition, the functional effects of ROR2 overexpression were investigated in Ishikawa and ARK-1 cells following ectopic ROR2 expression. ROR2 promoter methylation or reduced ROR2 expression were both found to correlate with shorter OS, high grade and serous subtype in the TCGA-UCEC and GEO datasets. ROR2 was epigenetically silenced by promoter methylation in both patient samples and cell lines. A significant correlation between ROR2 expression levels and promoter methylation was observed in patient samples (r = −0.797, p = 0.018). ROR2 restoration in ARK-1 significantly decreased invasion ability, with associated changes in epithelial-mesenchymal transition (EMT) markers. ROR2 plays a tumour-suppressor role in EC and is epigenetically suppressed with the development of disease. It may represent a diagnostic or therapeutic candidate for EC.
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selective modulation of wnt binding receptor tyrosine kinase ROR2 expression by human cytomegalovirus regulates trophoblast migration
Journal of General Virology, 2019Co-Authors: Kim T Huynh, Caroline E. Ford, Wendy J Van Zuylen, William D RawlinsonAbstract:Cytomegalovirus (CMV) infection during pregnancy may lead to adverse pregnancy outcomes and permanent neurological disabilities in infants infected in utero. Congenital CMV disease of the foetus and neonate results from both direct viral cytopathic damage and indirect effects through placental dysfunction. Infection specifically alters Wnt signalling, an essential pathway involved in trophoblast migration and placental development. We examined CMV regulation of trophoblast migration. This virus controls expression of Wnt-binding receptor tyrosine kinase ROR2, but not alternate receptor tyrosine kinases ROR1 or RYK. Ectopic expression of ROR2 reduced Wnt5a-induced trophoblast migration, whilst overexpression of ROR1 or RYK did not affect trophoblast migration. CMV infection increased ROR2 protein expression in trophoblasts, with no effect on ROR1 and RYK expression. These data further support the proposal that specific inhibition of this mechanism may be a target for therapeutic intervention to reduce placental damage and consequent foetal disease due to congenital CMV infection.
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Abstract 106: Shock and ROR! Targeting ROR1 and ROR2 in a preclinical patient-derived model of ovarian cancer
Tumor Biology, 2018Co-Authors: Claire Henry, Neville F. Hacker, Caroline E. FordAbstract:BACKGROUND: New targets for ovarian cancer treatment are critically needed. The Wnt receptors ROR1 and ROR2 are overexpressed in all histotypes of ovarian cancer and appear to play a role in both the tumour and the surrounding microenvironment. We have previously conducted an extensive suite of in vitro experiments, exploring the functional role of ROR1 and ROR2 in ovarian cancer. Silencing either receptor using siRNA inhibited ovarian cancer cell migration and invasion, and silencing both receptors had an even stronger inhibitory effect on the metastatic potential of ovarian cancer cellS. We have also shown that silencing ROR1 and ROR2 sensitises chemoresistant ovarian cancer cells to chemotherapy. Combined, these studies have confirmed the importance of RORs in ovarian cancer, and provided a strong argument for these receptors potential as clinical targets. However, these previous studies have utilised simple 2D in vitro models to investigate cancer cell growth and migration, which does not allow investigation of stromal involvement in ROR driven metastasis. AIM: To investigate targeting ROR1 and ROR2 in a 3D primary co-culture model of epithelial ovarian cancer dissemination to the omentum. METHODS: Primary fibroblasts (NOF) and mesothelial (HPMC) cells were isolated from fresh samples of omentum collected from women with benign or non-metastatic conditions and cultured with collagen to produce a organotypic 3D model. Stable shRNA knockdown of ROR1, ROR2 and double ROR1/ROR2 in OVCAR4 ovarian cancer cells were incorporated into the 3D model to measure adhesion, or using a transwell to measure invasion. Gene expression changes in primary cells upon OVCAR4 interaction was evaluated using indirect transwell co-culture. RESULTS: Double knockdown of ROR1 and ROR2 strongly inhibited cell adhesion (p CONCLUSION: The combination of ROR1 and ROR2 signalling influences ovarian cancer dissemination to the omentum, however ROR2 may also play a specific role in stromal activation during metastasis. Therefore, targeting both ROR1 and ROR2 may be a powerful approach to treating ovarian cancer. The development of a number of monoclonal antibodies targeting ROR1 currently in phase 1 trials for other tumour types makes this clinically feasible in the near future. Citation Format: Claire E. Henry, Neville F. Hacker, Caroline E. Ford. Shock and ROR! Targeting ROR1 and ROR2 in a preclinical patient-derived model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 106.
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ROR1 and ROR2 play distinct and opposing roles in endometrial cancer.
Gynecologic oncology, 2018Co-Authors: Claire Henry, Estelle Llamosas, Benjamin Daniels, A. Coopes, K. Tang, Caroline E. FordAbstract:Abstract Objective In recent years, the Wnt signalling pathway and the ROR1 and ROR2 receptors have been implicated in a range of gynecological cancers. These receptors have been described as prospective therapeutic targets, and this study investigated such potential in an endometrial cancer context. Method Immunohistochemistry for ROR1 and ROR2 was performed in a patient cohort, and expression was correlated with clinicopathological parameters including type, stage, grade, myometrial invasion, lymphovascular involvement, patient age and survival. The functional role of these receptors in endometrial cancer was investigated via siRNA knockdown of ROR1 and ROR2 in three cell line models (KLE, RL95-2 and MFE-319). Effects on proliferation, adhesion, migration and invasion were measured. Results High ROR1 expression in patient samples correlated with worse overall survival (p = 0.0169) while high ROR2 expression correlated with better overall survival (p = 0.06). ROR1 knockdown in KLE cells significantly decreased proliferation (p = 0.047) and reduced migration and invasion. ROR2 knockdown in RL95-2 cells increased cell migration and invasion (p = 0.011). Double ROR1 and ROR2 knockdown in MFE-319 cells decreased adhesion and significantly increased cell migration (P = 0.008) and invasion (p Conclusion ROR1 and ROR2 play distinct roles in endometrial cancer. ROR1 may promote tumor progression, similar to its role in ovarian cancer, while ROR2 may act as a tumor suppressor in endometrioid endometrial cancer, similar to its role in colorectal cancer. With several ROR-targeting therapies currently in development and phase I clinical trials for other tumor types, this study supports the potential of these receptors as therapeutic targets for women with endometrial cancer.
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silencing ror1 and ROR2 inhibits invasion and adhesion in an organotypic model of ovarian cancer metastasis
Oncotarget, 2017Co-Authors: Claire Henry, Neville F. Hacker, Caroline E. FordAbstract:// Claire Henry 1 , Neville Hacker 2 and Caroline Ford 1 1 Gynaecological Cancer Research Group, Lowy Cancer Research Centre and School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, Sydney, Australia 2 Gynaecological Cancer Centre, Royal Hospital for Women, Sydney, Australia Correspondence to: Caroline Ford, email: caroline.ford@unsw.edu.au Keywords: ROR2; ROR1; epithelial ovarian cancer; omentum; metastasis Received: August 29, 2017 Accepted: October 27, 2017 Published: November 20, 2017 ABSTRACT OBJECTIVE: Elevated expression of the ROR1 and ROR2 Wnt receptors has been noted in both the tumour and stromal compartments of ovarian cancer patient tissue samples. In vitro studies have suggested these receptors play a role in ovarian cancer metastasis. However, these previous studies have utilised simple 2D in vitro models to investigate cancer cell growth and migration, which does not allow investigation of stromal involvement in Wnt driven metastasis. AIM: To investigate targeting ROR1 and ROR2 using a primary co-culture 3D model of epithelial ovarian cancer dissemination to the omentum. METHODS: Primary fibroblasts (NOF) and mesothelial (HPMC) cells were isolated from fresh samples of omentum collected from women with benign or non-metastatic conditions and cultured with collagen to produce a organotypic 3D model. Stable shRNA knockdown of ROR1, ROR2 and double ROR1/ROR2 in OVCAR4 cells were plated onto the 3D model to measure adhesion, or using a transwell to measure invasion. Gene expression changes in primary cells upon OVCAR4 interaction was evaluated using indirect transwell co-culture. RESULTS: Double knockdown of ROR1 and ROR2 strongly inhibited cell adhesion (p<0.05) and invasion (P<0.05) to the omentum model. ROR2 was up regulated in primary fibroblasts when cultured with OVCAR4 (P=0.05) and ectopic overexpression of ROR2 in NOFs inhibited cell proliferation (P<0.01) but increased cell migration. CONCLUSION: The combination of ROR1 and ROR2 signalling influences ovarian cancer dissemination to the omentum, however ROR2 may also play a role in stromal activation during metastasis. Therefore, targeting both ROR1 and ROR2 may be a powerful approach to treating ovarian cancer.
Mitsuharu Endo - One of the best experts on this subject based on the ideXlab platform.
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e2f1 ROR2 signaling mediates coordinated transcriptional regulation to promote g1 s phase transition in bfgf stimulated nih 3t3 fibroblasts
The FASEB Journal, 2020Co-Authors: Mitsuharu Endo, Yuki Tanaka, Mako Otsuka, Yasuhiro MinamiAbstract:ROR2 signaling has been shown to regulate the cell cycle progression in normal and cancer cells. However, the molecular mechanism of the cell cycle progression upon activation of ROR2 signaling still remains unknown. Here, we found that the expression levels of ROR2 in G1-arrested NIH/3T3 fibroblasts are low and are rapidly increased following the cell cycle progression induced by basic fibroblast growth factor (bFGF) stimulation. By expressing wild-type or a dominant negative mutant of E2F1, we show that E2F1 mediates bFGF-induced expression of ROR2, and that E2F1 binds to the promoter of the ROR2 gene to activate its expression. We also found that G1/S phase transition of bFGF-stimulated NIH/3T3 cells is delayed by the suppressed expression of ROR2. RNA-seq analysis revealed that the suppressed expression of ROR2 results in the decreased expression of various E2F target genes concomitantly with increased expression of Forkhead box O (FoxO) target genes, including p21Cip1 , and p27Kip1 . Moreover, the inhibitory effect of ROR2 knockdown on the cell cycle progression can be restored by suppressed expression of p21Cip1 , p27Kip1 ,or FoxO3a. Collectively, these findings indicate that E2F1-ROR2 signaling mediates the transcriptional activation and inhibition of E2F1-driven and FoxO3a-driven cell cycle-regulated genes, respectively, thereby promoting G1/S phase transition of bFGF-stimulated NIH/3T3 cells.
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the ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle
Journal of Biological Chemistry, 2017Co-Authors: Koki Kamizaki, Mitsuharu Endo, Ryosuke Doi, Makoto Hayashi, Takeshi Saji, Motoi Kanagawa, Tatsushi Toda, Soichiro Fukada, Michael E Greenberg, Yasuhiro MinamiAbstract:The Ror family receptor tyrosine kinases, Ror1 and ROR2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and ROR2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and ROR2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and ROR2 in injured muscles. Importantly, expression of Ror1, but not ROR2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.
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critical role of ROR2 receptor tyrosine kinase in regulating cell cycle progression of reactive astrocytes following brain injury
Glia, 2017Co-Authors: Mitsuharu Endo, Guljahan Ubulkasim, Chiho Kobayashi, Reiko Onishi, Atsu Aiba, Yasuhiro MinamiAbstract:ROR2 receptor tyrosine kinase plays crucial roles in developmental morphogenesis and tissue-/organo-genesis. In the developing brain, ROR2 is expressed in neural stem/progenitor cells (NPCs) and involved in the regulation of their stemness. However, it remains largely unknown about its role in the adult brain. In this study, we show that ROR2 is up-regulated in reactive astrocytes in the neocortices within 3 days following stab-wound injury. Intriguingly, ROR2-expressing astrocytes were detected primarily at the area surrounding the injury site, where astrocytes express Nestin, a marker of NPCs, and proliferate in response to injury. Furthermore, we show by using astrocyte-specific ROR2 knockout (KO) mice that a loss of ROR2 in astrocytes attenuates injury-induced proliferation of reactive astrocytes. It was also found that basic fibroblast growth factor (bFGF) is strongly up-regulated at 1 day post injury in the neocortices, and that stimulation of cultured quiescent astrocytes with bFGF restarts their cell cycle and induces expression of ROR2 during the G1 phase predominantly in proliferating cells. By using this culture method, we further show that the proportions of ROR2-expressing astrocytes increase following treatment with the histone deacetylases inhibitors including valproic acid, and that bFGF stimulation increases the levels of ROR2 expression within the respective cells. Moreover, we show that bFGF-induced cell cycle progression into S phase is inhibited or promoted in astrocytes from ROR2 KO mice or NPCs stably expressing ROR2-GFP, respectively. Collectively, these findings indicate that ROR2 plays a critical role in regulating the cell cycle progression of reactive astrocytes following brain injury, GLIA 2016. GLIA 2017;65:182-197.
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activation of wnt5a ROR2 signaling associated with epithelial to mesenchymal transition of tubular epithelial cells during renal fibrosis
Genes to Cells, 2013Co-Authors: Kaoru Yamagata, Michiru Nishita, Mitsuharu Endo, Nur Arfian, Yoshiyuki Rikitake, Noriaki Emoto, Ken-ichi Hirata, Yoshiya Tanaka, Yasuhiro MinamiAbstract:Activation of Wnt5a-ROR2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-ROR2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and ROR2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that ROR2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these ROR2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that ROR2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at ROR2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from ROR2(+/-) mice compared with those from wild-type ROR2(+/+) mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from ROR2(+/-) mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-ROR2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.
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Activation of Wnt5a‐ROR2 signaling associated with epithelial‐to‐mesenchymal transition of tubular epithelial cells during renal fibrosis
Genes to cells : devoted to molecular & cellular mechanisms, 2013Co-Authors: Kaoru Yamagata, Michiru Nishita, Mitsuharu Endo, Nur Arfian, Yoshiyuki Rikitake, Noriaki Emoto, Ken-ichi Hirata, Yoshiya Tanaka, Yasuhiro MinamiAbstract:Activation of Wnt5a-ROR2 signaling has been shown to be associated with epithelial-to-mesenchymal transition (EMT) of epidermoid carcinoma cells via induction of matrix metalloproteinase-2 (MMP-2). Because EMT has also been implicated in the progression of tissue fibrosis, we examined the possible association of Wnt5a-ROR2 signaling with renal fibrosis. Here, we show that expression of Wnt5a and ROR2 is induced in a damaged mouse kidney after unilateral ureteral obstruction (UUO) treatment. Immunofluorescent analysis showed that ROR2 expression is clearly induced in tubular epithelial cells during renal fibrosis, and these ROR2-expressing cells also express Snail and vimentin, markers of mesenchymal cells, suggesting that ROR2 might be induced in epithelial cells undergoing EMT. We also found that MMP-2 expression is induced at ROR2-positive epithelium adjacent to significantly disrupted tubular basement membrane (TBM). Interestingly, reduced expression of MMP-2 is detected at epithelium in damaged kidneys from ROR2(+/-) mice compared with those from wild-type ROR2(+/+) mice. Importantly, extents of TBM disruption are apparently reduced in damaged kidneys from ROR2(+/-) mice compared with those from wild-type mice. Collectively, these findings indicate that activation of Wnt5a-ROR2 signaling in epithelial cells undergoing EMT may play an important role in disrupting TBM via MMP-2 induction during renal fibrosis.
