Kim Y, etal., Pathol Res Pract. 2015 Dec;211(12):963-72. doi: 10.1016/j.prp.2015.10.001. Epub 2015 Oct 26.
BACKGROUND: Wnt5a, a non-canonical Wnt ligand, has been shown to play tumor-promoting or tumor-suppressive roles in different neoplasms. Increased Wnt5a expression and Wnt5a-dependent invasive activity that is mediated by one of its receptors, Ryk, have been reported in glioblastomas. METHODS: We in
vestigated the protein expression of Wnt5a, its receptors Ryk and Ror2, and the canonical Wnt pathway marker beta-catenin in 186 cases of glioblastoma and its variants. Associations with clinicopathological and molecular variables and prognosis were analyzed. RESULTS: All glioblastoma cases expressed Wnt5a, Ryk and Ror2 with a different grade. The expression of both Ryk and Ror2 correlated with that of Wnt5a in glioblastomas. The expression of beta-catenin did not correlate with any of Wnt5a, Ryk or Ror2. Wnt5a expression was significantly different among subgroups of the glioblastoma. However, none of Wnt5a, Ryk or Ror2 had a prognostic impact on glioblastoma. For beta-catenin, a shorter progression-free survival was noted in the glioblastoma with oligodendroglioma component (GBMO) subgroup. CONCLUSIONS: Our results corroborated previous findings of Ryk-mediated Wnt5a effect, and suggested a role for Ror2 in the Wnt5a machinery in glioblastoma.
Huang D, etal., Gene. 2014 Aug 15;547(1):106-10. doi: 10.1016/j.gene.2014.06.035. Epub 2014 Jun 19.
Brachydactyly type B, an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails, can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is caused by mutations in the receptor tyrosine kinase gene ROR2
;'>ROR2, which maps to chromosome 9q22, whereas BDB2 is caused by point mutations in the bone morphogenetic protein antagonist NOGGIN. Here, we report a three-generation Chinese family with dominant inheritance of the BDB1 limb phenotype. Sequence analysis identified a novel heterozygous base deletion (c.1396-1398delAA) in the gene ROR2 in all affected family members. This new deletion is expected to produce a truncated Ror2 protein with a new polypeptide of 57 amino acids at the C-terminal.
Lima AR, etal., Hum Mutat. 2022 Jul;43(7):900-918. doi: 10.1002/humu.24375. Epub 2022 May 10.
Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as th
e main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.
Yang T, etal., J Dent Res. 2015 Jun;94(6):803-12. doi: 10.1177/0022034515576051. Epub 2015 Mar 6.
Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblas
t and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)-positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca(2+)/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.
OBJECTIVE: Robinow syndrome (RS) is an extremely rare genetic disorder characterized by short-limbed dwarfism, defects in vertebral segmentation and abnormalities in the head, face and external genitalia. Mutations in the ROR2 gene cause autosomal recessive RS (
RRS) whereas mutations in WNT5A are responsible for the autosomal dominant (AD) form of RS. In AD Robinow patients, oral manifestations are more prominent, while hemivertebrae and scoliosis rarely occur and facial abnormalities tend to be milder. METHODS: Three unrelated patients from different parts of India were studied. These patients were diagnosed as RRS due to presence of characteristic fetal facies, mesomelia, short stature, micropenis, hemivertebrae and rib abnormalities. One of the patients had fetal facies and micropenis but unusually mild skeletal features. This patient's mother had mild affection in the form of short stature and prominent eyes. Testosterone response to human chorionic gonadotropin was investigated in two patients and were normal. The exons and exon-intron boundaries of the ROR2 gene were sequenced for all probands. Bioinformatics analysis was done for putative variants using SIFT, PolyPhen2 and Mutation Taster. RESULTS: Patients 1, 2 and 3 were homozygous for c.G545A or p.C182Y in exon 5, c.227G>A or p.G76D in exon 3 and c.668G>A or p.C223Y in exon 6 respectively. Prenatal diagnosis could be performed in an ongoing pregnancy in one family and the fetus was confirmed to be unaffected. CONCLUSION: ROR2 mutations were documented for the first time in the Indian population. Knowledge of the molecular basis of the disorder served to provide accurate counseling and prenatal diagnosis to the families.
Wang H, etal., Chin Med J (Engl). 2012 Feb;125(3):476-80.
BACKGROUND: The receptor tyrosine kinase-like orphan receptor 2 (ROR2) gene has been recently shown to play important roles in palatal development in animal models and resides in the chromosomal region linked to non syndromic cleft lip with or without cleft pala
te in humans. The aim of this study was to investigate the possible association between ROR2 gene and non-syndromic oral clefts. METHODS: Here we tested 38 eligible single-nucleotide polymorphisms (SNPs) in ROR2 gene in 297 non-syndromic cleft lip with or without cleft palate and in 82 non-syndromic cleft palate case parent trios recruited from Asia and Maryland. Family Based Association Test was used to test for deviation from Mendelian inheritance. Plink software was used to test potential parent of origin effect. Possible maternally mediated in utero effects were assessed using the TRIad Multi-Marker approach under an assumption of mating symmetry in the population. RESULTS: Significant evidence of linkage and association was shown for 3 SNPs (rs7858435, rs10820914 and rs3905385) among 57 Asian non-syndromic cleft palate trios in Family Based Association Tests. P values for these 3 SNPs equaled to 0.000068, 0.000115 and 0.000464 respectively which were all less than the significance level (0.05/38 = 0.0013) adjusted by strict Bonferroni correction. Relevant odds ratios for the risk allele were 3.42 (1.80 - 6.50), 3.45 (1.75 - 6.67) and 2.94 (1.56 - 5.56), respectively. Statistical evidence of linkage and association was not shown for study groups other than non-syndromic cleft palate. Neither evidence for parent-of-origin nor maternal genotypic effect was shown for any of the ROR2 markers in our analysis for all study groups. CONCLUSION: Our results provided evidence of linkage and association between the ROR2 gene and a gene controlling risk to non-syndromic cleft palate.
Brachydactyly type B (BDB) is an autosomal dominant skeletal disorder characterized by hypoplasia/aplasia of distal phalanges and nails. Recently, heterozygous mutations of the orphan receptor tyrosine kinase (TK) ROR2, located within a distinct segment directly
after the TK domain, have been shown to be responsible for BDB. We report four novel mutations in ROR2 (two frameshifts, one splice mutation, and one nonsense mutation) in five families with BDB. The mutations predict truncation of the protein within two distinct regions immediately before and after the TK domain, resulting in a complete or partial loss of the intracellular portion of the protein. Patients affected with the distal mutations have a more severe phenotype than do those with the proximal mutation. Our analysis includes the first description of homozygous BDB in an individual with a 5-bp deletion proximal to the TK domain. His phenotype resembles an extreme form of brachydactyly, with extensive hypoplasia of the phalanges and metacarpals/metatarsals and absence of nails. In addition, he has vertebral anomalies, brachymelia of the arms, and a ventricular septal defect-features that are reminiscent of Robinow syndrome, which has also been shown to be caused by mutations in ROR2. The BDB phenotype, as well as the location and the nature of the BDB mutations, suggests a specific mutational effect that cannot be explained by simple haploinsufficiency and that is distinct from that in Robinow syndrome.
Brachydactyly type B1 (BDB1), an autosomal dominant condition characterized by terminal deficiency of the fingers and toes, results from mutations in the gene ROR2 encoding a receptor tyrosine kinase. In addition to BDB1, mutations in the gene ROR2
weight:700;'>ROR2 also cause a more severe form of skeletal dysplasia, autosomal recessive Robinow syndrome. The present study reports on a large Punjabi-speaking Pakistani family segregating autosomal dominant BDB1. In total, 34 individuals in this family showed features of BDB1. Sequence analysis of the gene ROR2 identified a previously reported nonsense mutation (c.2278C>T, p.Q760X) in all affected individuals of the family.
He L, etal., Clin Transl Oncol. 2015 Jun;17(6):438-45. doi: 10.1007/s12094-014-1254-y. Epub 2014 Nov 12.
PURPOSE: Chondrosarcoma is a malignant bone tumor with poor prognosis. Surgical treatment is the first choice for chondrosarcomas. Chondrosarcoma is not sensitive to chemotherapy and radiotherapy. Identification of biological markers is important for the early diagnosis and targeted treatment of ch
ondrosarcoma. This study investigated the protein expression and clinicopathological significance of ROR2 and FRAT1 in 59 chondrosarcomas and 33 osteochondromas. METHODS: ROR2 and FRAT1 protein expression in tissues was measured by immunohistochemistry. RESULTS: The percentage of positive ROR2 and FRAT1 expression was significantly higher in patients with chondrosarcoma than in patients with osteochondroma (P < 0.01). The percentage of positive ROR2 and FRAT1 expression was significantly lower in patients with histological grade I, AJCC stage I/II stage, Enneking stage I, non-metastatic and invasive chondrosarcoma than patients with histological grade III, AJCC stage III/IV, Enneking stage II + III, metastatic and invasive chondrosarcoma (P < 0.05 or P < 0.01). ROR2 expression was positively correlated with FRAT1 expression in chondrosarcoma. Kaplan-Meier survival analysis demonstrated that histological grade, AJCC stage, Enneking stage, metastasis, invasion, and ROR2 and FRAT1 expression significantly correlated with a short mean survival time of patients with chondrosarcoma (P < 0.05 or P < 0.01). Cox multivariate analysis showed that positive ROR2 and FRAT1 expression was an independent prognostic factor that negatively correlated with postoperative survival and positively correlated with mortality. CONCLUSION: Positive ROR2 and FRAT1 expression is associated with the progression and poor prognosis of chondrosarcoma.
Evolutionarily conserved receptor tyrosine kinase-like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocyt
ic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation.
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.
Wnt signaling regulates synaptic development and function and contributes to the fine-tuning of the molecular and morphological differentiation of synapses. We have shown previously that Wnt5a activates non-canonical Wnt signaling to stimulate postsynaptic differentiation in excitatory hippocampal
neurons promoting the clustering of the postsynaptic scaffold protein PSD-95 and the development of dendritic spines. At least three different kinds of Wnt receptors have been associated with Wnt5a signaling: seven trans-membrane Frizzled receptors and the tyrosine kinase receptors Ryk and ROR2. We report here that ROR2 is distributed in the dendrites of hippocampal neurons in close proximity to synaptic contacts and it is contained in dendritic spine protrusions. We demonstrate that ROR2 is necessary to maintain dendritic spine number and morphological distribution in cultured hippocampal neurons. ROR2 overexpression increased dendritic spine growth without affecting the density of dendritic spine protrusions in a form dependent on its extracellular Wnt binding cysteine rich domain (CRD) and kinase domain. Overexpression of dominant negative ROR2 lacking the extracellular CRD decreased spine density and the proportion of mushroom like spines, while ROR2 lacking the C-terminal and active kinase domains only affected spine morphology. Our results indicate a crucial role of the ROR2 in the formation and maturation of the postsynaptic dendritic spines in hippocampal neurons.
Cerpa W, etal., Proc Natl Acad Sci U S A. 2015 Apr 14;112(15):4797-802. doi: 10.1073/pnas.1417053112. Epub 2015 Mar 30.
Wnt signaling has a well-established role as a regulator of nervous system development, but its role in the maintenance and regulation of established synapses in the mature brain remains poorly understood. At excitatory glutamatergic synapses, NMDA receptors (NMDARs) have a fundamental role in synap
togenesis, synaptic plasticity, and learning and memory; however, it is not known what controls their number and subunit composition. Here we show that the receptor tyrosine kinase-like orphan receptor 2 (RoR2) functions as a Wnt receptor required to maintain basal NMDAR-mediated synaptic transmission. In addition, RoR2 activation by a noncanonical Wnt ligand activates PKC and JNK and acutely enhances NMDAR synaptic responses. Regulation of a key component of glutamatergic synapses through RoR2 provides a mechanism for Wnt signaling to modulate synaptic transmission, synaptic plasticity, and brain function acutely beyond embryonic development.
Mutations in ROR2, encoding the receptor tyrosine kinase-like orphan receptor 2, cause two distinct skeletal diseases: autosomal dominant brachydactyly type B1 (BDB1) and autosomal recessive Robinow syndrome. In a large Chinese family with a limb phenotype, con
sisting of atypical BDB1 and cutaneous syndactyly of varying degrees, we performed a two-point linkage analysis using microsatellite markers on 2q33-q37 and 9q22.31, and found a significant linkage to the ROR2 locus. We identified a novel single-base deletion in ROR2, c.2243delC (p.W749fsX24), and confirmed its segregation with the limb phenotype in the family. This deletion is predicted to produce a truncated ROR2 protein with an additional C-terminal polypeptide of 24 amino-acid residues. To the best of our knowledge, the deletion represents the second ROR2 mutation associated with a BDB1-syndactyly phenotype.
The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac meso
derm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications.
Aglan M, etal., Am J Med Genet A. 2015 Dec;167A(12):3054-61. doi: 10.1002/ajmg.a.37287. Epub 2015 Aug 18.
Robinow syndrome (RS) is a rare genetic disorder characterized by limb shortening, genital hypoplasia, and craniofacial/orodental abnormalities. The syndrome follows both autosomal dominant and recessive patterns of inheritance with similar phenotypic presentation and overlapping features. Autosomal
recessive Robinow syndrome (ARRS) is caused by mutations in the ROR2 gene. Here, we present the clinical, radiological and molecular findings of 11 Egyptian patients from 7 unrelated consanguineous families with clinical features of ARRS. Mutation analyses of ROR2 gene identified five pathogenic mutations distributed all over the gene. The identified mutations included four novel (G326A, D166H, S677F, and R528Q) and one previously reported (Y192D). Our results extend the number of ROR2 mutations identified so far, suggest a founder effect in the Egyptian population, and emphasize the important role of genetic testing in proper counseling and patients' management.
Huang J, etal., Sci Rep. 2015 Aug 11;5:12991. doi: 10.1038/srep12991.
RTK-like orphan receptor 2 (ROR2) is overexpressed in several cancers and has tumorigenic activity. However, the expression of ROR2 and its functional and prognostic significance have yet to be evaluated in pancreatic ductal
adenocarcinoma (PDAC). Quantitative real-time polymerase chain reaction was used to characterize the expression of ROR2 mRNA in PDAC, corresponding peritumoral tissues, and PDAC cell lines. Immunohistochemical analysis with tissue microarrays was used to evaluate ROR2 expression in PDAC and to investigate the relationship of this expression to clinicopathological factors and prognosis. The expression of ROR2 mRNA and protein was significantly higher in PDAC than in normal pancreatic tissues. High cytoplasmic ROR2 expression in cancer cells was significantly associated with a primary tumor, distant metastasis, and TNM stage, and high stromal ROR2 expression was significantly associated with regional lymph node metastasis and TNM stage. The Kaplan-Meier method and Cox regression analyses showed that high ROR2 expression in tumor cytoplasm or stromal cells was significantly associated with malignant attributes and reduced survival in PDAC. We present strong evidence that ROR2 could be used as an indicator of poor prognosis and could represent a novel therapeutic target for PDAC.
van Zuylen WJ, etal., J Virol. 2015 Nov 11;90(2):1108-15. doi: 10.1128/JVI.02588-15.
Maternal primary cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates that fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus but also
from indirect effects through placental infection and dysfunction. CMV alters Wingless (Wnt) signaling, an essential cellular pathway involved in placentation, as evidenced by reduced transcription of canonical Wnt target genes and decreased Wnt3a-induced trophoblast migration. Whether CMV affects the noncanonical Wnt signaling pathway has been unclear. This study demonstrates for the first time that CMV infection inhibits Wnt5a-stimulated migration of human SGHPL-4 trophoblasts and that inhibition of the pathway restores normal migration of CMV-infected cells. Western blot and real-time PCR analyses show increased expression of noncanonical Wnt receptor ROR2 in CMV-infected trophoblasts. Mimicking the CMV-induced ROR2 protein expression via ectopic expression inhibited Wnt5a-induced trophoblast migration and reduced T cell-specific factor (TCF)/lymphoid enhancer-binding factor (LEF)-mediated transcription as measured using luciferase reporter assays. Gene silencing using small interfering RNA (siRNA) duplexes decreased ROR2 transcript and protein levels. In contrast, proliferation of SGHPL-4 trophoblasts, measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was not affected. The siRNA-mediated downregulation of ROR2 in trophoblasts rescued CMV-induced reduction in trophoblast migration. These data suggest a mechanism where CMV alters the expression of the Wnt receptor ROR2 to alter Wnt5a-mediated signaling and inhibit trophoblast motility. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage and consequent fetal damage in congenital CMV infections. IMPORTANCE: Maternal primary cytomegalovirus (CMV) infection, reactivation, or reinfection with a different viral strain may cause fetal injury and adverse pregnancy outcomes. Increasing evidence indicates that fetal injury results not only from direct viral cytopathic damage to the CMV-infected fetus but also from indirect effects through placental infection and placental dysfunction. No effective therapy is currently proven to prevent or treat congenital CMV infection. Understanding the molecular underpinnings of CMV infection of the placenta is essential for therapeutic innovations and vaccine design. CMV alters canonical Wingless (Wnt) signaling, an essential cellular pathway involved in placental development. This study suggests a mechanism in which CMV alters the expression of noncanonical Wnt receptor ROR2 to alter motility of placental cells, which has important implications in the pathogenesis of CMV-induced placental dysfunction. Inhibition of this mechanism may be a target for therapeutic intervention for CMV-induced placental damage and consequent fetal damage in congenital CMV infection.
ROR2 is a member of the cell surface receptor tyrosine kinase (RTKs) family of proteins and is involved in the developmental morphogenesis of the skeletal, cardiovascular and genital systems. Mutations in ROR2 have been show
n to cause two distinct human disorders, autosomal recessive Robinow syndrome and dominantly inherited Brachydactyly type B. The recessive form of Robinow syndrome is a disorder caused by loss-of-function mutations whereas Brachydactyly type B is a dominant disease and is presumably caused by gain-of-function mutations in the same gene. We have previously established that all the missense mutations causing Robinow syndrome in ROR2 are retained in the endoplasmic reticulum and therefore concluded that their loss of function is due to a defect in their intracellular trafficking. These mutations were in the distal portion of the frizzled-like cysteine rich domain and kringle domain. Here we report the identification of two novel mutations in the frizzled-like cysteine-rich domain of ROR2 causing Robinow syndrome. We establish the retention of the mutated proteins in the endoplasmic reticulum of HeLa cells and therefore failure to reach the plasma membrane. The clustering of Robinow-causing mutations in the extracellular frizzled-like cysteine-rich domain of ROR2 suggests a stringent requirement for the correct folding of this domain prior to export of ROR2 from the endoplasmic reticulum to the plasma membrane.
Lu C, etal., Oncotarget. 2015 Sep 22;6(28):24912-21. doi: 10.18632/oncotarget.4701.
We investigated the expression of receptor tyrosine kinase-like orphan receptor (ROR) 2 and Wnt5a and their prognostic significance in non-small cell lung cancer (NSCLC). Tissue microarray-based immunohistochemical analysis was performed to determine the expression of ROR2
ROR2 and Wnt5a in 219 patients. mRNA expression of ROR2 and Wnt5a was examined in 20 pairs of NSCLC and matched adjacent normal tissues by real-time PCR. Compared with non-tumorous tissues, both mRNA expression and protein product of ROR2 and Wnt5a genes were significantly increased in NSCLC. c2 analysis revealed that high ROR2 or Wnt5a expression in NSCLC was significantly associated with advanced TNM stage. High expression of both ROR2 and Wnt5a was also related to advanced TNM stage. Multivariate analyses suggested that ROR2, Wnt5a and TNM stage were independent prognostic factors in NSCLC. Our clinical findings suggest that high ROR2 or Wnt5a expression is associated with poor prognosis in NSCLC, and combined detection of ROR2 and Wnt5a is helpful in predicting the prognosis of NSCLC.
The autosomal recessive form of Robinow syndrome (RRS; MIM 268310) is a severe skeletal dysplasia with generalized limb bone shortening, segmental defects of the spine, brachydactyly and a dysmorphic facial appearance. We previously mapped the gene mutated in RRS to chromosome 9q22 (ref. 4), a regio
n that overlaps the locus for autosomal dominant brachydactyly type B (refs 5,6). The recent identification of ROR2, encoding an orphan receptor tyrosine kinase, as the gene mutated in brachydactyly type B (BDB1; ref. 7) and the mesomelic dwarfing in mice homozygous for a lacZ and/or a neo insertion into Ror2 (refs 8,9) made this gene a candidate for RRS. Here we report homozygous missense mutations in both intracellular and extracellular domains of ROR2 in affected individuals from 3 unrelated consanguineous families, and a nonsense mutation that removes the tyrosine kinase domain and all subsequent 3' regions of the gene in 14 patients from 7 families from Oman. The nature of these mutations suggests that RRS is caused by loss of ROR2 activity. The identification of mutations in three distinct domains (containing Frizzled-like, kringle and tyrosine kinase motifs) indicates that these are all essential for ROR2 function.
Schwabe GC, etal., Dev Dyn. 2004 Feb;229(2):400-10.
Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2(-/-) mice as a model for the developmental pathology of RS. Our results dem
onstrate that vertebral malformations in Ror2(-/-) mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2(-/-) mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2(-/-) mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2(-/-) mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome.
Henry C, etal., Oncotarget. 2015 Nov 24;6(37):40310-26. doi: 10.18632/oncotarget.5643.
AIM: In recent years, the Wnt signalling pathway has been implicated in epithelial ovarian cancer and its members have potential as diagnostic, prognostic and therapeutic targets. Here we investigated the role of two Wnt receptor tyrosine kinases (RTKs), ROR1 and ROR2
/span>, and their putative ligand, Wnt5a, in ovarian cancer. METHODS: Immunohistochemistry for ROR2 was performed in a large patient cohort, including benign controls, borderline tumours and epithelial ovarian cancer. In addition, siRNA was used to silence ROR1, ROR2 and Wnt5a individually, and together, in two ovarian cancer cell lines, and the effects on cell proliferation, adhesion, migration and invasion were measured. RESULTS: ROR2 expression is significantly increased in ovarian cancer patients compared to patients with benign disease. In vitro assays showed that silencing either receptor inhibits ovarian cancer cell migration and invasion, and concurrently silencing both receptors has an even stronger inhibitory effect on proliferation, migration and invasion. CONCLUSIONS: ROR2 expression is increased in epithelial ovarian cancer, and silencing ROR2 and its sister receptor ROR1 has a strong inhibitory effect on the ability of ovarian cancer cells to proliferate, migrate and invade through an extracellular matrix.
Sato A, etal., Sci Rep. 2015 Jun 1;5:10536. doi: 10.1038/srep10536.
Wnt5a, which regulates various cellular functions in Wnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2
700;'>Ror2. Removing Wnt5a or Ror2 in adult mice suppressed dextran sodium sulfate (DSS)-induced colitis. It also attenuated the DSS-dependent increase in inflammatory cytokine production and decreased interferon-gamma (IFN-gamma)-producing CD4(+) Th1 cell numbers in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients. Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naive CD4(+) T cells to IFN-gamma-producing CD4(+) Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-gamma, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-gamma signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.
Xin H, etal., Int J Mol Med. 2013 Mar;31(3):583-8. doi: 10.3892/ijmm.2013.1242. Epub 2013 Jan 15.
Accumulating evidence have demonstrated that mesenchymal stem cells (MSCs) are involved in the initiation and progression of various vascular diseases. Canonical Wnt signaling controls the fate of MSCs, and plays an important role in vascular calcification. However, vascular calcification can be inh
ibited by the non-canonical Wnt signaling pathway Wnt5a/Ror2. This study aimed to investigate whether the Wnt5a/Ror2 pathway is associated with determination of the differentiation fate of MSCs in vascular calcification. Direct co-cultures were established by seeding smooth muscle cells (SMCs) or calcified SMCs and MSCs together at ratios of SMCs or calcified SMCs 15x104; SMCs or calcified SMCs 5x104: MSCs 10x104, SMCs or calcified SMCs 10x104: MSCs 5x104. Osteosynthesis-inducing medium (OS) was added to the culture medium in the groups of MSCs with non-calcified SMCs. Cells were cultured for nine days. Osteoblastic differentiation was evaluated by cell morphology and the activity of alkaline phosphatase (ALP) in cell lysates and ALP staining. Furthermore, we investigated the inhibition of Wnt signaling, and observed that the members of the non-canonical signaling pathway Wnt5a/Ror2 were expressed in each group. Additionally, MSCs cultured in culture media with OS did not differentiate into an osteoblast phenotype when in direct contact with non-calcified SMCs, irrespective of the number of MSCs. However, an osteoblast phenotype was observed when MSCs were cultured in media without OS differentiation towards direct contact with calcified SMCs, and the levels of osteoblastic markers had a direct correlation with the number of MSCs. Of note, the Wnt5a protein was associated with the levels of calcification, thus, although rarely detected in non-calcification, Ror2 mRNA in the non-calcified groups was significantly higher compared to that in the calcified groups. Therefore, the Wnt5a/Ror2 pathway is associated with determination of the differentiation fate of bone marrow mesenchymal stem cells in vascular calcification.
Takiguchi G, etal., Cancer Sci. 2016 Mar;107(3):290-7. doi: 10.1111/cas.12871. Epub 2016 Feb 9.
Wnt5a-Ror2 signaling has been shown to play important roles in promoting aggressiveness of various cancer cells in a cell-autonomous manner. However, little is known about its function in cancer-associated stromal cells, including mesenchymal stem cells (MSCs).
Thus, we examined the role of Wnt5a-Ror2 signaling in bone marrow-derived MSCs in regulating proliferation of undifferentiated gastric cancer cells. Coculture of a gastric cancer cell line, MKN45, with MSCs either directly or indirectly promotes proliferation of MKN45 cells, and suppressed expression of Ror2 in MSCs prior to coculture inhibits enhanced proliferation of MKN45 cells. In addition, conditioned media from MSCs, treated with control siRNA, but not siRNAs against Ror2, can enhance proliferation of MKN45 cells. Interestingly, it was found that expression of CXCL16 in MSCs is augmented by Wnt5a-Ror2 signaling, and that recombinant chemokine (C-X-C motif) ligand (CXCL)16 protein can enhance proliferation of MKN45 cells in the absence of MSCs. In fact, suppressed expression of CXCL16 in MSCs or an addition of a neutralizing antibody against CXCL16 fails to promote proliferation of MKN45 cells in either direct or indirect coculture with MSCs. Importantly, we show that MKN45 cells express chemokine (C-X-C motif) receptor (CXCR)6, a receptor for CXCL16, and that suppressed expression of CXCR6 in MKN45 cells results in a failure of its enhanced proliferation in either direct or indirect coculture with MSCs. These findings indicate that Wnt5a-Ror2 signaling enhances expression of CXCL16 in MSCs and, as a result, enhanced secretion of CXCL16 from MSCs might act on CXCR6 expressed on MKN45, leading to the promotion of its proliferation.
Raz R, etal., Development. 2008 May;135(9):1713-23. doi: 10.1242/dev.015149. Epub 2008 Mar 19.
Mutations in ROR2 result in a spectrum of genetic disorders in humans that are classified, depending on the nature of the mutation and the clinical phenotype, as either autosomal dominant brachydactyly type B (BDB, MIM 113000) or recessive Robinow syndrome (RRS
, MIM 268310). In an attempt to model BDB in mice, the mutation W749X was engineered into the mouse Ror2 gene. In contrast to the human situation, mice heterozygous for Ror2(W749FLAG) are normal and do not develop brachydactyly, whereas homozygous mice exhibit features resembling RRS. Furthermore, both Ror2(W749FLAG/W749FLAG) and a previously engineered mutant, Ror2(TMlacZ/TMlacZ), lack the P2/P3 joint. Absence of Gdf5 expression at the corresponding interzone suggests that the defect is in specification of the joint. As this phenotype is absent in mice lacking the entire Ror2 gene, it appears that specification of the P2/P3 joint is affected by ROR2 activity. Finally, Ror2(W749FLAG/W749FLAG) mice survive to adulthood and exhibit phenotypes (altered body composition, reduced male fertility) not observed in Ror2 knockout mice, presumably due to the perinatal lethality of the latter. Therefore, Ror2(W749FLAG/W749FLAG) mice represent a postnatal model for RRS, provide insight into the mechanism of joint specification, and uncover novel roles of Ror2 in the mouse.
