Ror-family Receptor Tyrosine Kinases Research Articles

Role of noncanonical Wnt ligands and Ror-family receptor tyrosine kinases in the development, regeneration, and diseases of the musculoskeletal system.

The Ror-family receptor tyrosine kinases (RTKs), consisting of Ror1 and Ror2, play crucial roles in morphogenesis and formation of various tissues/organs, including the bones and skeletal muscles, the so-called musculoskeletal system, during embryonic development, by acting as receptors or coreceptors for a noncanonical Wnt protein Wnt5a. Furthermore, several lines of evidence have indicated that Ror1 and/or Ror2 play critical roles in the regeneration and maintenance of the musculoskeletal system in adults. Considering the anatomical and functional relationship between the skeleton and skeletal muscles, their structural and functional association might be tightly regulated during their embryonic development, development after birth, and their regeneration after injury in adults. Importantly, in addition to their congenital anomalies, much attention has been paid onto the age-related disorders of the musculoskeletal system, including osteopenia and sarcopenia, which affect severely the quality of life. In this article, we overview recent advances in our understanding of the roles of Ror1- and/or Ror2-mediated signaling in the embryonic development, regeneration in adults, and congenital and age-related disorders of the musculoskeletal system and discuss possible therapeutic approaches to locomotive syndromes by modulating Ror1- and/or Ror2-mediated signaling.

Critical role of the Ror-family of receptor tyrosine kinases in invasion and proliferation of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis and closely related to exposure to asbestos. MPM is a heterogeneous tumor with three main histological subtypes, epithelioid, sarcomatoid, and biphasic types, among which sarcomatoid type shows the poorest prognosis. The Ror-family of receptor tyrosine kinases, Ror1 and Ror2, is expressed in various types of tumor cells at higher levels and affects their aggressiveness. However, it is currently unknown whether they are expressed in and involved in aggressiveness of MPM. Here, we show that Ror1 and Ror2 are expressed in clinical specimens and cell lines of MPM with different histological features. Studies using MPM cell lines indicate that expression of Ror2 is associated tightly with high invasiveness of MPM cells, whereas Ror1 can contribute to their invasion in the absence of Ror2. However, both Ror1 and Ror2 promote proliferation of MPM cells. We also show that promoted invasion and proliferation of MPM cells by Ror signaling can be mediated by the Rho-family of small GTPases, Rac1, and Cdc42. These findings elucidate the critical role of Ror signaling in promoting invasion and proliferation of MPM cells.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle

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.

Diverse roles for the ror-family receptor tyrosine kinases in neurons and glial cells during development and repair of the nervous system.

The Ror-family of receptor tyrosine kinases (RTKs) are involved critically in tissue genesis and organogenesis during development. In mammals, Ror1 and Ror2, members of the Ror-family RTKs, have been shown to mediate cell polarity, migration, proliferation, and differentiation through the activation of noncanonical Wnt signaling by acting as receptors or co-receptors for Wnt5a. Nematodes bearing mutations within the cam-1 gene, encoding a Ror2 ortholog, exhibit defects in various developmental processes of the nervous system, including neuronal cell migration, polarization, axonal extension, and synaptic transmission. In mice, Ror2 and/or Ror1 are also shown to play roles in regulating neurite extension, synapse formation, and synaptic transmission of hippocampal neurons, indicating that the Ror-family RTKs have evolutionarily conserved functions at least in part in neurons during development. Furthermore, Ror2 and/or Ror1 are expressed in neural stem/progenitor cells of the developing brain and in astrocytes of the adult brain after injury, and they play important roles in regulating cell proliferation under these different contexts. In this article, we overview recent advances in our understanding of the roles of the Ror-family RTKs in the development and repair of the nervous system and discuss their potential for therapeutic targets to neurodegenerative diseases. Developmental Dynamics 247:24-32, 2018. © 2017 Wiley Periodicals, Inc.

ROR-Family Receptor Tyrosine Kinases.

ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients.

WNT5A-ROR2 is induced by inflammatory mediators and is involved in the migration of human ovarian cancer cell line SKOV-3.

BackgroundWnt5A, which is a member of the non-transforming Wnt protein family, is implicated in inflammatory processes. It is also highly expressed by ovarian cancer cells. ROR2, which is a member of the Ror-family of receptor tyrosine kinases, acts as a receptor or co-receptor for Wnt5A. The Wnt5A–ROR2 signaling pathway plays essential roles in the migration and invasion of several types of tumor cell and influences their cell polarity. We investigated the modulation of Wnt5A–ROR2 by inflammatory mediators and its involvement in the migration of the human ovarian cancer cell line SKOV-3.MethodsSKOV-3 cells were treated with LPS (lipopolysaccharide), LTA (lipoteichoic acid) and recombinant human IL-6 alone or in combination with STAT3 inhibitor (S1155S31-201) or NF-kB inhibitor (BAY11-7082) for 4, 8, 12, 24 and 48 h. The Wnt5A and ROR2 expression levels were determined at the gene and protein levels. Cells were transfected with specific siRNA against Wnt5A in the absence or presence of human anti-ROR2 antibody and cell migration was assessed using transwells.ResultsThere was a strong downregulation of Wnt5A expression in the presence of STAT3 or NF-kB inhibitors. Cell stimulation with LTA or IL-6 for 8 h led to significantly increased levels of Wnt5A (5- and 3-fold higher, respectively). LPS, LTA or IL-6 treatment significantly increased ROR2 expression (2-fold after 48 h). LPS- or LTA-induced Wnt5A or ROR2 expression was abrogated in the presence of STAT3 inhibitor (p < 0.001). IL-6-induced Wnt5A expression was abrogated by both STAT3 and NF-kB inhibitors (p < 0.001). Although not significant, IL-6-induced ROR2 expression showed a modest decrease when STAT3 inhibitor was used. Moreover, cell migration was decreased by 80 % in siRNA Wnt5A-transfected cells in the presence of anti-human ROR2 antibody (p < 0.001).ConclusionsThis study revealed for the first time that inflammatory mediators modulate Wnt5A and ROR2 through NF-kB and STAT3 transcription factors and this may play a role in ovarian cancer cell migration. The results described here provide new insight into the role of the Wnt5A–ROR2 complex in ovarian cancer progression in relation to inflammation.

Essential role of Wnt5a-Ror1/Ror2 signaling in metanephric mesenchyme and ureteric bud formation.

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.

Insight into the role of Wnt5a-induced signaling in normal and cancer cells.

Wnt5a is involved in the activation of noncanonical Wnt signaling, including planar cell polarity (PCP) and Wnt-Ca(2+) pathways. The Ror-family of receptor tyrosine kinases is composed of Ror1 and Ror2 in mammals. Ror2 acts as a receptor or coreceptor for Wnt5a and regulates Wnt5a-induced activation of PCP pathway, and Wnt5a-Ror2 axis indeed plays critical roles in the developmental morphogenesis by regulating cell polarity and migration. Furthermore, Wnt5a-Ror2 axis is constitutively activated in cancer cells and confers highly motile and invasive properties on cancer cells through the expression of matrix metalloproteinase genes and enhanced formation of invadopodia. Meanwhile, Wnt5a also exhibits a tumor-suppressive function in certain cancers, including breast and colorectal carcinomas. Thus, it is of great importance to understand the respective molecular mechanisms governing Wnt5a-mediated tumor-progressive and tumor-suppressive functions, in order to develop novel and proper diagnostic and therapeutic strategies targeting Wnt5a signaling for human cancers.

Abstract B206: Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers.

Abstract Protein kinases play key roles in defining the transformation of many solid tumors, including renal cell carcinoma (RCC), and have become attractive drug targets. The Ror-family receptor tyrosine kinases (RTKs) are transmembrane proteins with putative tyrosine kinase activities that play crucial roles during the development of various organs and tissues. One of these receptors, the RTK-like orphan receptor 2 (Ror2) is known as a developmentally regulated receptor that enhances tumor cell migration and tumor invasiveness. Recently, our lab reported on the expression of Ror2 in human RCC tumors and cell lines, and that its expression is correlated with invasive growth in culture. In mammals, Ror2 has been shown to act as a receptor or co-receptor for Wnt5a, a member of the Wnt family, inducing a noncanonical Wnt signaling cascade. We have found that Ror2 is expressed in various cell lines, including 786-O, HEK293, HeLa, SaOS2, and U2OS. Cell migration analysis using single cell tracking confirmed that Ror2 promotes cell migration, further enhanced by Wnt5a stimulation. Separately, we have shown that Ror2 expression correlates with enhanced canonical Wnt-signaling through an increased pool of downstream stable β-catenin in RCC and activation of canonical targets. However, the kinase activity of Ror2 has been controversial. Using 786-O Ror2 overexpressing cells (786-O/Ror2), we detected that Ror2 becomes phosphorylated upon Wnt5a treatment. Based on a report of antibody induced homodimerization of Ror2 necessary for stimulation, we treated 786-O/Ror2 cells with Ror2 antibody and verified a significantly enhanced phosphorylation. Based on these findings, we hypothesize that receptor dimerization via Wnt ligand engagement or antibody treatment is necessary for effective signal transduction. We have thus utilized the PathHunter Ror2 activity assay developed by DiscoveRX, to use blockade of dimerization as an assay for Ror2 targeted drug development. This system utilizes an EGFR/Ror2 chimera cell line that expresses the cytosolic portion of ROR2 containing the kinase domain tagged with a ProLink tag at the C-terminus and fused to the extracellular and transmembrane domains of EGFR. Receptor activation is mediated by EGF addition, which results in a dose-dependent increase in signal caused by complementation of the SH2 tagged with the complementary EA enzyme fragment binding to the phosphorylated receptor. Thus, activation reads out in dimerization and phosphorylation which in turn, results in enzyme fragment complementation in this assay. Our data using this EGFR/Ror2 chimera U2OS cell line show that stimulation of these cells with EGF induces phosphorylation to a great extent both by IP/western, and chimeric signal. We believe these tools are useful in screening compounds in search of Ror2 inhibitors for RCC or other cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B206. Citation Format: Zufan Debebe, Melissa Porter, Emily E. Hull-Ryde, Neal Rasmussen, Adam Sendor, Jacqueline Norris-Drouin, Keefe Chan, James E. Bear, William P. Janzen, Kimryn Rathmell. Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B206.

Ror-family receptor tyrosine kinases regulate maintenance of neural progenitor cells in the developing neocortex

The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of βIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

Mice lacking the orphan receptor ror1 have distinct skeletal abnormalities and are growth retarded

Ror1 is a member of the Ror-family receptor tyrosine kinases. Ror1 is broadly expressed in various tissues and organs during mouse embryonic development. However, so far little is known about its function. The closely related family member Ror2 was shown to play a crucial role in skeletogenesis and has been shown to act as a co-receptor for Wnt5a mediating non-canonical Wnt-signaling. Previously, it has been shown that during embryonic development Ror1 acts in part redundantly with Ror2 in the skeletal and cardiovascular systems. In this study, we report that loss of the orphan receptor Ror1 results in a variety of phenotypic defects within the skeletal and urogenital systems and that Ror1 mutant mice display a postnatal growth retardation phenotype.

Ror‐family receptor tyrosine kinases in noncanonical Wnt signaling: Their implications in developmental morphogenesis and human diseases

The Ror-family receptor tyrosine kinases (RTKs) play crucial roles in the development of various organs and tissues. In mammals, Ror2, a member of the Ror-family RTKs, has been shown to act as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Ror2- and Wnt5a-deficient mice exhibit similar abnormalities during developmental morphogenesis, reflecting their defects in convergent extension movements and planar cell polarity, characteristic features mediated by noncanonical Wnt signaling. Furthermore, mutations within the human Ror2 gene are responsible for the genetic skeletal disorders dominant brachydactyly type B and recessive Robinow syndrome. Accumulating evidence demonstrate that Ror2 mediates noncanonical Wnt5a signaling by inhibiting the beta-catenin-TCF pathway and activating the Wnt/JNK pathway that results in polarized cell migration. In this article, we review recent progress in understanding the roles of noncanonical Wnt5a/Ror2 signaling in developmental morphogenesis and in human diseases, including heritable skeletal disorders and tumor invasion.

The Receptor Tyrosine Kinase Ror2 Associates with the Melanoma-associated Antigen (MAGE) Family Protein Dlxin-1 and Regulates Its Intracellular Distribution

The mammalian Ror family receptor tyrosine kinases, Ror1 and Ror2, play crucial roles in developmental morphogenesis. Although the functions of Ror1 and Ror2 are redundant, Ror2 exhibits more specific functions during development. We show that when expressed in mammalian cells, Ror2, but not Ror1, associates with the melanoma-associated antigen (MAGE) family protein, Dlxin-1, which is known to bind to the homeodomain proteins Msx2 and Dlx5 and regulate their transcriptional functions. This association requires the cytoplasmic C-terminal region of Ror2, containing proline-rich and serine/threonine-rich domains, and the C-terminal necdin homology domain of Dlxin-1. Interestingly, the cytoplasmic C-terminal region of Ror2 is missing in patients with brachydactyly type B. Interestingly, transient expression and immunohistochemical analyses reveal that both Dlxin-1 and Msx2 are co-localized in the nuclei in the absence of Ror2. In the presence of Ror2, Dlxin-1 is co-localized with Ror2 at the membranous compartments and Msx2 is retained in the nuclei. It was also found that the majority of cellular Dlxin-1 is retained in the membrane fractions of wild-type but not Ror2-/- mouse embryonic fibroblasts. Furthermore, we show that transcriptional activity of Msx2, irrespective of Ror2 kinase activity, is regulated by ectopic expression of Ror2 using a reporter plasmid containing the WIP element. Thus, Ror2 sequesters Dlxin-1 in membranous compartments, thereby affecting the transcriptional function of Msx2.

Expression of the receptor tyrosine kinase genes, Ror1 and Ror2, during mouse development

In mammals, the Ror-family receptor tyrosine kinases consist of two structurally related proteins, Ror1 and Ror2, characterized by the extracellular Frizzled-like cysteine-rich domain and membrane proximal kringle domains. As an attempt to gain insights into their roles in mouse development, expression patterns of Ror1 and Ror2 during early embryogenesis were examined and compared. Interestingly, at early stages, Ror1 and Ror2 exhibit similar expression patterns in the developing face, including the frontonasal process and pharyngeal arches, which are derived from cephalic neural crest cells. On the other hand, they exhibit different expression patterns in the developing limbs and brain, where the expression of Ror2 was detected broadly compared with that of Ror1. At a later stage, both genes are expressed in a similar fashion in the developing heart and lung, yet in a distinct manner in the brain and eye.