Search

Protein R-spondin 2, which is known as roof plate-specific spondin 2, is an extracellular matrix secreted protein that participates in a wide range of biological processes. However, the expression of R-spondin 2 in triple negative breast cancer (TNBC) and its specific mechanism have not been reported. In this study, RT-qPCR and western blot were used to detect the expression of R-spondin 2 and Axin2 in cells. Cell transfection techniques were used to overexpress Axin2 and interfere with the expression of R-spondin 2. CCk-8 and clone formation assay were used to detect cell viability. Wound healing and Traswell techniques were used to test the rate of invasion and migration of TNBC cells. Western blot was used to detect the expression of related proteins. The results showed that the expression of R-spondin 2 in TNBC cell lines was significantly increased compared with normal breast cancer cells. After interfering with the expression of R-spondin 2 in TNBC cell lines, the rate of cell viability, invasion and migration were decreased. It was also found that the expressions of Axin2 and β-catenin and Cyclin-D1, which are wnt/β-catenin pathway related proteins, were significantly decreased. Subsequently, the overexpression of Axin2 can inhibit the proliferation, invasion and migration that were ever promoted by R-spondin 2 of TNBC cells. Moreover, the overexpression of Axin2 inhibited the activation of wnt/β-catenin signaling pathway, which was also activated by R-spondin 2 in TNBC cells. In a word, R-spondin 2 promoted proliferation, invasion and migration of triple negative breast cancer cells through activating wnt/β-catenin signaling pathway after Axin2 inhibition.

The R-spondin family of proteins are Wnt agonists, and the complete embryonic disruption of Rspo2 results in skeletal developmental defects that recapitulate the phenotype observed with Lrp5/6 deficiency. Previous work has shown that R-spondin-2 (Rspo2, RSPO2) is both highly expressed in Wnt-stimulated pre-osteoblasts and its overexpression induces osteoblast differentiation in the same cells, supporting its putative role as a positive autocrine regulator of osteoblastogenesis. However, the role of Rspo2 in regulating osteoblastogenesis and bone formation in postnatal bone has not been explored. Here we show that limb-bud progenitor cells from Rspo2 knockout mice undergo reduced mineralization during osteoblastogenesis in vitro and have a corresponding alteration in their osteogenic gene expression profile. We also generated the first Rspo2 conditional knockout (Rspo2floxed) mouse and disrupted Rspo2 expression in osteoblast-lineage cells by crossing to the Osteocalcin-Cre mouse line (Ocn-Cre + Rspo2f/f). Ocn-Cre + Rspo2f/f male and female mice at 1, 3, and 6 months were examined. Ocn-Cre + Rspo2f/f mice are decreased in overall body size compared to their control littermates and have decreased bone mass. Histomorphometric analysis of 1-month-old mice revealed a similar number of osteoblasts and mineralizing surface per bone surface with a simultaneous decrease in mineral apposition and bone formation rates. Consistent with this observation, serum osteocalcin in 3-month-old Ocn-Cre + Rspo2f/f was reduced, and bone marrow-mesenchymal stem cells from Ocn-Cre + Rspo2f/f mice undergo less mineralization in vitro. Finally, gene expression analysis and immunohistochemistry of mature bone shows reduced beta-catenin signaling in Ocn-Cre + Rspo2f/f. Overall, RSPO2 reduces osteoblastogenesis and mineralization, leading to reduced bone mass., Bone Physiology: R-spondin-2 reduces mineralization, leading to decreased bone mass A loss of R-spondin-2 reduces osteoblastogenesis (production of osteoblasts, the cells from which bone develops) and mineralization, thereby leading to decreased bone mass in adults. R-spondin-2 is one of a family of four proteins that are expressed in the developing mouse limb as well as other tissues; each R-spondin family member exerts a different functional effect. R-spondins clearly influence several aspects of skeletal biology, but their specific roles—especially in postnatal bone—remained to be elucidated. A team headed by Kurt Hankenson at the University of Michigan Medical School investigated the role of R-spondin-2 in osteoblastogenesis, both in vitro and in vivo, using a mouse model. For the first time, the team was able to demonstrate that R-spondin-2 promotes osteoblastogenesis, bone development, and consequent bone mass growth in adult mice.

R-spondin proteins are identified as secreted agonists of the canonical Wnt/β-catenin signaling pathway, and leucine-rich repeat-containing G-protein-coupled receptors (LGR) are recognized as R-spondin receptors. The potential role of R-spondin 2 (Rspo2) and LGR4 in mediating osteogenesis remains poorly understood. In our in vitro experiments, we found that Rspo2 could promote osteogenesis through activating the Wnt signaling pathway in MC3T3-E1 cells. However, this effect of Rsop2 disappeared in the cells with functional disruption of LGR4. Meanwhile, Rspo2 significantly inhibited osteoclastogenesis and this effect of Rspo2 was dependent on the presence of osteoblasts with normal function of LGR4. In our in vivo experiments, we found that application of exogenous Rspo2 rescued the bone loss and improved the microarchitecture of bone in OVX mice. Rspo2 could be a positive regulator of bone metabolism through activating the canonical Wnt/β-catenin signaling, and LGR4 acted as a key receptor for Rspo2 to promote osteogenesis.

Aberrant activation of the Wnt/β-catenin signaling pathway promotes the progression of osteoarthritis (OA). We previously reported that R-spondin 2 (Rspo2), an activator of the Wnt/β-catenin signaling, facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification. However, the role of Rspo2 in OA remains elusive. Here, we showed that the amounts of Rspo2 protein in synovial fluid were increased in OA patients. We searched for a preapproved drug that suppresses Rspo2-induced Wnt/β-catenin signaling in chondrogenic cells and reduces joint pathology in a rat model of OA. In Rspo2-treated ATDC5 cells, mianserin, a tetracyclic antidepressant, inhibited Wnt/β-catenin signaling, increased proteoglycan production, and upregulated chondrogenic marker genes. Mianserin suppressed Rspo2-induced accumulation of β-catenin and phosphorylation of Lrp6. We identified that mianserin blocked binding of Rspo2 to its receptor Lgr5. We also observed that intraarticular administration of mianserin suppressed β-catenin accumulation and prevented OA progression in a rat model of OA. We conclude that mianserin suppresses abnormally activated Wnt/β-catenin signaling in OA by inhibiting binding of Rspo2 to Lgr5.

BACKGROUND: R-spondins (Rspo) and leucine-rich repeat-containing G-protein-coupled receptors (LGR) play important roles in development, stem cells survival, and tumorigenicity by activating Wnt signaling pathway. Whether R-spondins-LGR signaling affects the progression of squamous cell carcinoma (SCC) remain unknown. This study aims to uncover the role of R-spodin2/LGR4 in tongue SCC (TSCC). METHODS: The expression of Rspo2 in TSCC specimens and its correlation with TSCC clinical outcome were evaluated. Levels of Rspo2 or LGR4 were altered by pharmacological and genetic approaches, and the effects on TSCC progression were assessed. FINDINGS: Aberrantly high levels of Rspo2 were detected in TSCC specimens. Its levels were closely related with lymph node metastasis, clinical stage and survival rate in patients with tongue SCC. Exogenous Rspo2 or overexpression of Rspo2 promoted growth, migration and invasion, epithelial-mesenchymal transition (EMT) and stem-like properties in SCC both in vivo and in vitro. Silence of Rspo2 abolished these phenotypes. LGR4 was functionally upregulated by Rspo2 in TSCC. Overexpression of Rspo2 increased, whereas Rspo2 silencing decreased the expression of LGR4, leading to subsequent phosphorylation of LRP6 and nuclear translocation of β-catenin in TSCC cell lines. This nuclear translocation of β-catenin was associated with a significant alteration in TCF-1, a downstream nuclear transcription factor of β-catenin, as well as its target genes: CD44, CyclinD1 and c-Myc. INTERPRETATION: Rspo2-LGR4 system regulates growth, migration and invasion, EMT and stem-like properties of TSCC via Wnt/β-catenin signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblast population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondin family (RSPO) comprises a group of proteins essential for development. Among them, RSPO2 is expressed primarily in the lungs, and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical Wingless/int1 pathway, a critical route in the pathogenesis of IPF. Thus, the aim of this study was to examine the expression and putative role of RSPO2 in this disease. We found that RSPO2 and its receptor leucine-rich G protein-coupled receptor 6 were upregulated in IPF lungs, where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant human RSPO2 resulted in the deregulation of numerous genes, although the transcriptional response was essentially distinct. In IPF fibroblasts, RSPO2 stimulation induced the up- or downregulation of several genes involved in the Wingless/int1 pathway (mainly from noncanonical signaling). In both normal and IPF fibroblasts, RSPO2 modifies the expression of genes implicated in several pathways, including the cell cycle and apoptosis. In accordance with gene expression, the stimulation of normal and IPF fibroblasts with RSPO2 significantly reduced cell proliferation and induced cell death. RSPO2 also inhibited collagen production and increased the expression of matrix metalloproteinase 1. Silencing RSPO2 with shRNA induced the opposite effects. Our findings demonstrate, for the first time to our knowledge, that RSPO2 is upregulated in IPF, where it appears to have an antifibrotic role.

Background: In the cochlea, patterning of the organ of Corti is tightly regulated to produce a single row of sound-detecting inner hair cells and three rows of outer hair cells, which amplify and refine the signal. The recently identified R-Spondin family of signaling molecules usually act as co-activators of Wnt signaling; it is thought that they regulate turnover of Wnt receptors at the membrane. We sought to test whether R-Spondins function in the developing cochlea. Results: Expression analysis of all four members of the R-Spondin family showed that only R-Spondin2 (Rspo2) is expressed in the cochlea during development of the sensory epithelium. Examination of an Rspo2−/− mouse showed that loss of Rspo2 results in an additional single row of outer hair cells and disruption of peripheral innervation pattern. Addition of Rspo2 recombinant protein to organotypic cochlear cultures resulted in a small but significant decrease in the number of outer hair cells. Conclusions: Rspo2 is required to limit the number of outer hair cells to three rows and for optimal arrangement of peripheral nerve fibers. The Rspo2 gain- and loss-of-function studies show that in the ear, Rspo2 function is not consistent with its assigned role as a Wnt potentiator. Developmental Dynamics 242:179–188, 2013. © 2012 Wiley Periodicals, Inc.

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2Tg). The predicted protein encoded by Rspo2Tg neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2Tg/Tg mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2Tg/Tg lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2Tg and Lrp6- alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis. © 2013 Macmillan Publishers Limited. All rights reserved.

Background: R-spondins are secreted agonists of Wnt signaling that function in development and promote tissue stem cell proliferation. Rspo2 was identified as a candidate oncogene in intestinal tumors via a Sleeping Beauty transposon insertional mutagenesis screen in mice (1). Further, oncogenic activation of RSPO2 and RSPO3 mediated by recurrent genomic rearrangements has been identified in human colorectal cancer (2). The purpose of the current study was to determine if R-spondins function as oncogenes in other cancer types characterized by active Wnt signaling. Methods: R-spondin mRNA expression was determined in primary human breast tumors and adjacent normal tissue by quantitative RT-PCR. Affymetrix microarrays were used to assay gene expression and classify by molecular subtype human hepatocellular carcinomas compared to pre-malignant lesions and normal livers. RSPO2 was depleted or overexpressed in breast cell lines by stable transduction with anti-RSPO2 shRNA or RSPO2 cDNA. Resulting changes in gene expression and proliferation were measured by qRT-PCR and MTS assay respectively. RSPO2 was somatically overexpressed in murine liver by hydrodynamic injection and Fah selection in an Fah-null model. Subsequent gene expression studies were conducted by qRT-PCR and immunohistochemistry. Results: RSPO2 was highly expressed in 12% of primary human breast tumors compared to adjacent normal tissue. Similarly, RSPO2 expression was elevated in the subset of primary human liver cancers with activated Wnt/beta-catenin signaling. In human breast cancer cells with elevated RSPO2 expression, RSPO2 knockdown decreased Wnt signaling and proliferation, while RSPO2 overexpression in a non-tumorigenic breast epithelial cell line potentiated Wnt signaling. Overexpression of RSPO2 in the mouse liver increased Wnt signaling and promoted an enlarged liver phenotype and tumor formation. Conclusions: These data strongly suggest that RSPO2 is a driver of human breast and liver cancer. Future work will further characterize signaling pathways implicated in RSPO-driven phenotypes, and develop targeted therapy to inhibit RSPO signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A242. Citation Format: Caitlin B. Conboy, Bobbi R. Tschida, Hsiangyu Hu, Michael B. Burns, Nuri A. Temiz, Timothy Kuka, Vincent W. Keng, Sara Toffanin, Reuben S. Harris, Josep Llovet, Timothy K. Starr, David A. Largaespada. R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer. [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 A242.

Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis on brain sections, confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Subsequent analysis of Rspo2-/- embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. To conclude, our study revealed that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the Wnt activator Rspo2.

R-spondin (Rspo) encodes a multi-domain protein that modulates the Wnt-signaling pathway. Two distinct rspo2 zebrafish mutants were generated by TALEN-mediated mutagenesis: a null mutant, rspo2(null), lacking all functional domains, and a hypomorphic mutant, rspo2(tsp), lacking the two N-terminal domains. Mutants were analyzed mainly for abnormalities in the skeletal system. Fin ray skeletons were formed normally in the rspo2(tsp) mutants, but were absent from the rspo2(null) mutants. Hypoplasia of the neural/hemal arches and ribs was observed in both mutants. Thus, the two rspo2 mutants help to identify the functions of Rspo2 in skeletogenesis, as well as functional differences among multiple Rspo2 domains.