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R-spondins are secretory proteins localized in the endoplasmic reticulum and Golgi bodies and are processed through the secretory pathway. Among the R-spondin family, RSPO2 has emanated as a novel regulator of Wnt signaling, which has now been acknowledged in numerous in vitro and in vivo studies. Cancer is an abnormal growth of cells that proliferates and spreads uncontrollably due to the accumulation of genetic and epigenetic factors that constitutively activate Wnt signaling in various types of cancer. Colorectal cancer (CRC) begins when cells in the colon and rectum follow an indefinite pattern of division due to aberrant Wnt activation as one of the key hallmarks. Decades-long progress in research on R-spondins has demonstrated their oncogenic function in distinct cancer types, particularly CRC. As a critical regulator of the Wnt pathway, it modulates several phenotypes of cells, such as cell proliferation, invasion, migration, and cancer stem cell properties. Recently, RSPO mutations, gene rearrangements, fusions, copy number alterations, and altered gene expression have also been identified in a variety of cancers, including CRC. In this review, we addressed the recent updates regarding the recurrently altered R-spondins with special emphasis on the RSPO2 gene and its involvement in potentiating Wnt signaling in CRC. In addition to the compelling physiological and biological roles in cellular fate and regulation, we propose that RSPO2 would be valuable as a potential biomarker for prognostic, diagnostic, and therapeutic use in CRC.

Abstract Background The aggressive phenotype of fibroblast-like synoviocytes (FLS) has been identified as a contributing factor to the exacerbation of rheumatoid arthritis (RA) through the promotion of synovitis and cartilage damage. Regrettably, there is currently no effective therapeutic intervention available to address this issue. Recent research has shed light on the crucial regulatory role of R-spondin-2 (Rspo2) in cellular proliferation, cartilage degradation, and tumorigenesis. However, the specific impact of Rspo2 on RA remains poorly understood. We aim to investigate the function and mechanism of Rspo2 in regulating the aggressive phenotype of FLS and maintaining chondrocyte homeostasis in the context of RA. Methods The expression of Rspo2 in knee joint synovium and cartilage were detected in RA mice with antigen-induced arthritis (AIA) and RA patients. Recombinant mouse Rspo2 (rmRspo2), Rspo2 neutralizing antibody (Rspo2-NAb), and recombinant mouse DKK1 (rmDKK1, a potent inhibitor of Wnt signaling pathway) were used to explore the role and mechanism of Rspo2 in the progression of RA, specifically in relation to the aggressive phenotype of FLS and chondrocyte homeostasis, both in vivo and in vitro. Results We indicated that Rspo2 expression was upregulated both in synovium and articular cartilage as RA progressed in RA mice and RA patients. Increased Rspo2 upregulated the expression of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), as the ligand for Rspo2, and β-catenin in FLS and chondrocytes. Subsequent investigations revealed that intra-articular administration of rmRspo2 caused striking progressive synovitis and articular cartilage destruction to exacerbate RA progress in mice. Conversely, neutralization of Rspo2 or inhibition of the Wnt/β-catenin pathway effectively alleviated experimental RA development. Moreover, Rspo2 facilitated FLS aggressive phenotype and disrupted chondrocyte homeostasis primarily through activating Wnt/β-catenin pathway, which were effectively alleviated by Rspo2-NAb or rmDKK1. Conclusions Our data confirmed a critical role of Rspo2 in enhancing the aggressive phenotype of FLS and disrupting chondrocyte homeostasis through the Wnt/β-catenin pathway in the context of RA. Furthermore, the results indicated that intra-articular administration of Rspo2 neutralizing antibody or recombinant DKK1 might represent a promising therapeutic strategy for the treatment of RA.

This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7–21.6%], P < 0.001), decreased cell apoptosis (mean difference −15.6% [−8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD. [ABSTRACT FROM AUTHOR]

Wnt and R-spondin (Rspo) proteins are two major types of endogenous Wnt/β-catenin signaling agonists. While Wnt/β-catenin signaling is greatly diminished in Alzheimer's disease (AD), it remains to be elucidated whether the inhibition of this pathway is associated with dysregulation of Wnt and Rspo proteins. By analyzing temporal cortex RNA-seq data of the human postmortem brain samples, we found that WNT1 and RRPO2 were significantly downregulated in human AD brains. In addition, the expression of Wnt acyltransferase porcupine (PORCN), which is essential for Wnt maturation and secretion, was greatly deceased in these human AD brains. Interestingly, the lowest levels of WNT1, PORCN, and RSPO2 expression were found in human AD brains carrying two copies of APOE4 allele, the strongest genetic risk factor of late-onset AD. Importantly, there were positive correlations among the levels of WNT1, PORCN, and RSPO2 expression in human AD brains. Supporting observations in humans, Wnt1, PORCN, and Rspo2 were downregulated and Wnt/β-catenin signaling was diminished in the 5xFAD amyloid model mice. In human APOE-targeted replacement mice, downregulation of WNT1, PORCN, and RSPO2 expression was positively associated with aging and APOE4 genotype. Finally, WNT1 and PORCN expression and Wnt/β-catenin signaling were inhibited in human APOE4 iPSC-derived astrocytes when compared to the isogenic APOE3 iPSC-derived astrocytes. Altogether, our findings suggest that the dysregulations of Wnt1, PORCN, and Rspo2 could be coordinated together to diminish Wnt/β-catenin signaling in aging- and APOE4-dependent manners in the AD brain. [ABSTRACT FROM AUTHOR]

In vertebrates, the three orthogonal body axes, anteroposterior (AP), dorsoventral (DV) and left-right (LR) are determined at gastrula and neurula stages by the Spemann-Mangold organizer and its equivalents. A common feature of AP and DV axis formation is that an evolutionary conserved interplay between growth factors (Wnt, BMP) and their extracellular antagonists (e.g. Dkk1, Chordin) creates signaling gradients for axial patterning. Recent work showed that LR patterning in Xenopus follows the same principle, with R-spondin 2 (Rspo2) as an extracellular FGF antagonist, which creates a signaling gradient that determines the LR vector. That a triad of anti-FGF, anti-BMP, and anti-Wnt governs LR, DV, and AP axis formation reveals a unifying principle in animal development. We discuss how cross-talk between these three signals confers integrated AP-DV-LR body axis patterning underlying developmental robustness, size scaling, and harmonious regulation. We propose that Urbilateria featured three orthogonal body axes that were governed by a Cartesian coordinate system of orthogonal Wnt/AP, BMP/DV, and FGF/LR signaling gradients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/β-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability. [ABSTRACT FROM AUTHOR]

Qian Xu,1 Zhenwu Xu2 1Department of Oncology Medicine, Fujian Medical University Union Hospital, Fuzhou, 350001, People’s Republic of China; 2Department of Thoracic Medical Oncology, Fujian Provincial Cancer Hospital, The Affiliated Hospital of Fujian Medical University, Fuzhou, 350014, People’s Republic of ChinaCorrespondence: Zhenwu XuDepartment of Thoracic Medical Oncology, Fujian Provincial Cancer Hospital, The Affiliated Hospital of Fujian Medical University, No. 420 Fuma Road, Fuzhou 350014, People’s Republic of ChinaTel +86-13906931946Email xuzhenwu32540@163.comObjective: To explore the biological role of miR-196b-5p/RSPO2 in the occurrence and development of lung adenocarcinoma (LUAD) and to provide a basis for finding new therapeutic targets for LUAD.Methods: Differentially expressed genes were analyzed based on LUAD microarray, and the target gene of the target miRNA was predicted. qRT-PCR was used to detect the expression levels of miR-196b-5p and RSPO2 mRNA in normal human bronchial epithelial cell line BEAS-2B and LUAD cell lines A549, NCI-H1792 and NCI-H226. Western blot was used to evaluate protein expression. Cell proliferative, migratory and invasive abilities were detected by CCK-8 and transwell assays. Dual-luciferase assay was conducted to verify the targeting relationship between miR-196b-5p and RSPO2.Results: The results of qRT-PCR showed that miR-196b-5p was significantly highly expressed in LUAD cells, and the expression level of its downstream target gene RSPO2 was significantly decreased. The results of CCK-8 and transwell assays exhibited that miR-196b-5p promoted proliferation, migration and invasion of LUAD cells, while RSPO2 inhibited the malignant progression of LUAD cells. Dual-luciferase assay confirmed the targeted binding relationship between miR-196b-5p and RSPO2. Overexpression of RSPO2 partially reversed the promotion of miR-196b-5p on proliferation, migration and invasion of LUAD cells.Conclusion: miR-196b-5p promoted proliferation, migration and invasion of LUAD cells by targeting and down-regulating RSPO2, which provided ideas for searching new targets for the diagnosis and treatment of LUAD.Keywords: RSPO2, miR-196b-5p, lung adenocarcinoma, proliferation, invasion

Mechanisms of WNT and bone morphogenetic protein (BMP) signaling crosstalk is in the focus of multiple biological studies, and it also has been discovered to play important roles in human mesenchymal stromal cells (MSC) that are of great interest for neocartilage engineering due to their high chondrogenic differentiation potential. However, MSC-derived chondrocytes undergo hypertrophic degeneration that impedes their clinical application for cartilage regeneration. In our previous study, we established that several microRNAs (miRs) are differentially expressed between articular chondrocytes (AC) – and MSC-derived neocartilage, with miR-181a being the most prominent candidate as key microRNA involved in the regulation of a balance between chondral and endochondral differentiation. The aim of this study was the identification of precise mRNA targets and signaling pathways regulated by miR-181a in MSC during chondrogenesis. MiR-181a was upregulated during chondrogenesis of MSC, along with an increase of the hypertrophic phenotype in resulting cartilaginous tissue. By in silico analysis combined with miR reporter assay, the WNT signaling activator and BMP signaling repressor RSPO2 was suggested as a target of miR-181a. Further validation experiments confirmed that miR-181a targets RSPO2 mRNA in MSC. It was found that in human MSC miR-181a activated BMP signaling manifested by the accumulation of SOX9 protein and increased phosphorylation of SMAD1/5/9. These effects, together with the concomitant reduction of canonical WNT signaling induced by miR-181a mimic, were in accordance with the effects expected by the loss of RSPO2, thus indicating the causative link between miR-181a and RSPO2. Moreover, we observed that a tight correlation between miR-181a and miR-218 expression levels in healthy human cartilage tissue was disrupted in osteoarthritis (OA) highlighting the importance of the WNT-BMP signaling crosstalk for preventing OA.

Aims Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis. Methods and results The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells (LEC) in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signalling in LEC. In vivo low-density lipoprotein (LDL) tracking and perivascular blockade of RSPO2–LGR4 signalling using LGR4-extracellular domain (ECD) pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2–LGR4 signalling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 suppresses PI3K-AKT-endothelial nitric oxide synthase (eNOS) signalling via LGR4 and inhibits activation of the canonical Wnt-β-catenin pathway. ApoE−/− mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently labelled LDL to deep cervical LNs were observed in LGR4-ECD-treated mice. Conclusion These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-nitric oxide signalling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries. [ABSTRACT FROM AUTHOR]

Wnt signaling is activated in many cancer types, yet targeting the canonical Wnt pathway has been challenging for cancer therapy. The pathway might be effectively targeted at many levels depending on the mechanism by which it has become hyperactive. Recently, mouse genetic screens have found that R-spondins (RSPOs) act as oncogenes. Evidence includes recurrent genomic rearrangements that led to increased RSPO2 or RSPO3 expression in human colorectal adenocarcinomas, exclusive of APC mutations. RSPOs modulate Wnt signaling to promote epithelial cell proliferation and survival. These secreted proteins modulate Wnt signaling by binding to G-coupled receptors LGR4/5/6, ultimately inhibiting frizzled membrane clearance by RNF43 and ZNRF3. They also exert their function independent of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) by binding to ZNRF3 and RNF43. This results in increased β-catenin concentration that, after translocation to the nucleus, acts as a transcriptional coactivator of genes necessary for proliferation and cell survival. In this article, we aimed to identify the role of RSPOs in colon and breast cancers by using in silico and in vitro studies. We found that expression of RSPO2 and RSPO3 at high levels characterized a subset of colorectal cancers (CRCs). RSPO2 expression was found to characterize a subset of triple-negative breast cancers. In both instances, increased expression of RSPOs was associated with an activated Wnt signaling gene expression profile. Furthermore, knockdown of RSPO2 decreased Wnt signaling and proliferation in human breast cancer cells. Our findings show and confirm that RSPO2 and RSPO3 expression is upregulated in a subset of colorectal adenocarcinomas and breast cancers and that both are attractive druggable oncoprotein targets against such cancers. We also describe novel fusion transcripts that occur in CRC. [ABSTRACT FROM AUTHOR]

Background: Esophageal cancer is a highly aggressive malignancy with limited treatment options and poor prognosis. The identification of novel molecular subtypes and therapeutic targets is crucial for improving clinical outcomes., Method: In this study, we investigated the role of R-spondin 2 (RSPO2) in esophageal cancer and its association with mitochondrial metabolism. Using bioinformatics analysis of publicly available datasets, we identified a panel of RSPO2-related mitochondrial metabolism genes and their expression patterns in esophageal cancer. Based on these genes, we stratified esophageal cancer patients into distinct molecular subtypes with different survival rates, immune cell infiltration profiles, and drug sensitivities., Results: Our findings suggest that RSPO2-related mitochondrial metabolism genes may serve as potential therapeutic targets and prognostic markers for esophageal cancer. These genes play an important role in the prognosis, immune cell infiltration and drug sensitivity of esophageal cancer., Conclusion: The identified molecular subtypes provide valuable insights into the underlying molecular mechanisms of esophageal cancer and could guide personalized treatment strategies in the future., (© 2024 Wiley Periodicals LLC.)

Background: The distribution of sensory organs is important for detecting environmental signals efficiently. The mechanosensory receptors of the lateral line system, neuromasts, are stereotypically distributed over the head and body surface of fish, although how neuromasts arise in these predetermined positions during development remains unclear. Results: We investigated the development of the anterior lateral line (ALL) system in zebrafish head. The ALL neuromasts formed in the predetermined positions through proliferation and differentiation of (a) nonmigratory lateral line primordia, (b) migratory primordia, (c) interneuromast cells connecting preexisting neuromasts, and (d) budding primordia. We demonstrated that R‐spondin2 (Rspo2), an activator of Wnt/β‐catenin signaling, is required for the development of a particular set of neuromasts associated with hyomandibular cartilage. Further genetic analyses suggested that Rspo2, which emanates from the hyoid mesenchyme, acts on the adjacent neuromast progenitor cells to stimulate their proliferation through activating Wnt/β‐catenin signaling. Conclusion: This study has revealed novel mechanisms for neuromast positioning through local tissue‐tissue interactions, providing insights into the development and evolution of the vertebrate head. Key Findings: Development of the anterior lateral line system in the zebrafish head is described.Rspo2 is required for the development of a particular set of neuromasts associated with hyomandibular cartilage.Rspo2, which emanates from the head mesenchyme, acts on adjacent neuromast progenitor cells to stimulate their proliferation.Novel mechanisms for neuromast positioning provide insights into the development and evolution of the vertebrate head. [ABSTRACT FROM AUTHOR]

Study Design: Immunohistochemical and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis.Objective: The aim of this study was to analyze the expression of five susceptibility genes (RSPO2, HAO1, CCDC91, RHPH9, and STK38L) for human ossification of the posterior longitudinal ligaments (OPLL) identified in a genome-wide association study.Summary Of Background Data: Detailed expression and functional studies for the five susceptibility genes are needed to aid in clarification of the etiology and pathogenesis of OPLL.Methods: Immunostaining, cell culture, and real-time RT-PCR were performed on ossified ligament samples collected during anterior cervical decompression for symptomatic OPLL (n = 39 patients) and on control non-OPLL samples (n = 8 patients). Immunohistochemical analysis in spinal hyperostotic mice (ttw/ttw) (n = 25) was also performed. The sample sections were stained for RSPO2, HAO1, CCDC91, RHPH9, STK38L, Runx2, Sox9, and CD90. The mRNA expression levels of the five susceptibility genes were also analyzed in cultured human OPLL and non-OPLL cells subjected to cyclic tensile strain.Results: Immunoreactivity for RSPO2 and Sox9 was evident in proliferating chondrocytes in human OPLL tissues and ttw/ttw mice. Application of cyclic tensile strain to cultured human OPLL cells resulted in increases in mRNA levels for RSPO2, HAO1, and CCDC91. However, individual differences in expression in human OPLL-related samples were seen. HAO1-positive cells were detected only in 3- to 6-week-old ttw/ttw mice that did not simultaneously express RSPO2-positive samples.Conclusion: Among the five susceptibility genes, RSPO2, HAO1, and CCDC91 might be contributory factors in progression of OPLL. RSPO2 may be involved in endochondral ossification, especially in mixed or continuous type OPLL, HAO1 may be an initiation factor for OPLL that is rarely seen in mature human OPLL samples, and CCDC91 may be associated with progression of ossification caused by mechanical stress. These findings provide important insights into the pathogenesis and therapeutic targets for OPLL.Level Of Evidence: N/A. [ABSTRACT FROM AUTHOR]

Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.

Abstract Background R-spondin2 (Rspo2) is a secreted agonist of the canonical Wnt/β-catenin signaling pathway. Rspo2 plays a key role in development of limbs, lungs and hair follicles, and more recently during ovarian follicle development. Rspo2 heterozygous deficient female mice become infertile around 4 months of age mimicking primary ovarian insufficiency (POI). The study aimed to investigate the regulation of RSPO2 and its potential involvement in pathophysiology of POI. Methods We cloned the RSPO2 promoter and performed transcriptional assays to determine if RSPO2 can be regulated by NOBOX, an ovarian transcription factor. Then, we evaluated 100 infertile women after obtaining a detailed history of the disease and follicle-stimulating hormone measurements, besides karyotype determination and fragile-X premutation syndrome investigation. All exons, intron-exon boundaries and untranslated regions of the RSPO2 gene were identified by sequencing, and the results were statistically analyzed. Results We found that RSPO2 can be regulated by NOBOX via the presence of NOBOX Binding Element in its promoter. Among 9 identified variants in POI women, 4 of them were equally homozygous, 4 have never been described (c.-359C > G, c.-190G > A, c.-170 + 13C > T and c.-169-8 T > A), only one c.557 T > C was predicted to alter a single amino acid in the RSPO2 protein (p.Leu186Pro). Conclusions RSPO2 is a novel target gene of the NOBOX key transcription factor, confirming its important role during the follicular growth in ovary. However, RSPO2 mutations are rare or uncommon in women with POI.

RSPO2 protein may provide valuable insights into the mechanism underlying various types of tumorigenesis. The role of RSPO2 in pan-cancer has not been reported so far. Therefore, this study aimed to provide a comprehensive analysis of RSPO2 from a pan-cancer perspective employing multiomics data. The expression profile and function of RSPO2 across different tumors were investigated using various web-based tools UALCAN, GEPIA, TIMER, Human Protein Atlas, cBioPortal, TISIDB, STRING, and Metascape to interpret the expression profile, promoter methylation status, genomic alterations, survival analysis, protein-protein interaction, correlation with immune cell subtypes, tumor immune microenvironment and enrichment analysis. Comprehensive pan-cancer analysis indicated that RSPO2 was significantly downregulated in eleven and upregulated in five tumor types compared to normal tissues, validation results further suggest RSPO2 was downregulated in most of the tumors. The protein level expression of RSPO2 was mostly low in malignant tissues. We found that RSPO2 was significantly related to individual pathological stages in BLCA, COAD, LUAD and LUSC. Prognostic analysis indicates that the high RSPO2 expression was significantly correlated with the poor prognosis in BRCA, KICH, KIRP, READ, and UCES. Furthermore, RSPO2 is frequently amplified, exhibits hypermethylated promoter in most cancers, and is associated with immune subtypes, molecular subtypes and immune cell infiltration. Finally, enrichment analysis showed that RSPO2 is involved in the regulation of the canonical Wnt pathway and neuronal development. The overall comprehensive pan-cancer analysis affirms that RSPO2 could be a promising diagnostic and prognostic biomarker and latent therapy target in the future., (Copyright © 2024. Published by Elsevier Inc.)

Objective: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD)., Methods: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD., Results: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD., Conclusions: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The secretion of oocyte-derived paracrine factors, such as R-spondin2, is an essential mechanism for follicle growth by promoting the proliferation and differentiation of cumulus cells around oocytes. In the present study, we aimed to identify the effect of R-spondin2 during follicular development. First, R-spondin2-related factors (R-spondin2, CTNNB1, LGR4, and LGR5) were identified through immunofluorescence in porcine ovarian tissue. CTNNB1 was expressed in ooplasm, and CTNNB1 and LGR4 were expressed in granulosa cells. In addition, R-spondin2, LGR4, and LGR5 were expressed in the theca interna. These results imply that these proteins play a major role in porcine follicular development. In addition, the effects of R-spondin2 on the in vitro maturation process of porcine cumulus oocyte complexes and subsequent embryonic development were confirmed. A treatment of 100 ng/mL R-spondin2 in the in vitro maturation (IVM) process increased nuclear maturation and increased the expression of EGFR mRNA in cumulus cells. The EGFR-ERK signal is essential for oocyte maturation, ovulation, and luteinization. R-spondin2 treatment also increased the expression of CTNNB1 and EGFR in primary cultured cumulus cells. In conclusion, RSPO2 and WNT/CTNNB1 signaling pathways are required for porcine follicle development and are predicted to be involved in the EGFR-ERK signaling pathway.

Hepatocellular carcinoma (HCC) is known as a highly prevalent cancer with a poor prognosis and short survival time, despite intensive research and clinical efforts. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of hepatocellular carcinoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we report that the expression of microRNA-493 (miR-493) is decreased in HCC cell lines and in tumor tissues. Overexpression of miR-493 in HCC cells dramatically inhibited cell proliferation and colony-formation in vitro and inhibited tumor formation of HCC cell xenografts in vivo . miR-493 also suppressed cell migration and invasion in HCC cell lines. Novel targets ANTXR1 and RSPO2 were confirmed to be suppressed by miR-493 directly, and overexpression of ANTXR1 and RSPO2 could restore tumorigenesis in miR-493 treated HCC cell. Moreover, Wnt/β-catenin signaling pathway, which was reported to be activated by ANTXR1 and RSPO2, was also inhibited by miR-493 overexpression and might be involved in anti-tumor function of miR-493. These findings suggest that miR-493 acts as a negative regulator in hepatocellular carcinoma progression and may be a potential therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

R-spondins play critical roles in development, stem cell survival, and tumorigenicity by modulating Wnt/β-catenin signaling; however, the role of R-spondins in noncanonical Wnt signaling regulation remains largely unknown. We demonstrate here that R-spondin 2 (RSPO2) has an inhibitory effect on colorectal cancer (CRC) cell migration, invasion, and metastasis. Reduced RSPO2 expression was associated with tumor metastasis and poor survival in CRC patients. The metastasis-suppressive activity of RSPO2 was independent of the Wnt/β-catenin signaling pathway but dependent on the Fzd7-mediated noncanonical Wnt signaling pathway. The physical interaction of RSPO2 and Fzd7 increased the degradation of cell surface Fzd7 via ZNRF3-mediated ubiquitination, which led to the suppression of the downstream PKC/ERK signaling cascade. In late-stage metastatic cancer, Wnt5a promoted CRC cell migration by preventing degradation of Fzd7, and RSPO2 antagonized Wnt5a-driven noncanonical Wnt signaling activation and tumor cell migration by blocking the binding of Wnt5a to the Fzd7 receptor. Our study reveals a novel RSPO2/Wnt5a-competing noncanonical Wnt signaling mechanism that regulates cellular migration and invasion, and our data suggest that secreted RSPO2 protein could serve as a potential therapy for Wnt5a/Fzd7-driven aggressive CRC tumors. [ABSTRACT FROM AUTHOR]

Background: R-spondin2 (Rspo2) is a secreted agonist of the canonical Wnt/β-catenin signaling pathway. Rspo2 plays a key role in development of limbs, lungs and hair follicles, and more recently during ovarian follicle development. Rspo2 heterozygous deficient female mice become infertile around 4 months of age mimicking primary ovarian insufficiency (POI). The study aimed to investigate the regulation of RSPO2 and its potential involvement in pathophysiology of POI. Methods: We cloned the RSPO2 promoter and performed transcriptional assays to determine if RSPO2 can be regulated by NOBOX, an ovarian transcription factor. Then, we evaluated 100 infertile women after obtaining a detailed history of the disease and follicle-stimulating hormone measurements, besides karyotype determination and fragile-X premutation syndrome investigation. All exons, intron-exon boundaries and untranslated regions of the RSPO2 gene were identified by sequencing, and the results were statistically analyzed. Results: We found that RSPO2 can be regulated by NOBOX via the presence of NOBOX Binding Element in its promoter. Among 9 identified variants in POI women, 4 of them were equally homozygous, 4 have never been described (c.-359C > G, c.-190G > A, c.-170 + 13C > T and c.-169-8 T > A), only one c.557 T > C was predicted to alter a single amino acid in the RSPO2 protein (p.Leu186Pro). Conclusions: RSPO2 is a novel target gene of the NOBOX key transcription factor, confirming its important role during the follicular growth in ovary. However, RSPO2 mutations are rare or uncommon in women with POI. [ABSTRACT FROM AUTHOR]

Chondrocyte degeneration and classically activated macrophage (AM)-related inflammation play critical roles in osteoarthritis (OA). Here, we explored the effects of astaxanthin and Rspo2 on OA in vitro and in vivo . We observed that the Rspo2 gene was markedly elevated in synovial tissues of OA patients compared with healthy controls. In 2D cultures, Rspo2 and inflammatory factors were enhanced in AMs compared with nonactivated macrophages (NMs), and the protein expression levels of Rspo2, β-catenin, and inflammatory factors were increased, and anabolic markers were reduced in osteoarthritic chondrocytes (OACs) compared to normal chondrocytes (NCs). Astaxanthin reversed these changes in AMs and OACs. Furthermore, Rspo2 shRNA significantly abolished inflammatory factors and elevated anabolic markers in OACs. In NCs cocultured with AM, and in OACs cocultured with AMs or NMs, astaxanthin reversed these changes in these coculture systems and promoted secretion of Rspo2, β-catenin and inflammatory factors and suppressed anabolic markers compared to NCs or OACs cultured alone. In AMs, coculture with NCs resulted in a slight elevation of Rspo2 and AM-related genes, but not protein expression, compared to culture alone, but when cocultured with OACs, these inflammatory mediators were significantly enhanced at both the gene and protein levels. Astaxanthin reversed these changes in all the groups. In vivo , we observed a deterioration in cartilage quality after intra-articular injection of Rspo2 associated with medial meniscus (DMM)-induced instability in the OA group, and astaxanthin was protective in these groups. Our results collectively revealed that astaxanthin attenuated the process of OA by abolishing Rspo2 both in vitro and in vivo .

R-spondins are secretory proteins localized in the endoplasmic reticulum and Golgi bodies and are processed through the secretory pathway. Among the R-spondin family, RSPO2 has emanated as a novel regulator of Wnt signaling, which has now been acknowledged in numerous in vitro and in vivo studies. Cancer is an abnormal growth of cells that proliferates and spreads uncontrollably due to the accumulation of genetic and epigenetic factors that constitutively activate Wnt signaling in various types of cancer. Colorectal cancer (CRC) begins when cells in the colon and rectum follow an indefinite pattern of division due to aberrant Wnt activation as one of the key hallmarks. Decades-long progress in research on R-spondins has demonstrated their oncogenic function in distinct cancer types, particularly CRC. As a critical regulator of the Wnt pathway, it modulates several phenotypes of cells, such as cell proliferation, invasion, migration, and cancer stem cell properties. Recently, RSPO mutations, gene rearrangements, fusions, copy number alterations, and altered gene expression have also been identified in a variety of cancers, including CRC. In this review, we addressed the recent updates regarding the recurrently altered R-spondins with special emphasis on the RSPO2 gene and its involvement in potentiating Wnt signaling in CRC. In addition to the compelling physiological and biological roles in cellular fate and regulation, we propose that RSPO2 would be valuable as a potential biomarker for prognostic, diagnostic, and therapeutic use in CRC., (© 2023 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency. [Display omitted] • scRNA-seq of developing human distal lung mesenchyme identified cellular heterogeneity • RSPO2+ mesenchymal cells lie adjacent to LGR5+ epithelial bud tip progenitors • Blocking RSPO2/LGR5 in vitro reduced WNT signaling and led to airway differentiation • RSPO2+ mesenchyme provides a niche for bud tips in co-cultures Hein et al. present the scRNA-seq of the developing human distal lung, showing transcriptionally distinct populations of mesenchyme. Spatial profiling showed that RSPO2+ mesenchymal cells are physically adjacent to LGR5+ epithelial bud tips. Organoid experiments revealed that RSPO2+ cells create a high WNT signaling environment, supporting bud tip identity and multipotency. [ABSTRACT FROM AUTHOR]

R- spondin ( RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4 %) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population. [ABSTRACT FROM AUTHOR]