Leucine-rich Repeat-containing G Protein-coupled Receptor Research Articles

Evolutionary origin and distribution of leucine-rich repeat-containing G protein-coupled receptors in insects

Leucine-rich repeat-containing G protein-coupled receptors (LGRs) are crucial for animal growth and development. They were categorized into four types (A, B, C1, and C2) based on their sequence and domain structures. Despite the widespread distribution of LGRs across bilaterians, a thorough investigation of their distribution and evolutionary history remains elusive. Recent studies insect LGRs, especially the emergence of type C2 LGRs in various hemimetabolous insects, had prompted our study to address these problems. Initially, we traced the origins of LGRs by exploiting data from 99 species spanning 11 metazoan phyla, and discovered that type A and B LGRs originated from sponges, while type C LGRs originated from cnidarians. Subsequently, through comprehensive genomic and transcriptomic analyses across 565 species across 25 orders of insects, we found that both type A and C1 LGRs divided into two gene clusters. These clusters can be traced back to basal Insecta and an early ancestor of the Arthropoda, respectively. Furthermore, the absence of type B LGRs in wingless insects suggests a role in wing development, while the absence of type C2 LGRs in holometabolous insects hints at novel functions unrelated to insect metamorphosis. According to the origin of LGRs and the investigation of LGRs in insects, we speculated that type A and B LGRs appeared first among four types of LGRs, type A evolved into type C LGRs later, and type A and C1 LGRs independently duplicated during the evolutionary process. This study provides a more comprehensive view of the evolution of LGR genes than previously available, and sheds light on the evolutionary history and significance of LGRs in insect biology.

LGR signaling mediates muscle-adipose tissue crosstalk and protects against diet-induced insulin resistance

Obesity impairs tissue insulin sensitivity and signaling, promoting type-2 diabetes. Although improving insulin signaling is key to reversing diabetes, the multi-organ mechanisms regulating this process are poorly defined. Here, we screen the secretome and receptome in Drosophila to identify the hormonal crosstalk affecting diet-induced insulin resistance and obesity. We discover a complex interplay between muscle, neuronal, and adipose tissues, mediated by Bone Morphogenetic Protein (BMP) signaling and the hormone Bursicon, that enhances insulin signaling and sugar tolerance. Muscle-derived BMP signaling, induced by sugar, governs neuronal Bursicon signaling. Bursicon, through its receptor Rickets, a Leucine-rich-repeat-containing G-protein coupled receptor (LGR), improves insulin secretion and insulin sensitivity in adipose tissue, mitigating hyperglycemia. In mouse adipocytes, loss of the Rickets ortholog LGR4 blunts insulin responses, showing an essential role of LGR4 in adipocyte insulin sensitivity. Our findings reveal a muscle-neuronal-fat-tissue axis driving metabolic adaptation to high-sugar conditions, identifying LGR4 as a critical mediator in this regulatory network.

Abstract 2067: Myeloid Cell Lgr4 Contributes To Atherosclerotic Lesion Formation

Background: R-Spondin 2 (RSPO2), a matricellular protein, primarily functions via binding to cell surface receptors including leucine-rich repeat-containing G protein-coupled receptor (LGR) 4, 5, and 6. We previously identified elevated RSPO2 levels in both human and mouse atherosclerotic arteries. Our preliminary experiments demonstrated increased expression of LGR4 in human atherosclerotic aortic segments, identified Lgr4 as the major RSPO2’s receptor in primary murine macrophages, and atherogenic molecule oxLDL stimulated LGR4 levels in macrophages. However, the precise role of macrophage LGR4 in atherosclerosis development is unknown. Methods and Results: We generated novel myeloid cell-specific Lgr4 knockout mice ( Lgr4 fl/fl LysM Cre +/- , Lgr4 ΔM ) by breeding Lgr4 fl/fl mice with LysM Cre +/- mice. AAV8-h PCSK9 -injected Lgr4 ΔM and littermate control Lgr4 fl/fl ( Lgr4 WT ) were fed a Western diet (16 weeks) to investigate for atherosclerosis. Oil red O (ORO) staining of whole aortas (en face) and aortic root sections showed reduced atherosclerosis in male Lgr4 ΔM mice compared with sex-matched Lgr4 WT mice. Further histochemical staining performed on aortic root sections revealed smaller necrotic cores in Lgr4 ΔM mice. However, there were no significant differences in plasma total cholesterol and body weight. Interestingly, male Lgr4 ΔM mice had decreased fat mass percentage, blood glucose, and epididymal adipose tissue weight. Initial studies with female mice ( n = 4/group) demonstrated no differences in atherosclerosis and metabolic parameters. Additional in vitro studies exhibited that RSPO2 treatment induces oxLDL uptake, reduces efferocytic capacity, and promotes activation of VASP protein (inducer of actin aggregates) in macrophages. Further, the deletion of Lgr4 reduces lipid accumulation in macrophages. Conclusions: Taken together, these findings suggest that deletion of myeloid cell Lgr4 suppresses atherosclerotic lesion formation and identify Lgr4 as a novel therapeutic target for atherosclerosis.

Alterations in epidermal stem cells within the pilosebaceous unit in atrophic acne scars.

Atrophic acne scarring is a common sequela of inflammatory acne, causing significant problems for affected patients. Although prolonged inflammation and subsequent aberrant tissue regeneration are considered the underlying pathogenesis, the role of epidermal stem cells, which are crucial to the regeneration of pilosebaceous units, remains unknown. To examine the changes occurring in epidermal stem cells in atrophic acne scars. Changes in collagen, elastic fibre and human leukocyte antigen (HLA)-DR expression were analysed in normal skin and inflammatory acne lesions at days 1, 3 and 7 after development. The expression of epidermal stem cell markers and proliferation markers was compared between normal skin and mature atrophic acne scar tissue. In acne lesions, inflammation had invaded into pilosebaceous units over time. Their normal structure had been destructed and replaced with a reduced amount of collagen and elastic fibre. Expression of stem cell markers including CD34, p63, leucine-rich repeat-containing G protein-coupled receptor (LGR)6 and LGR5, which are expressed in the interfollicular epidermis, isthmus and bulge of hair follicles, significantly decreased in atrophic acne scar tissue compared to normal skin. Epidermal proliferation was significantly reduced in scar tissue. These findings suggest that as inflammatory acne lesions progress, inflammation gradually infiltrates the pilosebaceous unit and affects the resident stem cells. This disruption impedes the normal regeneration of the interfollicular epidermis and adnexal structures, resulting in atrophic acne scars.

DNA methylation and expression of LGR6 gene in ankylosing spondylitis: A case-control study

ObjectiveThe objectives of the present research were to ascertain the relationship of Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 6 (LGR6) methylation and transcript levels with ankylosing spondylitis (AS). MethodsTargeted bisulfite sequencing was applied to analyze LGR6 DNA methylation in 81 AS cases and 81 controls. Besides, the LGR6 transcription level of peripheral blood mononuclear cells (PBMCs) from 70 AS cases and 64 controls was measured utilizing quantitative real-time transcription-polymerase chain reaction (qRT-PCR). ResultsThe study detected the methylation levels of 43 sites in two CpG (cytosine-guanine dinucleotide) islands of LGR6 and found that LGR6 were significantly hypomethylated in AS patients (LGR6_1: P = 0.002; LGR6_2: P < 0.001). LGR6 transcript level was obviously reduced in AS (P = 0.001) and was positively related to DNA methylation level (CpG-1: P = 0.010; CpG-2: P = 0.007). Besides, the Receiver operating characteristic curve (ROC) exhibited good diagnostic performance of LGR6 methylation level (AUC = 0.676, 95% CI = 0.594–0.758, P < 0.001). Further subgroup analysis revealed that gender may affect the LGR6_1 methylation pattern. ConclusionThe present study revealed that LGR6 DNA methylation dysregulation may be involved in the pathogenesis of AS from an epigenetic perspective for the first time, with the aim of providing new directions for biomarker identification and treatment development for AS patients.

Crystal structure of LGR ligand α2/β5 from Caenorhabditis elegans with implications for the evolution of glycoprotein hormones

A family of leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) mediate diverse physiological responses when complexed with their cognate ligands. LGRs are present in all metazoan animals. In humans, the LGR ligands include glycoprotein hormones (GPHs) chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH). These hormones are αβ heterodimers of cystine-knot protein chains. LGRs and their ligand chains have coevolved. Ancestral hormone homologs, present in both bilaterian animals and chordates, are identified as α2β5. We have used single-wavelength anomalous diffraction and molecular replacement to determine structures of the α2β5 hormone from Caenorhabditis elegans (Ceα2β5). Ceα2β5 is unglycosylated, as are many other α2β5 hormones. Both Hsα2β5, the human homolog of Ceα2β5, and hTSH activate the same receptor (hTSHR). Despite having little sequence similarity to vertebrate GPHs, apart from the cysteine patterns from core disulfide bridges, Ceα2β5 is generally similar in structure to these counterparts; however, its α2 and β5 subunits are more symmetric as compared with α and β of hCG and hFSH. This quasisymmetry suggests a hypothetical homodimeric antecedent of the α2β5 and αβ heterodimers. Known structures together with AlphaFold models from the sequences for other LGR ligands provide representatives for the molecular evolution of LGR ligands from early metazoans through the present-day GPHs. The experimental Ceα2β5 structure validates its AlphaFold model, and thus also that for Hsα2β5; and interfacial characteristics in a model for the Hsα2β5:hTSHR complex are similar to those found in an experimental hTSH:hTSHR structure.

Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease

The roof plate-specific spondin-leucine-rich repeat-containing G-protein-coupled receptor 4/5 (Lgr4/5)-zinc and ring finger 3/ring finger protein 43 module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether hepatic epithelial cell-specific loss of the Wnt/β-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice on feeding with normal diet or high-fat diet (HFD). Livers of 6-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis compared with control mice. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with control mice. An exvivo primary hepatocyte culture assay and a comprehensive BA characterization in liver, plasma, bile, and feces demonstrated that normal diet-fed Lgr4/5dLKO mice revealed impaired BA secretion, predisposing to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.

The RSPO-LGR4/5-ZNRF3/RNF43 module in liver homeostasis, regeneration, and disease.

WNT/β-catenin signaling plays pivotal roles during liver development, homeostasis, and regeneration. Likewise, its deregulation disturbs metabolic liver zonation and is responsible for the development of a large number of hepatic tumors. Liver fibrosis, which has become a major health burden for society and a hallmark of NASH, can also be promoted by WNT/β-catenin signaling. Upstream regulatory mechanisms controlling hepatic WNT/β-catenin activity may constitute targets for the development of novel therapies addressing these life-threatening conditions. The R-spondin (RSPO)-leucine-rich repeat-containing G protein-coupled receptor (LGR) 4/5-zinc and ring finger (ZNRF) 3/ring finger 43 (RNF43) module is fine-tuning WNT/β-catenin signaling in several tissues and is essential for hepatic WNT/β-catenin activity. In this review article, we recapitulate the role of the RSPO-LGR4/5-ZNRF3/RNF43 module during liver development, homeostasis, metabolic zonation, regeneration, and disease. We further discuss the controversy around LGR5 as a liver stem cell marker.

Identification and Expression Analysis of G Protein-Coupled Receptors in the Miridae Insect Apolygus lucorum.

G protein-coupled receptors (GPCRs) are the largest and most versatile family of transmembrane receptors in the cell and they play a vital role in the regulation of multiple physiological processes. The family Miridae (Hemiptera: Heteroptera) is one of the most diverse families of insects. Until now, information on GPCRs has been lacking in Miridae. Apolygus lucorum, a representative species of the Miridae, is an omnivorous pest that occurs worldwide and is notorious for causing serious damage to various crops and substantial economic losses. By searching the genome, 133 GPCRs were identified in A. lucorum. Compared with other model insects, we have observed GPCR genes to be remarkably expanded in A. lucorum, especially focusing on biogenic amine receptors and neuropeptide receptors. Among these, there is a novel large clade duplicated from known FMRFamide receptors (FMRFaRs). Moreover, the temporal and spatial expression profiles of the 133 genes across developmental stages were determined by transcriptome analysis. Most GPCR genes showed a low expression level in the whole organism of A. lucorum. However, there were a few highly expressed GPCR genes. The highly expressed LW opsins in the head probably relate to nocturning of A. lucorum, and the expression of Cirl at different times and in different tissues indicated it may be involved in growth and development of A. lucorum. We also found C2 leucine-rich repeat-containing GPCRs (LGRs) were mainly distributed in Hemiptera and Phthiraptera among insects. Our study was the first investigation on GPCRs in A. lucorum and it provided a molecular target for the regulation and control of Miridae pests.

Expression and Significance of Leucine-rich Repeat-containing G-protein Coupled Receptor 5/6 in Wnt Pathway in Children with Acute Lymphoblastic Leukemia

Objective To study the expression and significance of leucine-rich repeat-containing G-protein coupled receptor(LGR)5/6 in childhood acute lymphoblastic leukemia(ALL). Methods A total of 39 children who had ALL and achieved complete remission on day 33 after induction therapy were enrolled.The children before induction therapy were considered as the incipient group,and those who achieved complete remission on day 33 by induction therapy were considered as the remission group.According to the degree of risk,they were assigned into 3 groups:low-risk(n=16),intermediate-risk(n=9),and high-risk(n=14)groups.A total of 30 children with immune thrombocytopenia were taken as the control group.From each child in the incipient group,remission group,and control group,3 ml bone marrow sample was collected.Real-time fluorescent quantitative polymerase chain reaction was conducted to measure the mRNA expression of LGR5 and LGR6 in the blood cells of bone marrow.Western blot was employed to measure the protein expression of LGR5 and LGR6 in blood cells of bone marrow. Results Compared with the control(mRNA:1.541±0.409,protein:0.138±0.041)and remission(mRNA:1.418±0.324,protein:0.130±0.033)groups,the incipient group had significantly lower mRNA(0.850±0.279)and protein(0.083±0.027)expression of LGR5(PmRNA=0.000,Pprotein=0.000).Compared with the control(mRNA:0.928±0.373,protein:0.094±0.037)and remission(mRNA:0.886±0.390,protein:0.111±0.039)groups,the incipient group had significantly higher mRNA(2.444±1.160)and protein(0.298±0.088)expression of LGR6(PmRNA=0.000,Pprotein=0.000).In the incipient groups,low-risk children showed significantly higher mRNA(1.004±0.284)and protein(0.097±0.030)expression of LGR5 than the intermediate-risk children(mRNA:0.728±0.239,protein:0.071±0.022)and high-risk children(mRNA:0.752±0.222,protein:0.074±0.020)(PmRNA=0.012,Pprotein=0.016);low-risk children showed significantly lower mRNA(1.822±0.979)and protein(0.245±0.077)expression of LGR6 than the intermediate-risk children(mRNA:2.954±1.039,protein:0.338±0.081)and high-risk children(mRNA:2.827±1.165,protein:0.333±0.075)(PmRNA=0.016,Pprotein=0.004).In the remission groups,low-risk children showed significantly higher mRNA(1.597±0.329)and protein(0.150±0.035)expression of LGR5 than the intermediate-risk children(mRNA:1.277±0.288,protein:0.117±0.029)and high-risk children(mRNA:1.305±0.253,protein:0.116±0.023)(PmRNA=0.012,Pprotein=0.006);low-risk children showed significantly lower mRNA(0.662±0.334)and protein(0.089±0.034)expression of LGR6 than the intermediate-risk children(mRNA:1.066±0.273,protein:0.130±0.033)and high-risk children(mRNA:1.027±0.405,protein:0.126±0.038)(PmRNA=0.007,Pprotein=0.007). Conclusion The expression of LGR5 and LGR6 are closely related to the occurrence and risk of childhood ALL,but its mechanism needs further study.

MiR-433-3p suppresses bone formation and mRNAs critical for osteoblast function in mice.

MicroRNAs (miRNAs) are key posttranscriptional regulators of osteoblastic commitment and differentiation. miR-433-3p was previously shown to target Runt-related transcription factor 2 (Runx2) and to be repressed by bone morphogenetic protein (BMP) signaling. Here, we show that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Multiple pathways regulate osteogenesis. Using luciferase-3' untranslated region (UTR) reporter assays, five novel miR-433-3p targets involved in parathyroid hormone (PTH), mitogen-activated protein kinase (MAPK), Wnt, and glucocorticoid signaling pathways were validated. We show that Creb1 is a miR-433-3p target, and this transcription factor mediates key signaling downstream of PTH receptor activation. We also show that miR-433-3p targets hydroxysteroid 11-β dehydrogenase 1 (Hsd11b1), the enzyme that locally converts inactive glucocorticoids to their active form. miR-433-3p dampens glucocorticoid signaling, and targeting of Hsd11b1 could contribute to this phenomenon. Moreover, miR-433-3p targets R-spondin 3 (Rspo3), a leucine-rich repeat-containing G-protein coupled receptor (LGR) ligand that enhances Wnt signaling. Notably, Wnt canonical signaling is also blunted by miR-433-3p activity. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433-3p tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating that miR-433-3p decreases osteoblast activity. Overall, we showed that miR-433-3p is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, Wnt, and endogenous glucocorticoids. Local delivery of miR-433-3p inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Leucine-rich repeat-containing G-protein-coupled receptor 2 (LGR2) can regulate PO activity and AMP genes expression in Macrobrachium nipponense

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.

Lgr4 Governs a Pro-Inflammatory Program in Macrophages to Antagonize Post-Infarction Cardiac Repair.

Macrophages are critically involved in wound healing following myocardial infarction (MI). Lgr4, a member of LGR (leucine-rich repeat-containing G protein-coupled receptor) family, is emerging as a regulator of macrophage-associated immune responses. However, the contribution of Lgr4 to macrophage phenotype and function in the context of MI remains unclear. To determine the role of macrophage Lgr4 in MI and to dissect the underlying mechanisms. During early inflammatory phase of MI, infarct macrophages rather than neutrophils expressed high level of Lgr4. Macrophage-specific Lgr4 knockout mice had no baseline cardiovascular defects but manifested improved heart function, modestly reduced infarct size, decreased early mortality due to cardiac rupture, and ameliorated adverse remodeling after MI. Improved outcomes in macrophage-specific Lgr4 knockout mice subjected to MI were associated with mitigated ischemic injury and optimal infarct healing, as determined by reduction of cardiac apoptosis in the peri-infarct zone, attenuation of local myocardial inflammatory response, decrease of matrix metalloproteinase expression in the infarct, enhancement of angiogenesis, myofibroblast proliferation, and collagen I deposition in reparative granulation tissue as well as formation of collagen-rich scar. More importantly, macrophage-specific Lgr4 knockout infarcts had reduced numbers of infiltrating leukocytes and inflammatory macrophages but harbored abundant reparative macrophage subsets. Lgr4-null infarct macrophages exhibited a less inflammatory transcriptional signature. These findings were further supported by transcriptomic profiling data showing repression of multiple pathways and broad-spectrum genes associated with proinflammatory responses in macrophage-specific Lgr4 knockout infarcts. Notably, we discovered that Lgr4-mediated functional phenotype programing in infarct macrophages was at least partly attributed to regulation of AP (activator protein)-1 activity. We further demonstrated that the synergistic effects of Lgr4 on AP-1 activation in inflammatory macrophages occurred via enhancing CREB (cAMP response element-binding protein)-mediated c-Fos, Fosl1, and Fosb transactivation. Together, our data highlight the significance of Lgr4 in governing proinflammatory phenotype of infarct macrophages and postinfarction repair.

Differential Expression Patterns of Rspondin Family and Leucine-Rich Repeat-Containing G-Protein Coupled Receptors in Chondrocytes and Osteoblasts.

Objective Rspondins (RSPOs) are regarded as the significant modulators of WNT signaling pathway and they are expressed dynamically during developmental stages. Since in osteoarthritis (OA) both cartilage and subchondral bone suffer damages and WNT signaling pathway has a crucial role in their maintenance, the objective of the study was to analyze expression profile of RSPO family and its receptors [leucine-rich repeat-containing G-protein coupled receptors (LGRs)] in OA tissue samples as well as in differentiating chondrocytes and osteoblasts. Materials and Methods:In this experimental study, human early and advanced stage of OA tissue samples were analyzed for the morphological changes of articular cartilage by hematoxylin and eosin (H&E) staining, safranin-O staining and lubricin immunostaining. RSPOs and LGRs expression were confirmed by immunohistochemistry. Human primary chondrocytes and human osteoblast cell line, SaOS-2, were cultured in differentiation medium till day 14 and they were analyzed in terms of expression of RSPOs, LGRs and specific marker for chondrogenesis and osteogenesis by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). ResultsAdvanced stage OA tissue samples showed increased expression of RSPO1 and LGR6 in a region close to subchondral bone. While RSPO2 and LGR5 expression were seen overlapping in the deep region of articular cartilage. Differentiating chondrocytes demonstrated elevated expression of RSPO2 and LGR5 from day 7 to day 14, whereas, osteoblasts undergoing differentiation showed enhanced expression of RSPO1 and LGR6 from day 2 to day 14. Under tumor necrosis factor alpha (TNFα) stimulatory conditions, RSPO2 and RSPO1 recovered the suppressed expression of inflammatory, chondrogenic and osteogenic markers, respectively. A recovery in the stability of β-catenin was also noticed in both cases. Conclusion:Spatial expression of RSPOs during progression of OA might be dynamically controlled by cartilage and subchondral bone. Interplay amid chondrocytes and osteoblasts, via RSPOs, might provide probable mechanisms for treating inflammatory pathogenic conditions like OA.

R-spondin-mediated WNT signaling potentiation in mammary and breast cancer development.

The mammary gland is a secretory organ, which develops as a network of growing epithelial ducts composed of luminal and basal cells that invade the surrounding adipose tissue through a series of developmental cycles. Mammary stem cells (MaSCs) maintain an accurate tissue homeostasis, and their proliferation and cell fate determination are regulated by multiple hormones and local factors. The WNT pathway plays a critical role in controlling the enormous tissue expansion and remodeling during mammary gland development through the maintenance and differentiation of MaSCs, and its deregulation has been implicated in breast cancer (BC) initiation and progression. The R-spondins (RSPOs) are four secreted proteins that strongly enhance target cell sensitivity to WNT ligands. Moreover, leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4-6 are considered obligate high-affinity receptors for RSPOs and have been described as stem cell markers. Importantly, elevated RSPO expression has been recently identified in several tumor types from patients, including BC, and it has been reported that they play a significant role in mammary tumor progression in experimental models. In this review, exploring our present knowledge, we summarize the role of the RSPO-LGR axis as a WNT-enhancing signaling cascade in the MaSC compartment and during the normal and neoplastic mammary gland development. In addition, we include an updated expression profile of the RSPOs and their action mediators at the cell membrane, the LGRs, and the ubiquitin-ligases ZNRF3/RNF43, in different BC subtypes. Finally and based on these data, we discuss the significance of tumor-associated alterations of these proteins and their potential use as molecular targets for detection and treatment of BC.

Epithelial stem cell marker LGR6 expression identifies a low-risk subgroup in human papillomavirus positive oropharyngeal squamous cell carcinoma.

R-Spondins (RSPOs) and leucine-rich repeat-containing G-protein coupled receptors (LGRs) play a critical role in embryonic and cancer development through potentiation of WNT/ß-catenin signaling, but their prognostic significance in head and neck squamous cell carcinoma (HNSCC) is still unclear. HNSCC is a group of neoplasms that include, amongst others, oropharyngeal squamous cell carcinoma (OPSCC), some of which are induced by human papillomavirus (HPV). We aimed to investigate the potential prognostic value of RSPO2 and LGR4/5/6 on overall survival (OS) and disease-free survival (DFS) in HNSCC patients. We examined RSPO and LGR expression by means of immunohistochemistry in 126 HNSCC patients. Furthermore, in order to validate our findings externally, we examined RSPO2 and LGR6 mRNA expression levels using independent secondary datasets. The five-year OS of our cohort was 59.6%. RSPO2 and LGR4/5/6 expression were not associated with OS or DFS in multivariable analyses. Within the HPV+cases (n=26, 33%), however, we observed a difference in OS by RSPO2 expression (5-year OS: RSPO+45.4% vs. RSPO2- 84.6%) and LGR6 expression (5-year OS: LGR6+52.9% vs. LGR6-100%). Evidence for an interaction of HPV status with RSPO2 and LGR6 was found for OS. Relative to HPV+/LGR6- patients, HPV+/LGR6+patients were 12 times more likely to die. These results were replicated in the second dataset. Our results indicated that the expression status of LGR6 had an influence on the aggressiveness of HPV+OPSCC, potentially making this receptor a useful marker for identifying patients with a high risk of death.

Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling.

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of ‘surrogate RSPOs’ that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

R-spondin 2-LGR4 system regulates growth, migration and invasion, epithelial-mesenchymal transition and stem-like properties of tongue squamous cell carcinoma via Wnt/β-catenin signaling

BackgroundR-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). MethodsThe 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. FindingsAberrantly 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. InterpretationRspo2-LGR4 system regulates growth, migration and invasion, EMT and stem-like properties of TSCC via Wnt/β-catenin signaling pathway.

Silencing LGR6 Attenuates Stemness and Chemoresistance via Inhibiting Wnt/β-Catenin Signaling in Ovarian Cancer

Leucine-rich-repeat-containing G protein-coupled receptors (LGRs) have been widely found to be implicated with development and progression in multiple cancer types. However, the clinical significance and biological functions of LGR6 in ovarian cancer remains unclear. In this study, LGR6 expression was mainly examined by immunohistochemistry. Functional assays in vitro and animal experiments in vivo were carried out to explore the effect of LGR6 on cancer stem cell (CSC) characteristics and chemotherapeutic responses in ovarian cancer cells. Luciferase assays and GSEA were used to discern the underlying mechanisms contributing to the roles of LGR6 in ovarian cancer. Here, we reported that LGR6 was upregulated in ovarian cancer, which positively correlated with poor chemotherapeutic response and progression survival in ovarian cancer patients. Loss-of-function assays showed that downregulating LGR6 abrogated the CSC-like phenotype and chemoresistance in vitro. More importantly, silencing LGR6 improved the chemoresistance of ovarian cancer cells to cisplatin in vivo. Mechanistic investigation further revealed that silencing LGR6 inhibited stemness and chemoresistance by repressing Wnt/β-catenin signaling. Collectively, our results uncover a novel mechanism contributing to LGR6-induced chemotherapeutic resistance in ovarian cancer, providing the evidence for LGR6 as a potential therapeutic target in ovarian cancer.