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

Loss of Lgr4 inhibits differentiation, migration and apoptosis, and promotes proliferation in bone mesenchymal stem cells.

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4+/- mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4-/- ) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4-/- ) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.

R-spondin 2 promotes osteoblastic differentiation of immature human periodontal ligament cells through the Wnt/β-catenin signaling pathway.

In this study, we measured the expression of R-spondin 2 (RSPO2) in periodontal ligament (PDL) tissue and cells. Further, we examined the effects of RSPO2 on osteoblastic differentiation of immature human PDL cells (HPDLCs). R-spondin (RSPO) family proteins are secreted glycoproteins that play important roles in embryonic development and tissue homeostasis through activation of the Wnt/β-catenin signaling pathway. RSPO2, a member of the RSPO family, has been reported to enhance osteogenesis in mice. However, little is known regarding the roles of RSPO2 in PDL tissues. Expression of RSPO2 in rat PDL tissue and primary HPDLCs was examined by immunohistochemical and immunofluorescence staining, as well as by semiquantitative RT-PCR. The effects of stretch loading on the expression of RSPO2 and Dickkopf-related protein 1 (DKK1) were assessed by quantitative RT-PCR. Expression of receptors for RSPOs, such as Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4, 5, and 6 in immature human PDL cells (cell line 2-14, or 2-14 cells), was investigated by semiquantitative RT-PCR. Mineralized nodule formation in 2-14 cells treated with RSPO2 under osteoblastic inductive condition was examined by Alizarin Red S and von Kossa stainings. Nuclear translocation of β-catenin and expression of active β-catenin in 2-14 cells treated with RSPO2 were assessed by immunofluorescence staining and Western blotting analysis, respectively. In addition, the effect of Dickkopf-related protein 1 (DKK1), an inhibitor of Wnt/β-catenin signaling, was also examined. Rat PDL tissue and HPDLCs expressed RSPO2, and HPDLCs also expressed RSPO2, while little was found in 2-14 cells. Expression of RSPO2 as well as DKK1 in HPDLCs was significantly upregulated by exposure to stretch loading. LGR4 was predominantly expressed in 2-14 cells, which expressed low levels of LGR5 and LGR6. RSPO2 enhanced the Alizarin Red S and von Kossa-positive reactions in 2-14 cells. In addition, DKK1 suppressed nuclear translocation of β-catenin, activation of β-catenin, and increases of Alizarin Red S and von Kossa-positive reactions in 2-14 cells, all of which were induced by RSPO2 treatment. RSPO2, which is expressed in PDL tissue and cells, might play an important role in regulating the osteoblastic differentiation of immature human PDL cells through the Wnt/β-catenin signaling pathway.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4

AimsDuring orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. Main methodsThe cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF—which was generated by increasing the volume of culture medium in the wells of a 96-well plate—in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. Key findingsIn the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4—another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. SignificanceContinuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.

R-spondins can potentiate WNT signaling without LGRs.

The WNT signaling pathway regulates patterning and morphogenesis during development and promotes tissue renewal and regeneration in adults. The R-spondin (RSPO) family of four secreted proteins, RSPO1-4, amplifies target cell sensitivity to WNT ligands by increasing WNT receptor levels. Leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4-6 are considered obligate high-affinity receptors for RSPOs. We discovered that RSPO2 and RSPO3, but not RSPO1 or RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. By mapping the domains on RSPO3 that are necessary and sufficient for this activity, we show that the requirement for LGRs is dictated by the interaction between RSPOs and the ZNRF3/RNF43 E3 ubiquitin ligases and that LGR-independent signaling depends on heparan sulfate proteoglycans (HSPGs). We propose that RSPOs can potentiate WNT signals through distinct mechanisms that differ in their use of either LGRs or HSPGs, with implications for understanding their biological functions.

Wnt, RSPO and Hippo Signalling in the Intestine and Intestinal Stem Cells.

In this review, we address aspects of Wnt, R-Spondin (RSPO) and Hippo signalling, in both healthy and transformed intestinal epithelium. In intestinal stem cells (ISCs), the Wnt pathway is essential for intestinal crypt formation and renewal, whereas RSPO-mediated signalling mainly affects ISC numbers. In human colorectal cancer (CRC), aberrant Wnt signalling is the driving mechanism initiating this type of neoplasia. The signalling role of the RSPO-binding transmembrane proteins, the leucine-rich-repeat-containing G-protein-coupled receptors (LGRs), is possibly more pleiotropic and not only limited to the enhancement of Wnt signalling. There is growing evidence for multiple crosstalk between Hippo and Wnt/β-catenin signalling. In the ON state, Hippo signalling results in serine/threonine phosphorylation of Yes-associated protein (YAP1) and tafazzin (TAZ), promoting formation of the β-catenin destruction complex. In contrast, YAP1 or TAZ dephosphorylation (and YAP1 methylation) results in β-catenin destruction complex deactivation and β-catenin nuclear localization. In the Hippo OFF state, YAP1 and TAZ are engaged with the nuclear β-catenin and participate in the β-catenin-dependent transcription program. Interestingly, YAP1/TAZ are dispensable for intestinal homeostasis; however, upon Wnt pathway hyperactivation, the proteins together with TEA domain (TEAD) transcription factors drive the transcriptional program essential for intestinal cell transformation. In addition, in many CRC cells, YAP1 phosphorylation by YES proto-oncogene 1 tyrosine kinase (YES1) leads to the formation of a transcriptional complex that includes YAP1, β-catenin and T-box 5 (TBX5) DNA-binding protein. YAP1/β-catenin/T-box 5-mediated transcription is necessary for CRC cell proliferation and survival. Interestingly, dishevelled (DVL) appears to be an important mediator involved in both Wnt and Hippo (YAP1/TAZ) signalling and some of the DVL functions were assigned to the nuclear DVL pool. Wnt ligands can trigger alternative signalling that directly involves some of the Hippo pathway components such as YAP1, TAZ and TEADs. By upregulating Wnt pathway agonists, the alternative Wnt signalling can inhibit the canonical Wnt pathway activity.

Leucine-rich Repeat-containing G-protein Coupled Receptor 5 Gene Overexpression of the Rat Small Intestinal Progenitor Cells in Response to Orally Administered Grape Exosome-like Nanovesicles.

Background:Grape exosome-like nanovesicles (GELNs) have the advantage of inherent biocompatibility and biodegradability, the potential to be used as oral delivery vehicles. The objective of this research was to evaluate the efficiency of Syrah GELN purification and their effects on the intestinal stem cells when orally administrated to the rats.Materials and Methods:In this experimental study, Syrah GELN isolated by differential centrifugation and sucrose gradient ultracentrifugation method, then the concentration of protein, size, and zeta potential were measured as well as nanoparticles morphology. The stability of nanoparticles was investigated in the solution that mimicked the condition encountered in the stomach and intestine. To demonstrate transfection efficiency of intestinal stem cells, real-time PCR was carried out using rat leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-specific primers on cDNA derived from total RNA extracted from the upper part of the small intestine of GELN-treated rats and their controls.Results:The mean size, zeta potential, and concentration of nanoparticles were 205.1 nm, −12.5 mV, and 250 μg/ml, respectively. The result of stability test demonstrated that Syrah GELN were resistant to the harsh environment of the stomach. Lgr5 gene expression was increased by tenfold in GELN-treated rats compared with the controls.Conclusions:As intestinal stem cells are poorly accessible by common exogenous agents in vivo, oral delivery of GELNs provides a new approach to modulate the stem cell microenvironment for intestinal remodeling. This novel and effective method would help to overcome conditions such as inflammatory bowel disease, colorectal cancer, and applicable in regenerative medicine.

Expression of Leucine-rich Repeat-containing G-protein Coupled Receptor 5 and CD44: Potential Implications for Gastric Cancer Stem Cell Marker.

BackgroundThe human leucine-rich repeat-containing G-protein coupled receptor (LGR) 5 and CD44 are one of the candidates for the marker of gastric cancer stem cells. We compared the expressions of two genes among control, dysplasia and cancer groups.MethodsWe compared the mRNA expression of LGR5, CD44 and CD44v8–10 and immunohistochemistry (IHC) of LGR5 and CD44 in gastric antral mucosa of 45 controls, 36 patients with gastric dysplasia, and 39 patients with early gastric cancer. Additionally, IHC of LGR5 in gastric body mucosa was analyzed. Normal mucosa adjacent to dysplastic or cancer lesions was used for the quantitative real-time–PCR and IHC.ResultsImmunoreactivity of LGR5 in base of antral mucosa was higher in non-cancerous tissues of cancer than those of control (P = 0.006), whereas the expression of LGR5 mRNA was not different among the three groups. Immunostaining of LGR5 was much stronger in the antrum than in the body of stomach (P < 0.001). Although there was no difference in antral immunointensity of LGR5 according to the severity of intestinal metaplasia, stronger immunostaining was found in the body with an aggravation of intestinal metaplasia (P trend < 0.001). The expression of CD44v8–10 mRNA was higher in cancer patients than control subjects and patients with dysplasia (P = 0.018 and 0.009) while the expression of CD44 mRNA was higher in the control groups than the others.ConclusionsIHC of LGR5 in crypt base and CD44 may be used for gastric CSC markers. LGR5 expression may be associated with the developing of corporal intestinal metaplasia. The expression of CD44v8–10 mRNA would be more suitable for gastric cancer stem cell marker than CD44 or LGR5 mRNA.

Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.

In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors.

LGR4 acts as a key receptor for R-spondin 2 to promote osteogenesis through Wnt signaling pathway

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.

Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles.

R-spondin proteins are novel Wnt/β-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/β-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/β-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes (Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/β-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/β-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.

MiR-218 impedes IL-6-induced prostate cancer cell proliferation and invasion via suppression of LGR4 expression.

Prostate cancer is the most common solid-organ malignancy and the second leading cause of cancer-related death in males. The oncogenic effect of leucine-rich repeat-containing G protein-coupled receptor (LGR) 4 has been recognized in the formation of various types of cancers, yet its regulatory mechanism in prostate cancer is still not fully understood. Previous study has shown that LGR4 may be a new responsive gene of interleukin-6 (IL-6) in cancer progression. In the present study, we established the LNCaP-IL-6+ cell subline by long-term incubation with a low concentration of IL-6 and explored the regulatory role of miR-218, a tumor-suppressing miRNA, in IL-6-induced LGR4 expression and LNCaP-IL-6+ cell proliferation and invasion. The results showed that miR-218 expression was gradually decreased and IL-6 expression was gradually increased in the process of prostate cancer progression from normal prostate, benign prostatic hyperplasia to prostate cancer, and from LNCaP to LNCaP-IL-6+ cells. Notably, we also found that miR-218 inhibited the expression of cell cycle regulatory protein cyclin A1 and invasion-related matrix metalloproteinase-9 protein induced by IL-6, and impeded the accelerative effect of IL-6 on LNCaP-IL-6+ cell proliferation, cell cycle progression and cell invasion. Moreover, our results confirmed that miR-218 directly targets LGR4 and modulated the PI3K/Akt and Wnt/β-catenin pathways in the LNCaP-IL-6+ cells. Taken together, these data clearly demonstrated the involvement of the miR-218/LGR4 regulatory pathway in IL-6-induced cell proliferation and invasion in LNCaP-IL-6+ cells via PI3K/Akt and Wnt/β-catenin signaling, providing new insight into therapeutics for inflammation-induced prostate cancer.

5-Fluorouracil induces apoptosis of colorectal cancer cells.

5-Fluorouracil (5-FU) is widely used in chemotherapy for treatment of colorectal cancer. Leucine-rich repeat-containing G protein-coupled receptor (LGR) is known to participate in the occurrence and development of breast cancer by regulating the rebirth of tumor vessels. This study aimed to explore the proliferation and apoptosis of HCT116 colorectal cancer cells treated with 5-FU and related molecular mechanisms. 5-FU (20 μg/mL) was used to treat cultured HCT116 cells. An MTT test, flow cytometry, and colony formation assays were used to examine the proliferation and apoptosis of HCT116 cells. Western blotting was applied to detect the expression of the LGR4 protein in HCT116 cells. Small interference RNA or over-expression techniques were used to manipulate LGR4 expression in HCT116 cells and describe the proliferation and apoptosis of HCT116 treated with 5-FU. A dosage of 20 μg/mL 5-FU resulted in a significant decrease in the proliferation and apoptosis of HCT116 cells and significantly decreased expression levels of LGR4. The specific gene silence or over-expression of LGR4 in HCT116 cells increased and decreased the levels of apoptosis in HCT116, respectively. 5-FU induces apoptosis of colorectal cancer cells and inhibits proliferation by suppressing LGR4 proteins.

In a Class of Their Own - RXFP1 and RXFP2 are Unique Members of the LGR Family.

The leucine-rich repeat-containing G protein-coupled receptors (LGRs) family consists of three groups: types A, B, and C and all contain a large extracellular domain (ECD) made up of the structural motif – the leucine-rich repeat (LRR). In the LGRs, the ECD binds the hormone or ligand, usually through the LRRs, that ultimately results in activation and signaling. Structures are available for the ECD of type A and B LGRs, but not the type C LGRs. This review discusses the structural features of LRR proteins, and describes the known structures of the type A and B LGRs and predictions that can be made for the type C LGRs. The mechanism of activation of the LGRs is discussed with a focus on the role of the low-density lipoprotein class A (LDLa) module, a unique feature of the type C LGRs. While the LDLa module is essential for activation of the type C LGRs, the molecular mechanism for this process is unknown. Experimental data for the potential interactions of the type C LGR ligands with the LRR domain, the transmembrane domain, and the LDLa module are summarized.

Effects of Weipixiao (胃痞消) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions.

To study the effects of Weipixiao (胃痞消, WPX) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions (GPL). Sprague Dawley rats were randomly divided into control, model, vitacoenzyme (0.2 g·kg(-1)·day(-1)), WPX high-dose (H-WPX, 15 g·kg(-1)·day(-1)), WPX medium-dose (M-WPX, 7.5 g·kg(-1)·day(-1)) and WPX low-dose (L-WPX, 3.75 g·kg(-1)·day(-1)) groups. After successfully establishing the GPL model, the rats were consecutively administered WPX or vitacoenzyme by gastrogavage for 10 weeks. Differential expression of Leucine-rich repeat-containing G-proteincoupled receptor 5 (Lgr5), matrix metalloproteinase-7 (MMP-7), Wnt1, Wnt3a, and β-catenin in gastric mucosal epithelial cells in all groups were immunohistochemically detected, and the images were taken and analyzed semiquantitatively by image pro plus 6.0 software. Gastric epithelium in the model group showed significantly higher expression levels of Lgr5, MMP-7, Wnt1, Wnt3a and β-catenin than those of the control group(P<0.01). Interestingly, we also observed Lgr5+ cells, which generally located at the base of the gastric glandular unit, migrated to the luminal side of gastric epithelium with GPL. The expression levels of Lgr5, MMP-7, Wnt1, and β-catenin were all down-regulated in the L-WPX group as compared with those of both model and vitacoenzyme groups (P<0.05). A similar, but nonsignificant down-regulation in expression level of Wnt3a was noted in all WPX groups (P>0.05). Our findings suggested that the therapeutic mechanisms of WPX in treating GPL might be related with its inhibitory effects on the expressions of Lgr5, MMP-7, Wnt1, β-catenin and the aberrant activation of Wnt/β-catenin pathway.

GPCR48/LGR4 promotes tumorigenesis of prostate cancer via PI3K/Akt signaling pathway.

G-protein-coupled receptor (GPCR) 48, also known as leucine-rich repeat-containing G-protein-coupled receptor (LGR) 4, is an orphan receptor belonging to the GPCR superfamily, which plays an important role in the development of various organs and multiple cancers. However, the function of GPCR48/LGR4 in prostate cancer has not been fully investigated. Herein, GPCR48/LGR4 was overexpressed and silenced in prostate cancer cells via plasmid and shRNA transfection, respectively. The expression of GPCR48/LGR4 in mRNA and protein levels was analyzed using RT-qPCR and Western blotting, respectively. Subsequently, we demonstrated the effects of GPCR48/LGR4 on the migration, invasion, proliferation and apoptosis of prostate cancer cells, including Du145 and PC-3 cells. Next, we investigated the relationship between GPCR48/LGR4 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway. The results showed that the overexpression of GPCR48/LGR4 was associated with the up-regulation of Akt, a key effector of PI3K/Akt signaling pathway, which meantime up-regulated the expression of mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK-3β), while down-regulated forkhead box, class O (FOXO), all of whom are the downstream targets of PI3K/Akt signaling pathway. Hence, the results suggested that GPCR48/LGR4 may regulate prostate cancer cells and tumor growth via the PI3K/Akt signaling pathway and could provide a better therapeutic target for the diagnosis and treatment of prostate cancer.

Structure and function of LGR5: An enigmatic G‐protein coupled receptor marking stem cells

G-protein coupled receptors (GPCRs) are an important class of membrane protein that transmit extracellular signals invoked by sensing molecules such as hormones and neurotransmitters. GPCR dysfunction is implicated in many diseases and hence these proteins are of great interest to academia and the pharmaceutical industry. Leucine-rich repeat-containing GPCRs contain a characteristic extracellular domain that is an important modulator of intracellular signaling. One member of this class is the leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a stem cell marker in intestinal crypts, and mammary glands. LGR5 modulates Wnt signaling in the presence of the ligand R-spondin (RSPO). The mechanism of activation of LGR5 by RSPO is not understood, nor is the intracellular signaling mechanism known. Recently reported structures of the extracellular domain of LGR5 bound to RSPO reveal a horseshoe-shaped architecture made up of consecutive leucine-rich repeats, with RSPO bound on the concave surface. This review discusses the discovery of LGR5 and the impact it is having on our understanding of stem cell and cancer biology of the colon. In addition, it covers functional relationships suggested by sequence homology and structural analyses, as well as some intriguing conundrums with respect to the involvement of LGR5 in Wnt signaling.

Leucine-rich repeat-containing G-protein coupled receptor 5/GPR49 activates G12/13-Rho GTPase pathway.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5/GPR49) is highly expressed in adult stem cells of various tissues, such as intestine, hair follicles, and stomach. LGR5 is also overexpressed in some colon and ovarian tumors. Recent reports show that R-spondin (RSPO) family ligands bind to and activate LGR5, enhancing canonical Wnt signaling via the interaction with LRP5/6 and Frizzled. The identity of heterotrimeric G-proteins coupled to LGR5, however, remains unclear. Here, we show that Rho GTPase is a downstream target of LGR5. Overexpression of LGR5 induced SRF-RE luciferase activity, a reporter of Rho signaling. RSPOs, ligands for LGR4, LGR5, and LGR6, however, did not induce SRF-RE reporter activity in the presence of LGR5. Consistently, LGR5-induced activity of the SRF-RE reporter was inhibited by Rho inhibitor C3 transferase and RhoA N19 mutant, and knockdown of Gα12/13 genes blocked the reporter activity induced by LGR5. In addition, focal adhesion kinase, NF-κB and c-fos, targets of Rho GTPase, were shown to be regulated by LGR5. Here, we have demonstrated, for the first time, that LGR5 is coupled to the Rho pathway through G12/13 signaling.

Abstract 3757: Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells.

Abstract Cancer stem cells (CSCs), also called tumor initiating cells, have gained substantial interest in the research field over the past few years. CSCs have been isolated from multiple tumor entities and were shown to play a crucial role during tumor growth and metastasis. However, there is still a major debate about specific cell surface markers capable of identifying CSCs in most tumor entities. The leucine-rich repeat-containing G-protein-coupled receptor (LGR) Lgr5 and its close homologues Lgr4 and Lgr6 associate with Wnt-receptors and act as R-spondin receptors thereby playing a central role in the modulation of Wnt/β-catenin signaling in normal and neoplastic stem cells (de Lau et al., 2011, Carmon et al., 2011). Initially described as a highly specific marker for stem cells in the small intestine, colon, hair follicle, stomach, and during kidney development (Barker et al., 2007, Jaks et al., 2008, Barker et al., 2010, Barker et al., 2012), Lgr5 positive cells were also shown to be crucial during the development and progression of cancer. It was shown that Lgr5 positive crypt stem cells are the cells-of-origin in intestinal cancer and that CSCs in human colorectal cancer can be identified and isolated based on Lgr5 expression (Barker et al., 2009, Kemper et al., 2012). However, the analysis of Lgr5 expressing cells is hampered by the lack of highly specific monoclonal antibodies. We have developed rabbit monoclonal antibodies against peptide sequences of Lgr5. After generating cell lines stably expressing Lgr5, we screened for high affinity antibodies by flow cytometry. Using stable transfectants for Lgr4 and Lgr6, we could prove that there is no cross reactivity with its close homologues. This is of particular importance for Lgr4, as this protein is also expressed on more differentiated progenitor cells. To allow for identification, analysis, and isolation of Lgr5 positive cells from solid tissues, automated and therefore user-independent methods for tissue dissociation were developed. This resulted in enzyme mixtures which allow dissociating different tissues without harming the target epitopes. To detect cells expressing Lgr5 at low levels in flow cytometry based assays we combined our novel high affinity antibodies with fluorescence amplification systems. In addition, we generated a new magnetic cell separation (MACS) protocol that can be used as an easy and fast method to isolate Lgr5 positive cells from primary human tissue. Taken together, we have developed highly specific monoclonal antibodies allowing for the analysis and isolation of Lgr5 positive cells from cell lines and primary tissues. Citation Format: David Agorku, Greg Parker, Nadine Chelius, Andreas Bosio, Olaf Hardt. Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3757. doi:10.1158/1538-7445.AM2013-3757

Troy, a Tumor Necrosis Factor Receptor Family Member, Interacts With Lgr5 to Inhibit Wnt Signaling in Intestinal Stem Cells

The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.

In Pursuit of Leucine-Rich Repeat-Containing G Protein-Coupled Receptor-5 Regulation and Function in the Uterus

Leucine-rich repeat-containing G protein-coupled receptor (LGR)-5 is a recently identified marker of stem cells in adult intestinal epithelium and hair follicles. Because of this characteristic, we studied the status of Lgr5 expression in the mouse uterus under various conditions. Lgr5 is highly expressed in the uterine epithelium of immature mice and is dramatically down-regulated after the mice resume estrous cycles. Surprisingly, whereas its expression is up-regulated in uteri of ovariectomized mice, the expression is down-regulated by estrogen and progesterone via their cognate nuclear receptors, estrogen receptor-alpha and progesterone receptor, respectively. Using a mouse endometrial cancer model, we also found that Lgr5 is highly expressed in the epithelium during the initial stages of tumorigenesis but is remarkably down-regulated in fully developed tumors. Lgr5 is a downstream target of Wnt signaling in the intestine. Genetic evidence shows that either excessive or absence of Wnt signaling dampens Lgr5 expression in the uterus. Collectively, our results show that Lgr5 expression in the mouse uterine epithelium is unique and dynamically regulated under various physiological and pathological states of the uterus, suggesting that this orphan receptor has important functions in uterine biology. However, identifying definitive uterine function of LGR5 will require further investigation using conditional deletion of uterine Lgr5 because systemic deletion of this gene is neonatally lethal.