The Experts below are selected from a list of 14613 Experts worldwide ranked by ideXlab platform

Mingyao Liu - One of the best experts on this subject based on the ideXlab platform.

  • LGR4 deletion delays the hair cycle and inhibits the activation of hair follicle stem cells
    Journal of Investigative Dermatology, 2020
    Co-Authors: Xiaolin Ren, Mingyao Liu, Weili Xia, Hongyang Shen, Xing Dai, Yuling Shi, Yongyan Dang
    Abstract:

    It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre–mediated skin epithelia–specific deletion of LGR4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although LGR4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in LGR4−/− mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.

  • LGR4 not lgr5 enhances hpsc hematopoiesis by facilitating mesoderm induction via tgf beta signaling activation
    Cell Reports, 2020
    Co-Authors: Yu Wang, Mingyao Liu, Hongtao Wang, Jiaojiao Guo, Jie Gao, Mengge Wang, Meijuan Xia, Yuqi Wen, Ming Yang, Lihong Shi
    Abstract:

    Summary Attempts to generate functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful, mainly due to the lack of understanding of the regulatory network of human hematopoiesis. In this study, we identified leucine-rich-repeat-containing G-protein-coupled receptor 4 (LGR4) as an essential regulator of early hematopoietic differentiation of hPSCs. The deletion of LGR4 severely impairs mesoderm development, thereby limiting hematopoietic differentiation both in vitro and in vivo. In contrast, LGR5 is dispensable for hPSC hematopoiesis. The four R-spondin proteins show differential activities and dependencies on LGR4 in hematopoietic differentiation. The deletion of LGR4 almost entirely abolishes the enhancement induced by R-spondin1 and R-spondin3, but not R-spondin2. In addition, ZNRF3 is required for the response of R-spondin1–R-spondin3. At the mechanistic level, LGR4 regulates transforming growth factor beta (TGF-beta) signaling to control hematopoietic differentiation. Together, our results reveal vital roles of LGR4 in hematopoietic development and uncover distinct functions and underlying mechanisms for R-spondins.

  • loss of LGR4 inhibits differentiation migration and apoptosis and promotes proliferation in bone mesenchymal stem cells
    Journal of Cellular Physiology, 2019
    Co-Authors: Peng Sun, Chunbing Zheng, Stefan Siwko, Mingyao Liu, Kunhang Jia, Xinlei Zhu, Feng Xue, Jian Luo
    Abstract:

    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.

  • leucine rich repeat containing g protein coupled receptor 4 LGR4 is necessary for prostate cancer metastasis via epithelial mesenchymal transition
    Journal of Biological Chemistry, 2017
    Co-Authors: Weijia Luo, Stefan Siwko, Mingyao Liu, Peng Tan, Melissa Rodriguez, Kunrong Tan, Li Zeng
    Abstract:

    Prostate cancer is a highly penetrant disease among men in industrialized societies, but the factors regulating the transition from indolent to aggressive and metastatic cancer remain poorly understood. We found that men with prostate cancers expressing high levels of the G protein–coupled receptor LGR4 had a significantly shorter recurrence-free survival compared with patients with cancers having low LGR4 expression. LGR4 expression was elevated in human prostate cancer cell lines with metastatic potential. We therefore generated a novel transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model to investigate the role of LGR4 in prostate cancer development and metastasis in vivo. TRAMP LGR4−/− mice exhibited an initial delay in prostate intraepithelial neoplasia formation, but the frequency of tumor formation was equivalent between TRAMP and TRAMP LGR4−/− mice by 12 weeks. The loss of LGR4 significantly improved TRAMP mouse survival and dramatically reduced the occurrence of lung metastases. LGR4 knockdown impaired the migration, invasion, and colony formation of DU145 cells and reversed epithelial–mesenchymal transition (EMT), as demonstrated by up-regulation of E-cadherin and decreased expression of the EMT transcription factors ZEB, Twist, and Snail. Overexpression of LGR4 in LNCaP cells had the opposite effects. Orthotopic injection of DU145 cells stably expressing shRNA targeting LGR4 resulted in decreased xenograft tumor size, reduced tumor EMT marker expression, and impaired metastasis, in accord with our findings in TRAMP LGR4−/− mice. In conclusion, we propose that LGR4 is a key protein necessary for prostate cancer EMT and metastasis.

  • LGR4 deficiency attenuates tumor formation by reprograming macrophage polarization in lung cancer tum6p 973
    Journal of Immunology, 2015
    Co-Authors: Binhe Tan, Yinglei Wei, Juliang Qin, Min Qian, Mingyao Liu
    Abstract:

    Macrophage polarization is increasingly recognized as an important pathogenetic factor in tumor formation. Our previous study has shown that LGR4 negatively regulates TLR2/4 associated pattern recognition in innate immune responses. However, the exact roles and mechanisms of LGR4 in macrophage polarization remain largely unknown. Here, we found that patients with LGR4-high lung cancers displayed much poorer survival than the rest of the cohorts. Meanwhile, LGR4 is also aberrantly expressed at high levels in macrophages from clinical lung adenocarcinomas (ADs), implying the potential role of LGR4 in tumor-associated macrophages (TAMs). In addition, the expression of LGR4 in bone marrow derived macrophages (BMMs) was reduced by IFN-γ and increased by IL-4 significantly. Interestingly, LGR4 deficiency BMMs showed a obviously increased M1 phenotypes and reduced M2 phenotypes. Accordingly, host deficiency in LGR4 resulted in defective and reduced growth of subcutaneously implanted Lewis lung carcinoma (LLC). Consistent with the defective growth, LLC cells implanted into LGR4 conditional knockout mice also displayed reduced TAMs and increased M1 like macrophages in tumor tissues, suggesting impaired TAMs polarization. These findings suggest that deletion of LGR4 may inhibits tumor growth via reprograming TAMs polarization. Thus, LGR4 expressed in TAMs may potentially become a novel target for cancer therapy.

Aaron J. W. Hsueh - One of the best experts on this subject based on the ideXlab platform.

  • a naturally occurring LGR4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of LGR4 in mammals
    PLOS ONE, 2014
    Co-Authors: Peijen Hsu, Masataka Kudo, Aaron J. W. Hsueh, Chihlun Hsiao, Chingwei Luo
    Abstract:

    Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring LGR4 splice variant encoding only the ectodomain of LGR4, which was named LGR4-ED. LGR4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of LGR4-ED in mammals. Recombinant LGR4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant LGR4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in LGR4-null mice. Administration of recombinant LGR4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed LGR4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated LGR4-Wnt signaling during gonadal development.

  • multi functional norrin is a ligand for the LGR4 receptor
    Journal of Cell Science, 2013
    Co-Authors: Cheng Deng, Pradeep Reddy, Yuan Cheng, Chingwei Luo, Chihlun Hsiao, Aaron J. W. Hsueh
    Abstract:

    Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

  • characterization of novel splice variants of lgr7 and lgr8 reveals that receptor signaling is mediated by their unique low density lipoprotein class a modules
    Journal of Biological Chemistry, 2006
    Co-Authors: Daniel Scott, Aaron J. W. Hsueh, Sharon Layfield, Satoko Sudo, Yan Yan, Geoffrey W Tregear
    Abstract:

    The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.

  • leucine rich repeat containing g protein coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality
    Molecular Endocrinology, 2004
    Co-Authors: Sabine Mazerbourg, Satoko Sudo, Donna M Bouley, Cynthia Klein, Jian Zhang, Kazuhiro Kawamura, Lisa V Goodrich, Helen Rayburn, Marc Tessierlavigne, Aaron J. W. Hsueh
    Abstract:

    Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven-transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4–6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene after the insertion of a β-galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistoch...

  • LGR4 receptor null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality
    2004
    Co-Authors: Sabine Mazerbourg, Satoko Sudo, Donna M Bouley, Jian Zhang, Kazuhiro Kawamura, Lisa V Goodrich, Helen Rayburn, Marc Tessierlavigne, Cindy Klein, Aaron J. W. Hsueh
    Abstract:

    ABSTRACT Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4-6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene following the insertion of a -galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistochemical studies in wild-type animals. Analysis of the viability of 250 newborn animals suggested a skewed inheritance pattern, indicating that only 40% of the expected LGR4 null mice were born. For the LGR4 null mice viable at birth, most of them died within two days. Furthermore, the LGR4 null mice showed intrauterine growth retardation as reflected by a 14% decrease in body weight at birth, together with 30% and 40% decreases in kidney and liver weights, respectively. The present findings demonstrated the widespread expression of LGR4, an essential role of LGR4 for embryonic growth as well as kidney and liver development. The observed pre- and postnatal lethality of LGR4 null mice underlies the importance of the LGR4 signaling system for the survival and growth of animals during the perinatal stage.

Marieisabelle Garcia - One of the best experts on this subject based on the ideXlab platform.

  • LGR4 and lgr5 regulate hair cell differentiation in the sensory epithelium of the developing mouse cochlea LGR4 5 in the developing cochlea
    Frontiers in Cellular Neuroscience, 2016
    Co-Authors: Magdalena Zak, Gilbert Vassart, Marieisabelle Garcia, Thys Van Oort, Ferry G J Hendriksen, Wilko Grolman
    Abstract:

    In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferationof precursors and promotes the formation of hair cells by up-regulating Atoh1expression. Not much, however, is known about the regulation of Wnt/β-catenin activityin the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated byan interaction between LGR receptors and their ligands from the R-spondin family. Thedeficiency in LGR4 and Lgr5 genes leads to developmental malformations and lethality.Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increasesWnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction ofinner and outer hair cells (OHC). Supernumerary hair cells are likely formed due toan up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3.Using a hypomorphic LGR4 mouse model we showed a mild overproduction of OHCs inthe heterozygous and homozygous LGR4 mice. The loss of LGR4 function prolongedthe proliferation in the mid-basal turn of E13 cochleae, causing an increase in thenumber of SOX2-positive precursor cells within the pro-sensory domain. The prematuredifferentiation of hair cells progressed in a medial to lateral gradient in LGR4 deficientembryos. No significant up-regulation of Atoh1 was observed following LGR4 deletion.Altogether, our findings suggest that LGR4 and LGR5 play an important role in theregulation of hair cell differentiation in the embryonic cochlea.

  • LGR4 and lgr5 regulate hair cell differentiation in the sensory epithelium of the developing mouse cochlea
    Frontiers in Cellular Neuroscience, 2016
    Co-Authors: Magdalena żak, Gilbert Vassart, Marieisabelle Garcia, Ferry G J Hendriksen, Thijs Van Oort, Wilko Grolman
    Abstract:

    In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in LGR4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells. Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic LGR4 mouse model we showed a mild overproduction of outer hair cells in the heterozygous and homozygous LGR4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in LGR4 deficient embryos. No significant up-regulation of Atoh1 was observed following LGR4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

  • identification of lgr5 independent spheroid generating progenitors of the mouse fetal intestinal epithelium
    Cell Reports, 2013
    Co-Authors: Roxana C Mustata, Gabriela Vasile, Valeria Fernandezvallone, Sandra Strollo, Anne Lefort, Frederick Libert, Daniel Monteyne, David Perezmorga, Gilbert Vassart, Marieisabelle Garcia
    Abstract:

    Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

  • LGR4 is required for paneth cell differentiation and maintenance of intestinal stem cells ex vivo
    EMBO Reports, 2011
    Co-Authors: Roxana C Mustata, Sandra Strollo, Anne Lefort, Frederick Libert, Gilbert Vassart, Marieisabelle Garcia
    Abstract:

    Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

Qingyun Liu - One of the best experts on this subject based on the ideXlab platform.

  • drug conjugates of antagonistic rspo4 mutant for simultaneous targeting of LGR4 5 6 for cancer treatment
    bioRxiv, 2021
    Co-Authors: Jie Cui, Kendra S. Carmon, Soohyun Park, Qingyun Liu
    Abstract:

    Abstract LGR4-6 (Leucine-rich repeating containing, G-protein-coupled receptors 4, 5, and 6) are three related receptors with distinct roles in organ development and stem cell survival. All three receptors are upregulated in gastrointestinal cancers to different levels, and LGR5 has been shown to be enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. The RSPO4 mutant was fused to the N-terminus of human IgG1-Fc to create a peptibody which was then conjugated with cytotoxins monomethyl auristatin or duocarmycin by site-specific conjugation. The resulting peptibody drug conjugates (PDCs) showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects. These results suggest that RSPO-derived PDCs may provide a novel approach to the treatment of cancers with high LGR expression.

  • unlike LGR4 lgr5 potentiates wnt β catenin signaling without sequestering e3 ligases
    Science Signaling, 2020
    Co-Authors: Soohyun Park, Kendra S. Carmon, Yukimatsu Toh, Qingyun Liu
    Abstract:

    LGR4 and LGR5 encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-β-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only LGR4 is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-β-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of LGR4 and LGR5 in organ development, as well as the self-renewal and fitness of adult stem cells.

  • rspo LGR4 functions via iqgap1 to potentiate wnt signaling
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Kendra S. Carmon, Xing Gong, Anthony Thomas, Qingyun Liu
    Abstract:

    R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO–LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO–LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO–LGR4’s interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO–LGR4 to the Wnt signaling complex through enhanced IQGAP1–DVL interaction following RSPO stimulation. In this configuration, RSPO–LGR4–IQGAP1 potentiates β-catenin–dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as β-catenin–independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO–LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.

  • analysis of LGR4 receptor distribution in human and mouse tissues
    PLOS ONE, 2013
    Co-Authors: Wei Xiong, Xing Gong, Seth Bellister, Lee M Ellis, Qingyun Liu
    Abstract:

    LGR4 is an R-spondin receptor with strong positive effect on Wnt signaling. It plays a critical role in development as its ablation in the mouse led to total embryonic/neonatal lethality with profound defects in multiple organs. Haplotype insufficiency of LGR4 in human was associated with several diseases, including increased risk of squamous cell carcinoma of the skin, reduced birth weights, electrolyte imbalance, and decreased levels of testosterone, which are similar to the phenotypes of LGR4-hypomorphic mice. Tissue distribution of LGR4 was extensively analyzed in the mouse using gene-trap reporter enzyme alleles. However, its expression pattern in human tissues remained largely unknown. We have developed LGR4-specific monoclonal antibodies and used them to examine the expression of LGR4 in selected adult human and mouse tissues by immunohistochemical analysis. Intense LGR4-like immunoreactivity was observed in the epidermis and hair follicle of the skin, pancreatic islet cells, and epithelial cells in both the male and female reproductive organs. Of particular interest is that LGR4 is highly expressed in germ cells and pancreatic islet cells, which have important implications given the role of R-spondin-LGR4 signaling in the survival of adult stem cells. In addition, the majority of colon tumors showed elevated levels of LGR4 receptor. Overall, the expression pattern of LGR4 in human tissues mapped by this IHC analysis is similar to that in the mouse as revealed from gene trap alleles. Importantly, the pattern lends strong support to the important role of LGR4 in the development and maintenance of skin, kidney, reproductive systems, and other organs.

  • Abstract 4900: Functional and mechanism analysis of LGR4 in human lung cancer cells.
    Tumor Biology, 2013
    Co-Authors: Xing Gong, Wei Xiong, Kendra S. Carmon, Qingyun Liu
    Abstract:

    LGR4 (leucine-rich repeat containing G protein-coupled receptor 4) and its two closely related homologues LGR5 and LGR6 (LGR4-6) are members of the seven transmembrane domain (7TM) receptors with critical functions in embryonic development and adult stem cells. Recently, we and others discovered that LGR4-6 function as receptors of R-spondins (RSPO) to potentiate Wnt signaling. RSPOs are a group of four secreted proteins (RSPO1-4) with essential roles in organ development, sex determination, carcinogenesis, etc. Particularly, RSPO2 is required for normal lung development through enhancement of Wnt signaling and LGR6 is expressed in adult lung stem cells in the mouse. Among the 900 or so 7TM receptors, LGR4 was found to be one of the few that were highly upregulated in both squamous cell carcinoma (SqCC) and adenocarcinoma (AC) of the lung. Alterations in the Wnt signaling pathways were found in a small percentage of lung cancer. Concurrent activation of β-catenin signaling enhances tumor growth and metastasis of lung cancer cells with KRAS mutation. Furthermore, RSPO3 and LGR4 are co-expressed at high levels in ∼20% of lung cancer cell lines. However, potential roles of RSPO3-LGR4 in lung carcinogenesis have not been investigated. We developed and validated a panel of LGR4 antibodies that can detect either native or denatured LGR4 with high specificity and sensitivity. Immunohistochemistry analyses confirmed that LGR4 was highly expressed in the majority of SqCC and AC of the lung. Knockdown of LGR4 in a lung cancer cell line with high levels of and LGR4 and RSPO3 leads to dramatic decrease in endogenous Wnt signaling and significant reduction in cell growth, migration, and colony formation in vitro. The effect of knockdown on tumor growth in vivo is being tested. Data from these expression and functional analyses of LGR4 in human lung cancer will be presented and discussed. Citation Format: Xing Gong, Wei Xiong, Jing Yi, Kendra S. Carmon, Qingyun Liu. Functional and mechanism analysis of LGR4 in human lung cancer 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 4900. doi:10.1158/1538-7445.AM2013-4900

Gilbert Vassart - One of the best experts on this subject based on the ideXlab platform.

  • LGR4 and lgr5 regulate hair cell differentiation in the sensory epithelium of the developing mouse cochlea LGR4 5 in the developing cochlea
    Frontiers in Cellular Neuroscience, 2016
    Co-Authors: Magdalena Zak, Gilbert Vassart, Marieisabelle Garcia, Thys Van Oort, Ferry G J Hendriksen, Wilko Grolman
    Abstract:

    In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferationof precursors and promotes the formation of hair cells by up-regulating Atoh1expression. Not much, however, is known about the regulation of Wnt/β-catenin activityin the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated byan interaction between LGR receptors and their ligands from the R-spondin family. Thedeficiency in LGR4 and Lgr5 genes leads to developmental malformations and lethality.Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increasesWnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction ofinner and outer hair cells (OHC). Supernumerary hair cells are likely formed due toan up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3.Using a hypomorphic LGR4 mouse model we showed a mild overproduction of OHCs inthe heterozygous and homozygous LGR4 mice. The loss of LGR4 function prolongedthe proliferation in the mid-basal turn of E13 cochleae, causing an increase in thenumber of SOX2-positive precursor cells within the pro-sensory domain. The prematuredifferentiation of hair cells progressed in a medial to lateral gradient in LGR4 deficientembryos. No significant up-regulation of Atoh1 was observed following LGR4 deletion.Altogether, our findings suggest that LGR4 and LGR5 play an important role in theregulation of hair cell differentiation in the embryonic cochlea.

  • LGR4 and lgr5 regulate hair cell differentiation in the sensory epithelium of the developing mouse cochlea
    Frontiers in Cellular Neuroscience, 2016
    Co-Authors: Magdalena żak, Gilbert Vassart, Marieisabelle Garcia, Ferry G J Hendriksen, Thijs Van Oort, Wilko Grolman
    Abstract:

    In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in LGR4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells. Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic LGR4 mouse model we showed a mild overproduction of outer hair cells in the heterozygous and homozygous LGR4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in LGR4 deficient embryos. No significant up-regulation of Atoh1 was observed following LGR4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

  • identification of lgr5 independent spheroid generating progenitors of the mouse fetal intestinal epithelium
    Cell Reports, 2013
    Co-Authors: Roxana C Mustata, Gabriela Vasile, Valeria Fernandezvallone, Sandra Strollo, Anne Lefort, Frederick Libert, Daniel Monteyne, David Perezmorga, Gilbert Vassart, Marieisabelle Garcia
    Abstract:

    Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

  • LGR4 is required for paneth cell differentiation and maintenance of intestinal stem cells ex vivo
    EMBO Reports, 2011
    Co-Authors: Roxana C Mustata, Sandra Strollo, Anne Lefort, Frederick Libert, Gilbert Vassart, Marieisabelle Garcia
    Abstract:

    Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

  • expression pattern of the orphan receptor LGR4 gpr48 gene in the mouse
    Histochemistry and Cell Biology, 2005
    Co-Authors: Gregory Van Schoore, Fernando Mendive, Roland Pochet, Gilbert Vassart
    Abstract:

    Leucine-rich G-protein-coupled Receptors (LGR) constitute a subfamily of receptors related to glycoprotein hormone receptors. Amongst them, LGR4, LGR5 and LGR6 form a cluster for which natural agonists are still unknown. By an extensive gene trapping approach, Leighton et al. (2001) obtained a mouse line in which the LGR4 gene is disrupted by a trap vector carrying two biological markers, beta-geo (a fusion between bacterial beta-galactosidase and neomycin phosphotransferase) and a placental alkaline phosphatase (PLAP). Due to perinatal lethality, characterization of adult mice homozygous for this insertion has been impaired. In the present study we have investigated LacZ and PLAP activity patterns in heterozygous mice as a marker for LGR4 natural expression at both macroscopic and histological levels. We present a detailed atlas of LGR4 expression, which displays very wide expression with particularly strong activity in cartilages, kidneys, reproductive tracts and nervous system cells.