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Alexander G. Robling - One of the best experts on this subject based on the ideXlab platform.
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Mechanical Loading-Driven Tumor Suppression Is Mediated by LRP5-Dependent and Independent Mechanisms.
Cancers, 2021Co-Authors: Yan Feng, Alexander G. Robling, Shengzhi Liu, Rongrong Zha, Xun Sun, Hiroki YokotaAbstract:Bone is mechanosensitive and lipoprotein receptor-related protein 5 (LRP5)-mediated Wnt signaling promotes loading-driven bone formation. While mechanical loading can suppress tumor growth, the question is whether LRP5 mediates loading-driven tumor suppression. Herein, we examined the effect of LRP5 using osteocyte-specific LRP5 conditional knockout mice. All mice presented noticeable loading-driven tumor suppression in the loaded tibia and non-loaded mammary pad. The degree of suppression was more significant in wild-type than knockout mice. In all male and female mice, knee loading reduced cholesterol and elevated dopamine. It reduced tumor-promoting nexin, which was elevated by cholesterol and reduced by dopamine. By contrast, it elevated p53, TNF-related apoptosis-inducing ligand (TRAIL), and chemerin, and they were regulated reversely by dopamine and cholesterol. Notably, LRP5 overexpression in osteocytes enhanced tumor suppression, and osteoclast development was inhibited by chemerin. Collectively, this study identified LRP5-dependent and independent mechanisms for tumor suppression. LRP5 in osteocytes contributed to the loaded bone, while the LRP5-independent regulation of dopamine- and cholesterol-induced systemic suppression.
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Co-deletion of LRP5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration.
Bone, 2020Co-Authors: Kyung-eun Lim, Bart O Williams, Whitney A. Bullock, Daniel J. Horan, Matthew L. Warman, Alexander G. RoblingAbstract:Abstract The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of LRP5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when LRP5 is suppressed), or through other yet undiscovered mechanisms independent of LRP5/6. To distinguish between these two possibilities, we generated mice with compound deletion of LRP5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both LRP5 and Lrp6 (LRP5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre−) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre− mice, as assessed by DXA, μCT, pQCT, and histology, indicating that LRP5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre− mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of LRP5 alone, the bone-selective late-stage co-deletion of LRP5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both LRP5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.
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New Insights into Wnt–LRP5/6–β-Catenin Signaling in Mechanotransduction
Frontiers in endocrinology, 2015Co-Authors: Kyung Shin Kang, Alexander G. RoblingAbstract:Mechanical loading is essential to maintain normal bone metabolism and the balance between bone formation and resorption. The cellular mechanisms that control mechanotransduction are not fully defined, but several key pathways have been identified. We discuss the roles of several components of the Wnt signaling cascade, namely LRP5, Lrp6, and β-catenin in mechanical loading-induced bone formation. LRP5 is an important Wnt co-receptor for regulating bone mass and mechanotransduction, and appears to function principally by augmenting bone formation. Lrp6 also regulates bone mass but its action might involve resorption as well as formation. The role of Lrp6 in mechanotransduction is unclear. Studies addressing the role of β-catenin in bone metabolism and mechanotransduction highlight the uncertainties in downstream modulators of LRP5 and Lrp6. Taken together, these data indicate that mechanical loading might affect bone regulation triggering the canonical Wnt signaling (and perhaps other pathways) not only via LRP5 but also via Lrp6. Further work is needed to clarify the role of the Wnt signaling pathway in LRP5 and/or Lrp6-mediated mechanotransduction, which could eventually lead to powerful therapeutic agents that might mimic the anabolic effects of mechanical stimulation.
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new insights into wnt LRP5 6 β catenin signaling in mechanotransduction
Frontiers in Endocrinology, 2015Co-Authors: Kyung Shin Kang, Alexander G. RoblingAbstract:Mechanical loading is essential to maintain normal bone metabolism and the balance between bone formation and resorption. The cellular mechanisms that control mechanotransduction are not fully defined, but several key pathways have been identified. We discuss the roles of several components of the Wnt signaling cascade, namely LRP5, Lrp6, and β-catenin in mechanical loading-induced bone formation. LRP5 is an important Wnt co-receptor for regulating bone mass and mechanotransduction, and appears to function principally by augmenting bone formation. Lrp6 also regulates bone mass but its action might involve resorption as well as formation. The role of Lrp6 in mechanotransduction is unclear. Studies addressing the role of β-catenin in bone metabolism and mechanotransduction highlight the uncertainties in downstream modulators of LRP5 and Lrp6. Taken together, these data indicate that mechanical loading might affect bone regulation triggering the canonical Wnt signaling (and perhaps other pathways) not only via LRP5 but also via Lrp6. Further work is needed to clarify the role of the Wnt signaling pathway in LRP5 and/or Lrp6-mediated mechanotransduction, which could eventually lead to powerful therapeutic agents that might mimic the anabolic effects of mechanical stimulation.
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Sclerostin inhibition reverses skeletal fragility in an LRP5-deficient mouse model of OPPG syndrome.
Science translational medicine, 2013Co-Authors: Rajendra Kedlaya, Daniel J. Horan, Matthew L. Warman, Shreya Veera, Rachel E. Moss, Ugur M. Ayturk, Christina M. Jacobsen, Margot E. Bowen, Chris Paszty, Alexander G. RoblingAbstract:Osteoporosis pseudoglioma syndrome (OPPG) is a rare genetic disease that produces debilitating effects in the skeleton. OPPG is caused by mutations in LRP5, a WNT co-receptor that mediates osteoblast activity. WNT signaling through LRP5, and also through the closely related receptor LRP6, is inhibited by the protein sclerostin (SOST). It is unclear whether OPPG patients might benefit from the anabolic action of sclerostin neutralization therapy (an approach currently being pursued in clinical trials for postmenopausal osteoporosis) in light of their LRP5 deficiency and consequent osteoblast impairment. To assess whether loss of sclerostin is anabolic in OPPG, we measured bone properties in a mouse model of OPPG (LRP5−/−), a mouse model of sclerosteosis (Sost−/−), and in mice with both genes knocked out (LRP5−/−;Sost−/−). LRP5−/−;Sost−/− mice have larger, denser, and stronger bones than do LRP5−/− mice, indicating that SOST deficiency can improve bone properties via pathways that do not require LRP5. Next, we determined whether the anabolic effects of sclerostin depletion in LRP5−/− mice are retained in adult mice by treating 17-week-old LRP5−/− mice with a sclerostin antibody for 3 weeks. LRP5+/+ and LRP5−/− mice each exhibited osteoanabolic responses to antibody therapy, as indicated by increased bone mineral density, content, and formation rates. Collectively, our data show that inhibiting sclerostin can improve bone mass whether LRP5 is present or not. In the absence of LRP5, the anabolic effects of SOST depletion can occur via other receptors (such as LRP4/6). Regardless of the mechanism, our results suggest that humans with OPPG might benefit from sclerostin neutralization therapies.
Bart O Williams - One of the best experts on this subject based on the ideXlab platform.
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Co-deletion of LRP5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration.
Bone, 2020Co-Authors: Kyung-eun Lim, Bart O Williams, Whitney A. Bullock, Daniel J. Horan, Matthew L. Warman, Alexander G. RoblingAbstract:Abstract The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of LRP5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when LRP5 is suppressed), or through other yet undiscovered mechanisms independent of LRP5/6. To distinguish between these two possibilities, we generated mice with compound deletion of LRP5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both LRP5 and Lrp6 (LRP5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre−) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre− mice, as assessed by DXA, μCT, pQCT, and histology, indicating that LRP5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre− mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of LRP5 alone, the bone-selective late-stage co-deletion of LRP5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both LRP5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.
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LRP5: From bedside to bench to bone
Bone, 2017Co-Authors: Bart O WilliamsAbstract:A role for low-density lipoprotein-related receptor 5 (LRP5) in human bone was first established by the identification of genetic alterations that led to dramatic changes in bone mass. Shortly thereafter, mutations that altered the function of the sclerostin (SOST) gene were also associated with altered human bone mass. Subsequent studies of LRP5 and sclerostin have provided important insights into the mechanisms by which these proteins regulate skeletal homeostasis. Sclerostin normally binds to LRP5 and the related LRP6 protein and prevents their activation by Wnts, the LRP5/LRP6 ligands. The interaction of sclerostin with LRP5 or LRP6 is facilitated by the LRP4 protein. Loss of LRP5 leads to defective osteoblast function and low bone mass, while loss of SOST or mutations in LRP5, which produce a protein that can no longer be bound by SOST, result in high bone mass. Insights gained from the use of genetically engineered mouse models are presented, as well as a brief summary of the status of antibodies in clinical trials that block the function of SOST as a mechanism to increase bone mass.
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structure and function of norrin in assembly and activation of a frizzled 4 LRP5 6 complex
Genes & Development, 2013Co-Authors: Kaleeckal G Harikumar, Clara Erice, Chen Chen, Liren Wang, Naomi R Parker, Zhihong Cheng, Bart O Williams, Karsten Melcher, Laurence J MillerAbstract:Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor LRP5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and LRP5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-LRP5/6 signaling complex.
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LRP5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition.
PloS one, 2013Co-Authors: Ryan C. Riddle, Cassandra R. Diegel, Julie M. Leslie, Kyle K. Van Koevering, Marie Claude Faugere, Thomas L. Clemens, Bart O WilliamsAbstract:The canonical Wnt signaling pathway is critical for skeletal development and maintenance, but the precise roles of the individual Wnt co-receptors, LRP5 and Lrp6, that enable Wnt signals to be transmitted in osteoblasts remain controversial. In these studies, we used Cre-loxP recombination, in which Cre-expression is driven by the human osteocalcin promoter, to determine the individual contributions of LRP5 and Lrp6 in postnatal bone acquisition and osteoblast function. Mice selectively lacking either LRP5 or Lrp6 in mature osteoblasts were born at the expected Mendelian frequency but demonstrated significant reductions in whole-body bone mineral density. Bone architecture measured by microCT revealed that Lrp6 mutant mice failed to accumulate normal amounts of trabecular bone. By contrast, LRP5 mutants had normal trabecular bone volume at 8 weeks of age, but with age, these mice also exhibited trabecular bone loss. Both mutants also exhibited significant alterations in cortical bone structure. In vitro differentiation was impaired in both LRP5 and Lrp6 null osteoblasts as indexed by alkaline phosphatase and Alizarin red staining, but the defect was more pronounced in Lrp6 mutant cells. Mice lacking both Wnt co-receptors developed severe osteopenia similar to that observed previously in mice lacking β-catenin in osteoblasts. Likewise, calvarial cells doubly deficient for LRP5 and Lrp6 failed to form osteoblasts when cultured in osteogenic media, but instead attained a chondrocyte-like phenotype. These results indicate that expression of both LRP5 and Lrp6 are required within mature osteoblasts for normal postnatal bone development.
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LRP5 and LRP6 in development and disease.
Trends in endocrinology and metabolism: TEM, 2013Co-Authors: Danese M. Joiner, Zhendong Zhong, Bart O WilliamsAbstract:Low-density lipoprotein-related receptors 5 and 6 (LRP5/6) are highly homologous proteins with key functions in canonical Wnt signaling. Alterations in the genes encoding these receptors or their interacting proteins are linked to human diseases, and as such they have been a major focus of drug development efforts to treat several human conditions including osteoporosis, cancer, and metabolic disease. Here, we discuss the links between alterations in LRP5/6 and disease, proteins that interact with them, and insights gained into their function from mouse models. We also highlight current drug development related to LRP5/6 as well as how the recent elucidation of their crystal structures may allow further refinement of our ability to target them for therapeutic benefit.
Matthew L. Warman - One of the best experts on this subject based on the ideXlab platform.
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Co-deletion of LRP5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration.
Bone, 2020Co-Authors: Kyung-eun Lim, Bart O Williams, Whitney A. Bullock, Daniel J. Horan, Matthew L. Warman, Alexander G. RoblingAbstract:Abstract The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of LRP5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when LRP5 is suppressed), or through other yet undiscovered mechanisms independent of LRP5/6. To distinguish between these two possibilities, we generated mice with compound deletion of LRP5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both LRP5 and Lrp6 (LRP5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre−) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre− mice, as assessed by DXA, μCT, pQCT, and histology, indicating that LRP5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre− mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of LRP5 alone, the bone-selective late-stage co-deletion of LRP5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both LRP5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.
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Critical Endothelial Regulation by LRP5 during Retinal Vascular Development.
PloS one, 2016Co-Authors: Wei Huang, Matthew L. Warman, Mahmood Amiry-moghaddam, Madoka Hokama, Sylvia H. Sardi, Masashi Nagao, Björn OlsenAbstract:Vascular abnormalities in the eye are the leading cause of many forms of inherited and acquired human blindness. Loss-of-function mutations in the Wnt-binding co-receptor LRP5 leads to aberrant ocular vascularization and loss of vision in genetic disorders such as osteoporosis-pseudoglioma syndrome. The canonical Wnt-β-catenin pathway is known to regulate retinal vascular development. However, it is unclear what precise role LPR5 plays in this process. Here, we show that loss of LRP5 function in mice causes retinal hypovascularization during development as well as retinal neovascularization in adulthood with disorganized and leaky vessels. Using a highly specific Flk1-CreBreier line for vascular endothelial cells, together with several genetic models, we demonstrate that loss of endothelium-derived LRP5 recapitulates the retinal vascular defects in LRP5-/- mice. In addition, restoring LRP5 function only in endothelial cells in LRP5-/- mice rescues their retinal vascular abnormalities. Furthermore, we show that retinal vascularization is regulated by LRP5 in a dosage dependent manner and does not depend on LRP6. Our study provides the first direct evidence that endothelium-derived LRP5 is both necessary and sufficient to mediate its critical role in the development and maintenance of retinal vasculature.
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Sclerostin inhibition reverses skeletal fragility in an LRP5-deficient mouse model of OPPG syndrome.
Science translational medicine, 2013Co-Authors: Rajendra Kedlaya, Daniel J. Horan, Matthew L. Warman, Shreya Veera, Rachel E. Moss, Ugur M. Ayturk, Christina M. Jacobsen, Margot E. Bowen, Chris Paszty, Alexander G. RoblingAbstract:Osteoporosis pseudoglioma syndrome (OPPG) is a rare genetic disease that produces debilitating effects in the skeleton. OPPG is caused by mutations in LRP5, a WNT co-receptor that mediates osteoblast activity. WNT signaling through LRP5, and also through the closely related receptor LRP6, is inhibited by the protein sclerostin (SOST). It is unclear whether OPPG patients might benefit from the anabolic action of sclerostin neutralization therapy (an approach currently being pursued in clinical trials for postmenopausal osteoporosis) in light of their LRP5 deficiency and consequent osteoblast impairment. To assess whether loss of sclerostin is anabolic in OPPG, we measured bone properties in a mouse model of OPPG (LRP5−/−), a mouse model of sclerosteosis (Sost−/−), and in mice with both genes knocked out (LRP5−/−;Sost−/−). LRP5−/−;Sost−/− mice have larger, denser, and stronger bones than do LRP5−/− mice, indicating that SOST deficiency can improve bone properties via pathways that do not require LRP5. Next, we determined whether the anabolic effects of sclerostin depletion in LRP5−/− mice are retained in adult mice by treating 17-week-old LRP5−/− mice with a sclerostin antibody for 3 weeks. LRP5+/+ and LRP5−/− mice each exhibited osteoanabolic responses to antibody therapy, as indicated by increased bone mineral density, content, and formation rates. Collectively, our data show that inhibiting sclerostin can improve bone mass whether LRP5 is present or not. In the absence of LRP5, the anabolic effects of SOST depletion can occur via other receptors (such as LRP4/6). Regardless of the mechanism, our results suggest that humans with OPPG might benefit from sclerostin neutralization therapies.
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an rna seq protocol to identify mrna expression changes in mouse diaphyseal bone applications in mice with bone property altering LRP5 mutations
Journal of Bone and Mineral Research, 2013Co-Authors: Ugur M. Ayturk, Alexander G. Robling, Christina M. Jacobsen, Danos C Christodoulou, Joshua M Gorham, J G Seidman, Christine E Seidman, Matthew L. WarmanAbstract:Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring LRP5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with LRP5 wild-type (LRP5(+/+) ), knockout (LRP5(-/-) ), and high bone mass (HBM)-causing (LRP5(p.A214V/+) ) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same LRP5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, LRP5(+/+) and LRP5(p.A214V/+) ) and "insufficient" (LRP5(-/-) ) diaphyseal bone, and far fewer differentially expressed genes between LRP5(p.A214V/+) and LRP5(+/+) diaphyseal bone.
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Mechanotransduction in bone tissue: The A214V and G171V mutations in LRP5 enhance load-induced osteogenesis in a surface-selective manner.
Bone, 2012Co-Authors: Paul J. Niziolek, Matthew L. Warman, Alexander G. RoblingAbstract:Mechanotransduction in bone requires components of the Wnt signaling pathway to produce structurally adapted bone elements. In particular, the Wnt co-receptor LDL-receptor-related protein 5 (LRP5) appears to be a crucial protein in the mechanotransduction cascades that translate physical tissue deformation into new bone formation. Recently discovered missense mutations in LRP5 are associated with high bone mass (HBM), and the altered function of these proteins provide insight into LRP5 function in many skeletal processes, including mechanotransduction. We further investigated the role of LRP5 in bone cell mechanotransduction by applying mechanical stimulation in vivo to two different mutant mouse lines, which harbor HBM-causing missense mutations in LRP5. Axial tibia loading was applied to mature male LRP5 G171V and LRP5 A214V knock-in mice, and to their wild type controls. Fluorochrome labeling revealed that 3 days of loading resulted in a significantly enhanced periosteal response in the A214V knock in mice, whereas the G171V mice exhibited a lowered osteogenic threshold on the endocortical surface. In summary, our data further highlight the importance of LRP5 in bone cell mechanotransduction, and indicate that the HBM-causing mutations in LRP5 can alter the anabolic response to mechanical stimulation in favor of increased bone gain.
Caroline M. Alexander - One of the best experts on this subject based on the ideXlab platform.
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LRP5 Has a Wnt-Independent Role in Glucose Uptake and Growth for Mammary Epithelial Cells
Molecular and cellular biology, 2015Co-Authors: Emily N Chin, Soyoung Kim, Saja A Fakhraldeen, Joshua A. Martin, Caroline M. AlexanderAbstract:LRP5 is typically described as a Wnt signaling receptor, albeit a less effective Wnt signaling receptor than the better-studied sister isoform, Lrp6. Here we show that LRP5 is only a minor player in the response to Wnt3a-type ligands in mammary epithelial cells; instead, LRP5 is required for glucose uptake, and glucose uptake regulates the growth rate of mammary epithelial cells in culture. Thus, a loss of LRP5 leads to profound growth suppression, whether growth is induced by serum or by specific growth factors, and this inhibition is not due to a loss of Wnt signaling. Depletion of LRP5 decreases glucose uptake, lactate secretion, and oxygen consumption rates; inhibition of glucose consumption phenocopies the loss of LRP5 function. Both LRP5 knockdown and low external glucose induce mitochondrial stress, as revealed by the accumulation of reactive oxygen species (ROS) and the activation of the ROS-sensitive checkpoint, p38α. In contrast, loss of function of Lrp6 reduces Wnt responsiveness but has little impact on growth. This highlights the distinct functions of these two Lrp receptors and an important Wnt ligand-independent role of LRP5 in glucose uptake in mammary epithelial cells.
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both LRP5 and lrp6 receptors are required to respond to physiological wnt ligands in mammary epithelial cells and fibroblasts
Journal of Biological Chemistry, 2012Co-Authors: Shruti Goel, Emily N Chin, Saja A Fakhraldeen, Scott M Berry, David J Beebe, Caroline M. AlexanderAbstract:A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.
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The Wnt Receptor, LRP5, Is Expressed by Mouse Mammary Stem Cells and Is Required to Maintain the Basal Lineage
PloS one, 2009Co-Authors: Nisha M. Badders, Bart O Williams, Shruti Goel, Rod J. Clark, Kristine S. Klos, Soyoung Kim, Anna Bafico, Charlotta Lindvall, Caroline M. AlexanderAbstract:Background: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, LRP5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of LRP5 confers resistance to Wnt1-induced tumor development. Methodology/Principal Findings: Here, we show that all basal mammary cells express LRP5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of LRP5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by .200 fold (over 80% of activity), based on high LRP5 expression alone. Though LRP5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22%) and LRP52/2 mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16 Ink4a and TA-p63. Conclusions/Significance: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through LRP5 is an important component of normal mammary stem cell function.
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The role of LRP5 and Lrp6 in mouse mammary development and Wnt-1-induced carcinogenesis
Cancer Research, 2005Co-Authors: Charlotta Lindvall, Troy A. Giambernardi, Cassandra R. Zylstra, Caroline M. Alexander, Nicole Evans, Michael T. Lewis, Bart O WilliamsAbstract:Proc Amer Assoc Cancer Res, Volume 46, 2005 1075 LRP5 and LRP6 form a distinct subfamily of low-density lipoprotein receptor (LDLR)-like proteins. They are co-receptors for Wnt ligands, which are a large family of secreted glycoproteins that initiate signaling by binding to members of the Frizzled (Fz) family of seven transmembrane receptors and to either LRP5 or Lrp6. We have used mice carrying mutations in LRP5 and Lrp6 to gain insight into the normal roles of the two genes in mammary development and in Wnt1-induced mammary tumorigenesis. Our results show that loss of either LRP5 or Lrp6 impairs normal mammary development. The most severe phenotype is obtained in LRP5-/-;Lrp6+/− virgin mice, who exhibit mammary glands lacking a ductal network. Besides a delay in development, the ductal network of adult virgin LRP5 -mutant mice display a reduction in side branching. Because Lrp6 -mutant mice die shortly after birth, we have examined mammary tissue from 19-day-old embryos. The mammary glands of Lrp6 -mutant embryos contain an epithelial sprout, but in contrast to wild type the mutants lack side branching. Interestingly, Lrp6 -mutant embryos seem to lack mammary fat pads. Female mice expressing the Wnt-1 gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeats reproducibly develop mammary adenocarcinomas at approximately six months of age. To investigate the requirement of LRP5 and Lrp6 in Wnt-induced carcinogenesis we have generated MMTV-Wnt-1 transgenic female mice that are deficient for LRP5. These homozygotes exhibit an almost complete resistance to Wnt-1-induced mammary tumors and the heterozygotes (LRP5+/−) display a significant delay in tumorigenesis. After 300 days, less than 10% of the MMTV-Wnt-1;LRP5 -mutant and more than 90% of the MMTV-Wnt-1;LRP5 wild type mice had developed mammary tumors. The tumors that arise in a LRP5 -deficient background are more heterogeneous than normal Wnt-1 tumors, and frequently display a papillary growth pattern. Current work is aimed at characterizing the differences in the tumors at the cellular and molecular level.
Lina Badimon - One of the best experts on this subject based on the ideXlab platform.
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PCSK9 and LRP5 in macrophage lipid internalization and inflammation.
Cardiovascular research, 2020Co-Authors: Lina Badimon, Aureli Luquero, Javier Crespo, Esther Peña, Maria Borrell-pagesAbstract:AIMS Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-Density Lipoprotein Receptor (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation. METHODS AND RESULTS Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 hours of LDL incubation and lasts up to 24hours; however in the absence of both LRP5 and PCSK9 there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally PCSK9 participates in TLR4/NFkB signaling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB was detected in PCSK9 silenced cells after lipid loading, indicating a down-regulation of the TLR4/NFκB pathway. CONCLUSION Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5 there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favoring inflammation. TRANSLATIONAL PERSPECTIVE We demonstrate that PCSK9 and LRP5 contribute to lipid uptake. We also show that LRP5 participates in PCSK9 transport to the plasma membrane and that PCSK9 inhibition protects against agLDL-induced inflammation associated to the TLR4/NFκB pathway. These results offer new targets to prevent the progression of inflammation and hypercholesterolemia and their increased risk of cardiovascular events.
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LRP5/canonical Wnt signalling and healing of ischemic myocardium
Basic research in cardiology, 2016Co-Authors: Maria Borrell-pages, July Carolina Romero, Gemma Vilahur, Laura Casaní, Maria Teresa Bejar, Lina BadimonAbstract:LRP5 (low-density lipoprotein receptor-related protein 5) activates canonical Wnt signalling. LRP5 plays multiple roles including regulation of lipoprotein and cholesterol homeostasis as well as innate immunity cell function. However, it is not known whether LRP5 has a role in the myocardium. The aim of this study was to investigate LRP5 and Wnt signalling in myocardial remodelling after acute myocardial infarction (MI). Wnt protein levels were determined in a hypercholesterolemic porcine model of MI, in LRP5−/− C57Bl6 mice, in cultured cardiomyocytes and in human explanted hearts with previous MI episodes. 21 days post-MI, there was upregulation of LRP5 in the ischemic myocardium of hypercholesterolemic pigs as well as an upregulated expression of proteins of the Wnt pathway. We demonstrate via overexpression and silencing experiments that LRP5 induces Wnt pathway activation in isolated cardiomyocytes. Hypoxia and lipid-loading induced the expression of Wnt proteins, whereas this effect is blocked in LRP5-silenced cardiomyocytes. To characterize the function of the LRP5-Wnt axis upregulation in the heart, we induced MI in wild-type and LRP5−/− mice. LRP5−/− mice had significantly larger infarcts than Wt mice, indicating a protective role of LRP5 in injured myocardium. The LRP5 upregulation in post-MI hearts seen in pigs and mice was also evident in human hearts as dyslipidemic patients with previous episodes of ischemia have higher expression of LRP5 and Wnt-signalling genes than non-ischemic dilated hearts. We demonstrate an upregulation of LRP5 and the Wnt signalling pathway that it is a prosurvival healing response of cardiomyocytes upon injury.
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LRP5 associates with specific subsets of macrophages: Molecular and functional effects.
Journal of molecular and cellular cardiology, 2015Co-Authors: Maria Borrell-pages, July Carolina Romero, Javier Crespo, Oriol Juan-babot, Lina BadimonAbstract:Innate and acquired immunity is involved in the progression of atherosclerosis. The molecular mechanisms ruling monocyte to macrophage (Mo) differentiation are not yet fully understood. Different subtypes of plaque macrophages that have differentiated from monocytes recruited from circulating blood, have been characterized based on surface epitopes. We have recently shown that LRP5, a member of the LDL receptor superfamily supporting Wnt signalling, has an important role in monocyte to macrophage differentiation. The aim of this study was to investigate whether the CD16- and CD16+ macrophage subsets found in human atherosclerotic plaques have a differential LRP5 expression/function and Wnt signalling potential. We show for the first time that LRP5 expression is significantly higher in human CD16+Mo derived from CD14(+)CD16(+) monocytes than in CD16-Mo macrophages derived from CD14(+)CD16(-) monocytes. LRP5 is not found in human healthy vessel or arterial intimal thickening but is found in advanced human atherosclerotic lesions co-localizing only with the CD16+Mo macrophage subset. LRP5 expressing macrophages infiltrate the deep layers of atherosclerotic plaques towards the intima-media boundaries showing increased migratory activity and higher phagocytic activity. The equivalent for human patrolling CD14(+)CD16(+) monocytes in mice, CD115(+)GR1(low) monocytes, also show an increased expression of LRP5. In summary, classical CD14(+)CD16(-)monocytes that differentiate into CD16-Mo do not express LRP5. Instead, human monocytes expressing LRP5 differentiate into CD16+Mo antiinflammatory macrophages. These antiinflammatory macrophages are found in advanced atherosclerotic human plaques. Thus LRP5 is a signature of the anti-inflammatory defensive phenotype of macrophages.
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LRP5 and plasma cholesterol levels modulate the canonical Wnt pathway in peripheral blood leukocytes.
Immunology and cell biology, 2015Co-Authors: Maria Borrell-pages, July Carolina Romero, Lina BadimonAbstract:Inflammation is triggered after invasion or injury to restore homeostasis. Although the activation of Wnt/β-catenin signaling is one of the first molecular responses to cellular damage, its role in inflammation is still unclear. It was our hypothesis that the low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and the canonical Wnt signaling pathway are modulators of inflammatory mechanisms. Wild-type (WT) and LRP5(-/-) mice were fed a hypercholesterolemic (HC) diet to trigger dislipidemia and chronic inflammation. Diets were supplemented with plant sterol esters (PSEs) to induce LDL cholesterol lowering and the reduction of inflammation. HC WT mice showed increased serum cholesterol levels that correlated with increased LRP5 and Wnt/β-catenin gene expression while in the HC LRP5(-/-) mice Wnt/β-catenin pathway was shut down. Functionally, HC induced pro-inflammatory gene expression in LRP5(-/-) mice, suggesting an inhibitory role of the Wnt pathway in inflammation. Dietary PSE administration downregulated serum cholesterol levels in WT and LRP5(-/-) mice. Furthermore, in WT mice PSE increased anti-inflammatory genes expression and inhibited Wnt/β-catenin activation. Hepatic gene expression of Vldlr, Lrp2 and Lrp6 was increased after HC feeding in WT mice but not in LRP5(-/-) mice, suggesting a role for these receptors in the clearance of plasmatic lipoproteins. Finally, an antiatherogenic role for LRP5 was demonstrated as HC LRP5(-/-) mice developed larger aortic atherosclerotic lesions than WT mice. Our results show an anti-inflammatory, pro-survival role for LRP5 and the Wnt signaling pathway in peripheral blood leukocytes.
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LRP5 deficiency down-regulates Wnt signalling and promotes aortic lipid infiltration in hypercholesterolaemic mice
Journal of cellular and molecular medicine, 2015Co-Authors: Maria Borrell-pages, July Carolina Romero, Lina BadimonAbstract:Low-density lipoprotein receptor-related protein 5 (LRP5) is a member of the LDLR family that orchestrates cholesterol homoeostasis. The role of LRP5 and the canonical Wnt pathway in the vascular wall of dyslipidaemic animals remains unknown. In this study, we analysed the role of LRP5 and the Wnt signalling pathway in mice fed a hypercholesterolaemic diet (HC) to trigger dyslipidaemia. We show that LRP5−/− mice had larger aortic lipid infiltrations than wild-type mice, indicating a protective role for LRP5 in the vascular wall. Three members of the LDLR family, Lrp1, Vldlr and Lrp6, showed up-regulated gene expression levels in aortas of LRP5−/− mice fed a hypercholesterolaemic diet. HC feeding in LRP5−/− mice induced higher macrophage infiltration in the aortas and accumulation of inflammatory cytokines in blood. Wnt/β-CATENIN signalling proteins were down-regulated in HC LRP5−/− mice indicating that LRP5 regulates the activation of Wnt signalling in the vascular wall. In conclusion, our findings show that LRP5 and the canonical Wnt pathway down-regulation regulate the dyslipidaemic profile by promoting lipid and macrophage retention in the vessel wall and increasing leucocyte-driven systemic inflammation.
