LRP6 Signaling Research Articles

Abstract 1053: Mouse Kremen 1 negatively regulates Wnt/β-catenin signaling in human breast cancer MDA-MB-231 cells

Abstract The canonical Wnt/β-catenin signaling pathway is a major developmental pathway that regulates embryonic development, cell motility, polarity, growth, and progression. Wnts are secreted glycoproteins that bind to the low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled, a seven pass transmembrane receptor protein, which increase cytosolic β-catenin levels and subsequent β-catenin transcriptional activity via the inhibition of glycogen synthase kinase-3β. Kremen receptors are single-pass transmembrane proteins that consist of two isoforms (Kremen 1 and 2) in mammals. Dickkopf (Dkk) proteins are secreted glycoproteins that inhibit Wnt signaling. Kremen antagonizes Wnt signaling by forming a ternary complex with Dkk1 and LRP5/6, while Kremen alone is able to promote LRP6 cell-surface localization and stimulate LRP6 signaling. Previous studies have shown that aberrant Wnt/β-catenin signaling promotes tumorigenesis in breast cancer and that LRP6 is an important mediator in mammary gland development and in breast cancer tumorigenesis. In the present studies, we found that breast cancer MDA-MB-231 cells express LRP6 and Dkk1, but not Kremen 1 and 2. Therefore, MDA-MB-231 breast cancer cells were used to make stable cell lines that conditionally express mouse Kremen 1 in the presence of doxycycline. We found that doxycycline-induced Kremen 1 expression resulted in the down-regulation of endogenous LRP6 expression and inhibition of Wnt/β-catenin signaling in MDA-MB-231 cells. Furthermore, we demonstrated that Kremen 1 expression blocked Wnt-3a-indced activation of Wnt/β-catenin signaling in MDA-MB-231 cells. Future studies will focus on the effects of Kremen 1 on tumor progression in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1053. doi:10.1158/1538-7445.AM2011-1053

Mutations in TSPAN12 Cause Autosomal-Dominant Familial Exudative Vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Although mutations in three genes (LRP5, FZD4, and NDP) are known to cause FEVR, these account for only a fraction of FEVR cases. The proteins encoded by these FEVR genes form part of a signaling complex that activates the Norrin-beta-catenin signaling pathway. Recently, through a large-scale reverse genetic screen in mice, Junge and colleagues identified an additional member of this signaling complex, Tspan12. Here, we report that mutations in TSPAN12 also cause autosomal-dominant FEVR. We describe seven mutations identified in a cohort of 70 FEVR patients in whom we had already excluded the known FEVR genes. This study provides further evidence for the importance of the Norrin-beta-catenin signaling pathway in the development of the retinal vasculature and also indicates that more FEVR genes remain to be identified.

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.

Norrin, Frizzled-4, and Lrp5 Signaling in Endothelial Cells Controls a Genetic Program for Retinal Vascularization

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

Expression, purification and functional characterization of Wnt signaling co-receptors LRP5 and LRP6

Activation of the Wnt signaling cascade plays a pivotal role during development and in various disease states. Wnt signals are transduced by seven-transmembrane Frizzled (Fz) proteins and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6). Genetic mutations resulting in a loss or gain of function of LRP5 in humans lead to osteopenia and bone formation, respectively. These findings demonstrate the genetic link between LRP5 signaling and the regeneration of bone mass. Herein we describe for the first time the production and characterization of soluble ectodomains of LRP5 and LRP6, (EC-LRP5, EC-LRP6). We have produced these proteins in amounts that are compatible with both in vitro and cell-based assays to study their binding properties. Purified EC-LRP5 and EC-LRP6 were able to interact with Wnt signaling components Dkk1 and Dkk2 and their functionality was confirmed in cell-based Wnt signaling assays. Hence, tools are now available to explore LRP5/6 interaction with other proteins and to screen for synthetic or natural compounds and biologics that might be novel therapeutics targeting the Wnt pathway.

Where Wnts went: the exploding field of Lrp5 and Lrp6 signaling in bone.

Wnt signaling has emerged as a central regulator of skeletal modeling and remodeling. Loss- or gain-of-function mutations in two Wnt co-receptors, Lrp5 and (more recently) Lrp6, have drawn attention to the importance of the Wnt pathway in bone biology. This review summarizes our current understanding of how the Wnt pathway operates on bone and the implications this has for skeletal physiology and drug discovery. Over the past 9 yr, rapid advances have been made in our understanding of the cellular targets for Wnt signaling and of the important regulatory molecules in this metabolic pathway. Both canonical and noncanonical signaling pathways seem to be important for mediating the effects of Wnt in bone. A rapidly expanding catalog of genetically engineered mice has been used to establish the importance of downstream effector molecules (such as β-catenin) in the Wnt pathway, as well as the critical role of endogenous inhibitors of Wnt signaling (such as Dkk1 and sclerostin) in bone metabolism. Indeed, regulation of sclerostin in osteocytes is emerging as an important final pathway for regulating bone anabolism in response to diverse trophic stimuli, from mechnotransduction to the anabolic actions of PTH. From the outset, it had been assumed that the effects of Wnt signaling in bone were caused by direct actions in osteoblast precursors, osteoblasts, and osteocytes. However, startling recent findings have challenged this view and suggest that a key target, at least in mice, is the duodenal enterochromaffin cell. There, Wnt signaling transduced by Lrp5 regulates serotonin synthesis, which acts in an endocrine fashion to regulate bone cell metabolism. It will take time to reconcile this new information with the considerable body of information we already have regarding the actions of Wnt in bone. The Wnt pathway has rapidly emerged as a therapeutic target for drug discovery. Neutralizing antibodies and small-molecule inhibitors of endogenous Wnt inhibitors have shown early promise as bone anabolic agents. However, given the central role of the Wnt pathway in regulating growth and development in extraskeletal tissues, as well as our still rudimentary understanding of how this signaling cascade actually affects bone metabolism, considerable work will be needed to ensure the safety of these new therapies.

Kremen is required for neural crest induction inXenopusand promotes LRP6-mediated Wnt signaling

Kremen 1 and 2 (Krm1/2) are transmembrane receptors for Wnt antagonists of the Dickkopf (Dkk) family and function by inhibiting the Wnt co-receptors LRP5/6. Here we show that Krm2 functions independently from Dkks during neural crest (NC) induction in Xenopus. Krm2 is co-expressed with, and regulated by, canonical Wnts. Krm2 is differentially expressed in the NC, and morpholino-mediated Krm2 knockdown inhibits NC induction, which is mimicked by LRP6 depletion. Conversely, krm2 overexpression induces ectopic NC. Kremens bind to LRP6, promote its cell-surface localization and stimulate LRP6 signaling. Furthermore, Krm2 knockdown specifically reduces LRP6 protein levels in NC explants. The results indicate that in the absence of Dkks, Kremens activate Wnt/beta-catenin signaling through LRP6.

The Wnt Co-receptor LRP5 Is Essential for Skeletal Mechanotransduction but Not for the Anabolic Bone Response to Parathyroid Hormone Treatment

The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass. Loss-of-function mutations in LRP5 cause the human skeletal disease osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by severely reduced bone mass and strength. We investigated the role of LRP5 on bone strength using mice engineered with a loss-of-function mutation in the gene. We then tested whether the osteogenic response to mechanical loading was affected by the loss of Lrp5 signaling. Lrp5-null (Lrp5-/-) mice exhibited significantly lower bone mineral density and decreased strength. The osteogenic response to mechanical loading of the ulna was reduced by 88 to 99% in Lrp5-/- mice, yet osteoblast recruitment and/or activation at mechanically strained surfaces was normal. Subsequent experiments demonstrated an inability of Lrp5-/- osteoblasts to synthesize the bone matrix protein osteopontin after a mechanical stimulus. We then tested whether Lrp5-/- mice increased bone formation in response to intermittent parathyroid hormone (PTH), a known anabolic treatment. A 4-week course of intermittent PTH (40 microg/kg/day; 5 days/week) enhanced skeletal mass equally in Lrp5-/- and Lrp5+/+ mice, suggesting that the anabolic effects of PTH do not require Lrp5 signaling. We conclude that Lrp5 is critical for mechanotransduction in osteoblasts. Lrp5 is a mediator of mature osteoblast function following loading. Our data suggest an important component of the skeletal fragility phenotype in individuals affected with osteoporosis-pseudoglioma is inadequate processing of signals derived from mechanical stimulation and that PTH might be an effective treatment for improving bone mass in these patients.

Human Degenerative Valve Disease Is Associated With Up-Regulation of Low-Density Lipoprotein Receptor-Related Protein 5 Receptor-Mediated Bone Formation

The goal of this research was to define the cellular mechanisms involved in myxomatous mitral valve disease and calcific aortic valve disease and to redefine the term degenerative valve disease in terms of an active cellular biology. "Degenerative" valvular heart disease is the primary cause of regurgitant and stenotic valvular lesion in the U.S. However, the signaling pathways are not known. We hypothesize that valve degeneration occurs due to an osteoblastic differentiation process mediated by the low-density lipoprotein receptor-related protein 5 (Lrp5) signaling pathway to cause valve thickening. We examined human diseased valves: myxomatous mitral valves (n = 23), calcified tricuspid aortic valves (n = 27), calcified bicuspid aortic valves (n = 23), and control tissue from mitral and aortic valves (n = 40). The valves were examined by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry for signaling markers important in osteoblast differentiation: Sox9 and Cbfa1 (transcription factors for osteoblast differentiation); Lrp5 and Wnt3 (osteoblast differentiation signaling marker), osteopontin and osteocalcin (osteoblast endochrondral bone matrix proteins), and proliferating cell nuclear antigen (a marker of cell proliferation). Cartilage development and bone formation was measured by Alcian blue stain and Alizarin red stain. Computed Scano MicroCT-40 (Bassersdorf, Switzerland) analysis measured calcium burden. Low-density lipoprotein receptor-related protein 5, osteocalcin, and other osteochrondrogenic differentiation markers were increased in the calcified aortic valves by protein and gene expression (p > 0.001). Sox9, Lrp5 receptor, and osteocalcin were increased in myxomatous mitral valves by protein and gene expression (p > 0.001). MicroCT was positive in the calcified aortic valves and negative in the myxomatous mitral valves. The mechanism of valvular heart disease involves an endochondral bone process that is expressed as cartilage in the mitral valves and bone in the aortic valves. Up-regulation of the Lrp5 pathway may play a role in the mechanism for valvular heart disease.

Kremen2 modulates Dickkopf2 activity during Wnt/lRP6 signaling

Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/β-catenin signaling pathway that acts by direct binding to and inhibiting the Wnt co-receptor LRP6. The related Dkk2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. We have recently identified the transmembrane proteins Kremen1 and -2 as additional Dkk receptors, which bind to both Dkk1 and Dkk2 with high affinity. Here we show that Kremen2 (Krm2) regulates Dkk2 activity during Wnt signaling. In human 293 fibroblasts transfected dkk2 activates LRP6 signaling. However, co-transfection of krm2 blocks the ability of Dkk2 to activate LRP6 and enhances inhibition of Wnt/Frizzled signaling. Krm2 also co-operates with Dkk4 to inhibit Wnt signaling, but not with Dkk3, which has no effect on Wnt signaling. Likewise, in Xenopus embryos, Dkk2 and Krm2 co-operate in Wnt inhibition leading to anteriorized embryos. Finally, we show that interaction with Krm2 is mediated by the second cysteine-rich domain of Dkks. These results suggest that Krm2 can function as a switch that turns Dkk2 from an activator into an inhibitor of Wnt/lRP6 signaling.