LRP6 Signaling Research Articles

LIM kinase 2 activates cardiac fibroblasts and exacerbates postinfarction left ventricular remodeling via crosstalk between the canonical and non-canonical Wnt pathways

Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of β-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-β treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-β-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.

Knockdown of LncRNA SBF2-AS1 Inhibited Gastric Cancer Tumorigenesis via the Wnt/LRP5 Signaling Pathway.

This investigation aimed to uncover the impact of a long noncoding RNA, SET-binding factor 2 antisense RNA1 (SBF2-AS1) on the malignant progression of gastric cancer (GC) and to further explore its underlying mechanism. SBF2-AS1 expression was quantified by qRT-PCR in GC cell lines and GC tissues. In vitro loss-of-function studies of SBF2-AS1, accompanied by flow cytometry, CCK-8, and cell invasion tests, were applied to elucidate the impact of SBF2-AS1 on the tumor progression of GC cells. Finally, Western blotting and a luciferase assay were used to detect WNT/LRP5 signaling pathway activation. SBF2-AS1 was aberrantly expressed in GC cell lines (p<0.05) and GC tissues (p<0.05). Cell invasive and proliferative capabilities were inhibited via SBF2-AS1 knockdown, resulting in apoptosis of NCI-N87 and MKN74 cells. Additionally, online database analysis uncovered a positive correlation between SBF2-AS1 and the Wnt/LRP5 signaling pathway (p<0.05). SBF2-AS1 knockdown blocked the Wnt/LRP5 signaling pathway, whereas the effects of SBF2-AS1 knockdown on the malignant genotype of MKN74 as well as NCI-N87 cells were partially restored by triggering the Wnt/ LRP5 signaling pathway. High expression of SBF2-AS1 was found in GC, the malignant progression of which was repressed via SBF2-AS1 knockdown by inhibiting the Wnt/LRP5 signaling pathway.

Zebrafish mutants reveal unexpected role of Lrp5 in osteoclast regulation.

Low-density Lipoprotein Receptor-related Protein 5 (LRP5) functions as a co-receptor for Wnt ligands, controlling expression of genes involved in osteogenesis. In humans, loss-of-function mutations in LRP5 cause Osteoporosis-Pseudoglioma syndrome, a low bone mass disorder, while gain-of-function missense mutations have been observed in individuals with high bone mass. Zebrafish (Danio rerio) is a popular model for human disease research, as genetic determinants that control bone formation are generally conserved between zebrafish and mammals. We generated lrp5- knock-out zebrafish to study its role in skeletogenesis and homeostasis. Loss of lrp5 in zebrafish leads to craniofacial deformities and low bone mineral density (total body and head) at adult ages. To understand the mechanism and consequences of the observed phenotypes, we performed transcriptome analysis of the cranium of adult lrp5 mutants and siblings. Enrichment analysis revealed upregulation of genes significantly associated with hydrolase activity: mmp9, mmp13a, acp5a. acp5a encodes Tartrate-resistant acid phosphatase (TRAP) which is commonly used as an osteoclast marker, while Matrix metalloprotease 9, Mmp9, is known to be secreted by osteoclasts and stimulate bone resorption. These genes point to changes in osteoclast differentiation regulated by lrp5. To analyze these changes functionally, we assessed osteoclast dynamics in mutants and observed increased TRAP staining, significantly larger resorption areas, and developmental skeletal dysmorphologies in the mutant, suggesting higher resorptive activity in the absence of Lrp5 signaling. Our findings support a conserved role of Lrp5 in maintaining bone mineral density and revealed unexpected insights into the function of Lrp5 in bone homeostasis through moderation of osteoclast function.

CTGF-D4 Amplifies LRP6 Signaling to Promote Grafts of Adult Epicardial-derived Cells That Improve Cardiac Function After Myocardial Infarction.

Transplantation of stem/progenitor cells holds promise for cardiac regeneration in patients with myocardial infarction (MI). Currently, however, low cell survival and engraftment after transplantation present a major barrier to many forms of cell therapy. One issue is that ligands, receptors, and signaling pathways that promote graft success remain poorly understood. Here, we prospectively isolate uncommitted epicardial cells from the adult heart surface by CD104 (β-4 integrin) and demonstrate that C-terminal peptide from connective tissue growth factor (CTGF-D4), when combined with insulin, effectively primes epicardial-derived cells (EPDC) for cardiac engraftment after MI. Similar to native epicardial derivatives that arise from epicardial EMT at the heart surface, the grafted cells migrated into injured myocardial tissue in a rat model of MI with reperfusion. By echocardiography, at 1 month after MI, we observed significant improvement in cardiac function for animals that received epicardial cells primed with CTGF-D4/insulin compared with those that received vehicle-primed (control) cells. In the presence of insulin, CTGF-D4 treatment significantly increased the phosphorylation of Wnt co-receptor LRP6 on EPDC. Competitive engraftment assays and neutralizing/blocking studies showed that LRP6 was required for EPDC engraftment after transplantation. Our results identify LRP6 as a key target for increasing EPDC engraftment after MI and suggest amplification of LRP6 signaling with CTGF-D4/insulin, or by other means, may provide an effective approach for achieving successful cellular grafts in regenerative medicine.

Combination therapy in the Col1a2G610C mouse model of Osteogenesis Imperfecta reveals an additive effect of enhancing LRP5 signaling and inhibiting TGFβ signaling on trabecular bone but not on cortical bone.

Enhancing LRP5 signaling and inhibiting TGFβ signaling have each been reported to increase bone mass and improve bone strength in wild-type mice. Monotherapy targeting LRP5 signaling, or TGFβ signaling, also improved bone properties in mouse models of Osteogenesis Imperfecta (OI). We investigated whether additive or synergistic increases in bone properties would be attained if enhanced LRP5 signaling was combined with TGFβ inhibition. We crossed an Lrp5 high bone mass (HBM) allele (Lrp5A214V) into the Col1a2G610C/+ mouse model of OI. At 6-weeks-of-age we began treating mice with an antibody that inhibits TGFβ1, β2, and β3 (mAb 1D11), or with an isotype-matched control antibody (mAb 13C4). At 12-weeks-old, we observed that combining enhanced LRP5 signaling with inhibited TGFβ signaling produced an additive effect on femoral and vertebral trabecular bone volumes, but not on cortical bone volumes. Although enhanced LRP5 signaling increased femur strength in a 3-point bending assay in Col1a2G610C/+ mice, femur strength did not improve further with TGFβ inhibition. Neither enhanced LRP5 signaling nor TGFβ inhibition, alone or in combination, improved femur 3-point-bending post-yield displacement in Col1a2G610C/+ mice. These pre-clinical studies indicate combination therapies that target LRP5 and TGFβ signaling should increase trabecular bone mass in patients with OI more than targeting either signaling pathway alone. Whether additive increases in trabecular bone mass will occur in, and clinically benefit, patients with OI needs to be determined.

Autosomal-Recessive Mutations in MESD Cause Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.

Lack of Lrp5 Signaling in Osteoblasts Sensitizes Male Mice to Diet-Induced Disturbances in Glucose Metabolism

Wnt signaling through the low-density lipoprotein-related receptor 5 (Lrp5) coreceptor regulates osteoblast maturation, matrix mineralization, and intermediary metabolism. In the mature osteoblast, signals downstream of Lrp5 are required for normal long-chain fatty acid β-oxidation. Mice rendered deficient for this coreceptor in osteoblasts and osteocytes accumulate body fat with elevated serum lipid levels but retain normal insulin sensitivity. In the present study, we challenged Lrp5-mutant mice with a high-fat diet (HFD) to determine whether they were more susceptible to diet-induced disturbances in glucose homeostasis. The HFD-fed Lrp5 mutant mice maintained a low bone mass phenotype with an increase in adipose tissue mass and hypertriglyceridemia and hypercholesterolemia. Examination of glucose metabolism revealed that Lrp5 deficiency in the osteoblast also resulted in hyperglycemia and hyperinsulinemia, with reductions in glucose tolerance, insulin sensitivity, and serum undercarboxylated osteocalcin. The results from in vivo genetic epistasis and in vitro studies suggest that this phenotype proceeds via the accumulation of diacylglycerol species and impaired insulin signaling in Lrp5-deficient osteoblasts. In turn, glucose uptake and osteocalcin production are diminished in mutant osteoblasts. Taken together, these data identify a link between Wnt-Lrp5 signaling and insulin signaling in the osteoblast that has the potential to influence energy balance and compound the detrimental effects of a HFD on whole-body metabolism.

Lipoprotein Receptor–related Protein 6 Signaling is Necessary for Vasculogenic Differentiation of Human Dental Pulp Stem Cells

The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor–related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)–tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor165 (rhVEGF165). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF165 induced expression of active β-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF165 induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.

Adiponectin inhibits Wnt co-receptor, Lrp6, phosphorylation and β-catenin signaling

Adiponectin is a pleiotropic adipokine implicated in obesity, metabolic syndrome and cardiovascular disease. Recent studies have identified adiponectin as a negative regulator of tissue fibrosis. Wnt/β-catenin signaling has also been implicated in metabolic syndrome and can promote tissue fibrosis, but the extent to which adiponectin cross-regulates Wnt/β-catenin signaling is unknown. Using primary human dermal fibroblasts and recombinant purified proteins, we show that adiponectin can limit β-catenin accumulation and downstream gene activation by inhibiting Lrp6 phosphorylation, a key activation step in canonical Wnt signaling. Inhibition of Wnt3a-mediated Lrp6 phospho-activation is relatively rapid (e.g., by 30 min), and is not dependent on established adiponectin G-protein coupled receptors, AdipoR1 and R2, suggesting a more direct relationship to Lrp6 signaling. In contrast, the ability of adiponectin to limit Wnt-induced and baseline collagen production in fibroblasts requires AdipoR1/R2. These results suggest the possibility that the pleiotropic effects of adiponectin may be mediated through distinct cell surface receptor complexes. Accordingly, we propose that the anti-fibrotic activity of adiponectin may be mediated through AdipoR1/R2 receptors, while the ability of adiponectin to inhibit Lrp6 phospho-activation may be relevant to other recently established roles for Lrp6 signaling in glucose metabolism and metabolic syndrome.

Serum levels of sclerostin, Dickkopf-1, and secreted frizzled-related protein-4 are not changed in individuals with high bone mass causing mutations in LRP5.

We compared circulating levels of Wnt inhibitors among patients with high bone mass mutations in LRP5, unaffected kindred, and unrelated normal controls. Inhibitors were unchanged in affected and unaffected kindred. We saw no meaningful differences between controls and affected individuals. LRP5 signaling may not influence circulating levels of these inhibitors. It is thought that gain-of-function mutations in LRP5 result in high bone mass syndromes because these allelic variants confer resistance to the actions of endogenous inhibitors of Wnt signaling. We therefore attempted to determine if circulating levels of Wnt inhibitors are altered in patients with gain-of-function mutations in LRP5. This is a cross-sectional study in a university research center. Serum was collected from consented volunteers known to have either the G171V or N198S gain-of-function mutations in LRP5, kindred members affected with either mutation, unrelated kindred, and unrelated normal age-matched controls. BMD was provided or measured on site. There were no significant differences found in the serum levels of sclerostin (SOST), Dickkopf-1 (Dkk-1), or secreted frizzled-related protein-4 (SFRP-4) in affected vs. unaffected individuals from different kindreds or when compared to age-matched unrelated normal individuals. Mean serum SOST values in affected and unaffected kindred members and unrelated normal controls were 52.7 ± 6.1, 36.5 ± 9.6, and 54.8 ± 5.4, respectively. For Dkk-1, the values were 25.9 ± 3.4, 25.7 ± 3.0, and 17.3 ± 2.3 and for SFRP-4, 38.1 ± 2.3, 39.8 ± 3.6, and 28.5 ± 1.7. Serum levels of RANKL and osteoprotegerin (OPG) were not different in the three groups. Circulating levels of endogenous Wnt inhibitors do not change in patients with gain-of-function mutations in LRP5 including Dkk1, which is suppressed by Wnt signaling. It may be that circulating levels of Wnt inhibitors do not reflect changes in target tissues. It is also possible that other mechanisms besides or in addition to resistance in Wnt inhibitors explains the skeletal effects of these mutations.

Cholesterol modulates LRP5 expression in the vessel wall

ObjectiveMacrophages are key players in atherosclerotic lesion formation and progression. We have recently demonstrated that lipid-loaded macrophages show activation of the canonical Wnt signaling pathway. MethodsTo test the in vivo role of the canonical Wnt pathway in atherosclerosis we used mice deficient in the Wnt signaling receptor LRP5 (LRP5−/−) fed a hypercholesterolemic diet (HC) to induce atherosclerosis. These dietary groups were further subdivided into two subgroups receiving their respective diets supplemented with 2% plant sterol esters (PSE). All mice remained on their assigned diets until age 18 weeks. ResultsHC WT mice had mildly increased non-HDL cholesterol levels, developed aortic atherosclerotic lesions and showed upregulated expression levels of aortic Lrp5. HC LRP5−/− mice develop larger aortic atherosclerotic lesions than WT mice indicating that LRP5 has a protective function in atherosclerosis progression. The oral administration of PSE, a dietary cholesterol-lowering agent, had an effect in the expression levels of the Wnt signaling receptor and in atherosclerosis progression. We found that PSE reduced serum total cholesterol levels, abolished HC-induced LRP5 overexpression and reduced aortic atherosclerotic plaques. ConclusionThe proatherogenic effects of the excess of plasma lipids are in part mediated by modulation of LRP5 in the aorta. LRP5 and canonical Wnt signaling exert a protective defense mechanism against hyperlipidemia and atherosclerosis lesion progression.

Toggling a conformational switch in Wnt/β‐catenin signaling: Regulation of Axin phosphorylation

The precise orchestration of two opposing protein complexes - one in the cytoplasm (β-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) - is critical for controlling levels of the transcriptional co-factor β-catenin, and subsequent activation of the Wnt/β-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area.

Reversing LRP 5-Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low-density lipoprotein receptor-related protein (LRP) 5/6 Wnt co-receptors, thereby inhibiting Wnt/β-catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis-pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost(-/-) ), Lrp5 (Lrp5(-/-) ), or both (Sost(-/-) ;Lrp5(-/-) ) to elucidate the mechanism of action of Sost in vivo. Sost deficiency-induced bone gain was significantly blunted in Sost(-/-) ;Lrp5(-/-) mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost(-/-) ;Lrp5(-/-) mice and most bone parameters were elevated relative to wild-type. To test whether the remaining bone increases in Sost(-/-) ;Lrp5(-/-) animals depend on Lrp6, we treated wild-type, Sost(-/-) , and Sost(-/-) ;Lrp5(-/-) mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class-mediated Lrp6 signaling reduced cancellous bone mass and density in wild-type mice. Surprisingly, it reversed the abnormal bone gain in Sost(-/-) and Sost(-/-) ;Lrp5(-/-) mice to wild-type levels irrespective of enhancement or blockage of Wnt3a class-mediated Lrp6 activity. Thus, whereas Sost deficiency-induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class-induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss-of-function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options.

Phosphorylation-Dependent Regulation of AXIN Modulates WNT Activity

Abstract WNT–LRP6 signaling promotes β-catenin stabilization via inactivation of the AXIN scaffold.

Atherosclerosis and Vascular Biology

Atherosclerosis and Vascular Biology

New Avenues in the LRP5-mediated Bone Mass Acquisition

Lipoprotein receptor-related protein (LRP5) signaling is well correlated with the bone mass in both human and mice. Loss-of-function mutations of LRP5 result in osteopenia or osteoporosis. In contrast, gain-of-function mutations show high bone mass phenotype. To elucidate the molecular mechanism of the LRP5-mediated bone mass acquisition, several groups have genetically dissected the Wingless and Int-1 (Wnt) -catenin signaling pathway using osteoblast-lineage specific Cre mice. Key players for LRP5-mediated bone mass acquisition turn out to be different molecules with respect to the expressing tissue and action mode of these molecules. One is serotonin, a tryptophan metabolite that originates from duodenum, which acts as a negative regulator for bone formation. LRP5 suppresses serotonin biosynthesis by inhibiting the expression of tryptophan hydroxylase 1 in the gut. The other is sclerostin, an osteocyte- producing antagonist for LRP5 signaling. Here is a summary of recent findings about these two molecules, providing a chance to speculate new avenues in the LRP5-mediated bone mass acquisition.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy

Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy. In this study, we show that Wnt receptors (Frizzled4 and low-density lipoprotein receptor-related protein5 [Lrp5]) and activity are significantly increased in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt coreceptor Lrp5 and downstream signaling molecule dishevelled2 significantly decreases the formation of pathological retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood-retinal barrier, which is linked to significant downregulation of tight junction protein claudin5 in Lrp5(-/-) vessels. Blocking claudin5 significantly suppresses Wnt pathway-driven endothelial cell sprouting in vitro and developmental and pathological vascular growth in retinopathy in vivo. These results demonstrate an important role of Wnt signaling in pathological vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.

Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening “gap4” region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.

Lrp5 functions in bone to regulate bone mass

The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.