Distinct roles of LRP5 and LRP6 in Wnt signaling regulation in the retina.

Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.

Cardiac-specific LRP6 knockout induces lipid accumulation through Drp1/CPT1b pathway in adult mice.

We recently reported low-density lipoprotein receptor-related protein 6 (LRP6) decreased in dilated cardiomyopathy hearts, and cardiac-specific knockout mice displayed lethal heart failure through activation of dynamin-related protein 1 (Drp1). We also observed lipid accumulation in LRP6 deficiency hearts, but the detailed molecular mechanisms are unclear. Here, we detected fatty acids components in LRP6 deficiency hearts and explored the potential molecular mechanisms. Fatty acid analysis by GC-FID/MS revealed cardiac-specific LRP6 knockout induced the higher level of total fatty acids and some medium-long-chain fatty acids (C16:0, C18:1n9 and C18:2n6) than in control hearts. Carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of mitochondrial ß-oxidation in adult heart, was sharply decreased in LRP6 deficiency hearts, coincident with the activation of Drp1. Drp1 inhibitor greatly improved cardiac dysfunction and attenuated the increase in total fatty acids and fatty acids C16:0, C18:1n9 in LRP6 deficiency hearts. It also greatly inhibited the decrease in the cardiac expression of CPT1b and the transcriptional factors CCCTC-binding factor (CTCF) and c-Myc induced by cardiac-specific LRP6 knockout in mice. C-Myc but not CTCF was identified to regulate CPT1b expression and lipid accumulation in cardiomyocytes in vitro. The present study indicated cardiac-specific LRP6 knockout induced lipid accumulation by Drp1/CPT1b pathway in adult mice, and c-Myc is involved in the process. It suggests that LRP6 regulates fatty acid metabolism in adult heart.

LRP6 acts as a scaffold protein in cardiac gap junction assembly.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/ß-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/ß-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking.

Wnt/ß-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/ß-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either ß-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of ß-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that ß-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/ß-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

Vascular smooth muscle LRP6 limits arteriosclerotic calcification in diabetic LDLR-/- mice by restraining noncanonical Wnt signals.

RATIONALE: Wnt signaling regulates key aspects of diabetic vascular disease. OBJECTIVE: We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis. METHODS AND RESULTS: As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation. CONCLUSIONS: LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.

Deficiency in LRP6-mediated Wnt signaling contributes to synaptic abnormalities and amyloid pathology in Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurological disorder characterized by synaptic loss and dementia. The low-density lipoprotein receptor-related protein 6 (LRP6) is an essential coreceptor for Wnt signaling, and its genetic variants have been linked to AD risk. Here we report that neuronal LRP6-mediated Wnt signaling is critical for synaptic function and cognition. Conditional deletion of Lrp6 gene in mouse forebrain neurons leads to age-dependent deficits in synaptic integrity and memory. Neuronal LRP6 deficiency in an amyloid mouse model also leads to exacerbated amyloid pathology due to increased APP processing to amyloid-ß. In humans, LRP6 and Wnt signaling are significantly downregulated in AD brains, likely by a mechanism that depends on amyloid-ß. Our results define a critical pathway in which decreased LRP6-mediated Wnt signaling, synaptic dysfunction, and elevated Aß synergistically accelerate AD progression and suggest that restoring LRP6-mediated Wnt signaling can be explored as a viable strategy for AD therapy.

Hepatocyte growth factor (Hgf) stimulates low density lipoprotein receptor-related protein (Lrp) 5/6 phosphorylation and promotes canonical Wnt signaling.

While Wnt and Hgf signaling pathways are known to regulate epithelial cell responses during injury and repair, whether they exhibit functional cross-talk is not well defined. Canonical Wnt signaling is initiated by the phosphorylation of the Lrp5/6 co-receptors. In the current study we demonstrate that Hgf stimulates Met and Gsk3-dependent and Wnt-independent phosphorylation of Lrp5/6 at three separate activation motifs in subconfluent, de-differentiated renal epithelial cells. Hgf treatment stimulates the selective association of active Gsk3 with Lrp5/6. In contrast, Akt-phosphorylated inactive Gsk3 is excluded from this association. Hgf stimulates ß-catenin stabilization and nuclear accumulation and protects against epithelial cell apoptosis in an Lrp5/6-dependent fashion. In vivo, the increase in Lrp5/6 phosphorylation and ß-catenin stabilization in the first 6-24 h after renal ischemic injury was significantly reduced in mice lacking Met receptor in the renal proximal tubule. Our results thus identify Hgf as an important transactivator of canonical Wnt signaling that is mediated by Met-stimulated, Gsk3-dependent Lrp5/6 phosphorylation.

Lipoprotein receptor-related protein-6 protects the brain from ischemic injury.

BACKGROUND AND PURPOSE: Loss-of-function mutations of the lipoprotein receptor-related protein-6 (LRP6), a coreceptor in the Wingless-related integration site-ß-catenin prosurvival pathway, have been implicated in myocardial ischemia and neurodegeneration. However, it remains to be established whether LRP6 is also involved in ischemic brain injury. We used LRP6+/- mice to examine the role of this receptor in the mechanisms of focal cerebral ischemia. METHODS: Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery. Motor deficits and infarct volume were assessed 3 days later. Glycogen-synthase-kinase-3ß (GSK-3ß) phosphorylation was examined by Western blotting with phosphospecific antibodies, and the mitochondrial membrane potential changes induced by Ca2+ were also assessed. RESULTS: LRP6+/- mice have larger stroke and more severe motor deficits, effects that were independent of intraischemic cerebral blood flow, vascular factors, or cytosolic ß-catenin levels. Rather, LRP6 haploinsufficiency increased the activating phosphorylation and decreased the inhibitory phosphorylation of GSK-3ß, a kinase involved in proinflammatory signaling and mitochondrial dysfunction. Accordingly, postischemic inflammatory gene expression was enhanced in LRP6+/- mice. Furthermore, the association of mitochondria with activated GSK-3ß was increased in LRP6+/- mice, resulting in a reduction in the Ca2+ handling ability of mitochondria. The mitochondrial dysfunction was reversed by pharmacological inhibition of GSK-3ß. CONCLUSIONS: LRP6 activates an endogenous neuroprotective pathway that acts independently of ß-catenin by controlling GSK-3ß activity and preventing its deleterious mitochondrial and proinflammatory effects. The findings raise the possibility that emerging treatment strategies for diseases attributable to LRP6 loss-of-function mutations could also lead to new therapeutic avenues for ischemic stroke.

FGF23-induced hypophosphatemia persists in Hyp mice deficient in the WNT coreceptor Lrp6.

Deregulated phosphate homeostasis can lead to a wide range of disorders, including myopathy, cardiac dysfunction, and skeletal abnormalities. Therefore, characterization of the molecular regulation of phosphate metabolism is of pathophysiological and clinical significance. Hyp mouse is the model for human X-linked hypophosphatemia which is due to mutations that inactivate the endopeptidases of the X chromosome (PHEX). PHEX inactivation leads to increased serum levels of fibroblast growth factor 23 (FGF23), a phosphaturic hormone that induces excessive renal phosphate excretion and severe hypophosphatemia. The expression of WNT signaling components is increased in Hyp mice. To determine the potential role of WNT signaling in FGF23-mediated hypophosphatemia, we cross-bred Hyp mice with mice deficient in the WNT coreceptor low-density lipoprotein receptor-related protein 6 (Lrp6) to generate Hyp and Lrp6 double mutant mice (Hyp/Lrp6). Like Hyp mice, Hyp/Lrp6 double mutants maintained high serum levels of FGF23, and accordingly exhibited hypophosphatemia to the same degree as the Hyp mice did, indicating that genetically reducing WNT signaling does not impact FGF23-induced phosphaturia. Moreover, similar to Hyp mice, the Hyp/Lrp6 double mutants also exhibited reduced mineralization of the bone, further supporting that reduced WNT signaling does not affect the chronic phosphate wasting caused by excess FGF23 in these mice. In further support of our finding, injection of bioactive FGF23 protein into Lrp6 mutant mice reduced serum phosphate levels to a similar degree as FGF23 injection into wild-type mice. Our in vivo studies provide genetic and pharmacological evidence for a WNT-independent function of FGF23 in the regulation of phosphate homeostasis.

Disruption of LRP6 in osteoblasts blunts the bone anabolic activity of PTH.

Mutations in low-density lipoprotein receptor-related protein 6 (LRP6) are associated with human skeletal disorders. LRP6 is required for parathyroid hormone (PTH)-stimulated signaling pathways in osteoblasts. We investigated whether LRP6 in osteoblasts directly regulates bone remodeling and mediates the bone anabolic effects of PTH by specifically deleting LRP6 in mature osteoblasts in mice (LRP6 KO). Three-month-old LRP6 KO mice had a significant reduction in bone mass in the femora secondary spongiosa relative to their wild-type littermates, whereas marginal changes were found in femoral tissue of 1-month-old LRP6 KO mice. The remodeling area of the 3-month-old LRP6 KO mice showed a decreased bone formation rate as detected by Goldner's Trichrome staining and calcein double labeling. Bone histomorphometric and immumohistochemical analysis revealed a reduction in osteoblasts but little change in the numbers of osteoclasts and osteoprogenitors/osteoblast precursors in LRP6 KO mice compared with wild-type littermates. In addition, the percentage of the apoptotic osteoblasts on the bone surface was higher in LRP6 KO mice compared with wild-type littermates. Intermittent injection of PTH had no effect on bone mass or osteoblastic bone formation in either trabecular and cortical bone in LRP6 KO mice, whereas all were enhanced in wild-type littermates. Additionally, the anti-apoptotic effect of PTH on osteoblasts in LRP6 KO mice was less significant compared with wild-type mice. Therefore, our findings demonstrate that LRP6 in osteoblasts is essential for osteoblastic differentiation during bone remodeling and the anabolic effects of PTH.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation.

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in ß-catenin expression. ß-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.