Lipoprotein Receptor-related Proteins 5 And 6 Research Articles

Anti-colon cancer activities of green-synthesized Moringa oleifera–AgNPs against human colon cancer cells

Abstract The anticancer activity of silver nanoparticles (AgNPs) is well known to be synthesized using green-synthesized methods, although its mechanism of action is not understood fully. Moringa oleifera leaves were used as reducing and stabilizing agents to synthesize AgNPs. Green-synthesized AgNPs were characterized using ultraviolet-visible spectroscopy, dynamic light scattering, transmission electronic microscopy, scanning electronic microscopy, Fourier transform infrared, and energy-dispersive X-ray spectroscopy analyses. The synthesized nanoparticles were then characterized by their anticancer properties by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The real-time polymerase chain reaction was used to check the expression levels of the four genes (β-catenin, adenomatous polyposis coli (APC), and lipoprotein receptor-related proteins 5 and 6 (LRP5/6)). The synthesized nanoparticles were 25 nm on average and spherical in shape and aggregated form. Noteworthy cytotoxicity is how green-synthesized M. oleifera–AgNPs were observed in comparison with the M. oleifera leaf extract against a cancerous cell line. The M. oleifera–AgNPs decreased the expression of CTNNB1 and LRP6 genes, while the LRP5 gene expression increased in both cell lines. With treatment, the APC gene expression decreased in SW480 but increased in HTC116. Our results imply that AgNPs synthesized by M. oleifera extract could be an ideal strategy to combat colon cancer.

The Wnt-β-catenin-IL-10 signaling axis in intestinal antigen-presenting cells protects mice from colitis-associated colon cancer in response to gut microbiota

Abstract Loss of immune tolerance to gut microflora is inextricably linked to inflammatory bowel disease (IBD) and colitis-associated colon cancer (CAC). Intestinal antigen presenting cells (APCs) play a pivotal role in regulating intestinal inflammation and CAC, but underlying signaling pathways and molecular mechanisms remain largely unknown. In the present study, using a mouse model of CAC, we show that the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6)-β-catenin-IL-10 signaling axis in CD11c+ APCs suppresses CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of Wnt co-receptors LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of tumor-promoting pro-inflammatory factors and decreased expression of the immunosuppressive cytokine, IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor promoting inflammatory factors in CD11c+ APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin in CD11c+ APCs or in vivo administration of IL-10 in LRP5/6ΔCD11c mice markedly reduced intestinal inflammation and CAC by suppressing the expression of inflammatory factors. In summary, here we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in regulating CAC in response to the commensal flora and suggest potential means for therapeutic intervention in inflammatory intestinal diseases.

Wnt5a promotes Frizzled-4 signalosome assembly by stabilizing cysteine-rich domain dimerization.

Wnt/β-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal "thumb" and a disulfide-stabilized C-terminal "index finger," yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD-CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

Opposing Roles of Wnt Inhibitors IGFBP-4 and Dkk1 in Cardiac Ischemia by Differential Targeting of LRP5/6 and β-catenin.

Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/β-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/β-catenin pathway activation. However, their roles in the adult heart remain unexplored. To understand the role of LRP5/6 and β-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and β-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and β-catenin. Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of β-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/β-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting β-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and β-catenin signaling within the Wnt/β-catenin pathway.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.

Insight into the WNT system and its drug related response

The WNT signalling pathway is a complex system for transferring information for DNA expression from the cell surface receptors to cytoplasm and then to the nucleus. It is based on several proteins that work together as agonists and antagonists in order to maintain homeostasys and to promote anabolic processes. The WNT system acts on all cellular lines involved in bone resorption and formation. WNT pathway can mainly be triggered by two different signalling cascades. The first is well known and is the so-called WNT-beta catenin system (or the canonical pathway), the second is known as the non canonical WNT pathway. WNT proteins form a superfamily of secreted glycoproteins. The association with surface receptors, called Frizzled, that are members of the G protein-coupled receptors superfamily and co receptors like low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) complete the WNT system. LRP5/6 show high affinity for WNT antagonists that modulate the activity of this pathway: DKK1 and sclerostin (SCL), that play a crucial role in modulating the WNT system. The WNT-pathway and in particular its antagonists SCL and DKK1 seems to play a key role in the regulation of bone remodeling during treatment with bone active agents such as bisphosphonates, but not only. Their effects become relevant especially in the course of long-term treatments.

G Protein-coupled Receptor Kinases Phosphorylate LRP6 in the Wnt Pathway

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Restricted Expression of the Orphan Tyrosine Kinase Receptor ROR1 in Chronic Lymphocytic Leukemia.

Gene expression analyses of isolated chronic lymphocytic leukemia (CLL) cells have identified gene expression profiles for CLL that are distinct from those of other B lymphoid malignancies or normal B cells. These signature genes may play a role in disease pathogenesis or progression and/or may encode proteins that can be targeted by immune therapy for patients with this disease. One such gene encodes ROR1, a surface receptor tyrosine kinase that ordinarily is expressed by a subset of developing neurons during embryogenesis but not by cells of normal adults, including blood and tissue lymphocytes. The extracellular domain of ROR1 contains a frizzled (FRZ) module that was first defined in G-protein-coupled receptors of the frizzled and smoothened families and that has been found in several frizzled-related proteins involved in Wnt signaling. Because our recent studies found that Wnt signaling genes are over-expressed and active in CLL (PNAS10:3118, 2004), we examined CLL B cells for expression of ROR1 protein and studied the capacity of this orphan receptor tyrosine kinase to mediate signaling in response to stimulation by any one of the multiple members of the Wnt factor family. Immunoblot analyses with antibodies raised against ROR1-peptides revealed expression of full-length ROR1 of appropriate size in each of the CLL samples examined (n = 12). However, we did not detect expression of ROR1 protein by other tissues, including blood or splenic lymphocytes of non-leukemic patients or normal adults donors. Moreover, we generated mouse antisera against full-length human ROR1 via DNA immunization with ROR1 cDNA expression vectors. These antisera reacted specifically with CHO cells transfected to express ROR1, but not with non-transfected CHO cells. Anti-ROR1 antisera, but not control sera, reacted specifically with intact CLL B cells, but not with lymphocytes of healthy adult donors, revealing that expression of surface-membrane ROR1 may be unique to leukemia B lymphocytes. We studied the functional significance of ROR1 by transfecting HEK293 cells with expression constructs encoding ROR1, one of several Wnt factors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), and/or reporter constructs. We observed that expression of ROR1 with any Wnt factor did not activate T-cell transcription factor (TCF), nuclear factors of activated T cells (NFAT), or AP-1 dependent gene expression, suggesting that ROR1 does not signal via the canonical Wnt-signaling pathway. However, we observed that co-expression of ROR1 in HEK293 cells with Wnt5a, but not with any other Wnt factor, induced activation of NF-κB. Induction of NF-κB was dose dependent on expression of ROR1 and Wnt5a, but independent of expression of LPR5/6 that ordinarily serve as co-receptors for the frizzled family of Wnt receptors. We conclude that CLL cells have distinctive expression of a surface tyrosine kinase that ordinarily is expressed during early development. Moreover, this tyrosine kinase may serve as a receptor for Wnt5a, a factor expressed in stromal microenvironments that can trigger activation of NF-κB in CLL cells via a non-canonical Wnt-signaling pathway. Because constitutive activation of NF-κB is implicated in oncogenesis of many types of cancers, the capacity of ROR1 to induce activation of NF-κB suggests that the unusual expression of this developmentally-regulated tyrosine kinase may play an important role in the pathogenesis of this disease.

Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.