PTK7 proteolytic fragment proteins function during early Xenopus development.

Protein Tyrosine Kinase 7 (PTK7) is as a critical regulator of canonical and non-canonical Wnt-signaling during embryonic development and cancer cell formation. Disrupting PTK7 activity perturbs vertebrate nervous system development, and also promotes human cancer formation. Observations in different model systems suggest a complex cross-talk between PTK7 protein and Wnt signaling. During Xenopus laevis nervous system development, we previously showed that PTK7 protein positively regulates canonical Wnt signaling by maintaining optimal LRP6 protein levels, but PTK7 also acts in concert with LRP6 protein to repress non-canonical Wnt activity. PTK7 is a transmembrane protein, but studies in cancer cells showed that PTK7 undergoes "shedding" by metalloproteases to different proteolytic fragments. Some PTK7 proteolytic fragments are oncogenic, being localized to alternative cytoplasmic and nuclear cell compartments. In this study we examined the biological activity of two proteolytic carboxyl-terminal PTK7 proteolytic fragments, cPTK7 622-1070 and cPTK7 726-1070 during early Xenopus nervous system development. We found that these smaller PTK7 proteolytic fragments have similar activity to full-length PTK7 protein to promote canonical Wnt-signaling via regulation of LRP6 protein levels. In addition to cancer systems, this study shows in vivo proof that these smaller PTK7 proteolytic fragments can recapitulate full-length PTK7 protein activity in diverse systems, such as vertebrate nervous system development.

PTK7 modulates Wnt signaling activity via LRP6.

Protein tyrosine kinase 7 (PTK7) is a transmembrane protein expressed in the developing Xenopus neural plate. PTK7 regulates vertebrate planar cell polarity (PCP), controlling mesodermal and neural convergent-extension (CE) cell movements, neural crest migration and neural tube closure in vertebrate embryos. Besides CE phenotypes, we now show that PTK7 protein knockdown also inhibits Wnt/ß-catenin activity. Canonical Wnt signaling caudalizes the neural plate via direct transcriptional activation of the meis3 TALE-class homeobox gene, which subsequently induces neural CE. PTK7 controls meis3 gene expression to specify posterior tissue and downstream PCP activity. Furthermore, PTK7 morphants phenocopy embryos depleted for Wnt3a, LRP6 and Meis3 proteins. PTK7 protein depletion inhibits embryonic Wnt/ß-catenin signaling by strongly reducing LRP6 protein levels. LRP6 protein positively modulates Wnt/ß-catenin, but negatively modulates Wnt/PCP activities. The maintenance of high LRP6 protein levels by PTK7 triggers PCP inhibition. PTK7 and LRP6 proteins physically interact, suggesting that PTK7 stabilization of LRP6 protein reciprocally regulates both canonical and noncanonical Wnt activities in the embryo. We suggest a novel role for PTK7 protein as a modulator of LRP6 that negatively regulates Wnt/PCP activity.

Systems Genetics Analysis of a Recombinant Inbred Mouse Cell Culture Panel Reveals Wnt Pathway Member Lrp6 as a Regulator of Adult Hippocampal Precursor Cell Proliferation.

In much animal research, genetic variation is rather avoided than used as a powerful tool to identify key regulatory genes in complex phenotypes. Adult hippocampal neurogenesis is one such highly complex polygenic trait, for which the understanding of the molecular basis is fragmented and incomplete, and for which novel genetic approaches are needed. In this study, we aimed at marrying the power of the BXD panel, a mouse genetic reference population, with the flexibility of a cell culture model of adult neural precursor proliferation and differentiation. We established adult-derived hippocampal precursor cell cultures from 20 strains of the BXD panel, including the parental strains C57BL/6J and DBA/2J. The rates of cell proliferation and neuronal differentiation were measured, and transcriptional profiles were obtained from proliferating cultures. Together with the published genotypes of all lines, these data allowed a novel systems genetics analysis combining quantitative trait locus analysis with transcript expression correlation at a cellular level to identify genes linked with the differences in proliferation. In a proof-of-principle analysis, we identified Lrp6, the gene encoding the coreceptor to Frizzled in the Wnt pathway, as a potential negative regulator of precursor proliferation. Overexpression and siRNA silencing confirmed the regulatory role of Lrp6. As well as adding to our knowledge of the pathway surrounding Wnt in adult hippocampal neurogenesis, this finding allows the new appreciation of a negative regulator within this system. In addition, the resource and associated methodology will allow the integration of regulatory mechanisms at a systems level.

Kallistatin as an inhibitory protein against colorectal cancer cells through binding to LRP6.

Kallistatin (KL) is a member of the serine proteinase inhibitor (serpin) family regulating oxidative stress, vascular relaxation, inflammation, angiogenesis, cell proliferation, and invasion. The heparin-binding site of Kallistatin has an important role in the interaction with LRP6 leading to the blockade of the Wnt signaling pathway. In this study, we aimed to explore the structural basis of the Kallistatin-LRP6E1E4 complex using in silico approaches and evaluating the anti-proliferative, apoptotic, and cell cycle arrest activities of Kallistatin in colon cancer lines. The molecular docking showed Kallistatin could bind to the LRP6E3E4 much stronger than LRP6E1E2. The Kallistatin-LRP6E1E2 and Kallistatin-LRP6E3E4 complexes were stable during Molecular Dynamics (MD) simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) showed that the Kallistatin-LRP6E3E4 has a higher binding affinity compared to Kallistatin-LRP6E1E2. Kallistatin induced higher cytotoxicity and apoptosis in HCT116 compared to the SW480 cell line. This protein-induced cell-cycle arrest in both cell lines at the G1 phase. The B-catenin, cyclin D1, and c-Myc expression levels were decreased in response to treatment with Kallistatin in both cell lines while the LRP6 expression level was decreased in the HCT116 cell line. Kallistatin has a greater effect on the HCT116 cell line compared to the SW480 cell line. Kallistatin can be used as a cytotoxic and apoptotic-inducing agent in colorectal cancer cell lines.

In-silico identification of new inhibitors for Low-density lipoprotein receptor-related protein6 (LRP6).

Low-density lipoprotein receptor-related protein 6 (LRP6) is an important therapeutic target for diseases such as osteoporosis, Alzheimer, cancer, and neurodegenerative disease. Computational methods such as ligand-based and structure-based virtual screening have been introduced as an extremely efficient and accurate tool for finding new drug targets and candidates. In this study, we aimed to screen the National Cancer Institute (NCI) Diversity Set II and parts of the ZINC database by virtual screening to identify potential and safe compounds that can inhibit the LRP6 protein. By utilizing various screening methods such as rigid and flexible molecular docking and Lipinski's rule of five, we identified 10 potential compounds. Then, their validity was further tested by molecular dynamics simulation and MMPBSA binding free energy calculations. Eventually, it was concluded that ZINC03954520, ZINC01729523, ZINC03898665, ZINC13152226, ZINC26730911 and ZINC01069082 compounds can be potential drug compounds for inhibiting LRP6 protein. These compounds in complex with ß-propeller domains of LRP6 showed that they are capable of altering the backbone of these domains and interfere with their structural dynamics which may lead to the inhibition of the signal transmission. Communicated by Ramaswamy H. Sarma.

Pattern of LRP6 gene expression in tumoral tissues of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is known as one of the most leading causes of death from cancer in the world. Wnt signaling pathway is the most important signaling pathway activated in this type of cancer. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) functions as a co-receptor in order to trigger this signaling pathway. OBJECTIVE: The expression of LRP6 was studied in cell lines, tumoral and normal adjacent tissues from patients affected with colorectal cancer. MATERIALS AND METHODS: The expression of LRP6 was assayed by quantitative Real time PCR. RESULTS: LRP6 is overexpressed at the transcript and protein level in HCT116 and SW480 in comparison to HUVEC as the non-cancerous cell. Furthermore, LRP6 was significantly up-regulated in 61% of human malignant tissues (p-value = 0.0008). In inherited type of disease, this upregulation was lower than other types of colorectal cancer, while in rectal cancer the overexpression was significantly higher (p-value = 0.039). Gene expression was not correlated with the age, gender and the stage of disease. CONCLUSION: Due to the profound overexpression of LRP6 in sporadic and rectal types of cancer compared to normal colonic ones, antagonist related approaches can be promising for targeted therapies of cancer.