Bone density ligand, Sclerostin, directly interacts with LRP5 but not LRP5G171V to modulate Wnt activity.

We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway. INTRODUCTION: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6. MATERIALS AND METHODS: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections. Results: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5(G171V)) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6(G158V)) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of alpha-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6. CONCLUSIONS: Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.     

Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation

Wnt signaling plays an important role in skeletal biology and diseases. In osteoblasts, we recently showed that the cell-cell adhesion molecule N-cadherin interacts with the Wnt coreceptors LRP5/6 to regulate osteogenesis. In this study we investigated whether targeting the intracellular domain of N-cadherin that interacts with LRP5/6 may promote Wnt signaling and bone formation. By investigating the molecular interactions between the Wnt coreceptors LRP5/6 and N-cadherin, we identified specific LRP5/6- and N-cadherin–interacting intracellular domains that impact Wnt/β-catenin signaling in murine osteoblasts. We showed that truncated N-cadherin constructs that impair N-cadherin-LRP5/6 interactions promote Wnt/β-catenin signaling and osteoblast differentiation. Based on this finding, we developed a peptide-based approach targeting N-cadherin-LRP5 interaction for promoting Wnt signaling and osteoblast function. We found that a competitor peptide containing the 28 last amino acids of LRP5 disrupts LRP5/6-N-cadherin interaction and thereby enhances Wnt/β-catenin signaling in osteoblasts. We also show that the peptide-mediated disruption of N-cadherin-LRP5/6 interaction increases Wnt/β-catenin signaling and osteoblast function in vitro and promotes calvaria bone formation in vivo. The targeted competitor peptide-based strategy reported here may provide a novel approach to stimulate Wnt/β-catenin signaling that can be used for promoting osteoblast function and bone formation. © 2012 American Society for Bone and Mineral Research.     

Bone morphogenetic protein‐2 promotes osteosarcoma growth by promoting epithelial‐mesenchymal transition (EMT) through the Wnt/β‐catenin signaling pathway

The correlation between BMP‐2 and osteosarcoma growth has gained increased interest in the recent years, however, there is still no consensus. In this study, we tested the effects of BMP‐2 on osteosarcoma cells through both in vitro and in vivo experiments. The effect of BMP‐2 on the proliferation, migration and invasion of osteosarcoma cells was tested in vitro. Subcutaneous and intratibial tumor models were used for the in vivo experiments in nude mice. The effects of BMP‐2 on EMT of osteosarcoma cells and the Wnt/β‐catenin signaling pathway were also tested using a variety of biochemical methods. In vitro tests did not show a significant effect of BMP‐2 on tumor cell proliferation. However, BMP‐2 increased the mobility of tumor cells and the invasion assay demonstrated that BMP‐2 promoted invasion of osteosarcoma cells in vitro. In vivo animal study showed that BMP‐2 dramatically enhanced tumor growth. We also found that BMP‐2 induced EMT of osteosarcoma cells. The expression levels of Axin2 and Dkk‐1 were both down regulated by BMP‐2 treatment, while β‐catenin, c‐myc and Cyclin‐D1 were all upregulated. The expression of Wnt3α and p‐GSK‐3β were also significantly upregulated indicating that the Wnt/β‐catenin signaling pathway was activated during the EMT of osteosarcoma driven by BMP‐2. From this study, we can conclude that BMP‐2 significantly promotes growth of osteosarcoma cells (143B, MG63), and enhances mobility and invasiveness of tumor cells as demonstrated in vitro. The underlying mechanism might be that BMP‐2 promotes EMT of osteosarcoma through the Wnt/β‐catenin signaling pathway. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1638–1648, 2019.     

Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells.

Genetic mutations in the LRP5 gene affect Wnt signaling and lead to changes in bone mass in humans. Our in vivo and in vitro results show that activated mutation T253I of LRP5 enhances osteogenesis and inhibits adipogenesis. Inactivating mutation T244M of LRP5 exerts opposite effects. INTRODUCTION: Mutations in the Wnt co-receptor, LRP5, leading to decreased or increased canonical Wnt signaling, result in osteoporosis or a high bone mass (HBM) phenotype, respectively. However, the mechanisms whereby mutated LRP5 causes changes in bone mass are not known. MATERIALS AND METHODS: We studied bone marrow composition in iliac crest bone biopsies from patients with the HBM phenotype and controls. We also used retrovirus-mediated gene transduction to establish three different human mesenchymal stem cell (hMSC) strains stably expressing wildtype LRP5 (hMSC-LRP5(WT)), LRP5(T244) (hMSC-LRP5(T244), inactivation mutation leading to osteoporosis), or LRP5(T253) (hMSC-LRP5(T253), activation mutation leading to high bone mass). We characterized Wnt signaling activation using a dual luciferase assay, cell proliferation, lineage biomarkers using real-time PCR, and in vivo bone formation. RESULTS: In bone biopsies, we found increased trabecular bone volume and decreased bone marrow fat volume in patients with the HBM phenotype (n = 9) compared with controls (n = 5). The hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) transduced high level of Wnt signaling. Wnt3a inhibited cell proliferation in hMSC-LRP5(WT) and hMSC-LRP5(T253), and this effect was associated with downregulation of DKK1. Both hMSC-LRP5(WT) and hMSC-LRP5(T253) showed enhanced osteoblast differentiation and inhibited adipogenesis in vitro, and the opposite effect was observed in hMSC-LRP5(T244). Similarly, hMSC-LRP5(WT) and hMSC-LRP5(T253) but not hMSC-LRP5(T244) formed ectopic mineralized bone when implanted subcutaneously with hydroxyapatite/tricalcium phosphate in SCID/NOD mice. CONCLUSIONS: LRP5 mutations and the level of Wnt signaling determine differentiation fate of hMSCs into osteoblasts or adipocytes. Activation of Wnt signaling can thus provide a novel approach to increase bone mass by preventing the age-related reciprocal decrease in osteogenesis and increase in adipogenesis.     

BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop.

Wnt/beta-catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells. INTRODUCTION: Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology. MATERIALS AND METHODS: We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3-E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation. RESULTS: In mesenchymal cells, only Wnt's capable of stabilizing beta-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xddl, dickkopf 1 (dkk1), or LRP5deltaC, indicating that canonical beta-catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP-2 enhanced Wntl and Wnt3a expression in our cells. In MC3T3-E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkkl reduced extracellular matrix mineralization in a BMP-2-dependent assay. CONCLUSIONS: Our data strongly suggest that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.     

Eradication of osteosarcoma by fluorescence‐guided surgery with tumor labeling by a killer‐reporter adenovirus

In a previous study, we developed fluorescence‐guided surgery (FGS) for osteosarcoma using an orthotopic model with 143B human osteosarcoma cells expressing red fluorescent protein (RFP) implanted into the intramedullary cavity of the tibia in nude mice. The FGS‐treated mice had a significantly higher disease‐free survival (DFS) rate than the bright‐light surgery (BLS). However, although FGS significantly reduced the recurrence of the primary tumor, it did not reduce lung metastasis. In the present study, we utilized the OBP‐401 telomerase‐dependent killer‐reporter adenovirus, carrying green fluorescent protein (GFP), to label human osteosarcoma in situ in orthotopic mouse models. OBP‐401‐illuminated human osteosarcoma cell lines, 143B and MNNG/HOS cells in vitro and in vivo. OBP‐401 tumor illumination enabled effective FGS of the 143B‐derived orthotopic mouse model of human osteosarcoma model as well as FGS eradication of residual cancer cells after BLS. OBP‐401‐assisted FGS significantly inhibited local recurrence and lung metastasis after surgery, thereby prolonging DFS and overall survival (OS), achieving a very important improvement of therapeutic outcomes over our previously reported FGS study. These therapeutic benefits of FGS were demonstrated using a clinically‐viable methodology of direct labeling of human osteosarcoma in situ with the OBP‐401 killer‐reporter adenovirus in contrast with previous reports, which used genetically engineered labeled cells or antibody‐based fluorescent labels for FGS. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:836–844, 2016.     

Wnt signaling in bone metabolism

A variety of in vivo models have increased understanding of the role of Wnt signaling in bone since mutations in the LRP5 gene were found in human bone disorders. Canonical Wnt signaling encourages mesenchymal progenitor cells to differentiate into osteoblasts. In osteoblasts, Wnt pathway also promotes proliferation and mineralization, while blocks apoptosis and osteoclastogenesis by increasing the OPG/RANKL ratio. Lrp6-mediated signaling in osteoblasts may regulate osteoclastogenesis. However, the role of canonical Wnt signaling in osteoclasts remains unknown, and our understanding of the role of non-canonical Wnt signaling in bone biology is also not sufficient. As to pharmacological intervention, many levels may be considered to target in Wnt signaling pathway, although tumorigenicity and toxicity to other tissues are important. Mesd might be one of target molecules to increase the quantity of LRP5/6 in the plasma membrane. Since sclerostin is almost exclusively expressed in osteocytes, abrogating sclerostin is the most promising design. T. Kubota is a recipient of JSBMR Encouragement Award of 2006.     

Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration

Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β‐catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well‐organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research     

Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass.

Wnt/beta-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation. INTRODUCTION: The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism. MATERIALS AND METHODS: In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1(+/-) animals. RESULTS: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1(+/-) mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold. CONCLUSIONS: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.     

Dkk1-mediated inhibition of Wnt signaling in bone results in osteopenia

Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.     

Novel LRP5 missense mutation in a patient with a high bone mass phenotype results in decreased DKK1-mediated inhibition of Wnt signaling.

We found a novel heterozygous missense mutation (M282V) in the LRP5 gene in a patient with a high bone mass phenotype. In vitro studies suggest that a reduced antagonistic effect of DKK1 on canonical Wnt signaling contributes to the molecular effect of this mutation and its pathogenic consequence. INTRODUCTION: Gain-of-function mutations in the gene encoding LDL receptor-related protein 5 (LRP5) cause high bone mass. Recent studies revealed that a reduced inhibition of canonical Wnt signaling by Dickkopf 1 (DKK1) contributes to the pathophysiology of this disease phenotype. MATERIALS AND METHODS: We report on a 55-yr-old female patient with a high bone mass phenotype. Sequencing of exons 2-4 of the LRP5 gene was carried out to screen for disease-associated mutations in genomic DNA of the patient. The effect of the identified mutation on LRP5 membrane trafficking was studied by immunoblotting of a truncated form of LRP5. Additionally, Wnt signal activation in the absence and presence of DKK1 was assessed using a TCF4-based reporter gene assay in Saos-2 cells. RESULTS: Our patient presents with dense bones (Z-scores > +6), and radiographic examination showed a generalized thickening of the skeleton. BMD at the hip and lumbar spine significantly decreased through the passage to menopause, indicating no protection to bone loss. Further clinical evaluation revealed torus palatinus. Mutation analysis showed the presence of a novel heterozygous missense variant (844A-->G; M282V) in LRP5, located in the first beta-propeller domain of the extracellular portion. Although protein secretion seemed to be impaired, this mutant was able to transduce Wnt signals at levels comparable with wildtype LRP5. We additionally observed a less efficient inhibition of canonical Wnt signaling by DKK1. CONCLUSIONS: Like all high BMD-associated gain-of-function LRP5 mutations described thus far, the M282V variant affects an amino acid located in the first beta-propeller domain, underlining the functional importance of this region in the pathophysiology of these conditions. This mutation most likely alters a region important for LRP5 modulation by DKK.     

Wnt5a as an Effector of TGFβ in Mammary Development and Cancer

Wnt5a is a member of the Wingless-related/MMTV-integration family of secreted growth factors, which are involved in a wide range of cellular processes. Wnt signaling can be broadly divided into two categories the canonical, ß-catenin-dependent pathway and the non-canonical ß-catenin-independent pathway. Wnt5a is a non-canonical signaling member of the Wnt family. Loss of Wnt5a is associated with early relapse of invasive breast cancer, increased metastasis, and poor survival in humans. It has been shown that TGF-ß directly regulates expression of Wnt5a in mammary gland and that Wnt5a mediates the effects of TGF-ß on branching during mammary gland development. Here we review the evidence suggesting Wnt5a acts as an effector of TGF-ß actions in breast cancer. It is suggested that the tumor suppressive functions of TGF-ß involve Wnt5a-mediated antagonism of Wnt/ß-catenin signaling and limiting the stem cell population. Interactions between TGF-ß and Wnt5a in metastasis appear to be more complex, and may depend on specific cues from the microenvironment as well as activation of specific intracellular signaling pathways.     

The orally bioavailable met inhibitor PF‐2341066 inhibits osteosarcoma growth and osteolysis/matrix production in a xenograft model

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Ninety percent of patients who present with metastatic and 30% to 40% of patients with nonmetastatic disease experience relapse, creating an urgent need for novel therapeutic strategies. The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are important for mitosis, motility, and cell survival. Upregulation of Met/HGF signaling via receptor overexpression, amplification, or mutation drives the proliferation, invasiveness, and metastasis of a variety of cancer cells, including OS, prompting the development of Met/HGF inhibitors. OS cells depend on Met overexpression because introduction of dominant‐negative Met inhibits in vivo tumorigenicity. Despite the importance of Met/HGF signaling in the development and maintenance of OS, the potential efficacy of pharmacologic Met inhibition in OS has been addressed only in in vitro studies. PF‐2341066 is an orally bioavailable, selective ATP‐competitive Met inhibitor that showed promising results recently in a phase I clinical trial in non–small cell lung cancer (NSCLC) patients. We tested the ability of PF‐2341066 to inhibit malignant properties of osteosarcoma cells in vitro and orthotopic xenograft growth in vivo. In vitro, PF‐2341066 inhibited osteosarcoma behavior associated with primary tumor growth (eg, proliferation and survival) as well as metastasis (eg, invasion and clonogenicity). In nude mice treated with PF‐2341066 via oral gavage, the growth and associated osteolysis and extracortical bone matrix formation of osteosarcoma xenografts were inhibited by PF‐2341066. PF‐2341066 may represent an effective new systemic therapy for localized and potentially disseminated osteosarcoma. © 2011 American Society for Bone and Mineral Research.     

A Comprehensive Overview of Skeletal Phenotypes Associated with Alterations in Wnt/β-catenin Signaling in Humans and Mice

The Wnt signaling pathway plays key roles in differentiation and development and alterations in this signaling pathway are causally associated with numerous human diseases. While several laboratories were examining roles for Wnt signaling in skeletal development during the 1990s, interest in the pathway rose exponentially when three key papers were published in 2001–2002. One report found that loss of the Wnt co-receptor, Low-density lipoprotein related protein-5 (LRP5), was the underlying genetic cause of the syndrome Osteoporosis pseudoglioma (OPPG). OPPG is characterized by early-onset osteoporosis causing increased susceptibility to debilitating fractures. Shortly thereafter, two groups reported that individuals carrying a specific point mutation in LRP5 (G171V) develop high-bone mass. Subsequent to this, the causative mechanisms for these observations heightened the need to understand the mechanisms by which Wnt signaling controlled bone development and homeostasis and encouraged significant investment from biotechnology and pharmaceutical companies to develop methods to activate Wnt signaling to increase bone mass to treat osteoporosis and other bone disease. In this review, we will briefly summarize the cellular mechanisms underlying Wnt signaling and discuss the observations related to OPPG and the high-bone mass disorders that heightened the appreciation of the role of Wnt signaling in normal bone development and homeostasis. We will then present a comprehensive overview of the core components of the pathway with an emphasis on the phenotypes associated with mice carrying genetically engineered mutations in these genes and clinical observations that further link alterations in the pathway to changes in human bone.     

Usefulness of inhibiting the lymph node metastasis in human gastric carcinoma by B7-1 gene transfection

INTRODUCTION: Lymph node metastasis is one of the crucial prognostic factors in gastric cancer. We have reported that ICAM-1 gene transfection was effective against lymph node metastases of gastric cancer. B7-1, one of the co-stimulatory factors, was reported to induce cytotoxic T lymphocytes when using melanoma and bladder cancer cell lines, as well as ICAM-1. In this study, we investigated the inhibitory effect of B7-1 on lymph node metastasis by B7-1 gene transfection into gastric cancer cells. MATERIALS AND METHODS: We transfected B7-1 genes into a gastric cancer cell line (OCUM-2MLN) and analyzed the effect of B7-1 transduction on lymph node metastasis, the in vitro adhesiveness and cytotoxicity assay of mononuclear lymphocytes to cancer cells and lymph node metastatic ability after orthotopic implantation of gastric cancer cells in vivo. RESULTS: We revealed that mononuclear lymphocytes showed significantly stronger adherence and cytotoxicity to B7-1 transfected cells (2MLN/B7) than its parent OCUM-2MLN cells. The tumor growth rate of 2MLN/B7 xenograft was significantly slower than OCUM-2MLN xenograft in nude mice. In orthotopic implantation experiments for nude mice, 2MLN/B7 cells in stomach developed significantly less lymph node metastasis than OCUM-2MLN cells. Histologic findings showed that leukocytes were intensively infiltrated in both the 2MLN/B7 tumors and its metastatic lesions, however, were scarcely observed in the lesions associated with 2MLN cells. CONCLUSION: B7-1 may play an important role in inhibiting lymph node metastasis by the mechanism of enhanced immunogenicity, and that B7-1 gene transduction might be effective against lymph node metastases of gastric cancer.     

Genetic variation at the low-density lipoprotein receptor-related protein 5 (LRP5) locus modulates Wnt signaling and the relationship of physical activity with bone mineral density in men

Polymorphisms in the LRP5 gene have been associated with bone mineral density (BMD) in men and/or women. However, the functional basis for this association remains obscure. We hypothesized that LRP5 alleles could modulate Wnt signaling and the relationship between physical activity and BMD. This genetic association study was performed in the population-based Framingham Study Offspring Cohort, and included a subset of 1797 unrelated individuals who provided blood samples for DNA and who had BMD measurements of the hip and spine. Ten single-nucleotide polymorphisms (SNPs) spanning the LRP5 gene were genotyped and used for association and interaction analyses with BMD by regression methods. LRP5 haplotypes were transiently co-expressed with Wnt3a, MesD and Dkk1 in HEK293 cells and their activity evaluated by the TCF-Lef reporter assay. Six out of ten SNPs in LRP5 were associated with one or more of the femur or spine BMDs in men or women after adjustment for covariates, and these associations differed between genders. In men< or =age 60 years, 3 SNPs were significantly associated with BMD: rs2306862 on Exon 10 with femoral neck BMD (p=0.01) and Ward's BMD (p=0.01); rs4988321/p. V667M with Ward's BMD (p=0.02); and intronic rs901825 with trochanter BMD (p=0.03). In women, 3 SNPs in intron 2 were significantly associated with BMD: rs4988330 for trochanter (p=0.01) and spine BMD (p=0.003); rs312778 with femoral neck BMD (p=0.05); and rs4988331 with spine BMD (p=0.04). For each additional rare allele, BMD changed by 3-5% in males and 2-4% in females. Moreover, there was a significant interaction between physical activity and rs2306862 in exon 10 (p for interaction=0.02) and rs3736228/p. A1330V in exon 18 (p for interaction=0.05) on spine BMD in men. In both cases, the TT genotype was associated with lower BMD in men with higher physical activity scores, conversely with higher BMD in men with lower physical activity scores. In vitro, TCF-Lef activity in presence of Wnt3a was significantly reduced in cells expressing LRP5 haplotypes carrying the T allele of exon 10 and 18 compared to the wild-type allele, whereas co-expression of Dkk1 completely inhibited Wnt3a response through all LRP5 haplotypes. In summary, genetic variation in exons 10 and 18 of the LRP5 gene modulates Wnt signaling and the relationship between physical activity and BMD in men. These observations suggest that Wnt-LRP5 may play a role in the adaptation of bone to mechanical load in humans, and may explain some gender-related differences in bone mass.