Knockdown Of N-cadherin Research Articles

N-Cadherin promotes cardiac regeneration by potentiating pro-mitotic β-Catenin signaling in cardiomyocytes

Adult human hearts exhibit limited regenerative capacity. Post-injury cardiomyocyte (CM) loss can lead to myocardial dysfunction and failure. Although neonatal mammalian hearts can regenerate, the underlying molecular mechanisms remain elusive. Herein, comparative transcriptome analyses identify adherens junction protein N-Cadherin as a crucial regulator of CM proliferation/renewal. Its expression correlates positively with mitotic genes and shows an age-dependent reduction. N-Cadherin is upregulated in the neonatal mouse heart following injury, coinciding with increased CM mitotic activities. N-Cadherin knockdown reduces, whereas overexpression increases, the proliferation activity of neonatal mouse CMs and human induced pluripotent stem cell-derived CMs. Mechanistically, N-Cadherin binds and stabilizes pro-mitotic transcription regulator β-Catenin, driving CM self-renewal. Targeted N-Cadherin deletion in CMs impedes cardiac regeneration in neonatal mice, leading to excessive scarring. N-Cadherin overexpression, by contrast, promotes regeneration in adult mouse hearts following ischemic injury. N-Cadherin targeting presents a promising avenue for promoting cardiac regeneration and restoring function in injured adult human hearts.

N-cadherin facilitates trigeminal sensory neuron outgrowth and target tissue innervation.

During embryonic development, diverse cell types coordinate to form functionally complex tissues. Exemplifying this process, the trigeminal ganglion emerges from the condensation of two distinct precursor cell populations, cranial placodes and neural crest, with neuronal differentiation of the former preceding the latter. While its dual cellular origin has been understood for decades, the molecules orchestrating trigeminal ganglion formation remain relatively obscure. Initial assembly of the trigeminal ganglion is mediated by cell adhesion molecules, including neural cadherin (N-cadherin), which is first expressed by placodal neurons and is required for their proper condensation with other neurons and neural crest cells. Axon outgrowth first occurs from placodal neurons, but as gangliogenesis proceeds, neural crest cells also differentiate into N-cadherin-expressing neurons, and together both extend axons toward target tissues. However, a role for N-cadherin in regulating axon outgrowth and innervation of target tissues by trigeminal neurons has not been explored. To this end, we depleted N-cadherin from chick trigeminal placode cells and observed decreases in trigeminal ganglion size, nerve growth, and target innervation in vivo, phenotypes that could only partially be attributed to increased apoptosis early in gangliogenesis. Accordingly, neurite number and branching of neural crest-derived neurons was decreased in vitro in response to N-cadherin knockdown in placode cells, providing a novel non-cell autonomous explanation for these morphological changes. Inhibiting N-cadherin-mediated adhesion with a function-blocking antibody prevented axon extension in most, but not all, placode-derived trigeminal neurons in vitro, indicating potential unique requirements for N-cadherin in various neuronal subtypes. Collectively, these findings reveal persistent cell autonomous and non-cell autonomous functions for N-cadherin, thus highlighting the critical role of N-cadherin in mediating reciprocal interactions between neural crest and placode neuronal derivatives during trigeminal ganglion development.

Abstract P1001: IGFBP2 Overcomes Cell Contact-mediated Proliferation Inhibition In Human IPSC-derived Cardiomyocytes

Introduction and Hypothesis: Human induced pluripotent stem cells (hiPSCs) and their derivatives provide a promising cellular source for regenerative cardiac therapies. However, generating large-scale hiPSC-derived cardiomyocytes (hiPSC-CMs) remains a major hurdle, caused, in part, by cell contact-driven proliferation inhibition in dense culture conditions. We hypothesize that differentially-secreted factors in sparsely vs densely-cultured hiPSC-CMs are responsible for the increased proliferation rate in sparsely cultured hiPSC-CMs. Methods: We performed a high-throughput screening (HTS) assay to identify growth factors (GFs) released in the supernatant of sparsely- and densely-cultured hiPSC-CMs. Differential expression of candidate GFs in dense vs sparse culture conditions was confirmed by single cell RNA sequencing (scRNAseq), Western Blot (WB), and Immunocytochemistry (ICC) analyses. The confirmed GF hits were further validated by measuring the expression of the cell cycle marker Ki67 in hiPSC-CMs after GF treatment. The GF treatment effects on cell-cell contact was further explored mechanistically using siRNA-based N-cadherin (CDH2) knockdown (KD) model in vitro. Results: Our HTS assay identified IGFBP2, IGFBP6, PDGF-AA as top candidate GFs in the sparse culture supernatant. Subsequent WB, ICC, scRNAseq analyses confirmed 1.5, 3.5, 2.0-fold higher IGFBP2 expression in the sparse condition compared to the dense condition. Supplementation of recombinant IGFBP2 to densely cultured hiPSC-CMs showed a dosage-dependent increase in Ki67 expression level from 13.0 +/- 1.4% in control to 44.1 +/- 8.4% after 3nM IGFBP2 treatment, a level comparable to that achieved in sparse culture. Mechanistically, IGFBP2 treatment resulted in a shift of CDH2 localization from cell-cell junction to the cytoplasm. Reducing CDH2 expression by CDH2-KD to mimic loss of cell-cell contact promoted hiPSC-CM proliferation. Conclusion: We discovered an unexpected role of IGFBP2 to overcome cell contact-mediated inhibition of hiPSC-CM proliferation which provides a promising new approach to engineer in situ large-scale 3D cardiac tissues for regenerative applications.

Myostatin increases human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-β superfamily member well known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. Reverse transcription quantitative real-time polymerase chain reaction and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way analysis of variance followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time-dependent manner in both study models. These effects were blocked by inhibition of TGF-β type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3, or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling.

Bone Marrow-Derived Mesenchymal Stem Cells Migrate toward Hormone-Insensitive Prostate Tumor Cells Expressing TGF-β via N-Cadherin.

The prostate tumor microenvironment plays important roles in the metastasis and hormone-insensitive re-growth of tumor cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited into prostate tumors to facilitate tumor microenvironment formation. However, the specific intrinsic molecules mediating BM-MSCs’ migration to prostate tumors are unknown. BM-MSCs’ migration toward a conditioned medium (CM) of hormone-insensitive (PC3 and DU145) or hormone-sensitive (LNCaP) prostate tumor cells was investigated using a three-dimensional cell migration assay and a transwell migration assay. PC3 and DU145 expressed transforming growth factor-β (TGF-β), but LNCaP did not. Regardless of TGF-β expression, BM-MSCs migrated toward the CM of PC3, DU145, or LNCaP. The CM of PC3 or DU145 expressing TGF-β increased the phosphorylation of Smad2/3 in BM-MSCs. Inactivation of TGF-β signaling in BM-MSCs using TGF-β type 1 receptor (TGFBR1) inhibitors, SB505124, or SB431542 did not allow BM-MSCs to migrate toward the CM. The CM of PC3 or DU145 enhanced N-cadherin expression on BM-MSCs, but the LNCaP CM did not. SB505124, SB431542, and TGFBR1 knockdown prevented an increase in N-cadherin expression. N-cadherin knockdown inhibited the collective migration of BM-MSCs toward the PC3 CM. We identified N-cadherin as a mediator of BM-MSCs’ migration toward hormone-insensitive prostate tumor cells expressing TGF-β and introduced a novel strategy for controlling and re-engineering the prostate tumor microenvironment.

Transforming growth factor-\u03b21-induced N-cadherin drives cell\u2013cell communication through connexin43 in osteoblast lineage

Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

N-Cadherin Regulates the Odontogenic Differentiation of Dental Pulp Stem Cells via β-Catenin Activity.

Dental pulp stem cell (DPSC) transplantation has shown new prospects in dental pulp regeneration, and is of great significance in the treatment of pulpitis and pulp necrosis. The fate and regenerative potential of stem cells are dependent, to a great extent, on their microenvironment, which is composed of various tissue components, cell populations, and soluble factors. N-cadherin-mediated cell–cell interaction has been implicated as an important factor in controlling the cell-fate commitment of mesenchymal stem cells. In this study, the effect of N-cadherin on odontogenic differentiation of DPSCs and the potential underlying mechanisms, both in vitro and in vivo, was investigated using a cell culture model and a subcutaneous transplantation mouse model. It was found that the expression of N-cadherin was reversely related to the expression of odontogenic markers (dentin sialophosphoprotein, DSPP, and runt-related transcription factor 2, Runx2) during the differentiation process of DPSCs. Specific shRNA-mediated knockdown of N-cadherin expression in DPSCs significantly increased the expression of DSPP and Runx2, alkaline phosphatase (ALP) activity, and the formation of mineralized nodules. Notably, N-cadherin silencing promoted nucleus translocation and accumulation of β-catenin. Inhibition of β-catenin by a specific inhibitor XAV939, reversed the facilitating effects of N-cadherin downregulation on odontogenic differentiation of DPSCs. In addition, knockdown of N-cadherin promoted the formation of odontoblast-like cells and collagenous matrix in β-tricalcium phosphate/DPSCs composites transplanted into mice. In conclusion, N-cadherin acted as a negative regulator via regulating β-catenin activity during odontogenic differentiation of DPSCs. These data may help to guide DPSC behavior by tuning the N-cadherin-mediated cell–cell interactions, with implications for pulp regeneration.

Human giant larvae-1 promotes migration and invasion of malignant glioma cells by regulating N-cadherin.

Human giant larvae-1 (Hugl-1) is a human homologue of Drosophila tumor suppressor lethal (2)-giant larvae and has been reported to be involved in the development of human malignancies. Previous studies performed by our group demonstrated that Hugl-1 inhibits glioma cell proliferation in an intracranial model of nude mice. However, the exact molecular mechanisms underlying the participation of Hugl-1 in glioma invasion and migration, and in the depolarizing process remain largely unknown. Utilizing the U251-MG cells with stable expression of Hugl-1, the present study used wound healing, Transwell invasion and western blot assays to explore the role and specific mechanism of Hugl-1 in glioma invasion and migration. The results of the present study demonstrated that overexpression of Hugl-1 decreased cell-cell adhesion and increased cell-cell extracellular matrix adhesion. In addition, overexpression of Hugl-1 promoted pseudopodia formation, glioma cell migration and invasion. The molecular mechanism of action involved the negative regulation of N-cadherin protein levels by Hugl-1. Overexpression or knockdown of N-cadherin partially suppressed or enhanced the effects of Hugl-1 on glioma cell migration and invasion, respectively. Furthermore, Hugl-1 inhibited cell proliferation, while promoting cell migration, which suggests that it may serve a two-sided biological role in cellular processes. Taken together, these results suggest that Hugl-1 promotes the migration and invasion of malignant glioma cells by decreasing N-cadherin expression. Thus, Hugl-1 may be applied in the development of targeted and personalized treatment.

N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells

Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor β (TGF-β), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration.Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-β was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells.Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-β increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-β-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-β pathways, such as extracellular signal‐regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-β. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-β type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells.Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-β canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-β via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.

The impact of N-cadherin–β-catenin signaling on the analgesic effects of glial cell-derived neurotrophic factor in neuropathic pain

Long-term neuropathic pain can lead to anxiety, depression, and other issues, which seriously affect patients’ quality of life. For this reason, it is important to find effective treatments. Studies have shown that glial cell-derived neurotrophic factor (GDNF) can relieve neuropathic pain. However, its mechanism of action is unknown. Our previous study of GDNF suggested that the N-cadherin–β-catenin transmembrane signaling system might play a role in GDNF transmembrane signaling. Based on this, the current study aimed to produce a neuropathic pain model to confirm the activation of the N-cadherin–β-catenin signaling system in the spinal dorsal horn under pain conditions and to study the impact of GDNF intrathecal injection on central sensitization of dorsal horn neurons. The results showed that N-cadherin expression, as well as the expression of membrane-associated β-catenin, was reduced in the dorsal horn of the spinal cord in the chronic pain model. Intrathecal injection of GDNF could reactivate the N-cadherin–β-catenin system, improve central sensitization, and relieve pain. Knockdown of N-cadherin or β-catenin could significantly reduce the analgesic effect of GDNF. These results provide clear experimental evidence that the N-cadherin–β-catenin signaling system participates in the analgesic effect of GDNF in neuropathic pain and help identify transmembrane and intracellular signal transduction mechanisms associated with GDNF’s analgesic effects.

Netrin-1 promotes the collective cell migration of liver cancer cells in a 3D cell culture model.

Collective cell migration plays an important role in embryonic development, wound healing, and cancer metastasis. We aimed to investigate the expression, role, and mechanism of Netrin-1 in collective cell migration using a3D culture model. An immunohistochemical study showed that certain cells invaded surrounding tissue by collective migration and that Netrin-1 expression in these cells was increased, especially at the invasive front. In the 3D culture model, collective cell migration was clearly observed, as leader cells were followed by cells migrating along a canal. N-cadherin-mediated cell junctions were observed in collective cell migration, and Netrin-1 expression was elevated in these cells. Netrin-1 did not affect the expression of N-cadherin in 2D-cultured cells; however, in 3D culture, the overexpression of Netrin-1 increased N-cadherin and promoted the collective migration of Huh7 cells, while the knockdown of Netrin-1 decreased N-cadherin and inhibited collective migration in SK-Hep-1 cells. Interestingly, N-cadherin knockdown in Huh7 cells significantly diminished Netrin-1-promoted collective cell migration, while the overexpression of N-cadherin restored collective migration in Netrin-1-knockdown SK-Hep1 cells. These results suggest that Netrin-1 enhances N-cadherin junctions to promote liver cancer cell collective migration in 3D cell culture and may subsequently increase liver cancer metastasis.

Abstract 3928: N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity

Abstract Background: Radiation is a mainstay of treatment in patients diagnosed with glioblastoma. Despite maximal surgical and chemo-radiotherapeutic treatment, the disease remains uniformly fatal, with local disease recurrence within the previously radiated field comprising the major pattern of progression. According to the molecular subtyping of GBM, there is a predominance of mesenchymal GBM cells within the recurrent tumor mass, with characteristic increased expression of CD44, N-cadherin, L1CAM, TWIST1 and phosphorylated STAT3. Moreover, these populations of cells are known to be resistant to additional salvage radiation. The current study aims to evaluate the role of N-cadherin in the relative radiation resistance of mesenchymal subtype GBM. Methods: Overall survival data from the TCGA was analyzed using Cox regression hazard ratios based on N-cadherin expression across multiple molecular subtypes of GBM along with age and MGMT covariates. Using the U3035 and U87MG cells, the analyses of N-cadherin expression or exogenous exposure in the setting of external beam radiation were performed via stable shRNA knockdowns or the use of recombinant human N-cadherin ectodomain-Fc fusion proteins in clonogenic survival assays. Orthotopic implantations were performed in NSG mice with the endpoints of overall survival and tumor size, as determined by luciferase imaging. Subcellular localization of RAD51 was accomplished by confocal microscopy. Statistical analyses were performed using SPSS and GraphPad Prism with significance at p<0.05. Results: N-cadherin expression above the median among the mesenchymal subtype of GBM in the TCGA dataset correlated with a significant decrease in overall survival (HR 1.924, p=0.029). The elimination of N-cadherin expression in normally high expressing U3035 GBM cells led to a significant increase in the ability of radiation to induce cell death in clonogenic assay (17.2% U3035 vs 2% U3035 Δ N-cadherin at 8Gy; 12.7% U3035 vs 1.75% U3035 Δ N-cadherin at 20Gy) and an increase in the presence of double stranded DNA breaks (DSBs) on TUNEL assay. N-cadherin ectodomain-Fc fusion protein exposure in U87 cells resulted in a decrease in NSG mouse mean OS (17.6±0.25 days) compared to control radiated mice (22.3±1.1 days). Conversely, N-cadherin knockdown in U3035 cells resulted in further decrease in tumor bioluminescence beyond that seen in 4Gy x 4 radiation treatment of native U3035 xenografts (21% vs 4% of baseline). Conclusions: The reduction of N-cadherin expression in mesenchymal U3035 GBM cells increases the efficacy of radiation-induced cell death. This finding is accompanied by a decrease in the nuclear localization of RAD51 following DSBs induced by ionizing radiation, indicating a potential crosstalk between DNA damage repair mechanisms and calcium dependent cell-cell adhesion. Citation Format: Christopher P. Cifarelli, J. Austin Vargo, Ian MacFawn, Steven M. Frisch. N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3928.

Effects of N-cadherin on neuronal migration during chicken optic tectum development.

N-cadherin, a member of the cadherin family, plays an important role in neural development. In addition, N-cadherin has been reported to be crucial in neuronal migration, axonal outgrowth, and axonal path-finding. However, the mechanism underlying the effects of N-cadherin in neuronal migration is not entirely clear. In this study, we investigated the overexpression or knockdown of N-cadherin in the optic tectum during chicken embryo development, and then analyzed the effect of N-cadherin on neuronal migration. The results showed that compared with the control group, in the N-cadherin knockdown group, the neuronal migration of the optic tectum was significantly affected and could not arrive at destination. The stratum griseum central layer of the optic tectum mainly includes multipolar neurons, which could not be formed after the knockdown of N-cadherin, and more neurons form the bipolar or monopolar neurons compared with the control group. Compared with the control group, more cells stayed in the neuroepithelium layer. The axonal length in the optic tectum was significantly (P < 0.001) shorter in the N-cadherin knockdown group than in the control group. These results reveal that the knockdown of N-cadherin mainly affects the length of axons and formation of multipolar neurons in the development of the chicken optic tectum, which eventually results in the inhibition of neuronal migration.

The lncRNA small nucleolar RNA host gene 5 regulates trophoblast cell proliferation, invasion, and migration via modulating miR-26a-5p/N-cadherin axis.

Pre-eclampsia (PE) is a pregnancy-specific disease characterized by the occurrence of hypertension and proteinuria after two weeks of gestation. Long noncoding RNAs (lncRNAs) are emerging as key regulators in PE development. This study aims to investigate the role of lncRNA, small nucleolar RNA host gene 5 (SNHG5), in the pathogenesis of PE. The expression of SNHG5 was significantly downregulated in placental tissues from patients with severe PE compared normal controls. Overexpression of SNHG5 promoted trophoblast (HTR-8/SVneo) cell proliferation, invasion, and migration, and flow cytometry results showed that SNHG5 overexpression inhibited apoptosis and caused a decrease of cell population at the G 0 /G 1 phase and an increase of cell population at the S phase, while knockdown of SNHG5 had the opposite effects. The interaction between SNHG5 and miR-26a-5p was predicted by bioinformatics analysis and confirmed by luciferase reporter assay and RNA immunoprecipitation, and miR-26a-5p was negatively regulated by SNHG5; miR-26a-5p expression was upregulated in PE placental tissues and was inversely correlated with SNHG5 expression. Furthermore, miR-26a-5p was predicted to target the 3' untranslated region of N-cadherin, which was confirmed by luciferase reporter assay, and miR-26a-5p overexpression suppressed N-cadherin expression in HTR-8/SVneo cells. N-cadherin mRNA expression was downregulated in PE placental tissues and was positively correlated with SNHG5 expression. Both overexpression of miR-26a-5p and knockdown of N-cadherin suppressed HTR-8/SVneo cell invasion and migration, and also attenuated the effects of SNHG5 on the cellular functions of HTR-8/SVneo cells. In conclusion, our study suggested that SNHG5 promotes trophoblast cell proliferation, invasion, and migration at least partly via regulating the miR-26a-5p/N-cadherin axis.

The inhibition of invasion of human melanoma cells through N-cadherin knock-down

N-cadherin seems to promote cell migration and invasion in many types of cancers. The object of this study is recognition of the possible role of N-cadherin and selected downstream protein kinases: PI3K, ERK1/2, and mTOR in cell invasion in malignant melanoma. Melanoma cells were transfected with the small interfering RNA (siRNA) that targets human N–cadherin gene (CDH2). Inhibitors LY294002 (PI3K), U0126 (ERK1/2), and everolimus (mTOR) were used to inhibit selected kinases of signalling pathways. In vitro cell invasion was studied using Matrigel and an analysis of matrix metalloproteinases MMP-2 and MMP-9 activity by gelatinase zymogram assay. Treatment of melanoma cell with either siRNA against N-cadherin or protein kinase inhibitors led to significantly decreased MMPs expression and activity, as well as diminished invasion. Both the current and the former results suggest that activation of PI3/AKT, mTOR, and ERK kinase, following N-cadherin expression, contributes not only to increased proliferation but also invasive potential of melanoma cells. The results also indicate that N-cadherin, as well as the studied kinases, should be considered as a potential target in melanoma therapy.

Involvement of Nectin-Afadin in the Adherens Junctions of the Corneal Endothelium.

The cell-cell adhesion molecules, cadherins and nectins, are involved in the formation of adherens junctions. However, involvement of nectins in the corneal endothelium has not yet been established. This study investigated the involvement of nectins in adherens junctions of the corneal endothelium. Nectin and cadherin expression in the corneal endothelium was evaluated by real-time polymerase chain reaction. Colocalization and direct binding of nectin-1 and N-cadherin to anchoring proteins (afadin and β-catenin, respectively) were determined by immunostaining and immunoprecipitation. The effect of afadin and N-cadherin knockdown on apical junctions was evaluated by immunostaining. Real-time polymerase chain reaction confirmed nectin-1, nectin-2, nectin-3, nectin-4, and afadin expression in the corneal endothelium. Immunofluorescence staining showed colocalization of nectin and afadin at the basal side of the tight junction (where adherens junctions typically locate) and immunoprecipitation confirmed direct binding of nectin to afadin. N-cadherin, P-cadherin, VE-cadherin, and OB-cadherin messenger RNAs were expressed in the corneal endothelium. N-cadherin and β-catenin colocalized at the cell-cell border, where they directly bound and formed a cell-cell adhesion complex. N-cadherin knockdown disrupted the normal expression pattern of zonula occludens protein-1 and afadin, but afadin knockdown had no effect on the expression pattern of zonula occludens protein-1 and N-cadherin. We believe this to be the first report of conservation of the nectin-afadin system in the corneal endothelium and its involvement in the formation of adherens junctions. N-cadherin, as a member of the cadherin family, is also essential for the formation and maintenance of cell-cell adhesion mediated by nectins and tight junctions in the corneal endothelium.

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

N-cadherin knockdown leads to disruption of trophoblastic and endothelial cell interaction in a 3D cell culture model – New insights in trophoblast invasion failure

ABSTRACTIntroduction: Trophoblast homing to maternal spiral arteries is mandatory for successful placentation. Cell-cell adhesion molecules regulate this process and adhesion molecule expression is altered in impaired placentation. We hypothesize that, similar to immune cell recruitment, trophoblast cell adherence and rolling are primarily mediated by adhesion molecules like, cadherins, immunoglobulins, selectins and their partnering ligands. Here, the interdependence of adhesion molecule expression in trophoblastic cell lines of diverse origin was investigated in relation to their interaction with endothelial cell networks on Matrigel® co-cultures and the effect of specific adhesion molecule knockdown analyzed. Methods: Trophoblastic cells were labeled in red and co-cultured with green HUVEC networks on Matrigel®. Association was quantified after collection of fluorescence microscopy pictures using Wimasis® internet platform and software. Expression of adhesion molecules was analyzed by PCR and Western blot, immuno-fluorescence and flow cytometry. The impact of adhesion molecules on trophoblast-endothelial-cell interaction was investigated using siRNA technique. Results: N-cadherin and CD162 were specifically expressed in the trophoblast cell line HTR-8/SVneo, which closely adhere to and actively migrate toward HUVEC networks on Matrigel®. Suppression of N-cadherin led to a significant alteration in trophoblast-endothelial cell interaction. Expression of VE-cadherin in closely interacting trophoblast cells was not confirmed in vitro. Discussion: We identified N-cadherin to mediate specific interaction between HUVEC and the migrating trophoblast cells HTR-8/SVneo in a Matrigel® co-culture model. VE-cadherin contribution could not be confirmed in vitro. Our results support the hypothesis that impaired N-cadherin but not VE-cadherin expression is involved in trophoblast recruitment to the maternal endothelium.

Abstract 308: CTRP9 Regulates the Fate of Implanted Mesenchymal Stem Cells and Mobilizes Their Protective Effects Against Ischemic Heart Injury via Multiple Novel Signaling Pathways

Cell therapy remains the most promising approach against ischemic heart failure. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair post-MI. CTRP9 is a novel pro-survival cardiokine with significantly downregulated expression after MI. Here, we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting stem cell survival and cardioprotection. Mice were subjected to MI and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P&lt;0.05 vs. CTRP9 alone), suggesting a synergistic effect. CTRP9 significantly increased ADSCs survival and migration after implantation. Conversely, the number of engrafted ADSCs was significantly reduced in the CTRP9KO heart. CTRP9 promoted ADSCs proliferation and migration in vitro, and protected ADSCs against hydrogen peroxide-induced cellular death. Discovery-drive approaches followed by cause-effect analysis identified that CTRP9 enhances ADSCs proliferation/migration by ERK1/2-MMP-9 signaling. CTRP9 promotes anti-apoptotic/cell survival via ERK-Nrf2/anti-oxidative protein expression. Mass spectrometry, immunocytochemistry, and immunoprecipitation identified N-cadherin as the novel CTRP9 binding partner on ADSC. N-cadherin knockdown completely abolished the above noted CTRP9 biological effects. Finally, CTRP9 promotes Sod-3 expression and secretion from ADSCs, protecting cardiomyocytes against oxidative stress-induced cell death. We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms (N-cadherin-ERK/MMP-9 and N-cadherin-ERK/Nrf2-SOD). These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.

Cell segregation and border sharpening by Eph receptor-ephrin-mediated heterotypic repulsion.

Eph receptor and ephrin signalling has a major role in cell segregation and border formation, and may act through regulation of cell adhesion, repulsion or tension. To elucidate roles of cell repulsion and adhesion, we combined experiments in cell culture assays with quantitations of cell behaviour which are used in computer simulations. Cells expressing EphB2, or kinase-inactive EphB2 (kiEphB2), segregate and form a sharp border with ephrinB1-expressing cells, and this is disrupted by knockdown of N-cadherin. Measurements of contact inhibition of locomotion reveal that EphB2-, kiEphB2- and ephrinB1-expressing cells have strong heterotypic and weak homotypic repulsion. EphB2 cells have a transient increase in migration after heterotypic activation, which underlies a shift in the EphB2–ephrinB1 border but is not required for segregation or border sharpening. Simulations with the measured values of cell behaviour reveal that heterotypic repulsion can account for cell segregation and border sharpening, and is more efficient than decreased heterotypic adhesion. By suppressing homotypic repulsion, N-cadherin creates a sufficient difference between heterotypic and homotypic repulsion, and enables homotypic cohesion, both of which are required to sharpen borders.