N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3-75 h) after seeding and continued for 24 h. Stretch stimulation in 3 h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24-75 h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell-cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell-cell adhesion, cardiomyocytes cultured for 72 h were replated by trypsin treatment and exposed to stretch 3 h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.     

POPX2 phosphatase regulates cell polarity and centrosome placement

Proper centrosome positioning is critical for many cellular functions, such as cell migration and maintenance of polarity. During wound healing, fibroblasts orient their centrosomes such that they face the wound edge. The centrosome orientation determines the direction of cells’ migration so that they can close the wound effectively. In this study, we investigated the regulation of centrosome polarization and have identified the phosphatase POPX2 as an important regulator of centrosome orientation. We found that POPX2 inhibits centrosome centration, but not rearward nuclear movement, by regulating multiple proteins that function in centrosome positioning. High POPX2 levels result in reduced motility of the kinesin-2 motor, which, in turn, inhibits the transport of N-cadherin to the cell periphery and cell junctions. Loss of N-cadherin localization to the cell membrane affects the localization of focal adhesions and perturbs CDC42-Par6/PKCζ signaling. In addition, overexpression of POPX2 also results in a loss of Par3 localization to the cell periphery and reduced levels of LIC2 (dynein light intermediate chain 2), leading to defects in microtubule tethering and dynamics at cell-cell contacts. Therefore, POPX2 functions as a regulator of signaling pathways to modulate the positioning of centrosome in fibroblast during wound healing.     

Pigment epithelium-derived factor protects retinal pigment epithelium from oxidant-mediated barrier dysfunction

Retinal pigment epithelium (RPE) cells form a monolayer at the blood-retina barrier between the retina and choriocapillaries. The barrier function may be damaged by multiple stresses to the cell, including the repeated exposure to oxidants that are generated by photoreceptor cell turnover. The purpose of our study was to document the protective effect of pigment epithelium-derived factor (PEDF), a tropic factor produced by the RPE, on H(2)O(2)-induced RPE barrier dysfunction. When assayed by a FITC-labeled dextran transepithelial flux, the increased permeability of the RPE barrier (induced by H(2)O(2)) was prevented by PEDF pretreatment. To further explore the mechanism leading to this permeability change, we investigated the distribution of cytoskeleton and junctional proteins. The redistribution of the two junctional proteins occludin, and N-cadherin and actin reorganization in RPE, induced by H(2)O(2), can be prevented by PEDF pretreatment. PEDF can also prevent H(2)O(2)-induced stress kinase p38/27-kDa heat shock protein signaling which is known to mediate actin rearrangement. These findings indicated that PEDF can stabilize actin, maintain normal membrane occludin and N-cadherin structure, and preserve the barrier function of RPE cells against oxidative stress.     

Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation

Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.     

Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell–cell interactions in vivo

The list of transforming growth factor-beta (TGF-β)-related proteins in non-canonical TGF-β signaling is growing. Examples include receptor-Smads directing micro-RNA processing and inhibitory-Smads, e.g. Smad7, directing cell adhesion. Human skin grafts with fluorescently tagged melanoma cells revealed Smad7-expressing cells positioned themselves proximal to the dermal–epidermal junction and failed to form tumors, while control cells readily invaded and formed tumors within the dermis. Smad7 significantly inhibited β-catenin T41/S45 phosphorylation associated with degradation and induced a 4.5-fold increase in full-length N-cadherin. Cell adhesion assays confirmed a strong interaction between Smad7-expressing cells and primary dermal fibroblasts mediated via N-cadherin, while control cells were incapable of such interaction. Immunofluorescent analysis of skin grafts indicated N-cadherin homotypic interaction at the surface of both Smad7 cells and primary dermal fibroblasts, in contrast to control melanoma cells. We propose that Smad7 suppresses β-catenin degradation and promotes interaction with N-cadherin, stabilizing association with neighboring dermal fibroblasts, thus mitigating invasion.     

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca²⁺ ([Ca²⁺]_i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABA_AR and GABA_BR) with the specific agonists muscimol and baclofen, respectively induces [Ca²⁺]_i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca²⁺. Bicuculline did not change the GABA-evoked Ca²⁺ responses in Ca²-containing buffers, but suppressed them significantly in Ca²⁺-free buffers suggesting the two receptors couple to convergent Ca²⁺ mobilization mechanisms with different extracellular Ca²⁺ sensitivity. Prolonged activation of GABA_BR induced wave propagation of the Ca²⁺ signal and persistent oscillations. The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABA_AR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca²⁺ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca²⁺ signaling may be involved in a variety of Ca²⁺-dependent cellular processes during lens growth and epithelial cell differentiation.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

Regulation of cadherin expression in nervous system development

This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. While the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. With the growing understanding of the broader roles of cadherins in cell–cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context.     

HMGA2与胃癌上皮细胞间质转化的相关性及临床意义

探讨HMGA2、E-cadherin及N-cadherin在胃癌组织中差异性表达的临床意义及其与上皮细胞间质转化的关系.应用RT-PCR方法检测20例胃癌组织及对应的正常胃粘膜组织HMGA2 mRNA的表达,应用免疫组织化学法检测71例胃癌组织及癌旁组织中HMGA2、E-cadherin及N-cadherin的表达情况,与临床资料做统计学分析.HMGA2 mRNA在胃癌组织和癌旁组织中的表达存在显著性差异(P<0.01),在胃癌组织中HMGA2、E-cadherin及N-cadherin的阳性表达率分别为64.7%、29.6%和43.7%,HMGA2和E-cadherin的表达成负相关(P<0.05),和N-cadherin的表达成正相关(P<0.05),HMGA2表达和胃癌患者淋巴结转移及临床分期有显著相关性(P<0.05).HMGA2高表达的胃癌组织中有明显的上皮细胞间质转化表型,其表达与胃癌侵润转移有一定的相关性.     

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.