Dataset Information

Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

ABSTRACT: Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural analysis of Sphk1 KO mouse retinas aged to 15 months or raised with moderate light stress revealed a degenerated outer limiting membrane (OLM). This membrane is formed by adherens junctions between neighboring Müller glia and photoreceptor cells. We also show that Sphk1 KO mice have reduced retinal function in mice raised with moderate light stress. In vitro assays revealed that exogenous S1P modulated cytoskeletal rearrangement and increased N-cadherin production in human Müller glia cells. Aged mice also had morphological degeneration of the RPE, as well as increased lipid storage vacuoles and undigested phagosomes reminiscent of RPE in age-related macular degeneration. These findings show that SPHK1 and S1P play a vital role in the structural maintenance of the mammalian retina and retinal pigmented epithelium by supporting the formation of adherens junctions.

SUBMITTER: Wilkerson JL

PROVIDER: S-EPMC7425817 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

Wilkerson Joseph L JL Stiles Megan A MA Gurley Jami M JM Grambergs Richard C RC Gu Xiaowu X Elliott Michael H MH Proia Richard L RL Mandal Nawajes A NA

Molecular neurobiology 20190417 10

Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural ana ...[more]

PMID: 30997640

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Project description:The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotransduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized adherens junction (AJ) involving the cadherin super-family of Ca(2+)-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein ?-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein ?-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in ?-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that ?-catenin is the core mechanosensor that allows cells to locally sense, transduce and adapt to environmental mechanical constrains.

Project description:During endochondral ossification, the cartilage is surrounded by a layer of cells that constitute the perichondrium. Communication between osteoblasts in the perichondrium via N-cadherin adherens junctions is essential for endochondral bone growth. We observed that adherens junction molecule N-cadherin and its interacting partners p120, ?-catenin and PTEN are expressed by cells present in the perichondrium. To study if N-cadherin mediated adherens junctions play a role in mediating signal transduction events during bone development, we utilized MC3T3E1 preosteoblasts plated at sub confluent (low) and confluent (high) densities to mimic adherens junction formation. When MC3T3E1 cells were plated at high density we observed an increase in phosphorylation of AKTSer473 and its downstream target GSK3Ser9, which coincided with an increase in Osterix, Osteomodulin and Osteoglycin gene expression. Using immunofluorescence, we identified N-cadherin, p120 and ?-catenin localized at the membrane of MC3T3E1 cells. Treatment of confluent MC3T3E1 cells with an N-cadherin junction inhibitor-EGTA and a PI3K inhibitor LY294002 resulted in reduction of phosphorylation levels of AKT and GSK3 and expression of Osterix, Osteomodulin and Osteoglycin. Furthermore, utilizing an N-cadherin blocking antibody resulted in reduced AKT signaling and Osterix gene expression, suggesting that osteoblast junction formation is linked to activation of PI3K signaling, which leads to osteoblast differentiation. To further explore the strength of this linkage, we utilized a conditional knockout approach using Dermo1cre to delete ?-catenin and PTEN, two important proteins known to be essential for adherens junctions and PI3K signaling, respectively. In the absence of ?-catenin, we observed a decrease in adherens junctions and AKT signaling in the perichondrium. PTEN deletion, on the other hand, increased the number of cells expressing N-cadherin in the perichondrium. These observations show that N-cadherin mediated junctions between osteoblasts are needed for osteoblast gene transcription.

Dataset Information

Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

Publications

Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

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