scholarly journals Activation of the Sphingosine 1 Phosphate–Rho Pathway in Pterygium and in Ultraviolet-Irradiated Normal Conjunctiva

2019 ◽  
Vol 20 (19) ◽  
pp. 4670 ◽  
Author(s):  
Nozomi Igarashi ◽  
Megumi Honjo ◽  
Takashi Fujishiro ◽  
Tetsuya Toyono ◽  
Takashi Ono ◽  
...  
Keyword(s):  
Fibroblast Cell ◽  
Cellular Activity ◽  
Myosin Phosphatase ◽  
Potential Therapeutic Target ◽  
Interacting Protein ◽  
Cytoskeletal Organization ◽  
Rhoa Activity ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor

Sphingosine 1 phosphate (S1P) is a bioactive lipid that regulates cellular activity, including proliferation, cytoskeletal organization, migration, and fibrosis. In this study, the potential relevance of S1P–Rho signaling in pterygium formation and the effects of ultraviolet (UV) irradiation on activation of the S1P/S1P receptor axis and fibrotic responses were investigated in vitro. Expressions of the S1P2, S1P4, and S1P5 receptors were significantly higher in pterygium tissue than in normal conjunctiva, and the concentration of S1P was significantly elevated in the lysate of normal conjunctival fibroblast cell (NCFC) irradiated with UV (UV-NCFCs). RhoA activity was significantly upregulated in pterygium fibroblast cells (PFCs) and UV-NCFCs, and myosin phosphatase–Rho interacting protein (MRIP) was upregulated, and myosin phosphatase target subunit 1 (MYPT1) was downregulated in PFCs. Fibrogenic changes were significantly upregulated in both PFCs and UV-NCFCs compared to NCFCs. We found that the activation of the S1P receptor–Rho cascade was observed in pterygium tissue. Additionally, in vitro examination showed S1P–rho activation and fibrogenic changes in PFCs and UV-NCFCs. S1P elevation and the resulting upregulation of the downstream Rho signaling pathway may be important in pterygium formation; this pathway offers a potential therapeutic target for suppressing pterygium generation.

scholarly journals CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function

2021 ◽  
Vol 14 (679) ◽  
pp. eabc0425
Author(s):  
De Yu Mao ◽  
Matthew L. Kleinjan ◽  
Irina Jilishitz ◽  
Bhairavi Swaminathan ◽  
Hideru Obinata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Development ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Rhoa Activity ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor ◽  
Guanosine Triphosphatase ◽  
And Function

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein–coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

2009 ◽  
Vol 297 (2) ◽  
pp. G361-G370 ◽  
Author(s):  
Eikichi Ihara ◽  
Lori Moffat ◽  
Meredith A. Borman ◽  
Jennifer E. Amon ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Conformational Changes ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

Sphingosine‐1‐phosphate (S1P) induces potent anti‐inflammatory effects in vitro and in vivo by S1P receptor 4‐mediated suppression of 5‐lipoxygenase activity

2018 ◽  
Vol 33 (2) ◽  
pp. 1711-1726 ◽  
Author(s):  
Jasmin Fettel ◽  
Benjamin Kühn ◽  
Nathalie A. Guillen ◽  
Duran Sürün ◽  
Marcus Peters ◽  
...  
Keyword(s):  
Lipoxygenase Activity ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor ◽  
Anti Inflammatory

Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions

2007 ◽  
Vol 292 (6) ◽  
pp. H2698-H2711 ◽  
Author(s):  
Lee K. Landeen ◽  
Nakon Aroonsakool ◽  
Jason H. Haga ◽  
Betty S. Hu ◽  
Wayne R. Giles
Keyword(s):  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Cardiac Fibroblasts ◽  
Culture Conditions ◽  
Receptor Expression ◽  
Receptor Subtypes ◽  
Essential Components ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor

The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P1–5) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P1–3; however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H2O2 had minimal effects on CFb S1P1–3 expression, whereas transforming growth factor-β1, TNF-α, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P2, whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P3 levels. S1P2 and S1P3 receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P3-null mice, we demonstrate that S1P3 is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs.

scholarly journals Signal Transduction of Sphingosine-1-Phosphate G Protein—Coupled Receptors

2006 ◽  
Vol 6 ◽  
pp. 946-966 ◽  
Author(s):  
Nicholas Young ◽  
James R. Van Brocklyn
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Cell Types ◽  
G Protein Coupled Receptors ◽  
Lymphocyte Trafficking ◽  
Cytoskeletal Organization ◽  
Cellular Effects ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor ◽  
G Protein Coupled

Sphingosine-1-phosphate (S1P) is a bioactive lipid capable of eliciting dramatic effects in a variety of cell types. Signaling by this molecule is by a family of five G protein—coupled receptors named S1P1–5that signal through a variety of pathways to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. These receptors are expressed in a wide variety of tissues and cell types, and their cellular effects contribute to important biological and pathological functions of S1P in many processes, including angiogenesis, vascular development, lymphocyte trafficking, and cancer. This review will focus on the current progress in the field of S1P receptor signaling and biology.

scholarly journals Sphingolipid signaling and treatment during remodeling of the uninfarcted ventricular wall after myocardial infarction

2009 ◽  
Vol 296 (4) ◽  
pp. H1193-H1199 ◽  
Author(s):  
Che-Chung Yeh ◽  
Hongzhe Li ◽  
Deepak Malhotra ◽  
Mei-Chuan Huang ◽  
Bo-Qing Zhu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiac Myocyte ◽  
Ex Vivo ◽  
Cell Types ◽  
Coronary Artery Ligation ◽  
Artery Ligation ◽  
Echocardiographic Measurement ◽  
Sphingosine 1 Phosphate ◽  
S1p Receptor

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P1 receptor agonism was tested via the administration of the S1P1-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P1 receptor mRNA also fell below baseline. The S1P1-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.

scholarly journals ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

2006 ◽  
Vol 282 (7) ◽  
pp. 4884-4893 ◽  
Author(s):  
Laura Hagerty ◽  
Douglas H. Weitzel ◽  
Jenica Chambers ◽  
Christopher N. Fortner ◽  
Matthew H. Brush ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Apoptotic Cell ◽  
Intracellular Localization ◽  
Stress Fiber ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca2+-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

scholarly journals Regulation of Rnd3 localization and function by protein kinase Cα-mediated phosphorylation

2009 ◽  
Vol 424 (1) ◽  
pp. 153-161 ◽  
Author(s):  
James P. Madigan ◽  
Brian O. Bodemann ◽  
Donita C. Brady ◽  
Brian J. Dewar ◽  
Patricia J. Keller ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Kinase Assay ◽  
Mobility Shift ◽  
Cytoskeletal Organization ◽  
Rhoa Activity ◽  
Rho Family

The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCα-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCα directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho–ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization.

scholarly journals MRIP Regulates the Myosin IIA Activity and DDR1 Function to Enable Collagen Tractional Remodeling

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1672
Author(s):  
Nuno M. Coelho ◽  
Andrew Wang ◽  
Petar Petrovic ◽  
Yongqiang Wang ◽  
Wilson Lee ◽  
...  
Keyword(s):  
Drug Target ◽  
Myosin Light Chain ◽  
Wound Closure ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Collagen Remodeling ◽  
Myosin Iia ◽  
Novel Drug ◽  
Muscle Myosin

DDR1 is a collagen adhesion-mechanoreceptor expressed in fibrotic lesions. DDR1 mediates non-muscle myosin IIA (NMIIA)-dependent collagen remodeling. We discovered that the myosin phosphatase Rho-interacting protein (MRIP), is enriched in DDR1-NMIIA adhesions on collagen. MRIP regulates RhoA- and myosin phosphatase-dependent myosin activity. We hypothesized that MRIP regulates DDR1-NMIIA interactions to enable cell migration and collagen tractional remodeling. After deletion of MRIP in β1-integrin null cells expressing DDR1, in vitro wound closure, collagen realignment, and contraction were reduced. Cells expressing DDR1 and MRIP formed larger and more abundant DDR1 clusters on collagen than cells cultured on fibronectin or cells expressing DDR1 but null for MRIP or cells expressing a non-activating DDR1 mutant. Deletion of MRIP reduced DDR1 autophosphorylation and blocked myosin light chain-dependent contraction. Deletion of MRIP did not disrupt the association of DDR1 with NMIIA. We conclude that MRIP regulates NMIIA-dependent DDR1 cluster growth and activation. Accordingly, MRIP may provide a novel drug target for dysfunctional DDR1-related collagen tractional remodeling in fibrosis.

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