Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

2003 ◽  
Vol 284 (4) ◽  
pp. C848-C859 ◽  
Author(s):  
Jaladanki N. Rao ◽  
Xin Guo ◽  
Lan Liu ◽  
Tongtong Zou ◽  
Karnam S. Murthy ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Epithelial Restitution ◽  
Mlc Phosphorylation

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca2+ concentration ([Ca2+]cyt) through controlling voltage-gated K+ channel expression and membrane potential ( E m) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca2+]cyt activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca2+]cyt, by either polyamine depletion or exposure to the Ca2+-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after wounding. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca2+]cytcan interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.

scholarly journals RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding

2015 ◽  
Vol 309 (9) ◽  
pp. G759-G767 ◽  
Author(s):  
Hee Kyoung Chung ◽  
Navneeta Rathor ◽  
Shelley R. Wang ◽  
Jian-Ying Wang ◽  
Jaladanki N. Rao
Keyword(s):  
Cell Migration ◽  
Transient Receptor Potential ◽  
Ectopic Expression ◽  
Receptor Potential ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
Epithelial Restitution ◽  
Epithelial Cell Migration ◽  
Transient Receptor ◽  
Wounded Area

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca2+ influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca2+ entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca2+ influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca2+ influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca2+ influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca2+ signaling.

scholarly journals Loss of PI3-kinase activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration and intestinal homeostasis

2020 ◽  
Author(s):  
Prasun Guha ◽  
Luke Reilly ◽  
Evan R. Semenza ◽  
Efrat Abramson ◽  
Subrata Mishra ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Membrane Localization ◽  
Intestinal Epithelial ◽  
Inositol Polyphosphate ◽  
Akt Activation ◽  
Inositol Polyphosphate Multikinase ◽  
Mlc Phosphorylation

AbstractInositol polyphosphate multikinase (IPMK) is a rate-limiting enzyme in the inositol phosphate (IP) pathway which converts IP3 to IP4 and IP5. In mammalian cells, IPMK can also act as a phosphoinositol-3-kinase (PI3-kinase). We previously found that IPMK is a critical PI3-kinase activator of AKT. Here, we show that IPMK mediates AKT activation by promoting membrane localization and activation of PDK1. The PI3-kinase activity of IPMK is dispensable for membrane localization of AKT, which is entirely controlled by classical PI3-kinase (p110α,ß, γ, δ). By contrast, we found that PDK1 membrane localization was largely independent of classical PI3-kinase. Membrane localization of PDK1 stimulates cell migration by dissociating ROCK1 from inhibitory binding to RhoE and promoting ROCK1-mediated myosin light chain (MLC) phosphorylation. Deletion of IPMK impairs cell migration associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent MLC phosphorylation. To investigate the physiological relevance of IPMK-mediated AKT activation, we generated mice selectively lacking IPMK in epithelial cells of the intestine, where IPMK is highly expressed. Deletion of IPMK in intestinal epithelial cells markedly reduced AKT phosphorylation and diminished numbers of Paneth cells – a crypt-resident epithelial cell type that generates the physiological niche for intestinal stem cells. Ablation of IPMK impaired intestinal epithelial cell regeneration basally and after; chemotherapy-induced damage, suggesting a broad role for IPMK in the activation of AKT and intestinal tissue regeneration. In summary, the PI3-kinase activity of IPMK promotes membrane localization of PDK1, a critical kinase whereby AKT maintains intestinal homeostasis.One Sentence SummaryPI3-kinase activity of IPMK is essential for activation of AKT.

scholarly journals Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2

2012 ◽  
Vol 303 (3) ◽  
pp. C308-C317 ◽  
Author(s):  
Jaladanki N. Rao ◽  
Navneeta Rathor ◽  
Ran Zhuang ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intestinal Epithelial Cell ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Intestinal Epithelial ◽  
Canonical Transient Receptor Potential ◽  
Pathological Conditions ◽  
Epithelial Restitution ◽  
Transient Receptor ◽  
Stimulation Of

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca2+ signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca2+ influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca2+ influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca2+ influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca2+ influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca2+ signaling and influencing cell migration after wounding.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

2021 ◽  
Author(s):  
Toshihiro Kobayashi ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Jingya Lyu ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Active Form ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Activation Mechanism ◽  
Dominant Negative Mutant ◽  
Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

2001 ◽  
Vol 114 (20) ◽  
pp. 3749-3757 ◽  
Author(s):  
Patrick Meraldi ◽  
Erich A. Nigg
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Microtubule Network ◽  
Similar Phenotype ◽  
Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

scholarly journals Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

1996 ◽  
Vol 7 (9) ◽  
pp. 1419-1427 ◽  
Author(s):  
B C Tilly ◽  
M J Edixhoven ◽  
L G Tertoolen ◽  
N Morii ◽  
Y Saitoh ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channels ◽  
Kinase Activity ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Cell Swelling ◽  
Phosphatidylinositol 3 Kinase ◽  
C3 Exoenzyme ◽  
Original Cell ◽  
Adp Ribosyltransferase

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

scholarly journals IGF1 suppresses cholesterol accumulation in the liver of growth hormone-deficient mice via the activation of ABCA1

2018 ◽  
Vol 315 (6) ◽  
pp. E1232-E1241 ◽  
Author(s):  
Kensaku Fukunaga ◽  
Hitomi Imachi ◽  
Jingya Lyu ◽  
Tao Dong ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Luciferase Assay ◽  
Dominant Negative Mutant ◽  
Chip Assay ◽  
Cholesterol Accumulation ◽  
Adult Growth ◽  
Abca1 Expression ◽  
Deficient Mice

Recently, several clinical studies have suggested that adult growth hormone (GH) deficiency that also has low concentration of IGF1 is associated with an increased prevalence of fatty liver (FL). ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role on formation of FL. In this study, we determined the effects of IGF1 on ABCA1 expression in GH-deficient mice to clarify its effects on FL. Western blotting, real-time PCR, and a luciferase assay were employed to examine the effect of IGF1. The binding of FoxO1 to the ABCA1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Cholesterol accumulation was analyzed by Oil Red O stain and cholesterol content measurement. We confirmed that IGF1 upregulated the ABCA1 expression. The activity of a reporter construct containing the ABCA1 promoter was induced by IGF1, and this effect was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Constitutively active Akt stimulated the ABCA1 promoter activity, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element abolished the effect of IGF1. A ChIP assay indicated that FoxO1 mediated IGF1 transcriptional activity by directly binding to the ABCA1 promoter region. For in vivo experiments, we used an inhibitor for the GH receptor (Pegvisomant) to reduce the IGF1 level. A high-fat diet induced FL in mice (C57BL/6J) given Pegvisomant treatment. IGF1 treatment stimulated ABCA1 expression to improve cholesterol accumulation in these mice. These results show that the PI3K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF1 stimulation that suppressed FL in GH-deficient mice.

Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway

2004 ◽  
Vol 287 (6) ◽  
pp. L1127-L1133 ◽  
Author(s):  
Jun Tamaoki ◽  
Kazuo Isono ◽  
Kiyoshi Takeyama ◽  
Etsuko Tagaya ◽  
Junko Nakata ◽  
...  
Keyword(s):  
Carbon Black ◽  
Airway Epithelium ◽  
Ultrafine Particles ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Dependent Manner

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione- S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52] Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.

Increased basal phosphorylation of detrusor smooth muscle myosin in alloxan-induced diabetic rabbit is mediated by upregulation of Rho-kinase β and CPI-17

2006 ◽  
Vol 290 (3) ◽  
pp. F650-F656 ◽  
Author(s):  
Shaohua Chang ◽  
Joseph A. Hypolite ◽  
Michael E. DiSanto ◽  
Arun Changolkar ◽  
Alan J. Wein ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Bladder Dysfunction ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Detrusor Smooth Muscle ◽  
Two Dimensional Gel Electrophoresis ◽  
Calcium Sensitization ◽  
Detrusor Contractility ◽  
Phosphorylation Level ◽  
Mlc Phosphorylation

Urinary bladder dysfunction caused by the alteration of detrusor smooth muscle (DSM) is one of the complications of diabetes. It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca2+ via myosin light chain (MLC) phosphorylation. However, recent studies have shown the modulation of MLC phosphorylation without a rise in Ca2+ in smooth muscle and that two key molecules (Rho-kinase and CPI-17) are involved in the regulation of calcium sensitization. This study investigates the effect of diabetes on DSM calcium sensitization. Diabetes was induced by alloxan in New Zealand White rabbits, and age-matched rabbits given 5% sucrose in the drinking water served as control for diuresis. Two-dimensional gel electrophoresis showed that basal MLC phosphorylation level was significantly higher in diabetic animals than normal or diuretic controls, and Rho-kinase-specific inhibitor, Y-27632, decreased MLC phosphorylation level. Adding Y-27632 to bethanechol-precontracted DSM strips can induce muscle relaxation, but it occurred much more slowly in diabetic samples compared with controls. RT-PCR, Western blot analysis, and immunohistochemistry revealed the overexpression of Rho-kinase β and CPI-17 at both mRNA and protein levels in response to diabetes. In conclusion, our results demonstrate that Rho-kinase contributes to DSM MLC phosphorylation and there is a higher basal MLC phosphorylation level in diabetic DSM. Our results also suggest that this high basal MLC phosphorylation may be due to the upregulation of Rho-kinase and CPI-17. Thus Rho-kinase- and CPI-17-mediated Ca2+ sensitization might play a role in diabetes-induced alteration of the detrusor contractility and bladder dysfunction.

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