CFTR Involvement in Cell Migration and Epithelial Restitution

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00120.2012 ◽

2012 ◽

Vol 303 (3) ◽

pp. C308-C317 ◽

Cited By ~ 42

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00371.2002 ◽

2003 ◽

Vol 284 (4) ◽

pp. C848-C859 ◽

Cited By ~ 41

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.

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TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00441.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. G782-G792 ◽

Cited By ~ 78

Author(s):

Jaladanki N. Rao ◽

Oleksandr Platoshyn ◽

Vera A. Golovina ◽

Lan Liu ◽

Tongtong Zou ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cells ◽

Transient Receptor Potential ◽

Intestinal Epithelial Cells ◽

Critical Role ◽

Receptor Potential ◽

Mucosal Injury ◽

Intestinal Epithelial ◽

Epithelial Restitution ◽

Transient Receptor

An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) results from Ca2+ release from intracellular stores and extracellular Ca2+ influx through Ca2+-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca2+ entry (CCE) induced by Ca2+ store depletion represents a major Ca2+ influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca2+-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca2+ currents and CCE generated by Ca2+ influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca2+ channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca2+]cyt.

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Respiratory Syncytial Virus Attenuates Epithelial Restitution by Inhibiting Actin Cytoskeleton-Dependent Cell Migration

10.1164/ajrccm-conference.2020.201.1_meetingabstracts.a6330 ◽

2020 ◽

Author(s):

C.C. Smallcombe ◽

G. Piedimonte ◽

F. Rezaee

Keyword(s):

Cell Migration ◽

Respiratory Syncytial Virus ◽

Actin Cytoskeleton ◽

Epithelial Restitution ◽

Dependent Cell ◽

Syncytial Virus

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Cycleoxygeases-2 (COX-2) accelerates gastric epithelial restitution via increasing cell preliferation and enhancement of cell migration

Gastroenterology ◽

10.1016/s0016-5085(08)82494-8 ◽

2001 ◽

Vol 120 (5) ◽

pp. A502

Author(s):

Miyoko Hirose ◽

Osamu Kobayashi ◽

Hanako Misawa ◽

Hiroto Miwa ◽

Yoshiyuki Takei ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Restitution ◽

Cox 2

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c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription

AJP Cell Physiology ◽

10.1152/ajpcell.00330.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. C367-C375 ◽

Cited By ~ 5

Author(s):

Peng-Yuan Wang ◽

Shelley R. Wang ◽

Lan Xiao ◽

Jie Chen ◽

Jian-Ying Wang ◽

...

Keyword(s):

Cell Migration ◽

Phospholipase C ◽

Proximal Region ◽

Intestinal Epithelial ◽

Cellular Functions ◽

Epithelial Restitution ◽

Enhancer Binding Protein ◽

Epithelial Cell Migration ◽

Response To Stress ◽

Cell Migration And Proliferation

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

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Nuclear factor-κB activation promotes restitution of wounded intestinal epithelial monolayers

AJP Cell Physiology ◽

10.1152/ajpcell.00167.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1028-C1035 ◽

Cited By ~ 71

Author(s):

Laurence J. Egan ◽

Ana de Lecea ◽

Evan D. Lehrman ◽

Gennett M. Myhre ◽

Lars Eckmann ◽

...

Keyword(s):

Cell Migration ◽

Intestinal Inflammation ◽

Healing Process ◽

Growth Factor Receptor ◽

Nuclear Factor ◽

Intestinal Epithelial ◽

Wound Edge ◽

Epithelial Restitution

Epithelial restitution, the movement of wound-edge cells into an area of epithelial cell denudation, is an important early step in the ulcer healing process. Growth factors regulate epithelial restitution, yet little is known about the transcriptional pathways that mediate their effects on cell migration. The transcription factor nuclear factor (NF)-κB is a master regulator of the host inflammatory response that is activated in the epithelium in intestinal inflammation, which often accompanies epithelial injury. We hypothesized that NF-κB may be an important transcriptional regulator of epithelial restitution. In an in vitro model of scrape-wounded monolayers of nontransformed rat intestinal epithelial (RIE-1) cells, NF-κB was activated in epithelial cells at the wound edge. Blocking of NF-κB activation by either pharmacological or genetic approaches inhibited intestinal epithelial restitution. Moreover, scrape wounding activated the epidermal growth factor receptor (EGFR) in cells at the wound edge, and, importantly, inhibiting EGFR tyrosine kinase activity decreased scrape wound-induced NF-κB activation and cell migration. These results indicate a novel role of NF-κB activation in a signaling pathway important for restitution and healing of intestinal epithelia. To the extent NF-κB may have parallel functions in vivo, they also suggest a need for caution in the proposed use of NF-κB inhibitors for the treatment of conditions associated with inflammation and injury of intestinal and other mucosal surfaces.

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