scholarly journals The P2Y2 receptor mediates the epithelial injury response and cell migration

2010 ◽  
Vol 299 (2) ◽  
pp. C411-C421 ◽  
Author(s):  
Ilene Boucher ◽  
Celeste Rich ◽  
Albert Lee ◽  
Meredith Marcincin ◽  
Vickery Trinkaus-Randall
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Wound Repair ◽  
Purinergic Receptor ◽  
Cell Damage ◽  
Wound Response ◽  
Injury Response ◽  
Corneal Injury ◽  
Repair Mechanisms ◽  
Interfering Rna

Injury to epithelial cells results in the release of ATP and stimulation of purinergic receptors and is thought to alter cell migration and wound repair. Medium from the injured cells triggers Ca2+ mobilization and phosphorylation of ERK, both of which are inhibited if the medium is pretreated with apyrase. To understand the wound repair mechanism that occurs with injury, our goal was to determine which purinergic receptor(s) was the critical player in the wound response. We hypothesize that the P2Y2 receptor is the key player in the response of corneal epithelial cells to cell damage and subsequent repair events. Cells transfected with short interfering RNA to either P2Y2 or P2Y4 were stimulated either by injury or addition of UTP and imaged using fluo 3-AM to monitor changes in fluorescence. When cells with downregulated P2Y2 receptors were injured or stimulated with UTP, the intensity of the Ca2+ release was reduced significantly. However, when cells with downregulated P2Y4 receptors were stimulated, only the UTP-induced Ca2+ response was reduced significantly. In addition, downregulation of the P2Y2 receptor inhibited wound closure compared with unstimulated cells or cells transfected with nontargeting sequence. This downregulation resulted also in an attenuation in phosphorylation of Src and ERK. Together, these data indicate that the P2Y2 receptor plays a major biological role in the corneal injury response and repair mechanisms.

scholarly journals cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells

2014 ◽  
Vol 307 (8) ◽  
pp. L643-L651 ◽  
Author(s):  
Todd A. Wyatt ◽  
Jill A. Poole ◽  
Tara M. Nordgren ◽  
Jane M. DeVasure ◽  
Art J. Heires ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Wound Repair ◽  
Bronchial Epithelial Cells ◽  
Dependent Protein Kinase ◽  
Bronchial Epithelial ◽  
Cyclic Monophosphate ◽  
Tnf Α ◽  
Dependent Protein

Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) before exposure to HDE ( P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP ( P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'- O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release ( P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices ( P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8.

scholarly journals Staphylococcus aureus β-toxin exerts anti-angiogenic effects by inhibiting re-endothelialization and neovessel formation

2021 ◽  
Author(s):  
Phuong M Tran ◽  
Sharon S Tang ◽  
Wilmara Salgado-Pabón
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Wound Repair ◽  
Cell Damage ◽  
Host Responses ◽  
Angiogenic Proteins ◽  
Invasive Infections ◽  
Repair Mechanisms ◽  
And Migration

Staphylococcus aureus is the causative agent of numerous severe human infections associated with significant morbidity and mortality worldwide. S. aureus often targets the vascular endothelium to interfere with proper host responses during invasive infections. In this study, we provide evidence that S. aureus β-toxin inhibits wound repair mechanisms in human endothelial cells by preventing cell proliferation and migration. These findings were confirmed in a rabbit aortic explant model where β-toxin impedes sprout formation. Decreased cell proliferation was accompanied by decreased production of the angiogenic proteins endothelin-1, IGFBP-3, thrombospondin-1, TIMP-1, and TIMP-4. Meanwhile, inhibited wound repair was marked by increased HGF secretion from endothelial cells, likely a marker of endothelial cell damage. Together, these findings establish a mechanistic role for β-toxin where it inhibits proper tissue repair processes that likely promote S. aureus infective niche.

scholarly journals Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased β1-integrin signaling and delayed wound repair

2014 ◽  
Vol 306 (9) ◽  
pp. C819-C830 ◽  
Author(s):  
Yutaka Itokazu ◽  
Richard E. Pagano ◽  
Andreas S. Schroeder ◽  
Scott M. O'Grady ◽  
Andrew H. Limper ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Migration ◽  
Epithelial Cells ◽  
Wound Repair ◽  
Airway Epithelial Cells ◽  
Integrin Signaling ◽  
Gm1 Ganglioside ◽  
Airway Epithelial ◽  
Hairpin Rna ◽  
Short Hairpin

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ∼60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated β1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated β1-integrin, pFAK, and pCAS to near control levels and partially restored (∼40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses β1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.

scholarly journals Hypoxia-induced changes in Ca2+ mobilization and protein phosphorylation implicated in impaired wound healing

2014 ◽  
Vol 306 (10) ◽  
pp. C972-C985 ◽  
Author(s):  
Albert Lee ◽  
Kelsey Derricks ◽  
Martin Minns ◽  
Sophina Ji ◽  
Cheryl Chi ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cells ◽  
Wound Repair ◽  
Purinergic Receptor ◽  
Sorting Algorithm ◽  
Impaired Wound Healing ◽  
Corneal Epithelial ◽  
Extracellular Milieu ◽  
Wound Model ◽  
Induced Changes

The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca2+ wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca2+ signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca2+ dynamic patterns were altered in response to decreased O2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca2+ mobilization and localization of N-methyl-d-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca2+-mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair.

scholarly journals Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells

2009 ◽  
Vol 20 (22) ◽  
pp. 4816-4825 ◽  
Author(s):  
Stefan Koch ◽  
Christopher T. Capaldo ◽  
Stanislav Samarin ◽  
Porfirio Nava ◽  
Irmgard Neumaier ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Leading Edge ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Interfering Rna

Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.

scholarly journals Identification of a meningococcal adhesin that inhibits epithelial cell migration

2021 ◽  
Author(s):  
◽  
Gabrielle Greig
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Host Cell ◽  
Wound Repair ◽  
Invasive Disease ◽  
Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Household Contacts ◽  
Cell Association

<p>Neisseria meningitidis virulence is polygenic, therefore comparing many genomes may not yield strictly disease-associated virulence factors. An alternative approach is comparing closely related isolates, such as those from household contacts. Disease isolates have been shown to inhibit epithelial cell wound repair, while many carriage isolates do not. In this study, bacteria collected from disease patients and healthy household contacts were compared to identify the meningococcal factor responsible for wound repair inhibition and investigate how it contributes to invasive disease. Host cell wound repair inhibition was compared between disease-associated meningococcal isolate, NZ97/052, and isolates NZCM111 and NZCM112, from asymptomatic household contacts. Migrating bronchial airway cells were infected with meningococcal isolates and wound closure was evaluated by microscopy. NZ97/052 and NZCM111 both inhibited wound repair, whereas NZCM112 did not. To investigate if this was due to bacterial consumption of an important nutrient, infected cells were supplemented with nutrients known to be important for meningococcal growth and cell migration. Iron supplementation resulted in carriage associated isolates gaining the ability to inhibit wound repair. Genome and transcriptome comparisons were completed between NZ97/052 and NZCM112, which differ in wound repair inhibition. This analysis identified a frameshift mutation in NZCM112 in the haptoglobin-haemoglobin utilization gene, hpuA, which caused complete loss of expression. The hpuA gene was deleted by allelic replacement from NZ97/052, nullifying its ability to inhibit host cell migration. Furthermore, bacterial association and fluorescence microscopy assays suggested that HpuA contributes to meningococcal attachment to bronchial epithelial cells, as the hpuA mutant had significantly lower cell association. Heterologous expression of HpuA in E. coli resulted in higher levels of cell association, indicating that HpuA is sufficient to mediate bacterial adhesion to human bronchial epithelial cells. This work reveals novel roles for HpuA as a meningococcal adhesin and a bacterial factor that inhibits host cell migration.</p>

scholarly journals Identification of a meningococcal adhesin that inhibits epithelial cell migration

2021 ◽  
Author(s):  
◽  
Gabrielle Greig
Keyword(s):  
Cell Migration ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Host Cell ◽  
Wound Repair ◽  
Invasive Disease ◽  
Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Household Contacts ◽  
Cell Association

<p>Neisseria meningitidis virulence is polygenic, therefore comparing many genomes may not yield strictly disease-associated virulence factors. An alternative approach is comparing closely related isolates, such as those from household contacts. Disease isolates have been shown to inhibit epithelial cell wound repair, while many carriage isolates do not. In this study, bacteria collected from disease patients and healthy household contacts were compared to identify the meningococcal factor responsible for wound repair inhibition and investigate how it contributes to invasive disease. Host cell wound repair inhibition was compared between disease-associated meningococcal isolate, NZ97/052, and isolates NZCM111 and NZCM112, from asymptomatic household contacts. Migrating bronchial airway cells were infected with meningococcal isolates and wound closure was evaluated by microscopy. NZ97/052 and NZCM111 both inhibited wound repair, whereas NZCM112 did not. To investigate if this was due to bacterial consumption of an important nutrient, infected cells were supplemented with nutrients known to be important for meningococcal growth and cell migration. Iron supplementation resulted in carriage associated isolates gaining the ability to inhibit wound repair. Genome and transcriptome comparisons were completed between NZ97/052 and NZCM112, which differ in wound repair inhibition. This analysis identified a frameshift mutation in NZCM112 in the haptoglobin-haemoglobin utilization gene, hpuA, which caused complete loss of expression. The hpuA gene was deleted by allelic replacement from NZ97/052, nullifying its ability to inhibit host cell migration. Furthermore, bacterial association and fluorescence microscopy assays suggested that HpuA contributes to meningococcal attachment to bronchial epithelial cells, as the hpuA mutant had significantly lower cell association. Heterologous expression of HpuA in E. coli resulted in higher levels of cell association, indicating that HpuA is sufficient to mediate bacterial adhesion to human bronchial epithelial cells. This work reveals novel roles for HpuA as a meningococcal adhesin and a bacterial factor that inhibits host cell migration.</p>

Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells

2001 ◽  
Vol 114 (23) ◽  
pp. 4185-4195 ◽  
Author(s):  
Veronica E. Klepeis ◽  
Ann Cornell-Bell ◽  
Vickery Trinkaus-Randall
Keyword(s):  
Wave Propagation ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Gap Junctions ◽  
Laser Scanning ◽  
Wound Repair ◽  
Cell Damage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
External Stimuli

This paper characterizes the early responses of epithelial cells to injury. Ca2+ is an important early messenger that transiently increases in the cytoplasm of cells in response to external stimuli. Its elevation leads to the regulation of signaling pathways responsible for the downstream events important for wound repair, such as cell migration and proliferation. Live cell imaging in combination with confocal laser scanning microscopy of fluo-3 AM loaded cells was performed. We found that mechanical injury in a confluent region of cells creates an elevation in Ca2+ that is immediately initiated at the wound edge and travels as a wave to neighboring cells, with [Ca2+]i returning to background levels within two minutes. Addition of epidermal growth factor (EGF), but not platelet-derived growth factor-BB, resulted in increased [Ca2+]i, and EGF specifically enhanced the amplitude and duration of the injury-induced Ca2+ wave. Propagation of the Ca2+ wave was dependent on intracellular Ca2+ stores, as was demonstrated using both thapsigargin and Ca2+ chelators (EGTA and BAPTA/AM). Injury-induced Ca2+ waves were not mediated via gap junctions, as the gap-junction inhibitors 1-heptanol and 18α-glycyrrhetinic acid did not alter wave propagation, nor did the cells recover in photobleaching experiments. Additional studies also demonstrated that the wave could propagate across an acellular region. The propagation of the injury-induced Ca2+ wave occurs via diffusion of an extracellular mediator, most probably via a nucleotide such as ATP or UTP, that is released upon cell damage. Movies available on-line

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