Rac1 promotes intestinal epithelial restitution by increasing Ca2+ influx through interaction with phospholipase C-γ1 after wounding

Intestinal mucosal restitution occurs as a consequence of epithelial cell migration and reseals superficial wounds after injury. This rapid reepithelialization is mediated in part by a phospholipase C-γ1 (PLC-γ1)-induced Ca2+ signaling, but the exact mechanism underlying such signaling and its regulation remains elusive. The small GTP-binding protein Rac1 functions as a pivotal regulator of several signaling networks and plays an important role in regulating cell motility. The current study tests the hypothesis that Rac1 modulates intestinal epithelial cell migration after wounding by altering PLC-γ1-induced Ca2+ signaling. Inhibition of Rac1 activity by treatment with its inhibitor NSC-23766 or Rac1 silencing with small interfering RNA decreased store depletion-induced Ca2+ influx and suppressed cell migration during restitution, whereas ectopic overexpression of Rac1 increased Ca2+ influx and promoted cell migration. Rac1 physically interacted with PLC-γ1 and formed Rac1/PLC-γ1 complex in intestinal epithelial cells. PLC-γ1 silencing in cells overexpressing Rac1 prevented stimulation of store depletion-induced Ca2+ influx and cell migration after wounding. Polyamine depletion inhibited expression of both Rac1 and PLC-γ1, decreased Rac1/PLC-γ1 complex levels, reduced Ca2+ influx, and repressed cell migration. Overexpression of Rac1 alone failed to rescue Ca2+ influx after store depletion and cell migration in polyamine-deficient cells, because it did not alter PLC-γ1 levels. These results indicate that Rac1 promotes intestinal epithelial cell migration after wounding by increasing Ca2+ influx as a result of its interaction with PLC-γ1.

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Polyamines are required for phospholipase C-γ1 expression promoting intestinal epithelial restitution after wounding

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00282.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. G335-G343 ◽

Cited By ~ 29

Author(s):

Jaladanki N. Rao ◽

Lan Liu ◽

Tongtong Zou ◽

Bernard S. Marasa ◽

Dessy Boneva ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Phospholipase C ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial ◽

Inositol 1 ◽

Store Depletion ◽

Epithelial Cell Migration ◽

Interfering Rna ◽

Stimulation Of

Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+]cyt) remains unclear. Phospholipase C (PLC)-γ1 catalyzes the formation of inositol ( 1 , 4 , 5 )-trisphosphate (IP3), which is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-γ1 activity, regulating [Ca2+]cyt and cell migration after wounding. Depletion of cellular polyamines by α-difluoromethylornithine inhibited PLC-γ1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-γ1 mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-γ1 by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-γ1 also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-γ1 by 2,4,6-trimethyl- N-( meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+]cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-γ1 expression in IECs and that polyamine-mediated PLC-γ1 signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.

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Attenuation of Intestinal Epithelial Cell Migration During Cryptosporidium parvum Infection Involves Parasite Cdg7_FLc_1030 RNA-Mediated Induction and Release of Dickkopf-1

The Journal of Infectious Diseases ◽

10.1093/infdis/jiy299 ◽

2018 ◽

Vol 218 (8) ◽

pp. 1336-1347 ◽

Cited By ~ 2

Author(s):

Zhenping Ming ◽

Yang Wang ◽

Ai-Yu Gong ◽

Xin-Tian Zhang ◽

Min Li ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Cryptosporidium Parvum ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial ◽

Epithelial Cell Migration ◽

Dickkopf 1

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UNC‐45A is a novel regulator of the intestinal epithelial cell migration

The FASEB Journal ◽

10.1096/fasebj.2021.35.s1.03484 ◽

2021 ◽

Vol 35 (S1) ◽

Author(s):

Susana Lechuga ◽

Andrei Ivanov

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial ◽

Epithelial Cell Migration

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Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.2.c303 ◽

2000 ◽

Vol 278 (2) ◽

pp. C303-C314 ◽

Cited By ~ 69

Author(s):

Jian-Ying Wang ◽

Jian Wang ◽

Vera A. Golovina ◽

Li Li ◽

Oleksandr Platoshyn ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Driving Force ◽

Intestinal Epithelial Cell ◽

Delayed Rectifier ◽

Intestinal Epithelial ◽

Voltage Gated ◽

Epithelial Cell Migration ◽

Channel Expression ◽

Exogenous Spermidine

Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K+ channels (Kv) controls membrane potential ( E m) that regulates cytoplasmic free Ca2+ concentration ([Ca2+]cyt) by governing the driving force for Ca2+ influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K+ channel gene expression, E m, and [Ca2+]cyt in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, α-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca2+ channels, the depolarized E m in DFMO-treated cells decreased [Ca2+]cyt as a result of reduced driving force for Ca2+ influx through capacitative Ca2+ entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E m, and [Ca2+]cyt but also restored cell migration to normal. Removal of extracellular Ca2+ or blockade of Kv channels (by 4-aminopyridine, 1–5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca2+]cyt by increasing the driving force (the electrochemical gradient) for Ca2+ influx and thus stimulates cell migration.

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Polyamines and cytoskeletal proteins in intestinal epithelial cell migration

Journal of Gastroenterology and Hepatology ◽

10.1111/j.1440-1746.1998.tb01888.x ◽

1998 ◽

Vol 13 (S3) ◽

pp. S257-S261

Author(s):

JIAN-YING WANG

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Intestinal Epithelial Cell ◽

Cytoskeletal Proteins ◽

Intestinal Epithelial ◽

Epithelial Cell Migration

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Ursodeoxycholic acid protects against intestinal barrier breakdown by promoting enterocyte migration via EGFR- and COX-2-dependent mechanisms

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00354.2017 ◽

2018 ◽

Vol 315 (2) ◽

pp. G259-G271 ◽

Cited By ~ 15

Author(s):

Jamie M. Golden ◽

Oswaldo H. Escobar ◽

Michelle V. L. Nguyen ◽

Michael U. Mallicote ◽

Patil Kavarian ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Ursodeoxycholic Acid ◽

Intestinal Epithelial Cell ◽

Growth Factor Receptor ◽

Intestinal Barrier ◽

Intestinal Epithelial ◽

Epithelial Cell Migration ◽

Cox 2

The intestinal barrier is often disrupted in disease states, and intestinal barrier failure leads to sepsis. Ursodeoxycholic acid (UDCA) is a bile acid that may protect the intestinal barrier. We hypothesized that UDCA would protect the intestinal epithelium in injury models. To test this hypothesis, we utilized an in vitro wound-healing assay and a mouse model of intestinal barrier injury. We found that UDCA stimulates intestinal epithelial cell migration in vitro, and this migration was blocked by inhibition of cyclooxygenase 2 (COX-2), epidermal growth factor receptor (EGFR), or ERK. Furthermore, UDCA stimulated both COX-2 induction and EGFR phosphorylation. In vivo UDCA protected the intestinal barrier from LPS-induced injury as measured by FITC dextran leakage into the serum. Using 5-bromo-2′-deoxyuridine and 5-ethynyl-2′-deoxyuridine injections, we found that UDCA stimulated intestinal epithelial cell migration in these animals. These effects were blocked with either administration of Rofecoxib, a COX-2 inhibitor, or in EGFR-dominant negative Velvet mice, wherein UDCA had no effect on LPS-induced injury. Finally, we found increased COX-2 and phosphorylated ERK levels in LPS animals also treated with UDCA. Taken together, these data suggest that UDCA can stimulate intestinal epithelial cell migration and protect against acute intestinal injury via an EGFR- and COX-2-dependent mechanism. UDCA may be an effective treatment to prevent the early onset of gut-origin sepsis. NEW & NOTEWORTHY In this study, we show that the secondary bile acid ursodeoxycholic acid stimulates intestinal epithelial cell migration after cellular injury and also protects the intestinal barrier in an acute rodent injury model, neither of which has been previously reported. These effects are dependent on epidermal growth factor receptor activation and downstream cyclooxygenase 2 upregulation in the small intestine. This provides a potential treatment for acute, gut-origin sepsis as seen in diseases such as necrotizing enterocolitis.

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