[Ca2cyt and Polyamine Dependent Cell Migration

GI mucosal injury occurs commonly from mild physical trauma during digestion, critical and chronic illnesses, various surgical conditions, the ingestion of alcohol, aspirin, nonsteroidal anti-inflammatory compounds, or Helicobacter pylori infection. Restoration of normal intestinal mucosal integrity—successful repair of wounds and ulcers—requires epithelial cell decisions that regulate signaling networks controlling gene expression, survival, migration, and proliferation. In the acute response to injury, damaged cells are sloughed, and remaining viable cells from areas adjacent to or just beneath the injured surface migrate to cover the denuded area. This early restitution refers to resealing of superficial wounds as a consequence of epithelial cell migration into the defect, a process independent from epithelial cell proliferation (3,20). This rapid repair appears to be an initial host response to prevent noxious agents from causing deeper tissue damage. The other repair process is the replacement of lost cells by cell proliferation and is much slower (21,22). Rapid epithelial restitution is regulated by numerous factors, including cellular polyamines. Cellular polyamine levels are dramatically increased during the process of early mucosal restitution in both in vivo and in vitro systems, and polyamine depletion through inhibition of ODC inhibits cell migration and delays mucosal restitution.

We have demonstrated that polyamines regulate intestinal epithelial cell migration by altering K+ channel activity, Em, and [Ca2+]cyt and that the resultant increase in [Ca2+]cyt exerts its regulatory effects on cell motility through interaction with specific targets during restitution (5,6,11,15). Because intestinal epithelial cells do not express VDCC, the depolarized Em by polyamine depletion decreases [Ca2+]cyt through the reduced driving force for Ca2+ influx (Fig. 2). Migration is reduced by 80% in the polyamine-deficient cells. Decreased [Ca2+]cyt by depolarization of Em by 4-aminopyridine also inhibits normal cell migration and prevents the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. In contrast, increased [Ca2+]cyt by the treatment with Ca2+ ionophore ionomycin stimulates cell migration in the absence of cellular polyamines (Fig. 2). These results indicate that polyamine-mediated intestinal epithelial cell migration is due partially to increase of Kv channel expression. The subsequent membrane hyperpolarization after increased levels of polyamines raises [Ca2+]cyt by increasing the driving force for Ca2+ influx and, thus, stimulates cell migration.

In another set of experiments we have further revealed that differentiated IEC-Cdx2L1 cells migrate over the wounded edge much faster than undifferentiated parental IEC-6 cells and that increased migration of differentiated IEC-Cdx2L1 cells after wounding results, at least partially, from the K+ channel activation and the increase in driving force for Ca2+ influx during restitution (11,20). Differentiated IEC-Cdx2L1 cells express higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than those observed in undifferentiated parental IEC-6 cells. Depletion of intracellular polyamines significantly decreases the expression of both Kv1.1 and Kv1.5 channel genes. Neither

Fig. 2. Effects of polyamine depletion and the Ca2+ ionophore ionomycin (Iono) on cytosolic free Ca2+ concentration ([Ca2+]cyt) and cell migration after wounding. (A) Summarized data showing [Ca2+]cyt measured in peripheral areas of cells that were grown in control cultures and in cultures containing 5 mM a-difluoromethylornithine (DFMO) with or without 5 ^M spermidine for 4 d, and cells treated with DFMO for 4 d and then exposed to Iono (1 ^M). (B) Summarized data showing cell migration after wounding in cells described in (A). Polyamine depletion reduced [Ca2+]cyt and inhibited cell migration (left), whereas increased [Ca2+]cyt by Iono stimulated cell migration in polyamine-deficient cells (right). *, + p < 0.05 compared with controls and DFMO alone.

Fig. 2. Effects of polyamine depletion and the Ca2+ ionophore ionomycin (Iono) on cytosolic free Ca2+ concentration ([Ca2+]cyt) and cell migration after wounding. (A) Summarized data showing [Ca2+]cyt measured in peripheral areas of cells that were grown in control cultures and in cultures containing 5 mM a-difluoromethylornithine (DFMO) with or without 5 ^M spermidine for 4 d, and cells treated with DFMO for 4 d and then exposed to Iono (1 ^M). (B) Summarized data showing cell migration after wounding in cells described in (A). Polyamine depletion reduced [Ca2+]cyt and inhibited cell migration (left), whereas increased [Ca2+]cyt by Iono stimulated cell migration in polyamine-deficient cells (right). *, + p < 0.05 compared with controls and DFMO alone.

IEC-Cdx2L1 cells nor parental IEC-6 cells express VDCC. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells is associated with an increase in whole cell K+ currents, membrane hyperpolarization, and rise of resting [Ca2+]cyt. The migration rates in differentiated IEC-Cdx2L1 cells are approx four times of parental IEC-6 cells. Inhibition of Kv channel expression by depletion of cellular polyamines reduced [Ca2+]cyt, resulted in cellular reorganization of cytoskeletal proteins, along with a marked reduction in actomyosin stress fiber formation, and inhibited epithelial cell migration. In contrast, elevation of [Ca2+]cyt by the Ca2+ ionophore, ionomycin, promoted formation of actomyosin stress fibers, and increased epithelial cell migration after wounding.

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