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IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.

Shiraha H, Glading A, Gupta K, Wells A - J. Cell Biol. (1999)

Bottom Line: These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility.To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases.IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.

ABSTRACT
During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor receptor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor-induced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- 15% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility. To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamma and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.

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The effects of IP-10 and calpain inhibitor I on EGF-induced cell detachment. Cells were plated into the 24-well Amgel coated plates with quiescent media and incubated for 12 h. After pretreatment with or without IP-10 (50 ng/ml for 4 h) or calpain inhibitor I (1 ng/ml for 30 min), cells were treated with or without EGF (1 nM) for 30 min. Before and after the centrifugation, the numbers of the cells on the plates were counted under the phase-contrast microscope observation. The data are the mean ± SEM of three independent studies each performed in at least 18 wells. Statistical analyses were performed by Student's t test.
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Figure 4: The effects of IP-10 and calpain inhibitor I on EGF-induced cell detachment. Cells were plated into the 24-well Amgel coated plates with quiescent media and incubated for 12 h. After pretreatment with or without IP-10 (50 ng/ml for 4 h) or calpain inhibitor I (1 ng/ml for 30 min), cells were treated with or without EGF (1 nM) for 30 min. Before and after the centrifugation, the numbers of the cells on the plates were counted under the phase-contrast microscope observation. The data are the mean ± SEM of three independent studies each performed in at least 18 wells. Statistical analyses were performed by Student's t test.

Mentions: Morphological analyses suggested that EGF-induced cell detachment from substratum is inhibited by IP-10 signaling. Therefore, we assessed the effect of IP-10 on cell adhesion to the human extracellular matrix Amgel by the centrifugation detachment method. Subjecting the cells to 2,920 g for 5 min resulted in negligible removal of nontreated control Hs68 cells (84 ± 3% remaining) but about half of the EGF-treated cells (53 ± 4% remaining) (Fig. 4). IP-10 by itself slightly diminished cell adhesiveness (80 ± 3% remaining), but significantly diminished EGF-induced detachment (77 ± 2% remaining, P < 0.01 vs. EGF treatment).


IP-10 inhibits epidermal growth factor-induced motility by decreasing epidermal growth factor receptor-mediated calpain activity.

Shiraha H, Glading A, Gupta K, Wells A - J. Cell Biol. (1999)

The effects of IP-10 and calpain inhibitor I on EGF-induced cell detachment. Cells were plated into the 24-well Amgel coated plates with quiescent media and incubated for 12 h. After pretreatment with or without IP-10 (50 ng/ml for 4 h) or calpain inhibitor I (1 ng/ml for 30 min), cells were treated with or without EGF (1 nM) for 30 min. Before and after the centrifugation, the numbers of the cells on the plates were counted under the phase-contrast microscope observation. The data are the mean ± SEM of three independent studies each performed in at least 18 wells. Statistical analyses were performed by Student's t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199733&req=5

Figure 4: The effects of IP-10 and calpain inhibitor I on EGF-induced cell detachment. Cells were plated into the 24-well Amgel coated plates with quiescent media and incubated for 12 h. After pretreatment with or without IP-10 (50 ng/ml for 4 h) or calpain inhibitor I (1 ng/ml for 30 min), cells were treated with or without EGF (1 nM) for 30 min. Before and after the centrifugation, the numbers of the cells on the plates were counted under the phase-contrast microscope observation. The data are the mean ± SEM of three independent studies each performed in at least 18 wells. Statistical analyses were performed by Student's t test.
Mentions: Morphological analyses suggested that EGF-induced cell detachment from substratum is inhibited by IP-10 signaling. Therefore, we assessed the effect of IP-10 on cell adhesion to the human extracellular matrix Amgel by the centrifugation detachment method. Subjecting the cells to 2,920 g for 5 min resulted in negligible removal of nontreated control Hs68 cells (84 ± 3% remaining) but about half of the EGF-treated cells (53 ± 4% remaining) (Fig. 4). IP-10 by itself slightly diminished cell adhesiveness (80 ± 3% remaining), but significantly diminished EGF-induced detachment (77 ± 2% remaining, P < 0.01 vs. EGF treatment).

Bottom Line: These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility.To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases.IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.

ABSTRACT
During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor receptor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor-induced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 +/- 15% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility. To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-gamma and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 +/- 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-gamma and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.

Show MeSH
Related in: MedlinePlus