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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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Morphology of EGF- and IP-10–treated cells. Cells were plated in 6-well tissue culture plates with DME containing 0.1% dialyzed FBS at the concentration of 105 cells/ml. After 12 h of incubation, cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 12 h, after which pictures were taken by phase-contrast microscopy. Pictures were representative fields from three independent experiments.
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Figure 3: Morphology of EGF- and IP-10–treated cells. Cells were plated in 6-well tissue culture plates with DME containing 0.1% dialyzed FBS at the concentration of 105 cells/ml. After 12 h of incubation, cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 12 h, after which pictures were taken by phase-contrast microscopy. Pictures were representative fields from three independent experiments.

Mentions: In the absence of effects on these signaling pathways, the target of signal attenuation is predicted to be downstream, and at or near the rate-limiting biophysical steps for motility. As an initial attempt to identify the affected biophysical process, cells were examined in the presence and absence of chemokine (Fig. 3). EGF-treated Hs68 cells presented a motile fusiform shape compared with control cells. IP-10 pretreatment prevented this conversion to a more contracted cell with shorter forward and rear projections. To determine if these morphological changes were significantly affected, two ratios were determined: extent of cell contraction and asymmetry of nuclear localization (Table ). Comparing the cell perimeter to surface contact area demonstrated that EGF-treated cells were significantly compacted compared with untreated cells, whereas IP-10 prevented this morphological change (P < 0.01), while not affecting basal cell morphometry. As this ratio may simply reflect centripetal contraction rather than motility-related changes, we also determined the asymmetry index of the nucleus localization; this should remain unchanged and central during retraction but be offset during locomotion. Again, EGF treatment resulted in a motile morphology which was abrogated by IP-10 pretreatment (P < 0.01). Thus, IP-10 inhibits EGF-induced cell compaction and results in elongated cell morphology, a phenotype reminiscent of cells that are unable to detach from substratum (Huttenlocher et al. 1997; Palecek et al. 1997).


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)

Morphology of EGF- and IP-10–treated cells. Cells were plated in 6-well tissue culture plates with DME containing 0.1% dialyzed FBS at the concentration of 105 cells/ml. After 12 h of incubation, cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 12 h, after which pictures were taken by phase-contrast microscopy. Pictures were representative fields from three independent experiments.
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Related In: Results  -  Collection

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Figure 3: Morphology of EGF- and IP-10–treated cells. Cells were plated in 6-well tissue culture plates with DME containing 0.1% dialyzed FBS at the concentration of 105 cells/ml. After 12 h of incubation, cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 12 h, after which pictures were taken by phase-contrast microscopy. Pictures were representative fields from three independent experiments.
Mentions: In the absence of effects on these signaling pathways, the target of signal attenuation is predicted to be downstream, and at or near the rate-limiting biophysical steps for motility. As an initial attempt to identify the affected biophysical process, cells were examined in the presence and absence of chemokine (Fig. 3). EGF-treated Hs68 cells presented a motile fusiform shape compared with control cells. IP-10 pretreatment prevented this conversion to a more contracted cell with shorter forward and rear projections. To determine if these morphological changes were significantly affected, two ratios were determined: extent of cell contraction and asymmetry of nuclear localization (Table ). Comparing the cell perimeter to surface contact area demonstrated that EGF-treated cells were significantly compacted compared with untreated cells, whereas IP-10 prevented this morphological change (P < 0.01), while not affecting basal cell morphometry. As this ratio may simply reflect centripetal contraction rather than motility-related changes, we also determined the asymmetry index of the nucleus localization; this should remain unchanged and central during retraction but be offset during locomotion. Again, EGF treatment resulted in a motile morphology which was abrogated by IP-10 pretreatment (P < 0.01). Thus, IP-10 inhibits EGF-induced cell compaction and results in elongated cell morphology, a phenotype reminiscent of cells that are unable to detach from substratum (Huttenlocher et al. 1997; Palecek et al. 1997).

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