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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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Effects of IP-10 on EGF-induced cell migration (a) and cell proliferation (b). Cells were grown to confluence and quiesced for 48 h in DME with 0.1% dialyzed FBS before treatment with or without IP-10 (1–50 ng/ml), EGF (1–10 nM), and/or HB-EGF (1 nM). Cell migration (a) and cell proliferation (b) assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative or cell proliferative activity. The data are the mean ± SEM of at least three independent studies each performed in triplicate. Statistical analyses were performed by Student's t test as compared with EGF- or HB-EGF–induced cell migrative or cell proliferative capacity in the absence of IP-10: *P < 0.05, **P < 0.01.
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Figure 1: Effects of IP-10 on EGF-induced cell migration (a) and cell proliferation (b). Cells were grown to confluence and quiesced for 48 h in DME with 0.1% dialyzed FBS before treatment with or without IP-10 (1–50 ng/ml), EGF (1–10 nM), and/or HB-EGF (1 nM). Cell migration (a) and cell proliferation (b) assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative or cell proliferative activity. The data are the mean ± SEM of at least three independent studies each performed in triplicate. Statistical analyses were performed by Student's t test as compared with EGF- or HB-EGF–induced cell migrative or cell proliferative capacity in the absence of IP-10: *P < 0.05, **P < 0.01.

Mentions: To determine whether the putative counterregulatory ELR-negative CXC chemokine IP-10 affects fibroblast functioning and responsiveness, we examined EGF-induced proliferation and migration in the presence of IP-10. Basal and EGF (1 nM)-induced cell migrative capacities were 452 ± 10 and 703 ± 26 μm/d, respectively. IP-10 was seen to inhibit EGF-induced cell migration (Fig. 1 a). IP-10 at 1 ng/ml had no effect, but 10 ng/ml and 50 ng/ml inhibited 1 nM EGF-induced cell migration 46% and 48%, respectively. This is not overcome by supersaturating doses of EGF, as IP-10 also inhibits 10 nM EGF-induced cell migration 43% (10 ng/ml) to 45% (50 ng/ml). On the other hand, no significant difference was found in basal cell migrative capacities which is signaled via adhesion receptors. These data suggest that IP-10 disrupts EGFR-mediated modulatory signals rather than the motility process per se.


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)

Effects of IP-10 on EGF-induced cell migration (a) and cell proliferation (b). Cells were grown to confluence and quiesced for 48 h in DME with 0.1% dialyzed FBS before treatment with or without IP-10 (1–50 ng/ml), EGF (1–10 nM), and/or HB-EGF (1 nM). Cell migration (a) and cell proliferation (b) assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative or cell proliferative activity. The data are the mean ± SEM of at least three independent studies each performed in triplicate. Statistical analyses were performed by Student's t test as compared with EGF- or HB-EGF–induced cell migrative or cell proliferative capacity in the absence of IP-10: *P < 0.05, **P < 0.01.
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Related In: Results  -  Collection

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Figure 1: Effects of IP-10 on EGF-induced cell migration (a) and cell proliferation (b). Cells were grown to confluence and quiesced for 48 h in DME with 0.1% dialyzed FBS before treatment with or without IP-10 (1–50 ng/ml), EGF (1–10 nM), and/or HB-EGF (1 nM). Cell migration (a) and cell proliferation (b) assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative or cell proliferative activity. The data are the mean ± SEM of at least three independent studies each performed in triplicate. Statistical analyses were performed by Student's t test as compared with EGF- or HB-EGF–induced cell migrative or cell proliferative capacity in the absence of IP-10: *P < 0.05, **P < 0.01.
Mentions: To determine whether the putative counterregulatory ELR-negative CXC chemokine IP-10 affects fibroblast functioning and responsiveness, we examined EGF-induced proliferation and migration in the presence of IP-10. Basal and EGF (1 nM)-induced cell migrative capacities were 452 ± 10 and 703 ± 26 μm/d, respectively. IP-10 was seen to inhibit EGF-induced cell migration (Fig. 1 a). IP-10 at 1 ng/ml had no effect, but 10 ng/ml and 50 ng/ml inhibited 1 nM EGF-induced cell migration 46% and 48%, respectively. This is not overcome by supersaturating doses of EGF, as IP-10 also inhibits 10 nM EGF-induced cell migration 43% (10 ng/ml) to 45% (50 ng/ml). On the other hand, no significant difference was found in basal cell migrative capacities which is signaled via adhesion receptors. These data suggest that IP-10 disrupts EGFR-mediated modulatory signals rather than the motility process per se.

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