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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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EGF-induced calpain activation. (a) Cells were grown to confluence in 10-cm tissue culture plates and quiesced for 48 h with DME containing 0.1% dialyzed FBS. Cells were treated with IP-10 (50 ng/ml) for 4 h or CPT-cAMP (20 μM) for 30 min before EGF (1 nM) for 30 min. Calpain activities were analyzed by measuring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein–labeled MAP2. The data are shown as a ratio to EGF-induced calpain activation. The data are the mean ± SEM of at least three independent studies. Statistical analyses were performed by Student's t test. (b) Cells were grown to confluence in 6-well tissue culture plates and quiesced for 48 h. Cells were treated with IP-10 (50 ng/ml) for 4 h before EGF (1 nM) for 30 min. Cell lysates were separated by SDS-PAGE, transferred to Immobilon-P (Millipore), and immunoblotted with an anti–calpain I or –calpain II antibody (Biomol).
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Figure 6: EGF-induced calpain activation. (a) Cells were grown to confluence in 10-cm tissue culture plates and quiesced for 48 h with DME containing 0.1% dialyzed FBS. Cells were treated with IP-10 (50 ng/ml) for 4 h or CPT-cAMP (20 μM) for 30 min before EGF (1 nM) for 30 min. Calpain activities were analyzed by measuring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein–labeled MAP2. The data are shown as a ratio to EGF-induced calpain activation. The data are the mean ± SEM of at least three independent studies. Statistical analyses were performed by Student's t test. (b) Cells were grown to confluence in 6-well tissue culture plates and quiesced for 48 h. Cells were treated with IP-10 (50 ng/ml) for 4 h before EGF (1 nM) for 30 min. Cell lysates were separated by SDS-PAGE, transferred to Immobilon-P (Millipore), and immunoblotted with an anti–calpain I or –calpain II antibody (Biomol).

Mentions: In parallel to its effect on EGF-induced cell adhesiveness and motility, IP-10 significantly inhibited EGF-induced calpain activity (by 71 ± 7%) (Fig. 6 a). If calpain activation was the point of signal convergence, then cAMP should also reduce calpain activity and de-adhesion. EGF-induced calpain activation in cells treated with CPT-cAMP was inhibited by 99 ± 2%. This decreased calpain activity was due to prevention of enzymatic activation and not a change in cellular levels of the calpain system as shown by relatively constant cellular levels of the calpains (Fig. 6 b) in the face of IP-10 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)

EGF-induced calpain activation. (a) Cells were grown to confluence in 10-cm tissue culture plates and quiesced for 48 h with DME containing 0.1% dialyzed FBS. Cells were treated with IP-10 (50 ng/ml) for 4 h or CPT-cAMP (20 μM) for 30 min before EGF (1 nM) for 30 min. Calpain activities were analyzed by measuring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein–labeled MAP2. The data are shown as a ratio to EGF-induced calpain activation. The data are the mean ± SEM of at least three independent studies. Statistical analyses were performed by Student's t test. (b) Cells were grown to confluence in 6-well tissue culture plates and quiesced for 48 h. Cells were treated with IP-10 (50 ng/ml) for 4 h before EGF (1 nM) for 30 min. Cell lysates were separated by SDS-PAGE, transferred to Immobilon-P (Millipore), and immunoblotted with an anti–calpain I or –calpain II antibody (Biomol).
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Figure 6: EGF-induced calpain activation. (a) Cells were grown to confluence in 10-cm tissue culture plates and quiesced for 48 h with DME containing 0.1% dialyzed FBS. Cells were treated with IP-10 (50 ng/ml) for 4 h or CPT-cAMP (20 μM) for 30 min before EGF (1 nM) for 30 min. Calpain activities were analyzed by measuring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein–labeled MAP2. The data are shown as a ratio to EGF-induced calpain activation. The data are the mean ± SEM of at least three independent studies. Statistical analyses were performed by Student's t test. (b) Cells were grown to confluence in 6-well tissue culture plates and quiesced for 48 h. Cells were treated with IP-10 (50 ng/ml) for 4 h before EGF (1 nM) for 30 min. Cell lysates were separated by SDS-PAGE, transferred to Immobilon-P (Millipore), and immunoblotted with an anti–calpain I or –calpain II antibody (Biomol).
Mentions: In parallel to its effect on EGF-induced cell adhesiveness and motility, IP-10 significantly inhibited EGF-induced calpain activity (by 71 ± 7%) (Fig. 6 a). If calpain activation was the point of signal convergence, then cAMP should also reduce calpain activity and de-adhesion. EGF-induced calpain activation in cells treated with CPT-cAMP was inhibited by 99 ± 2%. This decreased calpain activity was due to prevention of enzymatic activation and not a change in cellular levels of the calpain system as shown by relatively constant cellular levels of the calpains (Fig. 6 b) in the face of IP-10 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