Limits...
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.

Show MeSH

Related in: MedlinePlus

Tyrosine phosphorylation induced by EGF stimulation. Cells were grown to confluence and quiesced with the DME containing 0.1% dialyzed FBS for 48 h. (a and b) Cells then were treated with IP-10 (50 ng/ml) for 10 min before the 5 min of EGF (1 nM) stimulation. Cells were then lysed, and equal volumes of proteins were analyzed for phospho-tyrosine (a) and phospho-erk-MAPK (b) by immunoblotting. (c) For PLC-γ analyses, cells were treated with IP-10 (50 ng/ml) for 10 min or 5 h before the 5 min of EGF (1 nM) treatment. Cell lysates containing same amounts of proteins were immunoprecipitated with anti–PLC-γ antibody. Then immunoprecipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with anti–phospho-tyrosine antibody PY-20. Representative blots of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199733&req=5

Figure 2: Tyrosine phosphorylation induced by EGF stimulation. Cells were grown to confluence and quiesced with the DME containing 0.1% dialyzed FBS for 48 h. (a and b) Cells then were treated with IP-10 (50 ng/ml) for 10 min before the 5 min of EGF (1 nM) stimulation. Cells were then lysed, and equal volumes of proteins were analyzed for phospho-tyrosine (a) and phospho-erk-MAPK (b) by immunoblotting. (c) For PLC-γ analyses, cells were treated with IP-10 (50 ng/ml) for 10 min or 5 h before the 5 min of EGF (1 nM) treatment. Cell lysates containing same amounts of proteins were immunoprecipitated with anti–PLC-γ antibody. Then immunoprecipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with anti–phospho-tyrosine antibody PY-20. Representative blots of three independent experiments are shown.

Mentions: The fact that IP-10 blocks EGFR-mediated motility but not proliferation suggested that the point of signal disruption lies downstream of the receptor. This was confirmed by finding that IP-10 pretreatment had no effect on ligand activation of EGFR kinase as determined by whole cell tyrosyl-phosphorylation profiles in response to EGF (Fig. 2 a) (Chen et al. 1996). Even after 5 h of IP-10 exposure EGFR kinase was unaffected (data not shown).


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)

Tyrosine phosphorylation induced by EGF stimulation. Cells were grown to confluence and quiesced with the DME containing 0.1% dialyzed FBS for 48 h. (a and b) Cells then were treated with IP-10 (50 ng/ml) for 10 min before the 5 min of EGF (1 nM) stimulation. Cells were then lysed, and equal volumes of proteins were analyzed for phospho-tyrosine (a) and phospho-erk-MAPK (b) by immunoblotting. (c) For PLC-γ analyses, cells were treated with IP-10 (50 ng/ml) for 10 min or 5 h before the 5 min of EGF (1 nM) treatment. Cell lysates containing same amounts of proteins were immunoprecipitated with anti–PLC-γ antibody. Then immunoprecipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with anti–phospho-tyrosine antibody PY-20. Representative blots of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Tyrosine phosphorylation induced by EGF stimulation. Cells were grown to confluence and quiesced with the DME containing 0.1% dialyzed FBS for 48 h. (a and b) Cells then were treated with IP-10 (50 ng/ml) for 10 min before the 5 min of EGF (1 nM) stimulation. Cells were then lysed, and equal volumes of proteins were analyzed for phospho-tyrosine (a) and phospho-erk-MAPK (b) by immunoblotting. (c) For PLC-γ analyses, cells were treated with IP-10 (50 ng/ml) for 10 min or 5 h before the 5 min of EGF (1 nM) treatment. Cell lysates containing same amounts of proteins were immunoprecipitated with anti–PLC-γ antibody. Then immunoprecipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with anti–phospho-tyrosine antibody PY-20. Representative blots of three independent experiments are shown.
Mentions: The fact that IP-10 blocks EGFR-mediated motility but not proliferation suggested that the point of signal disruption lies downstream of the receptor. This was confirmed by finding that IP-10 pretreatment had no effect on ligand activation of EGFR kinase as determined by whole cell tyrosyl-phosphorylation profiles in response to EGF (Fig. 2 a) (Chen et al. 1996). Even after 5 h of IP-10 exposure EGFR kinase was unaffected (data not shown).

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