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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 calpain inhibitor I and CPT-cAMP on cell migration. 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), calpain inhibitor I (1 ng/ml), and/or CPT-cAMP (10 μM). Cell migration assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative 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.
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Figure 5: The effects of calpain inhibitor I and CPT-cAMP on cell migration. 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), calpain inhibitor I (1 ng/ml), and/or CPT-cAMP (10 μM). Cell migration assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative 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.

Mentions: According to the morphological and cell detachment analyses, we predicted that EGF-induced focal adhesion disassembly and cell de-adhesion (Xie et al. 1998) are affected by IP-10. Therefore, we focused on biochemical events which affect focal adhesion disassembly, with calpain being a prime candidate due to its recent association with adhesion disruption and reorganization (Huttenlocher et al. 1997; Stewart et al. 1998). In addition, an ancillary study in our lab has found that EGF induces calpain activity in fibroblasts and that this is required for cell de-adhesion from substratum (Glading, A., P. Chang, D.A. Lauffenburger, and A. Wells, manuscript in preparation). In agreement with the hypothesis that calpain modulation leads to de-adhesion, the pharmacological inhibitor, calpain inhibitor I, also prevented EGF-induced detachment by 68% (74 ± 3% remaining). That this is linked to cell motility is shown by the fact that calpain inhibitor I also diminished EGF-induced, but not basal, motility (Fig. 5). Interestingly, this partial inhibition of EGF-induced cell motility would be expected from the report that de-adhesion of the trailing edge of cells is rate limiting only on highly adhesive surfaces (Palecek et al. 1997); thus, on a mixed substratum such as the one used, Amgel, one might predict only a partial effect of diminishing de-adhesion.


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 calpain inhibitor I and CPT-cAMP on cell migration. 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), calpain inhibitor I (1 ng/ml), and/or CPT-cAMP (10 μM). Cell migration assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: The effects of calpain inhibitor I and CPT-cAMP on cell migration. 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), calpain inhibitor I (1 ng/ml), and/or CPT-cAMP (10 μM). Cell migration assays were performed as described in Materials and Methods. The data are shown as the ratio to the 1 nM EGF-induced cell migrative 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.
Mentions: According to the morphological and cell detachment analyses, we predicted that EGF-induced focal adhesion disassembly and cell de-adhesion (Xie et al. 1998) are affected by IP-10. Therefore, we focused on biochemical events which affect focal adhesion disassembly, with calpain being a prime candidate due to its recent association with adhesion disruption and reorganization (Huttenlocher et al. 1997; Stewart et al. 1998). In addition, an ancillary study in our lab has found that EGF induces calpain activity in fibroblasts and that this is required for cell de-adhesion from substratum (Glading, A., P. Chang, D.A. Lauffenburger, and A. Wells, manuscript in preparation). In agreement with the hypothesis that calpain modulation leads to de-adhesion, the pharmacological inhibitor, calpain inhibitor I, also prevented EGF-induced detachment by 68% (74 ± 3% remaining). That this is linked to cell motility is shown by the fact that calpain inhibitor I also diminished EGF-induced, but not basal, motility (Fig. 5). Interestingly, this partial inhibition of EGF-induced cell motility would be expected from the report that de-adhesion of the trailing edge of cells is rate limiting only on highly adhesive surfaces (Palecek et al. 1997); thus, on a mixed substratum such as the one used, Amgel, one might predict only a partial effect of diminishing de-adhesion.

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