Correspondence to: Alan Wells, Department of Pathology, Scaife 713, University of Pittsburgh, Pittsburgh, PA 15261., wellsa{at}msx.upmc.edu (E-mail), (412) 648-9550 (phone)
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Abstract |
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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 factorinduced 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- 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-
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.
Key Words: EGF receptor, cell motility, calpain, chemokine, fibroblasts
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Introduction |
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DURING normal skin wound healing, fibroblasts are recruited from the surrounding tissue (
Growth factors such as those that activate the EGF receptor (EGFR),1 including EGF, TGF-, and heparin-binding EGF-like growth factor (HB-EGF), are present at many stages of wound healing and have strong effects on both cell proliferation and cell migration (
In the search for counterstimulatory factors, select chemokines have been investigated. ELR-negative CXC chemokines, such as interferon inducible protein-10 (IP-10) and platelet factor 4 (PF4), have been shown to inhibit endothelial cell proliferation and migration and angiogenesis (
Cell motility is a biophysical process that is actively controlled by specific, if yet incompletely defined, biochemical signaling cascades that result in dissociable biophysical phenomena ( (PLC-
) as the immediate postreceptor effector, leads to cytoskeletal reorganization and possibly contributes to lamellipod protrusion (
A counterstimulatory factor would be predicted to interfere with a specific subset of these biophysical processes. Therefore, we investigated whether specific processes were targeted by the putative counterstimulatory chemokine IP-10. The foregoing data suggested a testable model in which IP-10 negatively affects cell detachment at the rear to prevent cell motility and thereby limit wound repair. In this paper we report that IP-10 inhibits EGF-induced cell migration but not proliferation. We find that only EGFR-induced motility, but not basal motility that is signaled by adhesion receptors, is diminished by IP-10. This occurs at a postreceptor level since upstream EGFR signaling pathways are unaffected by IP-10 exposure. EGFR-mediated cell de-adhesion and rear detachment are significantly impaired in the face of IP-10 signaling. Furthermore, our data suggest that this occurs via a cAMP-dependent protein kinase A (PKA) attenuation of calpain activation, as similar biochemical and biophysical readouts can be mimicked by directly modulating these biochemical signals and mediators.
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Materials and Methods |
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Reagents
Hs68 normal human diploid fibroblasts were purchased from American Type Culture Collection. Hs68 cells were used at passage 512. IP-10, monokine induced by IFN- (MIG), and PF4 were purchased from Peprotech. EGF was obtained from Collaborative Biomedical Products. HB-EGF and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was purchased from Sigma Chemical Co. Calpain inhibitor I was purchased from Biomol. PDGF-BB, Rp-8-Br-cAMPS, and Rp-8-Br-cGMPS were purchased from Calbiochem. Plastic dishes for cell culture were purchased from Becton Dickinson.
In Vitro Wound Healing Assay
EGF-induced migration was assessed by the ability of the cells to move into an acellular area (
Cell Proliferation Assay
EGF-induced proliferation was determined by the incorporation of [3H]thymidine (
cAMP Assay
Cells were plated in 10-cm culture plates and grown to confluence in DME with 10% FBS. Following the treatment of IP-10 for 2 or 4 h, ice cold extraction buffer (50% ethanol, 0.1 N HCl) were added and incubated on ice for 15 min. Extracts were lyophilized and resuspended in 100 µl of water. cAMP was quantitated using a cAMP assay kit (Amersham Life Science Inc.). After the extraction cells were lysed with 0.1 N NaOH and analyzed for protein content by Bradford protein assay.
Immunoblotting and Immunoprecipitation
Activation of EGFR, PLC-, and the erk MAPK were assessed by their tyrosyl-phosphorylation by immunoprecipitation and immunoblotting. Cells were treated with IP-10 (50 ng/ml) before EGF (1 nM) treatment. Cell lysates were separated on 7.5% SDS-PAGE and transferred to a PVDF membrane Immobilon-P (Millipore). Blots were probed by antiphospho-erk-MAPK (New England Biolabs) or anticalpain I or calpain II (Biomol) antibodies before visualizing with AP-conjugated secondary antibodies followed by development with a colorimetric method (Promega).
For immunoprecipitations, cells (2 x 107) were treated with IP-10 and EGF as described above. Cell lysates were incubated overnight at 4°C with the indicated antibody, mixed monoclonal antiPLC-1 (Upstate Biotechnology Inc.) or monoclonal anti-EGFR (Oncogene Science). Immunocomplexes were incubated with protein Gagarose and centrifuged. The pellets were washed three times with 20 nM Hepes buffer (pH 7.4) containing 10% glycerol, 0.1% Triton X-100, 500 mM sodium chloride, 1 mM sodium vanadate. Precipitated proteins were size-fractionated by SDS-PAGE and transferred to a PVDF membrane. Tyrosyl phosphorylation was determined by immunoblotting using the antiphospho-tyrosine PY-20 (Transduction Laboratories). By diluting test specimens, we empirically found that we could detect difference in the signal strength on the order of 10%.
Morphological Analysis
Cells were plated in 2 ml 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 at 37°C cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 24 h at 37°C. Cells were visualized by phase-contrast microscopy. Cell perimeter and cell surface area were analyzed by manually tracking cell edges on the computer-captured phase-contrast image using DIAS Dynamic image analyzing system (Solltech) and are expressed as a ratio of arbitrary units. Asymmetry index of nucleus localization was obtained by measuring the greatest cell length and the length between the nuclei and the tip of the longest projection, and calculating the deviation from equidistance (nucleus localization varies from central, a fraction of 0.5 and an index of 0, and at tip of a projection, for a fraction of 1.0 or an index of 100).
Adhesion Assay
Cell-substratum adhesiveness was quantitated using inverted centrifugation detachment. 24-well plates were coated with the human extracellular matrix Amgel (0.5 µg/ml) (
Calpain Activity Assay
Cells were grown to confluence in 10-cm tissue culture plates and quiesced in DME with 0.1% dialyzed FBS for 48 h. After 4 h of treatment of IP-10 (50 ng/ml) with or without Rp-8-Br-cAMPS (50 µM), cells were treated with EGF (1 nM) and/or CPT-cAMP (20 µM) for 30 min. Cells were washed twice with ice cold PBS and lysed with cell lysis buffer (20 nM Hepes, pH 7.4, 10% glycerol, 0.1% Triton X-100, 500 mM sodium chloride, 1 mM sodium vanadate). After removing the cell debris by centrifugation, dichlorotriazinylamino-fluoresceinlabeled microtubule-associated protein 2 (MAP2) (Cytoskeleton) (50 µg/ml) (
Levels of calpain I, calpain II, and calpastatin were assessed by immunoblotting using specific antibodies (Biomol).
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Results |
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IP-10 Inhibits EGFR-mediated Cell Migration but Not Proliferation
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 (Figure 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.
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IP-10 may diminish either EGFR signaling or specifically interrupt motility-enhancing pathways. To determine whether there was global abrogation of EGFR signaling, the effect of IP-10 on EGF-induced proliferation was examined (Figure 1 b). The presence of IP-10 diminished neither basal nor EGF-induced thymidine incorporation suggesting that IP-10 modulatory signals target motility-specific pathways.
To ascertain whether IP-10 affected EGF as a ligand rather than EGFR-mediated signals, we tested the cell response to HB-EGF. HB-EGFinduced cell migration was also found to be inhibited up to 47% by IP-10 (Figure 1 a). This was not unexpected as the pleiotropic nature of resultant cellular responses to EGF is thought to be due to intracellular signaling rather than multiple signals encoded in the ligand. Thus, these initial investigations pointed to a specific attenuation of the EGFR-mediated motility response.
IP-10 Does Not Inhibit EGFR, PLC-, or MAPK Phosphorylation
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 (Figure 2 a) (
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Two divergent pathways have been shown to be required for EGFR-mediated motility, those involving PLC- (
, nor dual phosphorylation of erk MAPK was diminished after either 10 min or 5 h of IP-10 exposure. These findings confirmed that IP-10 did not adversely impinge on EGFR signaling at the receptor or proximal postreceptor level.
IP-10 Exposure Results in an Elongated Cell Morphology
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 (Figure 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 1). 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 (
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IP-10 Inhibits EGF-induced Cell Detachment and Cell Migration by Inhibiting Calpain Activation
Morphological analyses suggested that EGF-induced cell detachment from substratum is inhibited by IP-10 signaling. Therefore, we assessed the effect of IP-10 on cell adhesion to the human extracellular matrix Amgel by the centrifugation detachment method. Subjecting the cells to 2,920 g for 5 min resulted in negligible removal of nontreated control Hs68 cells (84 ± 3% remaining) but about half of the EGF-treated cells (53 ± 4% remaining) (Figure 4). IP-10 by itself slightly diminished cell adhesiveness (80 ± 3% remaining), but significantly diminished EGF-induced detachment (77 ± 2% remaining, P < 0.01 vs. EGF treatment).
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According to the morphological and cell detachment analyses, we predicted that EGF-induced focal adhesion disassembly and cell de-adhesion (
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A major question is how this inhibition may be signaled. IP-10 is a member of the subfamily of ELR-negative CXC chemokines which bind to and activate the CXCR3 receptor, leading to an increase in cAMP levels (
IP-10 Inhibits Calpain Activation and Cell Motility through cAMP-PKA-Calpain Signaling Pathway
In parallel to its effect on EGF-induced cell adhesiveness and motility, IP-10 significantly inhibited EGF-induced calpain activity (by 71 ± 7%) (Figure 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 (Figure 6 b) in the face of IP-10 treatment.
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The above data suggested that IP-10 inhibits EGF-induced cell migration by inhibiting calpain activation through cAMP signaling. To determine whether cAMP-dependent PKA is involved in this IP-10 calpain signaling pathway, cell migration was performed in the presence of the cell permeant PKA inhibitor Rp-8-Br-cAMPS; the protein kinase G preferential inhibitor Rp-8-Br-cGMPS was used as a control. Rp-8-Br-cAMPS abrogated IP-10's inhibitory effect by 87%, but Rp-8-Br-cGMPS did not (Figure 7 a). Rp-8-Br-cAMPS, but not Rp-8-Br-cGMPS, also prevented IP-10 from inhibiting EGF-induced detachment (Figure 7 b). In support of our model that calpain activation is central to IP-10 inhibition of EGFR-mediated motility, the PKA preferential Rp-8-Br-cAMPS, but not the protein kinase G preferential inhibitor, abrogated the ability of IP-10 treatment to inhibit calpain activation almost completely (Figure 7 c). In a parallel study, NR6 fibroblasts expressing full-length EGFR were stimulated to migrate in the presence of CPT-cAMP; time lapse videomicroscopy revealed that they were retarded in the ability to retract the trailing edge (data not shown), a cell behavior similar to that noted with IP-10 (Figure 3).
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Other ELR-negative CXC Chemokines Also Increase Total Cellular cAMP Level and Inhibit EGF-induced Cell Migration by Inhibiting EGF-induced Calpain Activation
One of the predictions from our model of chemokine negative crossmodulation of growth factor receptormediated motility is that other chemokines that similarly lead to cAMP generation also would preferentially inhibit EGF-induced motility. The effects of two other ELR-negative CXC chemokines, MIG and PF4, were determined. These chemokines induced slow accumulation of intracellular cAMP (at 4 h: MIG, 1.92 ± 0.38-fold; PF4, 2.13 ± 0.49-fold) that was indistinguishable from IP-10. Both MIG (49 ± 4% inhibition) and PF4 (45 ± 10% inhibition) inhibited EGF-induced, but not basal motility (Figure 8 a), and had little effect on thymidine incorporation (data not shown). As per the proposed model, both MIG and PF4 inhibited EGF-induced calpain activity (Figure 8 b).
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A second prediction would be that IP-10 would prevent motility induced by other growth factors. We examined PDGF-induced motility of the Hs68 cells. Similar to its effect on EGF-induced motility, IP-10 partially blocked PDGF-induced motility by 46 ± 11% (Figure 8 c). That this is accomplished through the same signaling pathway as that which inhibits EGF-induced motility is demonstrated by IP-10 inhibition of PDGF-induced motility also being abrogated by Rp-8-Br-cAMPS (Figure 8 c). These findings support the model that growth factorinduced motility depends, at least in part, on a calpain-mediated de-adhesion step which may be negatively impacted by chemokine generation of cAMP.
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Discussion |
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We present here evidence that the counterstimulatory chemokine IP-10 affects dermal fibroblast cell responses to growth factors in addition to its known actions on hematopoietic and endothelial cells. We demonstrated that IP-10 inhibits EGFR-mediated motility specifically, likely via a cAMP/PKA-dependent inhibition of EGFR-mediated calpain activation. To our knowledge, this is the first report of the inhibitory effects of IP-10 on growth factorinduced fibroblast motility. These actions implicate a role for IP-10 in limiting fibroblast infiltration during wound healing in response to locally expressed growth factors.
We determined that IP-10 did not disrupt EGFR signaling at the ligand or receptor level. This was expected as (a) both EGF- and HB-EGFinduced motility was similarly inhibited; and (b) EGFR-mediated proliferation was unaffected by IP-10 pretreatment. As cellular response signaling diverges at the immediate postreceptor level with at least one pathway, via PLC-, being required for motility but not proliferation (
pathway. The activation status of this pathway and a second pathway required for both motility and mitogenesis, through erk MAPK, as mirrored by tyrosyl-phosphorylation, was investigated and found to be unaffected by IP-10 treatment. As the links between these and other postreceptor pathways and the biophysical events which actuate motility are still incompletely deciphered (
IP-10 could limit EGFR-mediated detachment, and thus motility, by either anchoring the adhesion sites or disrupting signaling pathways leading to de-adhesion. We did not favor the former, as IP-10 has no discernible effect on basal cell motility or morphometry; alterations in these assays due to inhibition of basal haptokinesis would be expected if IP-10 directly strengthened adhesive sites (
The molecular bases of both the calpain activation by EGFR signaling and its disruption by IP-10 receptor signaling are unknown. As IP-10 has been reported to bind to CXCR3 and elevate cAMP levels (
There appears to be a discrepancy between IP-10's predominant inhibition of calpain activation and only partial inhibition of cell motility and de-adhesion. This difference may suggest that IP-10 inhibition of EGF-induced de-adhesion and motility is independent of suppression of calpain activation. However, this is unlikely, as exposure of the cells to calpain inhibitor I presented a similar partial inhibition of motility and de-adhesion. While we and others have not yet determined the exact mechanism or precise molecular targets by which calpain modulates cell-substratum adhesion (
Two predictions derive from this model in which calpain serves as a point of convergence for pro-motility signals and their counterregulatory signals. The first is that other extracellular signals that increase cAMP in a similar fashion to IP-10 should also decrease EGF-induced motility. We determined that the other ELR-negative CXC chemokines, MIG and PF4, also inhibited EGF-induced cell migration. These chemokines induced cAMP generation and inhibited EGF-induced calpain activation indistinguishably from IP-10. The second prediction is that these chemokines should abrogate motility signaled by other growth factors. Motility induced by another growth factor present during wound healing, PDGF, was partially limited by IP-10. That these two postulates are supported by experimental data strongly supports our model of IP-10 limiting growth factorinduced cell migration by inhibiting calpain activation.
In summary, we have defined for the first time a regulatory crosstalk between counterstimulatory chemokines and growth factor receptors. That this is specific for one cellular response to a pleiotropic signal highlights the closely orchestrated control of wound healing. One could easily envision a function for IP-10 in this respect; IP-10 would limit cell infiltration late in the reparative phase while leaving untouched other EGFR-mediated responses, such as production of matrix components and remodeling enzymes ( and HB-EGF from platelets and macrophages, as well as other growth factors such as PDGF. As the wound healing progresses, the PF4 would be consumed and the motility block would be relieved with the fibroblasts then able to migrate to appropriate positions for the reparative phase of wound healing. While at present this is just one possible explanation for the specific counterregulatory effects of the ELR-negative CXC chemokines, it provides for a working model to test these cell behaviors and signaling pathways. Deciphering the precise molecular mechanisms will not only provide insight into the complex network of communications operational during organogenesis and repair, but also suggest targets for rational intervention.
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Footnotes |
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Hidenori Shiraha's and Angela Glading's present address is Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261.
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Acknowledgements |
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We thank Tom Lincoln, Anna Huttenlocher, Doug Lauffenburger, Kathryn Drabik, Jeffry Chou, Philip Chang, Jareer Kassis, Jose Souto, Scott Swindle, and Hyung Kim for valuable suggestions.
This work was supported by National Institutes of Health, National Institute of General Medical Sciences grant GM54739.
Submitted: December 14, 1998; Revised: June 3, 1999; Accepted: June 8, 1999.
1.used in this paper: CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; IP-10, interferon inducible protein-10; MAP2, microtubule-associated protein 2; MAPK, mitogen-activated protein kinase; MIG, monokine induced by IFN-; PF4, platelet factor 4; PKA, protein kinase A; PLC-
, phospholipase C-
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References |
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---|
Aronica, S.M., Mantel, C., Gonin, R., Marshall, M.S., Sarris, A., Cooper, S., Hague, N., Zhang, X.F., Broxmeyer, H.E. (1995) Interferon-inducible protein 10 and macrophage inflammatory protein-1 inhibit growth factor stimulation of Raf-1 kinase activity and protein synthesis in a human growth factordependent hematopoietic cell line. J. Biol. Chem. 270:21998-22007
Blotnick, S., Peoples, G.E., Freeman, M.R., Eberlein, T.J., Klagsbrun, M. (1994) T lymphocytes synthesize and export heparin-binding epidermal growth factorlike growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells. Proc. Natl. Acad. Sci. USA. 91:2890-2894[Abstract].
Brown, M.C., Perrotta, J.A., Turner, C.E. (1998) Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin. Mol. Biol. Cell. 9:1803-1816
Byzova, T.V., Plow, E.F. (1998) Activation of vß3 on vascular cells controls recognition of prothrombin. J. Cell Biol. 143:2081-2092
Chen, P., Gupta, K., Wells, A. (1994a) Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis. J. Cell Biol. 124:547-555[Abstract].
Chen, P., Xie, H., Sekar, M.C., Gupta, K., Wells, A. (1994b) Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but mitogen-activated protein kinase activity is not sufficient for induced cell movement. J. Cell Biol. 127:847-857[Abstract].
Chen, P., Xie, H., Wells, A. (1996) Mitogenic signaling from the EGF receptor is attenuated by a phospholipase C-/protein kinase C feedback mechanism. Mol. Biol. Cell. 7:871-881[Abstract].
Chen, W.T. (1981) Mechanism of retraction of the trailing edge during fibroblast movement. J. Cell Biol. 90:187-200[Abstract].
Du, X., Saido, T.C., Tsubuki, S., Indig, F.E., Williams, M.J., Ginsberg, M.H. (1995) Calpain cleavage of the cytoplasmic domain of the integrin ß3 subunit. J. Biol. Chem. 270:26146-26151
Engelhardt, E., Toksoy, A., Goebeler, M., Debus, S., Brocker, E.B., Gillitzer, R. (1998) Chemokines IL-8, GRO, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am. J. Pathol. 153:1849-1860
Farber, J.M. (1997) Mig and IP-10: CXC chemokines that target lymphocytes. J. Leukoc. Biol. 61:246-257[Abstract].
Han, J.D., Rubin, C.S. (1996) Regulation of cytoskeleton organization and paxillin dephosphorylation by cAMP. Studies on murine Y 1(adrenal cells. J. Biol. Chem. 271):29211-29215.
Huttenlocher, A., Palecek, S.P., Lu, Q., Zhang, W., Mellgren, R.L., Lauffenburger, D.A., Ginsberg, M.H., Horwitz, A.F. (1997) Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 272:32719-32722
Kiritsy, C.P., Lynch, A.B., Lynch, S.E. (1993) Role of growth factors in cutaneous wound healing. Crit. Rev. Oral Biol. Med. 4:729-760[Abstract].
Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis, I., Baggiolini, M., Moser, B. (1996) Chemokine receptor specific for IP10 and MIG: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184:963-969[Abstract].
Luster, A.D., Greenberg, S.M., Leder, P. (1995) The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J. Exp. Med. 182:219-231[Abstract].
Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J., Welgus, H.G., Kuwano, M. (1989) Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors, and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells. J. Immunol. 143:1619-1627
Palecek, S.P., Huttenlocher, A., Horwitz, A.F., Lauffenburger, D.A. (1998) Physical and biochemical regulation of integrin release during rear detachment of migrating cells. J. Cell Sci. 111:929-940
Pan, Z., Kravchenko, V.V., Ye, R.D. (1995) Platelet-activating factor stimulates transcription of the heparin-binding epidermal growth factor-like growth factor in monocytes. Correlation with an increased B binding activity. J. Biol. Chem. 270:7787-7790
Stewart, M.P., McDowall, A., Hogg, N. (1998) LFA-1mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J. Cell Biol. 140:699-707
Ware, M.F., Wells, A., Lauffenburger, D.A. (1998) Epidermal growth factor alters fibroblast migration speed and derectional persistence reciprocally and in a matrix-dependent manner. J. Cell Sci. 111:2423-2432
Xie, H., Pallero, M.A., Gupta, K., Chang, P., Ware, M.F., Witke, W., Kwiatkowski, D.J., Lauffenburger, D.A., Murphy-Ullrich, J.E., Wells, A. (1998) EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLC signaling pathway. J. Cell Sci. 111:615-624