Department of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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We have investigated cellular
Ca2+ regulation during A2058 human melanoma cell chemotaxis
to type IV collagen (CIV). We have identified
2
1-integrin as the primary mediator of
A2058 cell response to CIV in vitro. Integrin ligation initiated a
characteristic intracellular Ca2+ concentration
([Ca2+]i) response consisting of an
internal release and a receptor-mediated Ca2+ entry.
Thapsigargin (TG) pretreatment drained overlapping and CIV-inducible
internal Ca2+ stores while initiating a store-operated
Ca2+ release (SOCR). CIV-mediated Ca2+ entry
was additive to TG-SOCR, suggesting an independent signaling mechanism.
Similarly, ionophore application in a basal medium containing
Ca2+ initiated a sustained influx. Elevated
[Ca2+]i from TG-SOCR or ionophore
significantly attenuated cell migration to CIV by recruiting the
Ca2+/calcineurin-mediated signaling pathway. Furthermore,
low [Ca2+]i induced by EGTA application in
the presence of ionophore fully restored cell motility to CIV.
Together, these results suggest that [Ca2+]i
signaling accompanying A2058 cell response to
2
1-integrin ligation is neither necessary
nor sufficient and that elevated [Ca2+]i
downregulates cell motility via a calcineurin-mediated mechanism in
A2058 cell chemotaxis to CIV.
melanoma; chemotaxis; secondary messenger; signal transduction; intracellular Ca2+ concentration
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INTRODUCTION |
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CELL MOTILITY IS REGULATED by a multiplex of signal transduction mechanisms and is coordinated through intracellular second messengers. Phenomenological aspects of cell motility and migration in response to cytokines, chemokines, and extracellular matrix (ECM) proteins in a variety of cell systems have been well documented; however, biochemical signaling and regulation of these processes require further elucidation (2, 5, 9, 10, 19, 29). Cell chemotaxis is mediated by coupling an extracellular agonist to its transmembrane receptor that is capable of activating cascades of intracellular second messenger signaling pathways. Of those receptors, G protein-linked receptor activation and integrin-mediated cell signaling have been suggested to play significant roles in tumor cell adhesion and migration over ECM protein substrates (5, 18, 35, 39). Whereas the G protein-mediated signal transduction cascade has been well characterized (2, 5, 9), the signaling pathway that activates tumor cell response mediated by integrin ligation remains to be elucidated.
The integrin superfamily consists of a variety of cell surface
receptors, and those belonging to the VLA (very late antigen) subgroup
have been identified as de facto ECM receptors (1, 31, 32,
39). The VLA integrin complex consists of a heterodimer of -
and
-subunits. In cell interactions that involve ECM proteins, the
-subunit of the VLA integrin complex is conserved, while the
-subunit is variable. The cytoplasmic tails of
- and
-subunits have been shown to associate with intracellular actin cytoskeleton filaments through the formation of focal adhesion complex
(32). Cell attachment and spreading via the engagement of
these integrin receptors initiate signaling pathways that result in
mitotic and chemotactic cellular responses (11, 21, 22, 26, 32, 33, 36).
Recent findings indicated that the chemotaxis of A2058 human melanoma cells toward a member of ECM protein, type IV collagen (CIV), induced an intracellular Ca2+ second messenger pathway (28). The initial release of intracellular Ca2+ concentration ([Ca2+]i), which is triggered by a receptor engagement through the cell attachment and spreading, is followed by a prolonged period of Ca2+ influx from the extracellular compartments [store-operated Ca2+ release (SOCR) and receptor-mediated Ca2+ entry] (3, 4, 7, 27). However, the mechanism of initial [Ca2+]i release mediated by integrins remains poorly characterized in tumor cell chemotaxis. Evidently, A2058 cells do not significantly turn over phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2; PIP2] during CIV stimulation, ruling out the classic PIP2/D-myo-inositol 1,4,5-trisphosphate (IP3) [Ca2+]i release mechanism (28). Therefore, characterizing the source of cell Ca2+ will facilitate the elucidation of signaling events mediating tumor cell metastasis.
Here, we address the influence of Ca2+ signaling on
integrin-mediated A2058 human melanoma cell migration toward CIV. We
have identified a key integrin receptor initiating this specific
chemotactic response of A2058 cells to CIV as
2
1 (VLA-2). We have shown that the
initial [Ca2+]i release occurred from
thapsigargin (TG)-sensitive intracellular stores. However, the
receptor-mediated Ca2+ entry following the CIV stimulation
utilized a distinctly different set of influx channels from those
employed by TG-SOCR. TG, which specifically targets and irreversibly
inactivates Ca2+-ATPases [sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA)] on the endoplasmic reticulum
(17), is used to assess the importance of intracellular
store refilling during a cell response to collagen. Effects of the
depletion of extracellular Ca2+ source by EGTA, as well as
an enhancement of Ca2+ flux caused by Ca2+
ionophores, are addressed in this study and correlated to cellular chemotactic response toward CIV stimulation.
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MATERIALS AND METHODS |
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Cell culture and preparation. A2058 human melanoma cells were maintained in tissue culture, as described previously, detached when subconfluent by a brief trypsinization, and allowed to regenerate for 1 h in culture medium (5, 35). Cells were resuspended in serum-free DMEM (Biofluids, Rockville, MD) containing 0.1% wt/vol fraction V bovine serum albumin with 0.02 M HEPES (Sigma Chemical, St. Louis, MO) at 1.3 × 106 cells/ml concentration and allowed to regenerate for one additional hour before assays. Cells used were in passages 14-18 for all experiments.
Antibodies.
Integrin-specific monoclonal blocking antibodies (MAb) FB12
(anti-1; Chemicon International, Temecula, CA), Gi9
(anti-
2; Beckman Coulter, Fullerton, CA), and P1B5 and
6S6 (anti-
3 and anti-
1; Chemicon) were
obtained commercially. All antibodies are murine and of IgG1 isotype.
Isotype control experiments were also performed with nonspecific murine IgG.
Migration assay. A detailed procedure for chemotaxis assays using 48-well microchemotaxis chambers was described elsewhere (5, 13). In brief, 10-µm pore size polycarbonate filters (Neuro Probe, Cabin John, MD) were soaked overnight in 0.1% wt/vol poly-D-lysine solution (Sigma) to enhance cell adhesion. CIV (Becton Dickinson Labware, Bedford, MD) was dispersed into the experiment medium at a concentration of 100 µg/ml as the chemotactic solution. The pH of the chemotactic solution was adjusted to 7.4 immediately before the experiment. The chemotactic solutions were placed into the bottom wells of the 48-well chamber, and the cell suspension was placed into the top wells, separated by a filter. The chamber assembly was placed into a 5% CO2 37°C environment for 4 h. Upon completion of the experiment, the filter was stained with DiffQuik staining kit (Dade International, Miami, FL), and cells on the underside of the filter were visually counted under ×10 bright field magnification.
In cases involving EGTA (Sigma), the chemotactic solution contained EGTA at 3.2 mM to chelate 1.6 mM Ca2+ in basal medium. In this case, the medium additionally contained 3.2 mM Mg2+ to balance the depletion of Ca2+. Cells were transferred to medium that contained 3.2 mM EGTA 30 min before an assay and placed into top wells of the migration chamber at the specified cell concentration. EGTA (3.2 mM at pH 7.4) yielded extracellular free Ca2+ of ~65 nM
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Ratiometric measurements of intracellular free Ca2+ in adherent cells. A2058 cells were cultured onto 25-mm round glass coverslips (pretreated with 0.1% wt/vol poly-D-lysine solution overnight) and maintained under standard culture conditions. The fluorescence measurements were conducted in a temperature-, humidity-, and gas (37°C, 100%, and 5%, respectively)-controlled chamber mounted on top of the microscope stage. The procedure for a digital Ca2+ ratiometric assay is detailed elsewhere (12, 28, 38). A computer software package (Axon Imaging Workbench 2.1; Axon Instruments, Foster City, CA) was used to control the excitation light (340- and 380-nm band pass filters; Chroma Technology, Brattleboro, VT), sample, and record the emitted fluorescence (510 nm) images from the fura 2-AM (Molecular Probes)-loaded cells in the field of view once every 6 s (once every 2 min for longtime Ca2+ assays). The background fluorescence was subtracted from each image. Ratio images of the cells at rest were collected initially for the first minute to establish an [Ca2+]i baseline. After the establishment of a baseline [Ca2+]i, above mentioned experiment solutions were selectively perfused into the chamber, and raw images were collected for an additional 10-12 min or up to 4 h. A calibration curve was constructed by acquiring 340/380 values (background subtracted) of 50 µM fura 2 pentapotassium salt solution using the calcium calibration kit no. 1 (Molecular Probes) and using an off-line calibration of the ratio data.
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RESULTS |
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2
1-integrin mediates A2058 cell
chemotaxis to CIV.
To identify and characterize the receptors mediating cell response to
CIV, MAbs were used to functionally block the cell surface integrin
receptors during a chemotaxis assay. In the absence of integrin-specific MAbs, A2058 melanoma cells actively migrated in response to 100 µg/ml of CIV (Fig.
1). At saturating MAb concentrations of
10-25 µg/ml, antibodies to
1- and
3-integrins mildly attenuated the cell response to CIV,
while blocking
2 and
1 effectively abrogated the cell chemotaxis to CIV. These data indicate that A2058
cell chemotaxis to CIV is primarily mediated by
2
1-integrin.
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Cell migration abrogated during high
[Ca2+]i conditions.
To elucidate the regulatory role of Ca2+ in A2058 cell
chemotaxis to CIV, intra- and extracellular [Ca2+] levels
were controlled. As shown above, A2058 cells actively migrated to CIV
(100 µg/ml) in 1.6 mM Ca2+ basal medium, and this robust
response was used as the reference to normalize the migration assay
data (Table 1; 100%). Cellular [Ca2+]i response corresponding to this
control condition resulted in a peak [time (t) < 200 s] response and a plateau (t > 300 s)
[Ca2+]i that decayed with time to approach
the baseline level (100-150 nM), as previously reported
(28). [Ca2+]i assays were taken
to a duration of 4 h to reflect the time course of a typical
chemotaxis assay (Fig. 2). The data
indicate that a CIV stimulation in this control condition results in a singular [Ca2+]i peak, occurring at the
beginning, which subsides to the baseline by ~1,000-1,500 s.
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Subbasal [Ca2+]i was
sufficient for cell chemotaxis to CIV.
We wished to determine the effects of low Ca2+ on A2058
cell migration to CIV. Chelating the Ca2+ in basal medium
with 3.2 mM EGTA slightly reduced cell migration in response to CIV
(Table 2; 69 ± 3.8%). Baseline
[Ca2+]i and the peak
[Ca2+]i were similar to the control case,
while the plateau phase was eliminated due to lack of receptor-mediated
Ca2+ entry (Table 2).
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Elevated [Ca2+]i
downregulates cell motility via calcineurin-mediated signaling.
We have shown that a high [Ca2+]i is
inhibiting A2058 cell migration in response to CIV.
Calmodulin-dependent protein phosphatase IIB (calcineurin) has been
implicated in an [Ca2+]i signaling pathway
mediating neutrophil migration to ECM proteins fibronectin and
vitronectin (14). Similarly, TG- and A-23187-induced prostatic carcinoma cell apoptosis was mediated reversibly by Ca2+/calmodulin-activated calcineurin (37). We
therefore asked whether a signaling pathway involving calcineurin was
recruited when TG or A-23187 increased
[Ca2+]i in A2058 cells. Furthermore, we
wished to determine whether activation of this particular pathway may
downregulate cell chemotaxis in response to CIV. CsA specifically
inhibits calcineurin activation by formation of a complex with
cyclophilin A and then binds to calcineurin to inhibit the
Ca2+/calmodulin-dependent activation of calcineurin
(14, 20, 30, 37). Cells incubated with 1.5 µg/ml
of CsA (20 h) were pretreated with 1.0 µM TG or 1.0 µM A-23187 as
described and assayed for migration to 100 µg/ml of CIV in a 1.6 mM
Ca2+ basal medium. CsA incubation of 1-2 h resulted in
a negligible recovery of cell migration, while an overnight incubation
(15-20 h) maximally restored cell migration under these conditions
(Fig. 6). The recovery of cell migration
in the presence of an elevated [Ca2+]i due to
TG or A-23187 pretreatment indicated that
Ca2+/calcineurin-mediated signaling events were recruited
during an elevated [Ca2+]i to downregulate
A2058 cell chemotaxis to CIV.
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DISCUSSION |
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In this study, we examined the role of
[Ca2+]i in chemotactic response of A2058
cells to CIV. It was previously shown that A2058 cells respond to CIV
stimulation by a Ca2+ signal, and the source of internal
release was suggested to be from non-IP3-sensitive stores
(28). However, the causal relationship between the
recruitment of an [Ca2+]i signaling mechanism
and cell motility to CIV remained unclear. In this study, we attempted
to elucidate whether such a link between cell motility and
[Ca2+]i recruitment existed in A2058 cell
chemotaxis to CIV. We further wished to identify putative chemotaxis
receptors mediating this specific response to CIV. In our results, we
have identified 2
1-integrin as the
chemotaxis receptor of A2058 cells to soluble CIV. This CIV-mediated
response was also confirmed in other tumor cell lines such as C8161
melanoma, 293T embryonic kidney carcinoma, and MDA-MB435 breast
carcinoma (unpublished data).
Previously, Leavesley et al. (18) have shown that an
endothelial cell spreading over immobilized CIV was mediated by
2
1-integrin and that this process was
extracellular Ca2+ insensitive, while the same cell
spreading over immobilized vitronectin recruited
v
3 and was extracellular Ca2+
sensitive. Hendey and Maxfield (14) have shown that
neutrophil migration over fibronectin and vitronectin resulted in
[Ca2+]i transients, while inhibition caused
the neutrophils to remain anchored to the substrate, resulting in a
loss of motility. Whereas these studies focused on cell motility over
immobilized ECM proteins to result in a haptotactic response, our
system employed a polylysine-coated substrate with a soluble form of
CIV as the chemoattractant following protocols of previous studies
(5, 13). We have shown that chemotaxis of A2058 cells to
CIV was mediated by the same set of integrin receptors that induced a
haptotactic response in endothelial cells (18).
Unlike the neutrophils, A2058 cells did not exhibit repeated [Ca2+]i transients in our study. The cell response to CIV consisted of a singular [Ca2+]i peak followed by a decaying plateau [Ca2+]i that reached the baseline within 1,000-1,500 s. Hendey and Maxfield (14) have shown that cell spreading and pseudopodial extensions were not [Ca2+]i dependent in neutrophils. However, we have shown previously that A2058 cell pseudopodial activity to CIV was extracellular Ca2+ sensitive (15). Furthermore, neutrophil motility was mediated by calcineurin-calmodulin activation (14), whereas we have shown that tumor cells recruited a calcineurin signaling pathway only at an elevated [Ca2+]i to downregulate cell motility. Together, differential sensitivity of cell motility to [Ca2+]i in these cells was clearly identified and characterized in these cell types.
The most important finding we report is that A2058 cells migrated
significantly when [Ca2+]i was maintained at
a subbasal level throughout the duration of a chemotaxis assay. An
application of ionophore in the absence of extracellular
Ca2+ results in a complete efflux of
[Ca2+]i, because this is the basis for
obtaining the minimum ratio condition in an in situ ratiometric
microscopy calibration (12, 16, 38). We have used this
approach to deplete the [Ca2+]i instead of
using more invasive [Ca2+]i chelators, such
as BAPTA-AM or EGTA-AM. These chelating agents have been suggested to
cause affects of reduced cell viability and cell adhesion (8,
40). Because of the concentration difference driving the
[Ca2+]i drainage, the extent of extracellular
free Ca2+ sequestration was maintained to result in a
subresting baseline [Ca2+]i of ~65 nM. With
these conditions, the cell viability and motility were maintained
throughout the course of a migration assay. This demonstrated that the
method was less hazardous to cells while sufficiently removing the
cellular Ca2+. We have shown that abrogated
[Ca2+]i signaling did not affect cell
motility to CIV. This clearly indicates that A2058 cells do not require
an [Ca2+]i signaling mechanism during the
2
1-mediated chemotaxis over polylysine substrate.
As previously reported, ligation of
2
1-integrin resulted in an
[Ca2+]i response (28). The
induction of this response was neither necessary nor sufficient to
mediate cell migration to CIV in our results. The elevation of
[Ca2+]i via the SOCR influx was shown to be
inhibitory to cell motility via a calcineurin-mediated mechanism.
Furthermore, the role of SERCA and the refilling of intracellular
stores were insignificant during cell chemotaxis to CIV. These results
indicated that cells possess many redundant signaling mechanisms, and
[Ca2+]i induction in A2058 cells responding
to CIV may be one such mechanism.
In an intact cell, regulatory mechanisms function to control the
level and rate of Ca2+ flux. Evidently, Leavesley et al.
(18) suggested that highly localized
[Ca2+]i differences in a polarized cell
undergoing chemotaxis may be responsible for the topical
concentration-dependent differential role of Ca2+ in
promoting adhesion and deadhesion at the sites of focal contacts. Furthermore, it appears that different cell lineages and different ECM
chemotactic ligands present variable degrees of dependence on the
levels of cellular Ca2+ (3, 4, 18, 28). Our
results indicate that A2058 cell migration mediated by
2
1-integrin does not require the
associated internal [Ca2+]i release nor later
influx. Present work also demonstrates that the initial intracellular
Ca2+ release in response to CIV originates from
TG-sensitive stores, while the later receptor-mediated influx mechanism
utilizes a distinctly separate set of signaling pathways. There are
various theories that attempt to account for the nature of
Ca2+ regulation encountered by nonexcitable cells. The
likelihood of two distinctly separate signaling pathways recruited in
Ca2+ influx regulation may be attributed to the signaling
direction (23, 32). Depletion of intracellular stores is a
signal that cells transmit via either the cytoskeletal network
contraction or with yet another unidentified molecular signaling
mechanism (24, 25). Conversely, integrin engagement and an
ensuing focal contact formation with subsequent whole cell polarization
originate from an extracellular stimulus. Hence, the formation of a
focal adhesion complex initiated by an integrin receptor ligation may likely trigger different intracellular signaling intermediates than
those driven by TG-induced store drainage. In our results, the influx
Ca2+ amplitudes were additive to indicate that
2
1-integrin likely triggered its cognate
receptor-mediated Ca2+-influx mechanism.
In summary, we have shown that CIV-induced chemotaxis in the
A2058 human melanoma cell line was mediated by
2
1-integrin in vitro. The ligation of
integrin receptors induced an [Ca2+]i
response in these cells as previously reported
(28). We report that an elevated
[Ca2+]i, due to SOCR influx caused by an
application of TG or A-23187 ionophore, downregulated cell motility via
the calcineurin-mediated mechanism in A2058 cells. While an elevated
[Ca2+]i resulting from TG or ionophore
treatment in a 1.6 mM Ca2+ basal medium caused a
significant attenuation of cell motility, removing extracellular
Ca2+ with EGTA restored cell motility to near control
levels under these conditions. Most important, a sustained level of
subbaseline [Ca2+]i did not affect cell
motility to CIV via
2
1-integrin
signaling. Together, these observations suggested that
[Ca2+]i signaling recruited by
2
1-integrin ligation was neither
necessary nor sufficient to mediate A2058 cell chemotaxis to CIV.
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ACKNOWLEDGEMENTS |
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We acknowledge Andrew J. Henderson and Julie A. Cook at Penn State University, Veterinary Science Department, for reagents and Loretta L. Collins at University of Rochester, Department of Pathology, for reference work and helpful discussions.
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FOOTNOTES |
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This work was supported by National Cancer Institute Grant CA-76434 and National Science Foundation (NSF) Grant NSF-BES9502069. Cheng Dong is a recipient of the NSF Career Award.
Address for reprint requests and other correspondence: C. Dong, Dept. of Bioengineering, 229 Hallowell, Pennsylvania State Univ., University Park, PA 16802 (E-mail: cxd23{at}psu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 June 2000; accepted in final form 5 February 2001.
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