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Originally published In Press as doi:10.1074/jbc.M501625200 on May 23, 2005

J. Biol. Chem., Vol. 280, Issue 29, 27130-27137, July 22, 2005
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Ca2+ Influx through L-type Ca2+ Channels Controls the Trailing Tail Contraction in Growth Factor-induced Fibroblast Cell Migration*

Shengyu Yang and Xin-Yun Huang{ddagger}

From the Department of Physiology, Cornell University Weill Medical College, New York, New York 10021

Received for publication, February 11, 2005 , and in revised form, May 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth factor-induced cell migration plays essential roles in organism development, physiological functions, and pathological disease processes (1, 2). In wound healing, fibroblast cells migrate into the wound site. Once within the wound site, fibroblasts proliferate and lay down a new collagen-rich connective tissue matrix (3). Vascular smooth muscle cell migration induced by platelet-derived growth factor (PDGF)1 and other factors contributes to the pathophysiology of intimal hyperplasia and is an essential component of the intimal process that leads to recurrent stenosis (4). The invasion and metastasis of tumor cells also require cell migration (5). Solid tumors, which account for more than 85% of cancer mortality, require blood vessels for growth, and many new cancer therapies are directed against the tumor vasculature, a process involving growth factor-induced cell migration (6).

Ca2+ is a critical second messenger for a wide range of physiological processes, including the electrical excitability of neurons, muscle contraction, cellular secretion, and gene expression (7). Ca2+ also plays a regulatory role in cell migration such as in neutrophils, eosinophils, smooth muscle cells, and neurons (8-12). Cell migration is a sequential and interrelated multistep process (2). It involves the formation of lamellipodia/membrane protrusion at the front edge, cycles of adhesion and detachment, cell body contraction, and tail retraction. In migrating cells, Ca2+ concentration is high at the rear end and low at the front leading edge (9, 12-14).

Increase of cellular Ca2+ levels is brought about by either Ca2+ release from intracellular stores or Ca2+ influx through plasma membrane Ca2+ or other cation channels (7). Both Ca2+ release and Ca2+ influx have been linked to cell migration depending on cell-types and stimuli (10, 12, 15, 16). In some cases, it is the induction of a Ca2+ transient that is important for cell migration irrespective of the Ca2+ source (8, 16). Among the membrane channels are the voltage-gated L-type Ca2+ channels, which selectively allow the flow of Ca2+ ions down an electrochemical gradient, from a high concentration outside the cell to a low concentration inside the cell, to increase cytosolic Ca2+ level (17). These L-type Ca2+ channel proteins are expressed in excitable cells such as neurons and cardiac myocytes (17). Indeed, L-type Ca2+ channels have been genetically implicated in controlling neuronal migration in Caenorhabditis elegans (18) and axonal guidance in Xenopus spinal neurons (19). Although cellular Ca2+ is critical for fibroblast cell migration, the source of the Ca2+ increase and the mechanism by which Ca2+ plays its role in controlling fibroblast cell migration are not clear.

Here we use mouse embryonic fibroblasts (MEFs) to study the potential role of voltage-gated L-type Ca2+ channels in growth factor-induced cell migration. We found that Ca2+ release from intracellular stores is not essential for PDGF-induced MEF cell migration. Rather, PDGF-induced cell migration requires Ca2+ influx. This Ca2+ influx is mediated by L-type Ca2+ channels present in MEF cells. Furthermore, downstream of Ca2+, calmodulin and myosin light chain kinase (MLCK) mediate the phosphorylation of myosin light chain (MLC) at the trailing end of migrating fibroblasts, leading to the retraction of the rear edge during cell migration.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Reagents—MEF cells and human MDA-MB-231 breast tumor cells were cultured in DMEM supplemented with 10% FBS and 2 mM glutamine. Mouse 4T1 breast tumor cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Human umbilical vein endothelial cells were cultured in Medium 199 supplemented with 10% FBS. Phospholipase C (PLC){gamma}1-/- and PLC{gamma}1-/-+PLC{gamma}1 MEF cells were generously provided by Dr. G. Carpenter (Vanderbilt University) and were cultured in DMEM supplemented with 10% FBS and 2 mM glutamine (20). PDGF-BB was from Oncogene Research Products. BAPTA-AM (1,2-bis [aminophenoxy] ethane-N,N,N',N'-tetraacetic acid), W7, and ML-7 were from Calbiochem.

In Vitro Wound-healing Cell Migration Assay—Cell migration assays were performed as described previously (21). Cells were allowed to form a confluent monolayer in a 24-well plate coated with gelatin before wounding. The wound was made by scraping a conventional pipette tip across the monolayer. The migration was induced by adding medium supplemented with or without 10% FBS, 20 ng/ml PDGF or 20 ng/ml epidermal growth factor. For MEF cells, it typically took 8-10 h for the wound to close. When the wound for the positive control closed, cells were fixed with 3.7% formaldehyde and stained with crystal violet staining solution.

Boyden Chamber Cell Migration Assay—MEF cells (5 x 104) suspended in DMEM were added to the upper chamber of an insert coated with gelatin (6.5-mm diameter, 8-µm pore size, Becton Dickinson), and the chamber was placed in 24-well dishes containing DMEM with or without 10% FBS, 40 ng/ml PDGF or 40 ng/ml epidermal growth factor. When used, inhibitors were added to both chambers. Migration assays were carried out for 4 h and cells were fixed with 3.7% formaldehyde. Cells were stained with crystal violet staining solution, and cells on the upper side of the insert were removed with a cotton swab. Three randomly selected fields (10x objective) were photographed, and the migrated cells were counted. The migration was expressed as either the average number of migrated cells in a field or as percentage of migrated cells. Percentage was calculated with the formula P = 100 x (M - Mnc)/Mpc, where P is the percentage of migrated cells, M is the number of migrated cells, Mnc is the number of migrated cells in negative controls (DMEM only), and Mpc is the number of migrated cells in positive controls (with 10% FBS or PDGF).

Ca2+ Assay and Western Blots—The Ca2+ assay was conducted as previously described (22). Cells were plated onto gelatin-coated 96-well plates (~50,000 cells/well) 1 day before the experiments. The standard solution for calcium assays consisted of 20 mM HEPES (pH 7.4), 130 mM NaCl, 2 mM CaCl2, 5 mM KCl, 10 mM glucose, 0.45 mM KH2PO4, 0.4 mM Na2HPO4, 1.2 mM MgSO4, 1.2 mM MgCl2, 4.2 mM NaHCO3, 2.5 mM probenecid (Sigma), and 0.1% bovine serum albumin (Sigma). The calcium-sensitive dye, Fluo-3 (Molecular Probes), was dissolved in dimethyl sulfoxide (Me2SO), mixed with an equal volume of 20% Pluronic-127 (Sigma), and diluted in standard solution plus 1% bovine serum albumin to a final concentration of 4 µM. Cells were washed with standard solution and incubated with 100 µl of 4 µM Fluo-3/well for 45 min at 22 °C followed by 15 min at 37 °C. Cells were then washed three times with standard solution (200 µl/well) on ice. Finally, 100 µl of standard solution was added per well, and cells were incubated at 37 °C for 15 min before the assay. The change in fluorescence was monitored using a Fluorescent Ascent FL fluorometer. Western blots were performed as described previously (23).

Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as described previously (24). Cells cultured on gelatin-coated coverslips were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 3 min, and blocked with 1% bovine serum albumin in PBS for 1 h. Cells were then incubated with primary antibodies at appropriate dilutions (1:100 for rabbit anti-pSer-19 MLC (from Cell Signaling); 1:4000 for rabbit anti-{alpha}1c subunit of L-type Ca2+ channels (from Alomone Labs)) for 2 h. After washing with PBS, cells were incubated with Alexa 488-conjugated anti-rabbit IgG for 60 min. For visualization of actin filaments, Texas Red-conjugated phalloidin (Molecular Probes) was added during the incubation with secondary antibody. MEF cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were then incubated with 25 µg/ml Cy5 NHS (Amersham Biosciences) in PBS, pH 8.0, at room temperature for 30 min. After incubation, cells were washed with PBS, blocked with 1% bovine serum albumin, and stained with appropriate antibodies. pMLC/total protein ratio images were obtained with Metamoph software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ca2+ Release Is Not Essential for PDGF-induced MEF Cell Migration—In MEF cells, stimulation with PDGF induced cell migration, as measured by both in vitro wound-healing assay (Fig. 1A) and Boyden chamber assay (Fig. 1B). This migration was blocked by treatment with the membrane-permeable Ca2+ chelator, BAPTA-AM, indicating that Ca2+ plays an indispensable role in PDGF-induced MEF cell migration (Fig. 1B). However, Ca2+ elevation by itself was not sufficient because the Ca2+ ionophore, ionomycin, treatment did not induce cell migration (data not shown). To investigate the source of Ca2+ increase in fibroblast cells after PDGF treatment, we examined the significance of Ca2+ release and Ca2+ influx in fibroblast cell migration. Growth factor receptor tyrosine kinase-induced Ca2+ responses usually consist of two phases, an early transient peak that is due to Ca2+ release from internal stores, and a late sustained plateau that is due to Ca2+ influx (7). Ca2+ release is initiated by the binding of inositol 1,4,5-triphosphate to its receptor on the endoplasmic reticulum. Inositol 1,4,5-triphosphate is generated by the hydrolysis of phosphatidyl inositol 4,5-bisphosphate by PLC. In response to growth factors, PLC{gamma} is responsible for the Ca2+ release (25). In fibroblast cells, PLC{gamma}1 is the main expressed PLC{gamma} isoform (20). Indeed, in PLC{gamma}1-/- fibroblast cells, PDGF-induced Ca2+ release from internal stores was abolished, whereas Ca2+ influx was intact (Fig. 1, C and D). This Ca2+ response deficiency was because of the absence of PLC{gamma}1 because re-expression of PLC{gamma}1 in PLC{gamma}1-/- cells restored Ca2+ release after PDGF stimulation (Fig. 1E). If Ca2+ release is essential for PDGF-induced fibroblast cell migration, PLC{gamma}1-/- cells should be defective in migration. However, we observed that PLC{gamma}1-/- cells still migrate under the stimulation of PDGF (Fig. 1, F and G). Consistent with a previous report with epidermal growth factor (26), the PDGF-induced migration of PLC{gamma}1-/- cells and PLC{gamma}1-rescued cells was comparable (Fig. 1, H and I). These data demonstrate that Ca2+ release is not essential for PDGF-induced fibroblast cell migration.

Ca2+ Influx Is Required for Cell Migration—Next we investigated whether Ca2+ influx plays a role in PDGF-induced cell migration. Chelation of extracellular Ca2+ with EGTA completely blocked both PDGF and serum induced MEF cell migration (Fig. 2, A-D). This EGTA abolishment of cell migration was reversed by the addition of equal molar Ca2+ into the medium (data not shown). To further confirm the requirement for Ca2+ influx, we used the general Ca2+ channel blockers, Ni2+ and La3+, that block Ca2+ channels without affecting extracellular free Ca2+ concentrations. As shown in Fig. 2, E and F, both Ni2+ and La3+ blocked PDGF and serum-induced cell migration. Therefore, these data demonstrated that Ca2+ influx is critical for PDGF-induced cell migration. Furthermore, the abolishment of serum-induced MEF migration by EGTA, Ni2+, and La3+ indicated that Ca2+ influx might play a general regulatory role in cell migration. To test this hypothesis, the effect of Ni2+ and EGTA on cell migration was examined with mouse 4T1 breast tumor cells, human MDA-MB-231 breast tumor cells, and human umbilical vein endothelial cells. As shown in Fig. 2 G-I,Ni2+ and EGTA blocked serum-induced migration of all these three types of cells (data with EGTA not shown). These results suggest that Ca2+ influx is generally required for normal cell as well as cancer cell motility.

L-type Ca2+ Channels Mediate Ca2+ Influx in PDGF-induced MEF Cell Migration—In studying which type of Ca2+ channels were responsible for this Ca2+ influx, we found that the voltage-gated L-type Ca2+ channels were essential for PDGF-induced Ca2+ influx and cell migration of MEF cells. First, although L-type Ca2+ channel proteins are mainly expressed in excitable cells such as neurons and muscle cells, these channel proteins (the {alpha}1C subunits) are also present in MEF cells as shown by Western blot (Fig. 3A). Immunostaining of MEF cells with anti-L-type Ca2+ channel antibodies revealed membrane staining (Fig. 3B). L-type Ca2+ channel proteins are uniformly distributed on the plasma membrane (including the front as well as the tail of a migrating MEF cell). The staining at the tail membrane was overshadowed by the strong perinuclear staining (the nuclei in migrating cells are usually localized toward the tail). Second, not only are these L-type Ca2+ channels expressed in MEF cells, but they are functional also. Depolarization of MEF cell membrane potential with 150 mM KCl induced an immediate increase in [Ca2+]i, and this increase was abolished in the presence of EGTA or nimodipine, a specific L-type Ca2+ channel blocker (Fig. 3C). Third, L-type Ca2+ channels are responsible for PDGF-induced Ca2+ influx in MEF cells. We observed that treatment with L-type Ca2+ channel blocker nimodipine decreased the late sustained phase of the PDGF-induced Ca2+ elevation while having no effect on the initial Ca2+ peak (Fig. 3D). Fourth, L-type Ca2+ channels are required for PDGF-induced MEF cell migration. Pretreatment of MEF cells with nimodipine decreased PDGF-induced cell migration (Fig. 3, E and F). Furthermore, epidermal growth factor-induced MEF cell migration was also blocked by nimodipine, indicating that L-type Ca2+ channels are generally involved in growth factor-induced fibroblast migration (data not shown). Together, the above data strongly demonstrate that PDGF-induced fibroblast cell migration depends on L-type Ca2+ channel-mediated Ca2+ influx.



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  		FIG. 1.
Ca2+ release is not essential for PDGF-induced MEF cell migration. A, in vitro wound-healing assay showed that PDGF induced MEF cell migration. B, Boyden chamber assay showed that PDGF-induced MEF cell migration was blocked by BAPTA-AM pretreatment. C, PDGF-induced intracellular Ca2+ increase in MEF cells was monitored by relative fluorescent changes after cells were loaded with Ca2+-sensitive dye Fluo-3. D, in PLC{gamma}1-/- fibroblast cells, PDGF-induced Ca2+ release from internal stores was abolished while Ca2+ influx was intact. E, reexpression of PLC{gamma}1 in PLC{gamma}1-/- cells restored Ca2+ release after PDGF stimulation. F and G, in vitro wound-healing assay (F) and Boyden chamber assay (G) showed that PDGF induced PLC{gamma}1-/- fibroblast cell migration. H and I, in vitro wound-healing assay (H) and Boyden chamber assay (I) showed that PDGF induced PLC{gamma}1-reexpressed PLC{gamma}1-/- fibroblast cell migration. Data are representative of three experiments.

 
Calmodulin and MLCK Act Downstream of Ca2+ Influx in Phosphorylating MLC at the Trailing End of Migrating Fibroblasts—We next studied the mechanism by which Ca2+ influx controls MEF cell migration. Because the small GTPase Rac plays an essential role in controlling cell migration (27), we first examined whether Ca2+ acts upstream or downstream of, or in parallel to, Rac. MEF cells were infected with retroviruses carrying Rac1(G12V), a constitutively active mutant of Rac1. Compared with cells infected with the control retroviral vector, expression of Rac1(G12V) significantly promoted MEF cell migration (Fig. 4A). This Rac1(G12V)-promoted cell migration was abolished by Ca2+ influx blocker Ni2+ (Fig. 4B). Furthermore, 1 h after making the wound in an in vitro wound-healing assay, lamellipodia (a Rac-dependent phenomenon) were seen along the wound edge of EGTA-, Ni2+-, or nimodipine-treated cells in the presence of PDGF (Fig. 4C; data with Ni2+ not shown). These data suggested that the Ca2+ signal acts downstream of or in parallel to Rac and that Ca2+ is not required for the formation of lamellipodia and membrane protrusions at the leading edge of migrating fibroblasts.

Because calmodulin is a major mediator of Ca2+ signaling, we tested whether calmodulin mediates Ca2+ regulation of cell migration. Treatment of MEF cells with calmodulin antagonist W7 significantly inhibited PDGF- and serum-induced cell migration (Fig. 4, D and E). The role of calmodulin in cell migration was further confirmed by stable expression of a calmodulin binding peptide in MEF cells. Expression of a calmodulin binding peptide blocked PDGF- and serum-induced cell migration (Fig. 4, F and G). Among the calmodulin effectors, calcineurin was reported to be involved in integrin turnover. Inhibition of calcineurin blocked neutrophil migration, and this blockage could be reversed by the addition of integrin antibodies (28, 29). However, in MEF cells, calcineurin inhibitor cyclosporin A had no effect on PDGF-induced MEF migration, whereas control experiments showed that at this concentration cyclosporin A inhibited calcineurin activity effectively (data not shown).



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  		FIG. 2.
Ca2+ influx is required for cell migration. A and B, in vitro wound-healing assay (A) and Boyden chamber assay (B) showed that PDGF-induced MEF cell migration was blocked by addition of EGTA to the culture medium. C and D, the in vitro wound-healing assay (C) and Boyden chamber assay (D) showed that serum (10% FBS)-induced MEF cell migration was blocked by the addition of EGTA to the culture medium. E, the in vitro wound-healing assay showed that both PDGF- and serum-induced MEF cell migration was blocked by addition of Ni2+ to the culture medium. F, the in vitro wound-healing assay showed that both PDGF- and serum-induced MEF cell migration was blocked by addition of La3+. G-I, Ni2+ blocked serum-induced 4T1 mouse breast tumor cell migration (G), human MDA-MB-231 breast tumor cell migration (H), human umbilical vein endothelial cell migration (I). Data are representative of three experiments.

 
Another calmodulin target is MLCK, which phosphorylates the regulatory light chain of myosin II MLC and thus activates myosin II (30). The essential role of myosin II in actin cytoskeletal rearrangement and cell migration has long been appreciated (31). The contraction of actomyosin filaments contributes to the cell body movement and tail retraction in migrating cells (1, 32). Myosin II deficiency and myosin II inhibitors impaired Dictyostelium and neutrophil migration (33, 34). Moreover, in a number of cell types including leukocytes, lymphocytes, and Dictyostelium, myosin II has been reported to localize to the rear end of migrating cells (33, 34). Rho and its effector Rho kinase are considered to be the major regulators of myosin II in cell migration. Rho kinase could activate myosin II by either directly phosphorylating MLC, or by inhibiting MLC phosphatase thereby indirectly elevating the MLC phosphorylation level (35). However, the role of Rho and Rho kinase in cell migration is cell-type-dependent (36). In fast migrating cells such as macrophages and neutrophils, Rho and Rho kinase appear to be required for cell polarization and migration (35). In slow migrating cells, such as fibroblasts, Rho and Rho kinase appear to inhibit migration. Indeed, Rho inhibitor C3 toxin did not decrease MEF cell migration (data not shown), and Rho kinase inhibitor Y-27632 even promoted MEF cell migration (Fig. 5A), confirming that Rho and Rho kinase are not essential for promoting fibroblast migration. Therefore, we examined the role of MLCK in PDGF-induced cell migration. Treatment of MEF cells with MLCK inhibitor ML-7 markedly inhibited PDGF- and serum-induced migration (Fig. 5, B and C). These data suggested that MLCK acts downstream of calmodulin mediating Ca2+ control of cell migration.



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  		FIG. 3.
L-type Ca2+ channel mediates Ca2+ influx in PDGF-induced MEF cell migration. A, Western blot showed the presence of {alpha}1C L-type Ca2+ channel proteins in MEF cells. In lane 2, whole cell extracts from neuronal NG105 cells were used as a positive control. Western blot was probed with an antibody against {alpha}1c subunit of L-type Ca2+ channels. Bottom panel, the same filter was probed with anti-ERK1/2 antibodies to show similar amounts of protein samples were loaded in the two lanes. B, immunostaining of MEF cells with anti-L-type Ca2+ channel antibodies revealed membrane staining (indicated by an arrowhead). The strong staining inside the cell likely represented newly synthesized and trafficking channel proteins. C, depolarization of MEF cell membrane potential with 150 mM KCl induced an immediate increase in [Ca2+]i, and this increase was abolished in the presence of EGTA or nimodipine. D, nimodipine decreased the late sustained phase of the PDGF-induced Ca2+ response. E, in vitro wound-healing assay showed that nimodipine decreased PDGF-induced MEF cell migration. F, dosage-response curve of nimodipine inhibition of PDGF-induced MEF cell migration assayed by Boyden chamber assay. Data are representative of three experiments.

 
To further investigate the relationship between Ca2+ influx and MLC phosphorylation, we examined the distribution of phosphorylated MLC in migrating cells. As shown in Fig. 6A, there were two areas of phosphorylated-MLC staining in polarized migrating cells, postlamellipodia staining and trailing tail staining. Strong staining of phosphorylated-MLC was observed in the actin assembly immediately behind the lamellipodia of polarized migrating cells (Fig. 6A). The other area of phosphorylated-MLC staining was in the rear end of the cell body, especially the trailing tail of polarized cells (Fig. 6A). This staining pattern of phosphorylated-MLC was also confirmed by the ratio imaging of phosphorylated-MLC over total proteins (Fig. 6A). During migration, cells can adopt two morphologies. In the first morphology, membrane protrusion at the leading edge and strong adhesion at the rear force cells to adopt an elongated shape with a trailing tail (Fig. 6A). In these cells, extension of the front edge occurs as a distinct phase from retraction of the rear end (37). When elongation tension reaches a threshold, the trailing tail detaches from the substratum and retracts (37). After retraction, the cell adopts the second, fan-shaped morphology (Fig. 6B). The trailing tail phosphorylated-MLC staining was only observed in the elongated migrating cells. In the fan-shaped cells, only postlamellipodia staining of phosphorylated-MLC was observed (Fig. 6B). When Ca2+ influx was blocked with Ni2+, the strong postlamellipodia staining of phosphorylated-MLC remained, but no or weak trailing tail staining was observed in most cells (Fig. 6C). In the untreated control group (Fig. 6A), 87% (41 of 47) of the observed elongated migrating cells displayed strong trailing tail staining with anti-phosphorylated MLC antibody, whereas the remaining 6 cells showed weak trailing tail staining of phosphorylated-MLC. In Ni2+-treated group (Fig. 6C), 39 of the 47 observed elongated cells showed no or weak tail staining of phosphorylated-MLC, whereas the remaining 17% (8 of 47) of cells showed strong trailing edge staining. Moreover, treatment with MLCK inhibitor ML-7 reduced the staining of phosphorylated-MLC at the tail, without significant effect on the staining at the front (Fig. 6D). Taken together, our data demonstrated that Ca2+ influx regulates the phosphorylation of MLC at the trailing tail and thus contributes to the trailing tail contraction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results have demonstrated that L-type Ca2+ channels are required for MEF cell migration. There are two unexpected findings here. One is that voltage-gated L-type Ca2+ channels play an important role in fibroblast cells. Previously, voltage-gated L-type Ca2+ channel proteins were thought to be mainly expressed in excitable cells such as neurons and muscle cells (38). Our data revealed that these channel proteins are expressed in fibroblast cells and play an essential role in fibroblast cell migration. Recently the multiorgan disorder Timothy syndrome was identified to be caused by an L-type Ca2+ channel missense mutation, which leads to the loss of voltage-dependent channel inactivation (39). The dysfunction includes lethal arrhythmias, webbing of fingers and toes, congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism (39). Furthermore, another recent report has shown that L-type Ca2+ channels are essential for T lymphocyte functions in mice (40). These recent studies revealed a much wider expression pattern for L-type Ca2+ channel proteins, consistent with our observation here.



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  		FIG. 4.
Calmodulin acts downstream of Ca2+ influx in MEF cell migration. A-C,Ca2+ works either downstream of or in parallel to Rac. A, compared with cells infected with a control retroviral vector, expression of Rac1(G12V) significantly promoted MEF cell migration. B, Ni2+ blocked Rac1(G12V)-promoted cell migration. C, actin polymer staining of cells at the wound edge of control, EGTA-, and nimodipine-treated cells in the presence of PDGF. Although no lamellipodia were observed in control cells, lamellipodia were seen in cells treated with EGTA or nimodipine in the presence of PDGF or with PDGF alone (indicated by arrowheads). D, in vitro wound-healing assay showed that calmodulin antagonist W7 significantly inhibited PDGF- and serum-induced cell migration. E, dosage-response curve of W7 inhibition of PDGF-induced MEF cell migration assayed by Boyden chamber assay. F and G, Boyden chamber assay (F) and in vitro wound-healing assay (G) showed that serum- and PDGF-induced MEF cell migration was blocked by stable expression of a calmodulin binding peptide (CBP) in MEF cells. Data are representative of three experiments.

 



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  		FIG. 5.
MLCK acts downstream of Ca2+ influx in MEF cell migration. A, Boyden chamber assay showed that Rho kinase inhibitor Y-27632 did not inhibit PDGF- and serum-induced cell migration. B, in vitro wound-healing assay showed that serum- and PDGF-induced MEF cell migration was decreased by MLCK inhibitor ML-7 (30 µM for PDGF treatment and 60 µM for serum treatment). C, dosage-response curve of MLCK inhibitor ML-7 inhibition of PDGF-induced MEF cell migration assayed by Boyden chamber assay. Data are representative of three experiments.

 
The second unexpected finding reported here is that L-type Ca2+ channels, instead of Rho and Rho kinase, control the contraction of the trailing end of migrating fibroblasts. Rho and Rho kinase have long been suggested to be responsible for the actomyosin contraction at the trailing tail in fast migrating cells such as neutrophils and macrophages (35, 36). Our data clearly demonstrate that Ca2+ influx through L-type Ca2+ channels is responsible for the trailing tail MLC phosphorylation in slow moving fibroblast cells. It is possible that in other slow migrating cell types such as epithelial cells, Ca2+ influx rather than Rho and Rho kinase might be responsible for the rear end contraction. Consistent with this possibility, we have shown that mouse breast tumor cells and human breast tumor cells (both are epithelial origin) were sensitive to Ca2+ influx blockers (Fig. 2).

Through calmodulin and MLCK, Ca2+ influx modulates the phosphorylation of MLC at the trailing tail of migrating cells, and thus the actomyosin contractile force that is required for cell body movement and trailing tail detachment. MLC phosphorylation has also been proposed to control membrane protrusions (41). Furthermore, we have also found that Ca2+/calmodulin-dependent protein kinase II, another direct effector of calmodulin, is also required for PDGF-induced MEF cell migration (data not shown). Indeed, in vascular smooth muscle cells, it was claimed that Ca2+/calmodulin-dependent protein kinase II might be the major mechanism by which Ca2+ modulates cell migration (42). At the present time, it is not clear what the downstream substrate of Ca2+/calmodulin kinase II is in the MEF cell migration process. In addition to this increased cytoskeletal contractility, Ca2+ has also been implicated to induce the disassembly of cell substratum adhesion and to activate gelsolin and other Ca2+-sensitive actin-binding proteins (43, 44).

The connection between PDGF stimulation and the opening of L-type Ca2+ channels remains to be determined. Similar to those in T lymphocytes (40), L-type Ca2+ channels in fibroblast cells retain their voltage dependence (sensitive to membrane depolarization by KCl). However, the opening of these channels in T lymphocytes and fibroblast cells is controlled by receptor signals. In neurons, stimulation of insulin-like growth factor 1 activates L-type Ca2+ channels through a pathway involving phosphatidylinositol 3-kinase, Akt, and tyrosine kinase Src leading to channel protein phosphorylation (45). Whether such a pathway operates in MEF cells is not clear. Additionally, in Xenopus spinal neurons, cAMP and cGMP have been shown to modulate the activity of L-type Ca2+ channels in axonal guidance through protein kinases A and G (19). Moreover, because L-type Ca2+ channels are also directly or indirectly sensitive to mechanical stretching (46), tension could serve as a means to open L-type Ca2+ channels. It has been reported that tension generated in migrating cells could open Ca2+ channels and induce a transient increase in local Ca2+ concentration (16). Noticeably, Ca2+ influx-induced trailing tail phosphorylation of MLC was only observed in the migrating cells under high tension, i.e. elongated migrating cells, whereas in the cells with reduced tension, i.e. fan-shaped migrating cells, the trailing tail staining disappeared. Therefore, because of membrane protrusion at the leading edge and strong adhesion at the rear induced by PDGF, the tension increases during migration. Tension, possibly with other signals initiated from PDGF, activates Ca2+ channels, including L-type Ca2+ channels in MEF cells. When the tension reaches a threshold, the trailing tail detaches from the substratum and retracts. Consequently, the decrease in membrane tension leads to the closure of Ca2+ channels and the start of the next round of migration turnover. Alternatively, growth factor signals trigger the opening of Ca2+ channels that lead to the generation of tension.



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  		FIG. 6.
Role of Ca2+ influx in phosphorylating MLC at the trailing end of migrating fibroblasts. A, staining of phosphorylated MLC and actin polymers in elongated migrating MEF cells in the presence of serum. There were two areas of phosphorylated-MLC staining in polarized migrating cells (indicated by arrowheads), postlamellipodia staining and trailing tail staining. This distribution of phosphorylated MLC was also assessed by determining the ratio of phosphorylated MLC to total proteins (as measured by Cy5 NHS). B, staining of phosphorylated MLC and actin polymers in fan-shaped MEF cells. In these fan-shaped cells, only postlamellipodia staining of phosphorylated-MLC was observed. C, staining of phosphorylated MLC and actin polymers in Ni2+-treated elongated MEF cells in the presence of serum. The strong postlamellipodia staining of phosphorylated-MLC remained, whereas no trailing tail staining was observed. D, ML-7 (50 µM) treatment reduced the staining of phosphorylated MLC at the rear end but had no effect on the staining at the postlamellipodia. Data are representative of three experiments.

 
In migrating MEF cells, there are two areas with strong staining of phosphorylated MLC, post-lamellipodia and trailing tail. A similar bimodal staining pattern of phosphorylated MLC was observed in motile REF-2A fibroblast cells (47). We have shown that Ca2+ influx-activated MLCK is responsible for the phosphorylation of MLC at the trailing tail, without an effect on the phosphorylation of MLC at the postlamellipodia. Then what is responsible for phosphorylating MLC at the postlamellipodia? Surprisingly, we found that inhibition of Rho kinase blocked the phosphorylation of MLC at the post-lamellipodia, without affecting the phosphorylation of MLC at the trailing tail.2 As we showed in Fig. 5A, Rho kinase inhibitor Y-27632 actually increased MEF cell migration. These results indicate that the phosphorylation of MLC at the postlamellipodia does not play an essential role for MEF cell migration. It is possible that the phosphorylation of MLC at the postlamellipodia functions in focal adhesion formation (41). During cell migration, these focal adhesions need to be formed and detached repeatedly. Therefore, Rho kinase inhibitors might enhance this focal adhesion cycle (possibly through the activation of myosin phosphatase), thus leading to a slight increase of cell migration (41). Focal adhesion turnover is not solely controlled by MLC phosphorylation because abolishment of phosphorylation of MLC (by Rho kinase inhibitors) at the postlamellipodia did not block cell migration.

The fact that inhibition of Ca2+ influx blocked both PDGF- and serum-induced cell migration indicates that Ca2+ influx plays a general and essential role for cell migration. Most importantly, we have shown here that blocking Ca2+ influx inhibited the migration of tumor cells (Fig. 2, G and H). Therefore, inhibitors of Ca2+ influx should be explored as blockers of tumor metastasis in cancer therapies.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 212-746-6362; Fax: 212-746-8690; E-mail: xyhuang{at}med.cornell.edu.

1 The abbreviations used are: PDGF, platelet-derived growth factor; MEF, mouse embryonic fibroblast; MLC, myosin light chain; MLCK, MLC kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BAPTA-AM, 1,2-bis [aminophenoxy] ethane-N,N,N',N'-tetraacetic acid; PLC, phospholipase C. Back

2 S. Yang and X.-Y. Huang, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Olaf Andersen, Deirdre McGarrigle, Larry Palmer, and Xian Zhou for reading the manuscript and G. Carpenter for PLC{gamma}1-/- and PLC{gamma}1-/-+PLC{gamma}1 MEF cells.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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