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Am J Physiol Gastrointest Liver Physiol 293: G1272-G1280, 2007. First published October 18, 2007; doi:10.1152/ajpgi.00134.2007
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LIVER AND BILIARY TRACT

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Andrew C. Melton, Russell K. Soon, Jr., J. Genevieve Park, Luis Martinez, Gregory W. deHart, and Hal F. Yee, Jr.

Department of Medicine and Liver Center, University of California San Francisco, San Francisco, California

Submitted 22 March 2007 ; accepted in final form 15 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.

platelet-derived growth factor; migration; microtubules; myosin light chain kinase; fibronectin

HEPATIC STELLATE CELLS, the liver's resident pericyte, mediate the development of liver fibrosis (2, 13, 30). Following exposure to inflammatory cytokines and changes in the extracellular milieu, both in vivo and in vitro, stellate cells secrete type I collagen and other matrix proteins (16, 36, 38). Under normal wound healing conditions this matrix functions as a basement membrane for hepatocyte replenishment and is degraded when wound repair is complete (3). In contrast, chronic injury to the liver results in the accumulation of matrix and can lead to fibrosis, which is characterized by impaired blood flow to hepatocytes, abnormal hepatic architecture, and portal hypertension (5, 21). A requisite step in the response of stellate cells to injury, and the subsequent development of fibrosis, is the migration of these cells to areas of inflammation (13). Therefore, the signaling pathways that regulate stellate cell migration have been a subject of extensive investigation.

Several studies demonstrate that inflammatory mediators released during hepatic injury, such as platelet-derived growth factor-BB (PDGF), monocyte chemoattractant protein 1, and angiotensin II, function as chemokines that induce stellate cell migration (1, 15, 20). PDGF, the most potent stimulus of stellate cell movement, activates Ras, Rho, focal adhesion kinase (FAK), ERK, mitogen-activated protein (MAP) kinase, and PI3-kinase signaling proteins, and controls myosin regulatory light chain phosphorylation in a distinct spatial and temporal manner (6, 23, 26, 35). Since these signaling proteins are thought to regulate focal adhesions in other cell types, it has been proposed that induction of stellate cell migration by PDGF could be due to its effects on focal adhesion turnover. However, the role of focal adhesion turnover during stellate cell migration in response to PDGF is currently unknown.

Focal adhesions are large protein complexes that contain over 32 structural and signaling components, such as integrins, vinculin, talin, paxillin, FAK, PI3-kinase, Src kinases, MAP kinases, and Rho GTPases (24, 41). By linking intracellular cytoskeleton components with extracellular matrix they function to attach cells to their surrounding environment (40). We recently developed a live cell imaging technique specifically designed to examine chemotaxis (i.e., the directed migration of cells toward a chemoattractant) in stellate cells, which differ from most motile cells in that they exhibit a star-shaped morphology with elongate protrusions. To test the hypothesis that focal adhesion turnover is an essential early event for protrusion-mediated chemotaxis in stellate cells, we characterized the role of focal adhesion turnover during stellate cell migration toward PDGF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and fibronectin coating. Hepatic stellate cells were isolated from Sprague-Dawley rats as previously described (12). Fibronectin-coated substrates were made by adding fibronectin (diluted in PBS to the appropriate concentration, Sigma) directly to glass coverslips, dishes, or 96-well plates for 3 h at 37°C. Coverslips, dishes, or 96-well plates were then washed three times with PBS and incubated for 1 h at 37°C in growth media prior to the addition of cells.

Cell staining and measurement of adhesion fluorescence. Hepatic stellate cells were fixed, stained, and imaged as previously described with the following exception (23). To visualize focal adhesions, a monoclonal antibody directed against vinculin (clone hVIN-1, Sigma-Aldrich, St. Louis, MO) was used at a 1:400 dilution. Images of stellate cells stained with vinculin were acquired by using identical filter, microscope, and camera settings. Adhesion fluorescence was determined by using a graphics tablet (Graphire, Wacom, Vancouver, WA) and the trace region function within Metamorph (Universal Imaging, Downingtown, PA) to create a line around the tip of the protrusion. Before placement of a bead (i.e., time point 0), an area ~20 µm2 at the tip of the protrusion was chosen for study because this is the size of the area likely to come into contact with a PDGF-coated bead. Because the protrusion shape changes when the cell moves toward the bead, we evaluated a slightly larger region (~30 µm2) that included the area directly under the bead and nearby adhesions. Metamorph was used to calculate the fluorescence of the region and the fluorescence of a background region of the same size adjacent to the protrusion. After subtracting the background fluorescence signal, we determined adhesion fluorescence by dividing the protrusion fluorescence by the surface area of the region.

Stellate cell chemotaxis assay and protrusion retraction measurement. The stellate cell chemotaxis assay and protrusion retraction measurements were performed as previously described with the following exception (23). In certain experiments, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride; Sigma] or nocodazole (Sigma) were added directly to the cells at the time points indicated. Briefly, stellate cells in day 2 of primary culture were washed with HEPES-buffered serum-free media and placed into a 37°C chamber on an inverted microscope (Olympus). PDGF-coated beads (20-µm diameter) were then added directly to cells and a time-lapse series of images was acquired with a cooled charge-coupled device camera (Photometrics). Protrusion retraction was determined by using Metamorph software to measure the distance between the tip of the protrusion and its original location at the indicated time points.

Measurement of nonstimulated movement. To measure movement of stellate cells on fibronectin and glass in the absence of PDGF stimulation, we selected solitary cells that were not touching PDGF-coated beads from the chemotaxis time-lapse experiments described above. Time-lapse images of these cells were first processed by using the dilate filter within Metamorph to reduce the variation of pixel intensities within the cell body. A binary mask of the cell body region was then created and used to track the location of the cell body throughout the time-lapse series. The total distance traveled and velocity of the cell body was computed by using the track objects function within Metamorph. Values presented are means ± SE.

Adhesion assay. Following isolation, stellate cells in growth medium were added to a 96-well plate coated with varying concentrations of fibronectin and allowed to adhere for 1 h at 37°C in a humidified 5% CO2 incubator. In select experiments the H-Gly-Arg-Gly-Asp-Ser-Pro-OH peptide (RGD, 100 µg/ml, Calbiochem, La Jolla, CA) was added with the cells. Nonadherent cells were removed by washing the wells twice with warm PBS. The adherent cells in each well were treated with 100 µl of 50 mM sodium acetate (pH 5.0), 0.4% Triton X-100, and 3 mg/ml phosphatase substrate (nitrophenylphosphate, Sigma) for 16 h at 37°C (33). The phosphatase reaction was stopped by adding 50 µl of 1 N NaOH to each well, and the absorbance of the resulting solution was measured at 405 nm with a plate reader (BioTek, Winooski, VT). Data are presented as the absorbance from cells added to fibronectin-coated wells divided by the absorbance of cells added to noncoated wells and multiplied by 100.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PDGF stimulates the disassembly and reassembly of focal adhesions at the tips of stellate cell protrusions. To investigate the role of focal adhesion turnover, we employed a new method for directly visualizing stellate cell chemotaxis (23). As seen in Figure 1, in this assay placement of a PDGF-coated bead at the tip of a protrusion induced a series of morphological changes: 1) stellate cell protrusion spreading around a PDGF-coated bead, 2) translocation of the cell body toward the bead, and 3) retraction of trailing protrusions that define a chemotactic response (Fig. 1A and supplemental movie 1). Focal adhesion turnover during chemotaxis was examined by fixing and staining stellate cells for focal adhesions at 0, 6.25, 12.5, 25, 37.5, and 50 min after protrusion contact with a PDGF-coated bead. Prior to stimulation with PDGF, stellate cells exhibited characteristic focal adhesions within their protrusions when stained with an antibody directed against vinculin, an essential structural component of the adhesion complex (8) (Fig. 1B). Focal adhesions measured 3.7 ± 0.4 µm long by 1.9 ± 0.3 µm wide (n = 17 adhesions selected at random from the protrusions of 5 cells) and localized to the termini of actin stress fibers. Focal adhesions disappeared at the site of bead contact ~6.25 min after placement of a PDGF-coated bead at the tip of a protrusion (Fig. 1B). When the number and size of focal adhesions present at this site was determined by measuring the amount of fluorescent staining, adhesion fluorescence was reduced by 32 ± 7% (Fig. 1C). Focal adhesions inside the protrusion contacting the PDGF-coated bead returned ~12.5 min after placement of a PDGF-coated bead at the tip of a protrusion and were more numerous than those present prior to stimulation. When adhesion fluorescence was measured, we observed a 29 ± 8% increase above baseline levels (Fig. 1, B and C). Adhesions within the leading protrusion continued to assemble in concert with the spreading protrusion and adhesion fluorescence remained elevated until the completion of chemotaxis (Fig. 1, B and C). In contrast, adhesions within protrusions not in contact with a PDGF-coated bead did not change within the first 12.5 min of chemotaxis (Fig. 1D). Beginning at 15 min, trailing protrusions began to retract with coincident disappearance of adhesions (data not shown). Our observation that PDGF at the tip of a protrusion induces an early disassembly of focal adhesions that is followed by the rapid reappearance and growth of adhesions suggests that local focal adhesion turnover participates in stellate cell chemotaxis.


Figure 1
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  		Fig. 1. PDGF-coated beads placed at the tips of stellate cell protrusions induce chemotaxis and focal adhesion turnover. A: representative experiment in which a PDGF-coated bead was placed at the tip of a protrusion on a stellate cell in serum-free media. Phase-contrast images correspond to the time intervals after placement of the bead. White arrowhead indicates the bead in contact with the protrusion. Scale bar = 40 µm. B: stellate cells were fixed and stained for F-actin with rhodamine-phalloidin (top) and an antibody directed against vinculin (middle) at the indicated times after placement of a PDGF-coated bead at the tip of a protrusion. Images for each time point were taken from the same cell. The position of the bead is shown on the F-actin images ({circ}). Bottom: a magnified image of vinculin staining (vinculin mag) within the protrusion contacting the bead. Scale bar = 50 µm. C: time course for adhesion fluorescence within protrusion tips at the site of the PDGF-coated bead (n = 5 cells at each time point, P < 0.05 compared with values at 0 min). D: time course for adhesion fluorescence within protrusion tips not contacting a PDGF-coated bead in the same cells as in C. A.U. = arbitrary fluorescence units.

 
Stimulation of focal adhesion assembly with fibronectin prevents protrusion-mediated chemotaxis. To test whether focal adhesion turnover is required for protrusion-mediated chemotaxis, we modulated rates of focal adhesion disassembly and reassembly by growing cells on fibronectin. Fibronectin is an extracellular matrix component that promotes focal adhesion formation through engagement of {alpha}5β1 integrins on the cell surface (25). We verified that fibronectin engages integrins on stellate cells by comparing the ability of cells to adhere on fibronectin or plastic. Fibronectin (10 µg/ml) stimulated stellate cell adherence by 59 ± 12% compared with cells plated on plastic (Fig. 2A). To ensure that increased adherence was due to a fibronectin-integrin interaction, we employed a soluble RGD peptide to act as a competitive antagonist of fibronectin by mimicking the recognition site of {alpha}5β1 integrins (25). Indeed, adherence on fibronectin (10 µg/ml) was reduced by 46 ± 3% with RGD peptide (Fig. 2A), suggesting that integrins on stellate cells can be engaged by growing cells on fibronectin.


Figure 2
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  		Fig. 2. Fibronectin stimulates stellate cell adherence and focal adhesion assembly in protrusions. A: stellate cells plated onto varying concentrations of fibronectin (fn) in the absence (bullet) or presence of H-Gly-Arg-Gly-Asp-Ser-Pro-OH (RGD peptide, 100 µg/ml, {blacksquare}). Results are presented as the percentage of cells that adhered 1 h after plating relative to cells added to noncoated wells (n = 11). B: fluorescent images of representative stellate cells grown on glass (control), fibronectin (10 µg/ml), or fibronectin (10 µg/ml) + RGD peptide (100 µg/ml) and stained with an antibody directed against vinculin. Arrowheads indicate focal adhesions at the tips of protrusions. Scale bar = 40 µm. C: quantification of adhesion fluorescence in the protrusions of cells grown on glass, fibronectin (10 µg/ml), or fibronectin (10 µg/ml) + RGD peptide (100 µg/ml). P < 0.05 for adhesion fluorescence measured in cells on fibronectin compared with control cells and for cells on fibronectin + RGD peptide compared with cells on fibronectin (n = 5).

 
To determine whether stimulation of integrin engagement promotes focal adhesion assembly, we grew stellate cells on fibronectin and measured the size of adhesions. Growth of cells on fibronectin increased the size of focal adhesions at the tips of protrusions and elevated adhesion fluorescence at these sites by 72 ± 8% (Fig. 2, B and C). Focal adhesion growth was prevented following the addition of RGD peptide, indicating that the attachment of cells to fibronectin by integrins was responsible for stimulating focal adhesion size (Fig. 2, B and C). These data, together with the observation that fibronectin stimulates stellate cell adherence, indicate that fibronectin stabilizes focal adhesions and therefore impairs the ability of focal adhesions to turn over during chemotaxis.

To determine the effects of stabilizing focal adhesions on protrusion-mediated chemotaxis, we placed PDGF-coated beads at the tips of protrusions and measured the chemotactic response. Cells grown on fibronectin failed to show any signs of chemotaxis after 50 min of PDGF-coated bead contact with the tip of a protrusion (Fig. 3A and supplemental movie 2). However, movement toward PDGF-beads was initiated when RGD peptide was added to the media 45 min after bead placement (Fig. 3B and supplemental movie 3). Addition of RGD peptide triggered nearly all of the morphological changes associated with chemotaxis, including lamella formation around the PDGF-coated bead and movement of the cell body in the direction of the bead. These data suggest that blocking focal adhesion disassembly prevents stellate cell chemotaxis.


Figure 3
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  		Fig. 3. Growth of stellate cells on fibronectin prevents chemotaxis toward PDGF-coated beads. A: representative experiment in which a PDGF-coated bead was placed at the tip of a protrusion of a stellate cell grown on fibronectin (10 µg/ml). Phase-contrast images correspond to the time intervals after placement of the bead. White arrowhead indicates the bead in contact with the protrusion. Scale bar = 40 µm. B: representative experiment in which PDGF-coated beads were placed at the tips of protrusions on 2 cells grown on fibronectin (10 µg/ml) and treated with RGD peptide 45 min later. Phase-contrast images correspond to the time intervals after placement of the beads. Scale bar = 40 µm.

 
To establish whether fibronectin inhibited chemotaxis by stabilizing adhesions at the site of PDGF detection or impaired the general ability of cells to move, we compared the random movement of stellate cells grown on fibronectin or glass in serum-free media. Using Metamorph computer software to locate and track the centroid (geometric center) of the cell body during 50 min of random movement, we found no difference between the total distance traveled or speed of cell body movement for cells grown on glass or fibronectin (Fig. 4, A and B). Since changes in the shape of stellate cells grown on fibronectin could alter their response to PDGF-coated beads, we measured the morphology of cells grown on fibronectin or glass. We found no difference in stellate cell morphology, as cells grown on glass exhibited 3.9 ± 0.2 protrusions with a length of 48.6 ± 3.9 µm and midwidth of 5.2 ± 0.6 µm (n = 10 cells) compared with cells grown on fibronectin that displayed 4.1 ± 0.2 protrusions with a length of 48.5 ± 4.9 µm and midwidth of 5.2 ± 0.7 µm (n = 10 cells). These results indicate that fibronectin prevents stellate cell chemotaxis through the stabilization of focal adhesions at the site of PDGF detection, rather than by inhibiting the general ability of cells to move or altering cell morphology.


Figure 4
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  		Fig. 4. Growth of stellate cells on fibronectin does not inhibit random cell movement. A: measurement of the total distance traveled by cells grown on glass (control) or fibronectin (10 µg/ml) during 50 min in serum-free media (n = 10). B: measurement of cell body velocity for cells grown on glass (control, {blacklozenge}, n = 10) or fibronectin (10 µg/ml, {square}, n = 10) during 50 min in serum-free media.

 
Nocodazole stabilizes focal adhesions and prevents protrusion-mediated chemotaxis. To further investigate the role of focal adhesion dynamics in stellate cell chemotaxis, we stabilized focal adhesions with the small molecule inhibitor, nocodazole. Nocodazole prevents microtubules from elongating and subsequently triggers the enlargement of focal adhesions (4, 9, 11). Stellate cells treated with nocodazole for 30 min displayed an increase in the size of focal adhesions and demonstrated a 67 ± 7% elevation in adhesion fluorescence at the tips of protrusions (Fig. 5, A and B). When stellate cells were treated with nocodazole at the same time as PDGF-coated bead addition, cells failed to undergo any morphological changes associated with chemotaxis (Fig. 5C and supplemental movie 4). However, nocodazole-treated cells continued to move randomly, as shown by the displacement of the cell body from its starting location after 50 min of treatment (Fig. 5C and supplemental movie 4). This movement was similar to the random movement seen in control cells grown on glass and placed in serum-free medium (Fig. 4A). To ensure that nocodazole was disrupting focal adhesion turnover during chemotaxis and not impairing the general ability of cells to move, we added nocodazole 25 min after PDGF-coated bead placement. When nocodazole was added to cells at this time, cells moved normally toward PDGF-coated beads, with the exception that trailing protrusions did not retract (Fig. 5D and supplemental movie 5). These data suggest that stabilizing focal adhesion dynamics with nocodazole blocks the ability of stellate cells to initiate a chemotactic response toward PDGF-coated beads. Taken together with the observation that cells grown on fibronectin also fail to move toward PDGF-coated beads, these data indicate that focal adhesion turnover is a necessary early event for stellate cell chemotaxis.


Figure 5
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  		Fig. 5. Nocodazole triggers focal adhesion assembly and prevents stellate cell chemotaxis toward PDGF-coated beads. A: fluorescent images from a representative experiment in which stellate cells were treated with carrier (control) or nocodazole (10 µM) for 30 min and then fixed and stained with an antibody directed against vinculin. B: adhesion fluorescence measured in the protrusions of control cells or cells treated with nocodazole (10 µM, n = 16, P < 0.05). C: representative experiment in which a stellate cell grown on glass was treated with nocodazole (10 µM) at the same time as bead placement. Phase-contrast images correspond to the time intervals after placement of the bead. Scale bar = 40 µm. D: representative experiment in which a cell was treated with nocodazole (10 µM) 25 min after PDGF-coated bead placement. Phase-contrast images correspond to the time intervals after placement of the bead. Scale bar = 40 µm.

 
ML-7-induced focal adhesion disassembly augments morphological changes associated with protrusion-mediated chemotaxis. Since inhibition of focal adhesion disassembly blocked chemotaxis, we next tested whether stimulation of focal adhesion turnover could augment morphological changes associated with chemotaxis. To induce focal adhesion turnover, we employed ML-7, a selective small-molecule inhibitor of myosin light chain kinase (17, 37). Our earlier observations (Fig. 1B) indicated that protrusions are attached to their substrate by focal adhesions. Therefore, to test whether ML-7 could trigger focal adhesion disassembly in protrusions we measured protrusion retraction, since the length of protrusions should correlate with the number of adhesions present. ML-7 treatment induced retraction of protrusions within 6 min, and continued to facilitate retraction until most of the protrusion had incorporated into the cell body ~30 min later (Fig. 6, A and B; supplemental movie 6). To ensure that ML-7 was causing protrusions to retract through focal adhesion disassembly, we added nocodazole to stimulate focal adhesion assembly (11). Combined ML-7 and nocodazole treatment completely prevented protrusion retraction induced by ML-7 alone (Fig. 6B). We also fixed and stained cells treated with ML-7 to see whether we could detect a reduction in the size or number of focal adhesions. In accordance with the predicted effect of ML-7, focal adhesions were undetectable in cells treated for 30 min (Fig. 6C). Furthermore, combined ML-7 and nocodazole treatment prevented focal adhesion disassembly induced by ML-7 (Fig. 6C). These data suggest that ML-7 induces focal adhesion turnover by triggering focal adhesion disassembly.


Figure 6
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  		Fig. 6. ML-7 induces protrusion retraction through focal adhesion disassembly. A: representative experiment in which a stellate cell grown on glass was treated with ML-7 (50 µM) 15 min after being placed in serum-free media. Phase-contrast images correspond to the time intervals after the cell was placed into the imaging chamber and first imaged. White arrowheads denote a protrusion undergoing retraction. The background surface appears uneven due to the photoetched alphanumeric grid on the coverslip. Scale bar = 40 µm. B: time course for protrusion retraction in control cells ({blacklozenge}, n = 5) and after addition of ML-7 ({blacksquare}, 50 µM, n = 5) or ML-7 + nocodazole ({circ}, 50 µM ML-7, 10 µM nocodazole, n = 5). The arrow indicates the addition of ML-7 or ML-7 + nocodazole 15 min after the first image was acquired. The distance between the protrusion and its original location at the beginning of the experiment was measured every 2 min as described in MATERIALS AND METHODS. C: fluorescent images from a representative experiment where stellate cells were treated with carrier (control), ML-7 (50 µM), or ML-7 (50 µM) + nocodazole (10 µM) for 30 min and then fixed and stained with an antibody directed against vinculin.

 
If focal adhesion turnover allows stellate cell protrusions to spread toward PDGF-coated beads, it is expected that triggering focal adhesion disassembly after movement has begun would augment morphological changes associated with chemotaxis. To test this possibility, we added ML-7 to cells 15 min after the addition of PDGF-coated beads. ML-7 sped chemotaxis and augmented phenotypic changes associated with movement toward PDGF (Fig. 7, A and B; supplemental movie 7). Trailing protrusions retracted 33.7 ± 2.1 µm in ML-7-treated cells compared with 11.4 ± 2.2 µm in control cells and the protrusion surface area increased to 2,246.2 ± 82.7 µm compared with 1,165.7 ± 53.5 µm in nontreated cells (Fig. 7B). In addition to augmentation of chemotaxis, ML-7 treatment also induced movement of the bead toward the cell, which further suggests that ML-7 acts by disrupting focal adhesions (Fig. 7A and supplemental movie 7). It has been reported that high concentrations of ML-7 also inhibit protein kinase A (Ki = 21 µM, PKA) and protein kinase C (Ki = 42 µM, PKC) in vitro (31). To ensure that the effects of ML-7 were not from inhibition of these proteins, we measured protrusion retraction and chemotaxis with the selective PKA inhibitors H-89 and KT5720 and the selective PKC inhibitors Gö-6976 and Ro-31-8220 (10). None of these reagents induced protrusion retraction or sped migration in response to PDGF-bead stimulation (data not shown), indicating that ML-7 acted specifically on MLCK to trigger focal adhesion disassembly. These results suggest that focal adhesion turnover regulates protrusion spreading toward PDGF-coated beads and that focal adhesion disassembly may be a limiting step after the initiation of chemotaxis in stellate cells.


Figure 7
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  		Fig. 7. ML-7 addition after the initial focal adhesion disassembly and reassembly events augments phenotypic changes associated with chemotaxis toward PDGF. A: representative experiment in which a cell was treated with ML-7 (50 µM) 15 min after PDGF-coated bead placement. Phase-contrast images correspond to the time intervals after placement of the bead. White arrowhead indicates the complete retraction of a trailing protrusion following ML-7 treatment. Scale bar = 40 µm. B: time course for changes in trailing protrusion retraction (bullet, ML-7 treated cells; {circ} = control cells, top) and protrusion surface area ({blacksquare}, ML-7 treated cells; {square}, control cells, bottom) following PDGF-bead contact with the tip of a stellate cell protrusion. ML-7 was added 15 min after placement of the PDGF-bead (black arrow). Trailing protrusion retraction and protrusion surface area were measured every 2 min as described in MATERIALS AND METHODS. Each data point represents the mean ± SE (n = 5 cells).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Following the discovery that hepatic stellate cells are the predominant fibrogenic cell type involved in the development of cirrhosis, many studies have examined signaling pathways that control stellate cell migration (6, 7, 15, 18–20, 23, 34, 42). Using a combination of small molecule inhibitors and protein constructs, these studies demonstrated that Ras, Rho, FAK, ERK, MAP kinase, and PI3-kinase pathways are required for migration of stellate cells. Although the involvement of these pathways pointed toward a key role for focal adhesion turnover in migration, the contribution of adhesions has, until now, remained obscure. This study presents novel evidence indicating that focal adhesions disassemble at the tips of protrusions soon after detecting a chemoattractant and that this event is required for stellate cell chemotaxis. This finding is supported by the following observations: 1) the tips of stellate cell protrusions contained focal adhesions that resemble those described in other cell types, both in size and anatomic location at the ends of stress fibers (14, 32); 2) placement of a localized source of PDGF, a potent chemoattractant for stellate cells (15, 19), at the tips of protrusions rapidly induced focal adhesion disassembly and subsequent reassembly; 3) stabilization of adhesion turnover with fibronectin or nocodazole prevented membrane spreading and the initiation of chemotaxis toward localized sources of PDGF; and 4) induction of adhesion turnover with ML-7 triggered protrusion retraction and augmented morphological changes associated with chemotaxis. These results suggest that focal adhesion disassembly at the tips of protrusions is a necessary early event in stellate cell migration.

Focal adhesions have been proposed to play a critical role in stellate cell migration on the basis of studies showing that inhibition of pathways known to regulate adhesion turnover in other cell types also slow or prevent migration in stellate cells (6, 7, 28, 34). However, many of these pathways, such as those involving Ras, Rho, FAK, ERK, MAP kinase, and PI3-kinase, also control processes independent of stellate cell migration (19, 22, 26). For instance, PDGF stimulation of the PI3-kinase-Akt pathway triggers proliferation and type I collagen secretion (28). The secretion of collagen following exposure to PDGF could speed migration since stellate cells grown on collagen are known to migrate faster that those grown on glass or plastic (6, 42). Therefore, to elucidate the effects of PDGF specific to focal adhesion turnover we employed a recently developed technique that allows stellate cell migration to be examined with high spatial and temporal resolution (23). Using this single-cell migration assay, we found that local stimulation with PDGF at the tips of stellate cell protrusions induced rapid disassembly and subsequent reassembly of focal adhesions. Moreover, the initial disassembly of adhesions was required for the commencement of migration.

In most cell types, migration begins with the formation of a leading edge at the site of chemoattractant detection and focal adhesion disassembly is not believed to be a required early event (29, 39). The leading edge is created through actin polymerization and branching, which spreads the membrane into a thin structure termed a lamellipodia (27). Next, nascent focal adhesions form to stabilize the lamellipodia by attaching it to the substrate (29). Finally, preexisting focal adhesions disassemble at the leading edge only after the new adhesions have formed (39). Although this model faithfully reconstructs the early events in the migration of many cell types, several observations in this study suggest that focal adhesion disassembly must occur before lamellipodia formation in stellate cells. First, we observed that addition of PDGF-coated beads triggers a rapid disassembly of focal adhesions at the site of PDGF detection that was not observed in adjacent or trailing protrusions. Second, growing stellate cells on fibronectin at a concentration where adhesion growth is stimulated, but random movement is not impaired, prevented lamellipodia formation and the initiation of movement. Third, treatment with nocodazole, which prevents adhesion disassembly by inducing adhesion formation, also blocked PDGF migration. Taken together, these findings strongly suggest that focal adhesions at the tips of protrusions must disassemble before lamellipodia formation can occur.

Disparities between our observations here and those seen in other cell types could reflect differences in how cells with diverse shapes detect chemoattractants. Whereas most cells used to study migration are circular or spindle-shaped and detect chemoattractants at sites on or near the cell body, stellate cells detect chemoattractants distant from their cell body on elongate protrusions (23, 29). Therefore, the conversion of a stellate cell protrusion from a detecting structure to the leading edge of a migrating cell could involve unique signaling mechanisms and may present a specific target for blocking stellate cell migration in vivo.

Apart from this study, the regulation of focal adhesion disassembly has not been examined in stellate cells and is only beginning to be understood in other cell types. Several studies using fibroblasts indicate that microtubules and FAK are required for focal adhesion disassembly (11, 17). In support of this concept, we observed that nocodazole induced focal adhesion assembly within stellate cell protrusions and prevented migration when added at the initiation of chemotaxis. Preliminary observations from our laboratory have shown that stellate cells expressing the Y397F-FAK mutant fail to form protrusions, suggesting that FAK is an important regulator of adhesions within protrusions (A. Melton and H. Yee, unpublished observation). Although the signals transduced by PDGF to trigger focal adhesion disassembly in stellate cell protrusions await future study, several pathways that regulate adhesion assembly have been examined.

Based on the findings in this study and the work of our laboratory and others, we propose the following model for the early events of stellate cell chemotaxis. First, chemoattractant detection by the tip of a protrusion triggers focal adhesion disassembly through local regulation of microtubule dynamics and FAK. Next, actin polymerization drives lamellipodia formation and rho GTPase-mediated myosin-II activation produces the tension required for focal adhesion assembly. Nascent focal adhesions stabilize the lamellipodia and provide anchored sites for the production of force to pull the cell body forward. Finally, myosin-actin contraction between adhesions at the leading edge and the cell body continues until the cell body has translocated into the spread protrusion and the trailing protrusions have retracted. This model clarifies the role of focal adhesions in stellate cell migration and suggests that focal adhesion disassembly may present a unique target to selectively interrupt stellate cell migration during the development of fibrosis.


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H. F. Yee was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) R01 DK61532 and the William and Mary Ann Rice Memorial Distinguished Professorship. A. C. Melton was supported by a Hefni Fellowship from the Technical Training Foundation. This work was also supported by the Cell and Tissue Biology Core Facility of the University of California San Francisco Liver Center (NIDDK R01 P30 DK26743).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. F. Yee, Jr., San Francisco General Hospital, Bldg. 40, Rm. 4102, 1001 Potrero Ave., San Francisco, CA 94110 (e-mail: hyee{at}medsfgh.ucsf.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.


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  1. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, Qian T, Schoonhoven R, Hagedorn CH, Lemasters JJ, Brenner DA. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest 112: 1383–1394, 2003.[CrossRef][Web of Science][Medline]
  2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 115: 209–218, 2005.[CrossRef][Web of Science][Medline]
  3. Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 46: 443–446, 2000.[Free Full Text]
  4. Bershadsky A, Chausovsky A, Becker E, Lyubimova A, Geiger B. Involvement of microtubules in the control of adhesion-dependent signal transduction. Curr Biol 6: 1279–1289, 1996.[CrossRef][Web of Science][Medline]
  5. Bissell DM. Chronic liver injury, TGF-beta, and cancer. Exp Mol Med 33: 179–190, 2001.[Web of Science][Medline]
  6. Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini P. Focal adhesion kinase and phospholipase C gamma involvement in adhesion and migration of human hepatic stellate cells. Gastroenterology 112: 522–531, 1997.[CrossRef][Web of Science][Medline]
  7. Carloni V, Pinzani M, Giusti S, Romanelli RG, Parola M, Bellomo G, Failli P, Hamilton AD, Sebti SM, Laffi G, Gentilini P. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology 31: 131–140, 2000.[CrossRef][Web of Science][Medline]
  8. Critchley DR. Focal adhesions—the cytoskeletal connection. Curr Opin Cell Biol 12: 133–139, 2000.[CrossRef][Web of Science][Medline]
  9. Danowski BA. Fibroblast contractility and actin organization are stimulated by microtubule inhibitors. J Cell Sci 93: 255–266, 1989.[Abstract/Free Full Text]
  10. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][Web of Science][Medline]
  11. Ezratty EJ, Partridge MA, Gundersen GG. Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat Cell Biol 7: 581–590, 2005.[CrossRef][Web of Science][Medline]
  12. Friedman SL. Isolation and culture of hepatic nonparenchymal cells. Methods Toxicol 1A: 292–310, 1993.
  13. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 275: 2247–2250, 2000.[Free Full Text]
  14. Goffin JM, Pittet P, Csucs G, Lussi JW, Meister JJ, Hinz B. Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol 172: 259–268, 2006.[Abstract/Free Full Text]
  15. Ikeda K, Wakahara T, Wang YQ, Kadoya H, Kawada N, Kaneda K. In vitro migratory potential of rat quiescent hepatic stellate cells and its augmentation by cell activation. Hepatology 29: 1760–1767, 1999.[CrossRef][Web of Science][Medline]
  16. Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 117: 539–548, 2007.[CrossRef][Web of Science][Medline]
  17. Kaverina I, Krylyshkina O, Small JV. Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J Cell Biol 146: 1033–1044, 1999.[Abstract/Free Full Text]
  18. Kinnman N, Hultcrantz R, Barbu V, Rey C, Wendum D, Poupon R, Housset C. PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury. Lab Invest 80: 697–707, 2000.[Web of Science][Medline]
  19. Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H, Dianzani MU, Laffi G, Abboud HE, Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology 112: 1297–1306, 1997.[CrossRef][Web of Science][Medline]
  20. Marra F, Romanelli RG, Giannini C, Failli P, Pastacaldi S, Arrighi MC, Pinzani M, Laffi G, Montalto P, Gentilini P. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology 29: 140–148, 1999.[CrossRef][Web of Science][Medline]
  21. Marra F, Pinzani M. Role of hepatic stellate cells in the pathogenesis of portal hypertension. Nefrologia 22, Suppl 5: 34–40, 2002.[Web of Science][Medline]
  22. Melton AC, Datta A, Yee HF Jr. [Ca2+]i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1. Am J Physiol Gastrointest Liver Physiol 290: G7–G13, 2006.[Abstract/Free Full Text]
  23. Melton AC, Yee HF. Hepatic stellate cell protrusions couple platelet-derived growth factor-BB to chemotaxis. Hepatology 45: 1446–1453, 2007.[CrossRef][Web of Science][Medline]
  24. Miyamoto S, Akiyama SK, Yamada KM. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267: 883–885, 1995.[Abstract/Free Full Text]
  25. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309: 30–33, 1984.[CrossRef][Medline]
  26. Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci 7: d1720–d1726, 2002.[Web of Science][Medline]
  27. Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453–465, 2003.[CrossRef][Web of Science][Medline]
  28. Reif S, Lang A, Lindquist JN, Yata Y, Gabele E, Scanga A, Brenner DA, Rippe RA. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem 278: 8083–8090, 2003.[Abstract/Free Full Text]
  29. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science 302: 1704–1709, 2003.[Abstract/Free Full Text]
  30. Rockey DC. Vascular mediators in the injured liver. Hepatology 37: 4–12, 2003.[CrossRef][Web of Science][Medline]
  31. Saitoh M, Ishikawa T, Matsushima S, Naka M, Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem 262: 7796–7801, 1987.[Abstract/Free Full Text]
  32. Sastry SK, Burridge K. Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp Cell Res 261: 25–36, 2000.[CrossRef][Web of Science][Medline]
  33. Schwartz MA, Denninghoff K. Alpha v integrins mediate the rise in intracellular calcium in endothelial cells on fibronectin even though they play a minor role in adhesion. J Biol Chem 269: 11133–11137, 1994.[Abstract/Free Full Text]
  34. Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through rho-kinase-mediated alterations in the acto-myosin cytoskeleton. Hepatology 33: 74–80, 2001.[CrossRef][Web of Science][Medline]
  35. Tangkijvanich P, Santiskulvong C, Melton AC, Rozengurt E, Yee HF Jr. p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts. J Cell Physiol 191: 351–361, 2002.[CrossRef][Web of Science][Medline]
  36. Tangkijvanich P, Yee HF. Cirrhosis—can we reverse hepatic fibrosis? Eur J Surg Suppl 587: 100–112, 2002.[Medline]
  37. Thomas MG, Santa Coloma TA, Correale J, Boccacci GL. Myosin light chain kinase inhibitors induce retraction of mature oligodendrocyte processes. Neurochem Res 27: 1305–1312, 2002.[CrossRef][Web of Science][Medline]
  38. Tsukamoto H. Cytokine regulation of hepatic stellate cells in liver fibrosis. Alcohol Clin Exp Res 23: 911–916, 1999.[CrossRef][Web of Science][Medline]
  39. Webb DJ, Parsons JT, Horwitz AF. Adhesion assembly, disassembly and turnover in migrating cells—over and over and over again. Nat Cell Biol 4: E97–E100, 2002.[CrossRef][Web of Science][Medline]
  40. Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta 1692: 103–119, 2004.[Medline]
  41. Yamada KM, Miyamoto S. Integrin transmembrane signaling and cytoskeletal control. Curr Opin Cell Biol 7: 681–689, 1995.[CrossRef][Web of Science][Medline]
  42. Yang C, Zeisberg M, Mosterman B, Sudhakar A, Yerramalla U, Holthaus K, Xu L, Eng F, Afdhal N, Kalluri R. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology 124: 147–159, 2003.[CrossRef][Web of Science][Medline]




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