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First published online 8 April 2008
doi: 10.1242/jcs.021683

Journal of Cell Science 121, 1393-1402 (2008)
Published by The Company of Biologists 2008
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Fibroblast migration is mediated by CD44-dependent TGFβ activation

Pinak S. Acharya1,2,*, Sonali Majumdar2,*, Michele Jacob2, James Hayden2, Paul Mrass2, Wolfgang Weninger2, Richard K. Assoian3 and Ellen Puré2,4,{ddagger}

1 Department of Pulmonary and Critical Care Medicine, The University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA
2 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
3 Department of Pharmacology, The University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104, USA
4 Ludwig Institute of Cancer Research, 3601 Spruce Street, Philadelphia, PA 19104, USA


Figure 1
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  		Fig. 1. Altered cytoskeleton and decreased focal adhesion complex formation in CD44-deficient fibroblasts compared with wild-type fibroblasts. (A) Primary fibroblasts stained with phalloidin reveal that CD44WT cells have increased stress fibers compared with CD44KO fibroblasts. Scale bar: 50 µm. (B) Quantification of fluorescent intensity across the lines shown in the corresponding panels in A, indicative of stress fiber density, using Image Pro software. Asterisks demarcate cells being quantified in A and their corresponding line graphs in B. (C) Cells were grown to subconfluence on glass coverslips and stained for total F-actin (Rhodamine-phalloidin) and vinculin. Overlay images were obtained where yellow fluorescence represents focal adhesions in CD44WT (top panel) and CD44KO (bottom panel) fibroblasts. Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. CD44-deficient fibroblasts exhibit less directional migration despite an increased migrational velocity. Cells were grown in 10% FBS and wounded. (A) Efficiency of wound closure in CD44WT (left top) vs CD44KO (right top). Cells (10 CD44KO and 10 CD44WT) were tracked using ImagePro 5.0 software (lower images). Individual tracks represent direction of cell migration as determined by live microscopy. Arrows represent position of cells at zero timepoint. Scale bar: 500 µm. (B) Analysis of 10 representative CD44KO and CD44WT cells and their positional y-coordinates. Convergence of the tracks towards the same y-coordinate indicates that cells have closed the gap. Similarly, as a cell exhibits directional migration, the y-coordinate track is linear, whereas a directionless path reveals a `zig-zag' pattern. (C) Average cell velocity calculated over the course of wound closure. Error bars represent s.e.m. (*P<0.0001). These experiments were repeated three times for each group with similar results.

 

Figure 3
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  		Fig. 3. Reconstitution of CD44KO cells with CD44 rescues the migrational phenotype. CD44KO fibroblasts were transfected with recombinant CD44 (pRC/pCMV {Delta}1230) using nucleofection. (A) Following reconstitution, cells were stained with anti-CD44 (mAb) (left panel), and phalloidin (middle panel). Merge of CD44 and phalloidin stains (right panel). Arrow indicates CD44KO fibroblast and asterisk a reconstituted (CD44KO.CD44) fibroblast. Scale bar: 50 µm. (B) Reconstitution with CD44 (CD44KO.CD44) decreases the mean velocity. Error bars represent s.e.m. (P<0.002). (C) Measure of instantaneous velocity in CD44WT, CD44KO and reconstituted cells (CD44KO.CD44). (D) Measure of the confinement ratio (distance between start and end point divided by total length of track) in CD44WT, CD44KO and reconstituted (CD44KO.CD44) cells. A ratio of 1.0 indicates that the cell is moving in a straight line. Data in B, C and D are representative of one experiment performed three times with similar results.

 

Figure 4
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  		Fig. 4. CD44 mediates the activation of TGFβ in murine adult fibroblasts in response to wounding. CD44KO fibroblasts generate reduced levels of active TGFβ in response to wounding compared with CD44WT fibroblasts. Levels of total and active TGFβ in conditioned media from unwounded and wounded CD44WT and CD44KO fibroblasts were determined after 24 hours, in the presence or absence of anti-TGFβ antibody (5 µg/ml). Values were normalized to total protein concentration. Cell viability was comparable between CD44WT and CD44KO fibroblasts under each condition. (A) Levels of active (top) and total (bottom) TGFβ (pg/ml) produced in conditioned media under each condition, as measured in three independent experiments. Within each experiment, the conditions were done in triplicate and also assayed in triplicate. Error bars represent s.e.m. (B) CD44KO fibroblasts exhibit lower levels of nuclear Smad2-P (pSmad2). Immunoblot analysis and corresponding quantification of Smad2-P of nuclear lysates from CD44WT and CD44KO fibroblasts unwounded, wounded and treated with active TGFβ1 (0.02 nM), extracted after 1 hour. Immunoblot analysis was done with anti-Smad2-P (Ser465/467) and anti-Rb (loading control). Immunoblot is representative of one experiment performed three times with similar results. Corresponding quantification is an average of the three experiments normalized to the loading control. Error bars represent s.e.m.

 

Figure 5
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  		Fig. 5. MMP-dependent activation of TGFβ in response to wounding in CD44WT fibroblasts. Levels of total and active TGFβ in conditioned media from unwounded and wounded CD44WT and CD44KO fibroblasts, treated with or without the MMP inhibitor GM6001 were determined after 24 hours. (A) CD44WT fibroblasts, unwounded vs wounded, and in the absence or presence of GM6001 (1 µM). (B) CD44KO fibroblasts, unwounded vs wounded, and in the absence or presence of GM6001 (1 µM). Values were normalized to protein concentration. Cell viability and total levels of TGFβ were comparable between CD44WT and CD44KO fibroblasts under each condition. Data are presented as an average of the percentage of active TGFβ (active TGFβ divided by total TGFβ x 100), as measured in three independent experiments. Within each experiment, the conditions were applied in triplicate and also assayed in triplicate. Error bars represent s.e.m.

 

Figure 6
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  		Fig. 6. Active TGFβ partially rescues the phenotype of CD44KO fibroblasts. (A) CD44KO fibroblasts were grown on coverslips and treated with either latent or active TGFβ1 (0.02 nM) for 24 hours, then stained for F-actin and compared with untreated CD44KO and WT fibroblasts. Scale bar: 40 µm. (B) Quantification of fluorescent intensity across the lines shown in the corresponding panels in A (above), to indicate stress fiber density, using Image Pro software. Asterisks demarcate cells being quantified in A and their corresponding line graphs in B. (C) Instantaneous velocity of CD44WT, CD44KO and CD44KO cells treated with active TGFβ. (D) Measure of the confinement ratio (distance between start and end point divided by total length of track) in CD44WT, CD44KO and CD44KO cells treated with active TGFβ. A ratio of 1.0 indicates that the cell is moving in a straight line. Data in A, C and D are representative of one experiment performed three times with similar results.

 

Figure 7
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  		Fig. 7. Anti-TGFβ antibody reduces and alters stress fiber architecture in CD44WT fibroblasts. (A) CD44WT and CD44KO fibroblasts were grown on coverslips and treated with anti-TGFβ antibody (5 µg/ml) for 24 hours, then stained for F-actin and compared with untreated CD44WT and CD44KO fibroblasts. Scale bar: 20 µm. (B) Quantification of fluorescent intensity across the lines shown in the corresponding panels in A (above), to indicate stress fiber density, using Image Pro software. In the CD44KO and CD44KO + anti-TGFβ panels, two different cells are being quantified in each. Asterisks and triangular shapes demarcate cells being quantified in A and their corresponding line graphs in B. Data are representative of one experiment performed three times with similar results.

 

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© The Company of Biologists Ltd 2008